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B?cell translocation 1 gene inhibits cellular metastasis?associated behavior in breast cancer.
B?cell translocation geneĀ1 (BTG1) is a member of the BTG/transducer of ERBB2 family, which regulates cell cycle progression in a variety of cell types and may have a role in inhibiting proliferation, promoting apoptosis and stimulating cellular differentiation in numerous cell types. However, the role of BTG1 in cancer metastasis is yet to be elucidated. In the present study, analysis of clinical specimens revealed that BTG1 mRNA levels were lower in lymph node metastases than those in benign breast tumors and normal human breast tissue. The effect of BTG1 on the metastatic behavior of breast cancer cells following stable transfection with a BTG1 expression vector was also investigated. The overexpression of BTG1 was observed to inhibit cell adhesion, migration and invasion. Furthermore, the overexpression of BTG1 was found to be involved in the inhibition of the metastasis?related proteins matrix metalloproteinase?2 and ?9, as well as the promotion of the cell?cell adhesion?associated protein E?cadherin. In syngeneic nude mice breast tumor models, hepatic metastasis and angiogenesis were observed in the mice injected with the control cells, but not in those injected with pcDNA3?BTG1 cells. Immunohistochemistry revealed that overexpression of BTG1 decreased vascular endothelial growth factor expression in tumors. To the best of our knowledge, this is the first study to show that BTG1 overexpression decreases migration and invasion of breast cancer cells and thereby inhibits distant metastasis in mice breast tumor models. PMID:24714932
Li, Wei; Zou, Shi-Tao; Zhu, Ran; Wan, Jian-Mei; Xu, Yan; Wu, Hao-Rong
Topoisomerase inhibitors modulate gene expression of B-cell translocation gene 2 and prostate specific antigen in prostate carcinoma cells.
Camptothecin (CPT) and doxorubicin (DOX) have been demonstrated to have potent anti-tumor activity. The B-cell translocation gene 2 (BTG2) is involved in the regulation of cell cycle progression. We evaluated the molecular mechanisms of CPT and DOX on cell proliferation and the expressions of BTG2 and prostate specific antigen (PSA) in prostate carcinoma cells. Our results indicated that CPT or DOX treatments induced Go/G1 cell cycle arrest in LNCaP cells and apoptosis at higher dosage. Immunoblot and transient gene expression assay indicated that CPT or DOX treatments induced p53 and BTG2 gene expression, with the later effect dependent on the p53 response element within BTG2 promoter area since mutation of the p53 response element from GGGAAAGTCC to GGAGTCC or from GGCAGAGCCC to GGCACC by site-directed mutagenesis abolished the stimulation of CPT or DOX on the BTG2 promoter activity, which is also supported by our results that cotreatments of pifithrin-?, an inhibitor of p53 dependent transcriptional activation, blocked the induction of CPT or DOX on BTG2 gene expression. CPT or DOX also downregulated the protein expressions of androgen receptor (AR) and PSA. Transient gene expression assays suggested that CPT or DOX's attenuation of PSA promoter activity is dependent on both the androgen and p53 response elements within of the PSA promoter. Our results indicated that CPT and DOX attenuate cell proliferation via upregulation of BTG2 gene expression through the p53-dependent pathway. The CPT and DOX block the PSA gene expression by upregulation of p53 activity and downregulation of androgen receptor activity. PMID:24586533
Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Chang, Phei-Lang; Chen, Wen-Tsung; Juang, Horng-Heng
Loss of B-cell translocation gene 2 expression in estrogen receptor-positive breast cancer predicts tamoxifen resistance.
B-cell translocation gene 2 (BTG2), a gene suppressed in a subset of aggressive breast cancer, is repressed by estrogen. BTG2 inhibits the expression of HER ligands and promotes AKT activation, which plays an essential role in the tamoxifen resistance of estrogen receptor (ER)-positive breast cancer. To determine if BTG2 expression modifies tamoxifen efficacy, a cohort of 60 patients treated with adjuvant tamoxifen monotherapy was analyzed. We found that increased BTG2 expression showed better clinical survival and was the only independent prognostic factor for disease-free survival (hazard ratio, 0.691; 95% confidence interval, 0.495-0.963; PĀ=Ā0.029). Tamoxifen suppressed the human epidermal growth factor receptor 2 (HER2)-Akt signaling in BTG2 expressing ER-positive breast cancer cells with a correlated increase in sensitivity, whereas BTG2 knockdown abrogated this sensitivity. Consistent with this observation, tamoxifen significantly suppressed the growth ratio, tumor weight and Ki-67 expression in BTG2 expressing breast cancer xenografts in mice. These studies demonstrate that BTG2 is a significant factor in tamoxifen response, acting through modification of AKT activation in ER-positive/HER2-negative breast cancer. PMID:24698107
Takahashi, Maiko; Hayashida, Tetsu; Okazaki, Hiroshi; Miyao, Kazuhiro; Jinno, Hiromitsu; Kitagawa, Yuko
B cell translocation gene 2 enhances susceptibility of HeLa cells to doxorubicin-induced oxidative damage.
BTG2/TIS21/PC3 (B cell translocation gene 2) has been known as a p53 target gene and functions as a tumor suppressor in carcinogenesis of thymus, prostate, kidney, and liver. Although it has been known that the expression of BTG2/TIS21/PC3 is induced during chemotherapy-mediated apoptosis in cancer cells, a role of BTG2/TIS21/PC3 in cell death remains to be elucidated. In this study, the mechanism and role of BTG2 involved in the enhancement of doxorubicin (DOXO)-induced cell death were examined. Treatment of HeLa cells with DOXO revealed apoptotic phenomena, such as chromatin condensation and cleavage of poly(ADP-ribose) polymerase and lamin A/C with concomitant increase of BTG2/TIS21/PC3 expression. Employing infections of Ad-TIS21 virus and lentivirus with short hairpin RNA to BTG2, the effect of BTG2/TIS21/PC3 on the DOXO-induced apoptosis of HeLa cells and liver cancer cells was evaluated. Not only short hairpin RNA-BTG2 but also N-acetyl-L-cysteine significantly reduced the DOXO-induced HeLa cell death and generation of H2O2. Moreover, forced expression of BTG2/TIS21/PC3 using adenoviral vector augmented DOXO-induced cancer cell death concomitantly with increase of manganese-superoxide dismutase but not catalase, CuZnSOD, and glutathione peroxidase 1. The increased apoptosis by forced expression of BTG2/TIS21/PC3 could be inhibited by N-acetyl-L-cysteine and polyethylene glycol-catalase. These results therefore suggest that BTG2/TIS21/PC3 works as an enhancer of DOXO-induced cell death via accumulation of H2O2 by up-regulating manganese-superoxide dismutase without any other antioxidant enzymes. In summary, BTG2/TIS21/PC3 enhances cancer cell death by accumulating H2O2 via imbalance of the antioxidant enzymes in response to chemotherapy. PMID:18840609
Lim, Young-Bin; Park, Tae Jun; Lim, In Kyoung
1q12 chromosome translocations form aberrant heterochromatic foci associated with changes in nuclear architecture and gene expression in B cell lymphoma.
Epigenetic perturbations are increasingly described in cancer cells where they are thought to contribute to deregulated gene expression and genome instability. Here, we report the first evidence that a distinct category of chromosomal translocations observed in human tumours--those targeting 1q12 satellite DNA--can directly mediate such perturbations by promoting the formation of aberrant heterochromatic foci (aHCF). By detailed investigations of a 1q12 translocation to chromosome 2p, in a case of human B cell lymphoma, aberrant aHCF were shown to be localized to the nuclear periphery and to arise as a consequence of long range 'pairing' between the translocated 1q12 and chromosome 2 centromeric regions. Remarkably, adjacent 2p sequences showed increased levels of repressive histone modifications, including H4K20me3 and H3K9me3, and were bound by HP1. aHCF were associated to aberrant spatial localization and deregulated expression of a novel 2p gene (GMCL1) that was found to have prognostic impact in diffuse large B cell lymphoma. Thus constitutive heterochromatin rearrangements can contribute to tumourigenesis by perturbing gene expression via long range epigenetic mechanisms. PMID:20432501
Fournier, Alexandra; McLeer-Florin, Anne; Lefebvre, Christine; Duley, Samuel; Barki, Leila; Ribeyron, Juliana; Alboukadel, Kassambara; Hamaidia, Sieme; Granjon, Aurķlie; Gressin, Rķmy; Lajmanovich, Alicia; Bonnefoix, Thierry; Chauvelier, Stķphanie; Debernardi, Alexandra; Rousseaux, Sophie; de Fraipont, Florence; Figeac, Martin; Kerckaert, Jean-Pierre; De Vos, John; Usson, Yves; Delaval, Katia; Grichine, Alexei; Vourc'h, Claire; Khochbin, Saadi; Feil, Robert; Leroux, Dominique; Callanan, Mary B
1q12 chromosome translocations form aberrant heterochromatic foci associated with changes in nuclear architecture and gene expression in B cell lymphoma
Epigenetic perturbations are increasingly described in cancer cells where they are thought to contribute to deregulated gene expression and genome instability. Here, we report the first evidence that a distinct category of chromosomal translocations observed in human tumoursŚthose targeting 1q12 satellite DNAŚcan directly mediate such perturbations by promoting the formation of aberrant heterochromatic foci (aHCF). By detailed investigations of a 1q12 translocation to chromosome 2p, in a case of human B cell lymphoma, aberrant aHCF were shown to be localized to the nuclear periphery and to arise as a consequence of long range æpairingÆ between the translocated 1q12 and chromosome 2 centromeric regions. Remarkably, adjacent 2p sequences showed increased levels of repressive histone modifications, including H4K20me3 and H3K9me3, and were bound by HP1. aHCF were associated to aberrant spatial localization and deregulated expression of a novel 2p gene (GMCL1) that was found to have prognostic impact in diffuse large B cell lymphoma. Thus constitutive heterochromatin rearrangements can contribute to tumourigenesis by perturbing gene expression via long range epigenetic mechanisms.
Fournier, Alexandra; McLeer-Florin, Anne; Lefebvre, Christine; Duley, Samuel; Barki, Leila; Ribeyron, Juliana; Kassambara, Alboukadel; Hamaidia, Sieme; Granjon, Aurelie; Gressin, Remy; Lajmanovich, Alicia; Bonnefoix, Thierry; Chauvelier, Stephanie; Debernardi, Alexandra; Rousseaux, Sophie; de Fraipont, Florence; Figeac, Martin; Kerckaert, Jean-Pierre; De Vos, John; Usson, Yves; Delaval, Katia; Grichine, Alexei; Vourc'h, Claire; Khochbin, Saadi; Feil, Robert; Leroux, Dominique; Callanan, Mary B
B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells*
The B-cell translocation gene 2, BTG2, a member of the BTG/TOB (B-cell translocation gene/transducers of ErbB2) gene family, has been implicated in cell cycle regulation, normal development, and possibly tumor suppression. Previously, it was shown that BTG2 expression is lost or down-regulated in human breast cancers. We now report that BTG2 protects human mammary epithelial cells from oxidative stress due to hydrogen peroxide and other oxidants. BTG2 protection against oxidative stress is BRCA1-independent but requires the antioxidant transcription factor NFE2L2 and is associated with up-regulation of the expression of antioxidant enzymes, including catalase and superoxide dismutases 1 and 2. BTG2 stimulation of antioxidant gene expression is also NFE2L2-dependent. We further demonstrate that BTG2 is a binding partner for NFE2L2 and increases its transcriptional activity. In addition, BTG2 is detectable at the antioxidant response element (ARE) of several NFE2L2-responsive genes. Finally, we show that the ability of BTG2 to associate with NFE2L2, to protect cells against oxidative stress, and to stimulate antioxidant gene expression requires box B, a short highly conserved amino acid motif characteristic of BTG2/TOB family proteins, but does not require boxes A or C. These findings suggest a novel role for BTG2 as a co-activator for NFE2L2 in up-regulating cellular antioxidant defenses.
Karve, Tejaswita M.; Rosen, Eliot M.
Myc translocations in B cell and plasma cell neoplasms.
Chromosomal translocations that join the cellular oncogene Myc (c-myc) with immunoglobulin (Ig) heavy-chain (Igh) or light-chain (Igk, Igl) loci are widely believed to be the crucial initiating oncogenic events in the development of B cell and plasma cell neoplasms in three mammalian species: Burkitt lymphoma (BL) in human beings, plasmacytoma (PCT) in mice, and immunocytoma in rats. Among the Myc-Ig translocations found in these neoplasms, mouse PCT T(12;15)(Igh-Myc) is of special interest because it affords a uniquely useful model system to study the fundamental outstanding questions on the mechanisms, genetics, and biological consequences of Myc translocations. Mouse T(12;15) is the direct counterpart of the human BL t(8;14)(q24;q32) translocation and thus of great relevance for human cancer. Mouse T(12;15) is the only cancer-associated translocation in mice that occurs with high incidence, spontaneity, and cell-type specificity. Due to the development of PCR methods for the detection of the underlying reciprocal Myc-Igh junction fragments, it is now known that mouse T(12;15) can be a dynamic process that begins with the genetic exchange of Myc and the Igh switch mu region (Smu), progresses by class switch recombination (CSR) just 3' of the translocation break site, and then undergoes further clonal diversification by micro-deletions in the junction flanks. The molecular pathway that subverts CSR to mediate trans-chromosomal joining of Myc and Smu (translocation origin) and secondary modification of Myc-Igh junctions (translocation "remodeling") has not been elucidated, but recent evidence indicates that it includes CSR factors, such as the activation-induced cytidine deaminase (AID), that may also be involved in the ongoing neoplastic progression of the translocation-bearing tumor precursor. Transgenic mouse models of T(12;15)/t(8;14), including newly developed "iMyc" gene-insertion mice, will be useful in elucidating the role of these CSR factors in the progression of Myc-induced B cell tumors. PMID:16815105
Janz, Siegfried
B-Cell Translocation Gene 2 Regulates Hepatic Glucose Homeostasis via Induction of Orphan Nuclear Receptor Nur77 in Diabetic Mouse Model.
B-cell translocation gene 2 (BTG2) is a member of an emerging gene family that is involved in cellular functions. In this study, we demonstrate that BTG2 regulates glucose homeostasis via upregulation of Nur77 in diabetic mice. Hepatic BTG2 gene expression was elevated by fasting and forskolin. Overexpression of Btg2 increased the expression of hepatic gluconeogenic genes and blood glucose output and subsequently impaired glucose and insulin tolerance. Upregulation of the transcriptional activity of Nur77, gluconeogenic genes, and glucose production by forskolin was observed by Btg2 transduction, but not in Btg2 knockdown. BTG2-stimulated glucose production and glucose-6-phosphatase promoter activity were attenuated by dominant-negative Nur77. Coimmunoprecipitation and chromatin immunoprecipitation assays showed that BTG2 induced Nur77 occupancy on the glucose-6-phosphatase promoter via a physical interaction. Btg2 gene expression was increased in streptozotocin-treated and db/db mice. Finally, impairment of glucose homeostasis, such as the increase of blood glucose, glucose intolerance, and insulin intolerance, was elevated in diabetic mice, whereas this phenomenon was abolished in knockdown of Btg2. Together, these data suggest that BTG2 participates in the regulation of hepatic glucose homeostasis, which means that BTG2 might serve as a potential therapeutic target for combating metabolic dysfunction. PMID:24647738
Kim, Yong Deuk; Kim, Sun-Gyun; Hwang, Seung-Lark; Choi, Hueng-Sik; Bae, Jae-Hoon; Song, Dae-Kyu; Im, Seung-Soon
B-cell translocation gene 2 positively regulates GLP-1-stimulated insulin secretion via induction of PDX-1 in pancreatic ?-cells.
Glucagon-like peptide-1 (GLP-1) is a potent glucoincretin hormone and an important agent for the treatment of type 2 diabetes. Here we demonstrate that B-cell translocation gene 2 (BTG2) is a crucial regulator in GLP-1-induced insulin gene expression and insulin secretion via upregulation of pancreatic duodenal homeobox-1 (PDX-1) in pancreatic ?-cells. GLP-1 treatment significantly increased BTG2, PDX-1 and insulin gene expression in pancreatic ?-cells. Notably, adenovirus-mediated overexpression of BTG2 significantly elevated insulin secretion, as well as insulin and PDX-1 gene expression. Physical interaction studies showed that BTG2 is associated with increased PDX-1 occupancy on the insulin gene promoter via a direct interaction with PDX-1. Exendin-4 (Ex-4), a GLP-1 agonist, and GLP-1 in pancreatic ?-cells increased insulin secretion through the BTG2-PDX-1-insulin pathway, which was blocked by endogenous BTG2 knockdown using a BTG2 small interfering RNA knockdown system. Finally, we revealed that Ex-4 and GLP-1 significantly elevated insulin secretion via upregulation of the BTG2-PDX-1 axis in pancreatic islets, and this phenomenon was abolished by endogenous BTG2 knockdown. Collectively, our current study provides a novel molecular mechanism by which GLP-1 positively regulates insulin gene expression via BTG2, suggesting that BTG2 has a key function in insulin secretion in pancreatic ?-cells. PMID:23703573
Hwang, Seung-Lark; Kwon, Okyun; Kim, Sun-Gyun; Lee, In-Kyu; Kim, Yong Deuk
Differential nuclear organization of translocation-prone genes in nonmalignant B cells from patients with t(14;16) as compared with t(4;14) or t(11;14) myeloma.
Gene organization in nonmalignant B cells from t(4;14) and t(11;14) multiple myeloma (MM) patients differs from that of healthy donors. Among recurrent IGH translocations in MM, the frequency of t(4;14) (IGH and FGFR3) or t(11;14) (IGH and CCND1) is greater than the frequency of t(14;16) (IGH and MAF). Gene organization in t(14;16) patients may influence translocation potential of MAF with IGH. In patients, three-dimensional FISH revealed the positions of IGH, CCND1, FGFR3, and MAF in nonmalignant B cells that are likely similar to those when MM first arose, compared with B cells from healthy donors. Overall, IGH occupies a more central nuclear position while MAF is more peripherally located. However, for B cells from t(4;14) and t(11;14) patients, IGH and FGFR3, or IGH and CCND1 are found in spatial proximity: IGH and MAF are not. This differs in B cells from t(14;16) patients and healthy donors where IGH is approximately equidistant to FGFR3, CCND1, and MAF, suggesting that gene organization in t(14;16) patients is different from that in t(4;14) or t(11;14) patients. Translocations between IGH and MAF may arise only in the absence of close proximity to the more frequent partners, as appears to be the case for individuals who develop t(14;16) MM. PMID:23460268
Martin, Lorri D; Harizanova, Jana; Righolt, Christiaan H; Zhu, George; Mai, Sabine; Belch, Andrew R; Pilarski, Linda M
Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways
Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NF?B response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NF?B pathway.
Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Feng, Tsui-Hsia; Chen, Wen-Tsung; Chang, Phei-Lang; Chiang, Hou-Yu; Juang, Horng-Heng
Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways.
Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NF?B response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NF?B pathway. PMID:24981574
Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Feng, Tsui-Hsia; Chen, Wen-Tsung; Chang, Phei-Lang; Chiang, Hou-Yu; Juang, Horng-Heng
Identification of the gene associated with the recurring chromosomal translocations t(3;14)(q27;q32) and t(3;22)(q27;q11) in B-cell lymphomas.
Chromosomal translocations involving chromosome 3, band q27, are among the most common rearrangements in B-cell non-Hodgkin lymphoma. From a bacteriophage lambda library prepared from a lymphoma characterized by a t(3;14)(q27;q32), genomic clones were isolated using a probe from the immunoglobulin heavy chain locus (IGH) joining region. In addition to clones containing an apparently normal IGH rearrangement, others were found to contain one of the translocation breakpoint junctions. Normal chromosome 3 sequences and the reciprocal breakpoint junction were subsequently isolated. DNA probes on each side of the chromosome 3 breakpoint hybridized at high stringency to the DNA of various mammalian species, demonstrating evolutionary conservation. One such probe from the presumptive der(3) chromosome detected an 11-kilobase transcript when hybridized to RNA of B- and T-cell lines. A probe made from partial cDNA clones isolated from a T-cell line hybridized with genomic DNA from both sides of the chromosome 3 breakpoint, indicating that the t(3;14) is associated with a break within the gene on chromosome 3. In situ chromosomal hybridization revealed that the same gene is involved in the t(3;22)(q27;q11). Preliminary nucleotide sequencing shows no identity of the cDNA to gene sequences in available data banks. We propose the name BCL6 (B-cell lymphoma 6) for this gene, since it is likely to play a role in the pathogenesis of certain B-cell lymphomas. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5
Baron, B W; Nucifora, G; McCabe, N; Espinosa, R; Le Beau, M M; McKeithan, T W
Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy.
Abstract Recent findings have suggested biological classification of B-cell malignancies as exemplified by the "activated B-cell-like" (ABC), the "germinal-center B-cell-like" (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and "recurrent translocation and cyclin D" (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy. PMID:23998255
Johnsen, Hans Erik; Bergkvist, Kim Steve; Schmitz, Alexander; Kjeldsen, Malene Krag; Hansen, Steen M°ller; Gaihede, Michael; N°rgaard, Martin Agge; Bµch, John; Gr°nholdt, Marie-Louise; Jensen, Frank Svendsen; Johansen, Preben; B°dker, Julie St°ve; B°gsted, Martin; Dybkµr, Karen
Insulin Phosphorylates Tyrosine Residue 464 of Tub and Translocates Tubby into the Nucleus in HIRcB Cells
Background The tubby protein has a motif that might be relevant for its action in the insulin signaling pathway. Previous studies have indicated that tubby undergoes phosphorylation on tyrosine residues in response to several stimuli and is known to localize in the nucleus as well as in the plasma membrane. However, the relationship between phosphorylation and nuclear translocation is not well understood. Here, we report that insulin directly phosphorylates tubby, which translocates into the nucleus. Methods The effects of insulin on Tubby were performed with Western blot. The immunoprecipitation and confocal microscopy were performed to prove phosphorylation and nuclear translocation. Results Mutation study reveals that tyrosine residue 464 of tubby gene (Tub) is a phosphorylation site activated by insulin. In addition, major portions of tubby protein in the plasma membrane are translocated into the nucleus after insulin treatment. Tyrosine kinase inhibitor pretreatment blocked insulin-induced tubby translocation, suggesting that phosphorylation is important for nuclear translocation. Moreover, mutant tyrosine residue 464 did not translocate into the nucleus in respond to insulin. These findings demonstrate that insulin phosphorylates tyrosine residue 464 of Tub, and this event is important for insulin-induced tubby nuclear translocation. Conclusion Insulin phosphorylates tyrosine residue 464 of Tub and translocates tubby into the nuclei of HIRcB cells.
Kim, Jin Wook; Kim, Hyeon Soo; Kim, Sang Dae
MALT1is deregulated by both chromosomal translocation and amplification in B-cell non-Hodgkin lymphoma
The MALT1 gene was identified through its involvement in t(11;18)(q21;q21), seen in 30% of cases of mucosa-associated lymphoid tissue (MALT) lymphoma. Here, we show that deregulated MALT1 expres- sion may occur in B-cell non-Hodgkin lymphoma (B-NHL) of various histologic subtypes either through translocation to the immunoglobulin heavy chain (IGH) locus or by genomic amplification. First, 2 cases, one case of
Dolors Sanchez-Izquierdo; Gerard Buchonnet; Reiner Siebert; Randy D. Gascoyne; Joan Climent; Loraine Karran; Miguel Marin; David Blesa; Douglas Horsman; Andreas Rosenwald; Louis M. Staudt; Donna G. Albertson; Ming-Qing Du; Hongtao Ye; Peter Marynen; Javier Garcia-Conde; Daniel Pinkel; Martin J. S. Dyer; Jose Angel; Martinez-Climent
Characterization of IGH locus breakpoints in multiple myeloma indicates a subset of translocations appear to occur in pregerminal center B cells.
Translocations in myeloma are thought to occur solely in mature B cells in the germinal center through class switch recombination (CSR). We used a targeted captured technique followed by massively parallel sequencing to determine the exact breakpoints in both the immunoglobulin heavy chain (IGH) locus and the partner chromosome in 61 presentation multiple myeloma samples. The majority of samples (62%) have a breakpoint within the switch regions upstream of the IGH constant genes and are generated through CSR in a mature B cell. However, the proportion of CSR translocations is not consistent between cytogenetic subgroups. We find that 100% of t(4;14) are CSR-mediated; however, 21% of t(11;14) and 25% of t(14;20) are generated through DH-JH recombination activation gene-mediated mechanisms, indicating they occur earlier in B-cell development at the pro-B-cell stage in the bone marrow. These 2 groups also generate translocations through receptor revision, as determined by the breakpoints and mutation status of the segments used in 10% and 50% of t(11;14) and t(14;20) samples, respectively. The study indicates that in a significant number of cases the translocation-based etiological events underlying myeloma may arise at the pro-B-cell hematological progenitor cell level, much earlier in B-cell development than was previously thought. PMID:23435460
Walker, Brian A; Wardell, Christopher P; Johnson, David C; Kaiser, Martin F; Begum, Dil B; Dahir, Nasrin B; Ross, Fiona M; Davies, Faith E; Gonzalez, David; Morgan, Gareth J
Translocation (8;14)(q24;q32) as the sole cytogenetic abnormality in B-cell prolymphocytic leukemia
B-cell prolymphocytic leukemia is a relatively rare lymphoproliferative disorder. No specific cytogenetic abnormality has yet been associated with it. The most common translocation reported in patients with this disease is t(11;14)(q13;q32). We describe the case of a patient with B-cell prolymphocytic leukemia and a hitherto unreported genetic translocation (8;14)(q24;q32) as the sole genetic abnormality, classically seen in patients with B-cell
Philip Kuriakose; Nusrat Perveen; Koichi Maeda; Anne Wiktor; Daniel L Van Dyke
Biological characterization of adult MYC-translocation-positive mature B-cell lymphomas other than molecular Burkitt lymphoma.
Chromosomal translocations affecting the MYC oncogene are the biological hallmark of Burkitt lymphomas but also occur in a subset of other mature B-cell lymphomas. If accompanied by a chromosomal break targeting the BCL2 and/or BCL6 oncogene these MYC translocation-positive (MYC(+)) lymphomas are called double-hit lymphomas, otherwise the term single-hit lymphomas is applied. In order to characterize the biological features of these MYC(+) lymphomas other than Burkitt lymphoma we explored, after exclusion of molecular Burkitt lymphoma as defined by gene expression profiling, the molecular, pathological and clinical aspects of 80 MYC-translocation-positive lymphomas (31 single-hit, 46 double-hit and 3 MYC(+)-lymphomas with unknown BCL6 status). Comparison of single-hit and double-hit lymphomas revealed no difference in MYC partner (IG/non-IG), genomic complexity, MYC expression or gene expression profile. Double-hit lymphomas more frequently showed a germinal center B-cell-like gene expression profile and had higher IGH and MYC mutation frequencies. Gene expression profiling revealed 130 differentially expressed genes between BCL6(+)/MYC(+) and BCL2(+)/MYC(+) double-hit lymphomas. BCL2(+)/MYC(+) double-hit lymphomas more frequently showed a germinal center B-like gene expression profile. Analysis of all lymphomas according to MYC partner (IG/non-IG) revealed no substantial differences. In this series of lymphomas, in which immunochemotherapy was administered in only a minority of cases, single-hit and double-hit lymphomas had a similar poor outcome in contrast to the outcome of molecular Burkitt lymphoma and lymphomas without the MYC break. Our data suggest that, after excluding molecular Burkitt lymphoma and pediatric cases, MYC(+) lymphomas are biologically quite homogeneous with single-hit and double-hit lymphomas as well as IG-MYC and non-IG-MYC(+) lymphomas sharing various molecular characteristics. PMID:24179151
Aukema, Sietse M; Kreuz, Markus; Kohler, Christian W; Rosolowski, Maciej; Hasenclever, Dirk; Hummel, Michael; K³ppers, Ralf; Lenze, Dido; Ott, German; Pott, Christiane; Richter, Julia; Rosenwald, Andreas; Szczepanowski, Monika; Schwaenen, Carsten; Stein, Harald; Trautmann, Heiko; Wessendorf, Swen; Tr³mper, Lorenz; Loeffler, Markus; Spang, Rainer; Kluin, Philip M; Klapper, Wolfram; Siebert, Reiner
Distinctive growth requirements and gene expression patterns distinguish progenitor B cells from pre-B cells
Long-term bone marrow cultures have been useful in determining gene expression patterns in pre-B cells and in the identification of cytokines such as interleukin 7 (IL-7). We have developed a culture system to selectively grow populations of B lineage restricted progenitors (pro-B cells) from murine bone marrow. Pro-B cells do not grow in response to IL-7, Steel locus factor (SLF), or a combination of the two. c-kit, the SLF receptor, and the IL-7 receptor are both expressed by pro-B cells, indicating that the lack of response is not simply due to the absence of receptors. Furthermore, SLF is not necessary for the growth of pro-B cells since they could be expanded on a stromal line derived from Steel mice that produces no SLF. IL-7 responsiveness in pre-B cells is associated with an increase in n-myc expression and is correlated with immunoglobulin (Ig) gene rearrangements. Although members of the ets family of transcription factors and the Pim-1 kinase are expressed by pro-B cells, n-myc is not expressed. Pro-B cells maintain Ig genes in the germline configuration, which is correlated with a low level of recombination activating genes 1 and 2 (Rag-1 and 2) mRNA expression, but high expression of sterile mu and terminal deoxynucleotidyl transferase. Pro-B cells are unable to grow separated from the stromal layer by a porous membrane, indicating that stromal contact is required for growth. These results suggest that pro-B cells are dependent on alternative growth signals derived from bone marrow stroma and can be distinguished from pre-B cells by specific patterns of gene expression.
Developmental propagation of V(D)J recombination-associated DNA breaks and translocations in mature B cells via dicentric chromosomes
Mature IgM+ B-cell lymphomas that arise in certain ataxia telangiectasia-mutated (ATM)-deficient compound mutant mice harbor translocations that fuse V(D)J recombination-initiated IgH double-strand breaks (DSBs) on chromosome 12 to sequences downstream of c-myc on chromosome 15, generating dicentric chromosomes and c-myc amplification via a breakage-fusion-bridge mechanism. As V(D)J recombination DSBs occur in developing progenitor B cells in the bone marrow, we sought to elucidate a mechanism by which such DSBs contribute to oncogenic translocations/amplifications in mature B cells. For this purpose, we applied high-throughput genome-wide translocation sequencing to study the fate of introduced c-myc DSBs in splenic IgM+ B cells stimulated for activation-induced cytidine deaminase (AID)-dependent IgH class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-Mb region downstream of IgH in ATM-deficient, but not wild-type, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. Our findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via recombination-activating gene-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. We propose that dicentrics formed by joining V(D)J recombination¢associated IgH DSBs to DSBs downstream of c-myc in ATM-deficient B lineage cells similarly contribute to c-myc amplification and mature B-cell lymphomas.
Hu, Jiazhi; Tepsuporn, Suprawee; Meyers, Robin M.; Gostissa, Monica; Alt, Frederick W.
Developmental propagation of V(D)J recombination-associated DNA breaks and translocations in mature B cells via dicentric chromosomes.
Mature IgM(+) B-cell lymphomas that arise in certain ataxia telangiectasia-mutated (ATM)-deficient compound mutant mice harbor translocations that fuse V(D)J recombination-initiated IgH double-strand breaks (DSBs) on chromosome 12 to sequences downstream of c-myc on chromosome 15, generating dicentric chromosomes and c-myc amplification via a breakage-fusion-bridge mechanism. As V(D)J recombination DSBs occur in developing progenitor B cells in the bone marrow, we sought to elucidate a mechanism by which such DSBs contribute to oncogenic translocations/amplifications in mature B cells. For this purpose, we applied high-throughput genome-wide translocation sequencing to study the fate of introduced c-myc DSBs in splenic IgM(+) B cells stimulated for activation-induced cytidine deaminase (AID)-dependent IgH class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-Mb region downstream of IgH in ATM-deficient, but not wild-type, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. Our findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via recombination-activating gene-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. We propose that dicentrics formed by joining V(D)J recombination-associated IgH DSBs to DSBs downstream of c-myc in ATM-deficient B lineage cells similarly contribute to c-myc amplification and mature B-cell lymphomas. PMID:24982162
Hu, Jiazhi; Tepsuporn, Suprawee; Meyers, Robin M; Gostissa, Monica; Alt, Frederick W
High resolution copy number analysis of IRF4 translocation-positive diffuse large B-cell and follicular lymphomas.
Translocations affecting chromosome subband 6p25.3 containing the IRF4 gene have been recently described as characteristic alterations in a molecularly distinct subset of germinal center B-cell-derived lymphomas. Secondary changes have yet only been described in few of these lymphomas. Here, we performed array-comparative genomic hybridization and molecular inversion probe microarray analyses on DNA from 12 formalin-fixed paraffin-embedded and two fresh-frozen IRF4 translocation-positive lymphomas, which together with the previously published data on nine cases allowed the extension of copy number analyses to a total of 23 of these lymphomas. All except one case carried chromosomal imbalances, most frequently gains in Xq28, 11q22.3-qter, and 7q32.1-qter and losses in 6q13-16.1, 15q14-22.31, and 17p. No recurrent copy-neutral losses of heterozygosity were observed. TP53 point mutations were detected in three of six cases with loss of 17p. Overall this study unravels a recurrent pattern of secondary genetic alterations in IRF4 translocation-positive lymphomas. PMID:23073988
Salaverria, Itziar; Martin-Guerrero, Idoia; Burkhardt, Birgit; Kreuz, Markus; Zenz, Thorsten; Oschlies, Ilske; Arnold, Norbert; Baudis, Michael; Bens, Susanne; GarcĒa-Orad, Africa; Lisfeld, Jasmin; Schwaenen, Carsten; Szczepanowski, Monika; Wessendorf, Swen; Pfreundschuh, Michael; Tr³mper, Lorenz; Klapper, Wolfram; Siebert, Reiner
Nuclear translocation of B-cell-specific transcription factor, BACH2, modulates ROS mediated cytotoxic responses in mantle cell lymphoma.
BACH2, a B-cell specific transcription factor, plays a critical role in oxidative stress-mediated apoptosis. Bortezomib (Velcade(TM)) is widely used to treat relapsed mantle cell lymphoma (MCL) patients despite varying clinical outcomes. As one of the potential mechanisms of action, bortezomib was reported to elicit endoplasmic reticulum (ER) stress which triggers reactive oxygen species (ROS). In the present study, we investigated the redox-sensitive intracellular mechanism that might play a critical role in bortezomib response in MCL cells. We demonstrated that in MCL cells that are sensitive to bortezomib treatments, BACH2 was translocated to the nucleus in response to bortezomib and induced apoptotic responses through the modulation of anti-oxidative and anti-apoptotic genes. On the other hand, in bortezomib resistant cells, BACH2 expression was confined in the cytoplasm and no suppression of antiapoptotic or antioxidative genes, Nrf2, Gss, CAT, HO-1 and MCL1, was detected. Importantly, levels of BACH2 were significantly higher in bortezomib sensitive MCL patient cells, indicating that BACH2 levels could be an indicator for clinical bortezomib responses. BACH2 translocation to the cytoplasm after phosphorylation was inhibited by PI3K inhibitors and combinatory regimens of bortezomib and PI3K inhibitors sensitized MCL cells to bortezomib. These data suggest that cellular distribution of BACH2 in response to ROS determines the threshold for the induction of apoptosis. Therapies that inhibit BACH2 phosphorylation could be the key for increasing bortezomib cytotoxic response in patients. PMID:23936317
Chen, Zheng; Pittman, Eric F; Romaguera, Jorge; Fayad, Luis; Wang, Michael; Neelapu, Sattva S; McLaughlin, Peter; Kwak, Larry; McCarty, Nami
Nuclear Translocation of B-Cell-Specific Transcription Factor, BACH2, Modulates ROS Mediated Cytotoxic Responses in Mantle Cell Lymphoma
BACH2, a B-cell specific transcription factor, plays a critical role in oxidative stress-mediated apoptosis. Bortezomib (VelcadeTM) is widely used to treat relapsed mantle cell lymphoma (MCL) patients despite varying clinical outcomes. As one of the potential mechanisms of action, bortezomib was reported to elicit endoplasmic reticulum (ER) stress which triggers reactive oxygen species (ROS). In the present study, we investigated the redox-sensitive intracellular mechanism that might play a critical role in bortezomib response in MCL cells. We demonstrated that in MCL cells that are sensitive to bortezomib treatments, BACH2 was translocated to the nucleus in response to bortezomib and induced apoptotic responses through the modulation of anti-oxidative and anti-apoptotic genes. On the other hand, in bortezomib resistant cells, BACH2 expression was confined in the cytoplasm and no suppression of antiapoptotic or antioxidative genes, Nrf2, Gss, CAT, HO-1 and MCL1, was detected. Importantly, levels of BACH2 were significantly higher in bortezomib sensitive MCL patient cells, indicating that BACH2 levels could be an indicator for clinical bortezomib responses. BACH2 translocation to the cytoplasm after phosphorylation was inhibited by PI3K inhibitors and combinatory regimens of bortezomib and PI3K inhibitors sensitized MCL cells to bortezomib. These data suggest that cellular distribution of BACH2 in response to ROS determines the threshold for the induction of apoptosis. Therapies that inhibit BACH2 phosphorylation could be the key for increasing bortezomib cytotoxic response in patients.
Chen, Zheng; Pittman, Eric F.; Romaguera, Jorge; Fayad, Luis; Wang, Michael; Neelapu, Sattva S.; Mclaughlin, Peter; Kwak, Larry; McCarty, Nami
Interleukin 6 gene expression in normal and neoplastic B cells.
In the present report we demonstrate that the IL-6 gene is expressed in anti-Ig-activated and neoplastic B cells. After activation with anti-Ig, splenic B cells rapidly expressed IL-6 mRNA with peak expression occurring at 4 h and declining rapidly thereafter. In an attempt to exclude that the IL-6 mRNA expression was in non-B cells, T cells and monocytes were extensively depleted. In this highly purified B cell population, IL-6 mRNA was retained, whereas the expression of the T cell- and monocyte-restricted CD2 and CD14 genes was nearly undetectable. These results are consistent with the conclusion that activated B cells express IL-6 mRNA. Because we found IL-6 mRNA expression in normal activated B lymphocytes, we examined the expression of IL-6 mRNA in B cell neoplasms. 11 of 25 non-Hodgkins B cell lymphomas and 4 of 4 myelomas and plasma cell leukemias expressed IL-6 mRNA, whereas only 1 of 19 B cell leukemias was positive. To exclude that IL-6 mRNA expression in neoplastic B cells was the result of contaminating non-B cells, T cells and monocytes were extensively depleted from the tumor specimens. In the three IL-6-positive tumor samples depleted of T cells and monocytes, IL-6 mRNA expression was retained in all cases. These observations provide support for the idea that the IL-6 gene is expressed in normal activated and neoplastic B cells. Images
Freeman, G J; Freedman, A S; Rabinowe, S N; Segil, J M; Horowitz, J; Rosen, K; Whitman, J F; Nadler, L M
Transitional B cells Exhibit a BCR-specific Nuclear Defect In Gene Transcription
The signaling programs that enforce negative selection in early transitional (T1) B cells in response to B cell receptor (BCR) engagement remain poorly defined. We carried out a comprehensive comparison of BCR signaling in T1 vs. follicular mature (FM) splenic B cells. T1, in contrast to FM B cells, failed to express key NF-?B target genes in response to BCR engagement; and exhibited a striking defect in assembly of an active transcriptional complex at the promoter of the survival and proliferative genes, A1 and c-Myc. Surprisingly, and contrary to previous models, classical PKC and IKK activation, NF-?B nuclear translocation and DNA binding were intact in T1 B cells. Further, despite a marked reduction in NFAT1 expression, differential NFAT or AP-1 activation cannot explain this transcriptional defect. Our combined findings demonstrate that T1 B cells are programmed for signal- and stage-specific ænuclear non-responsivenessÆ upon encounter with self-antigens.
Andrews, Sarah F; Rawlings, David J
DNA repair genes are selectively mutated in diffuse large B cell lymphomas.
DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis. PMID:23960188
de Miranda, Noel F C C; Peng, Roujun; Georgiou, Konstantinos; Wu, Chenglin; Falk S÷rqvist, Elin; Berglund, Mattias; Chen, Longyun; Gao, Zhibo; Lagerstedt, Kristina; Lisboa, Susana; Roos, Fredrik; van Wezel, Tom; Teixeira, Manuel R; Rosenquist, Richard; Sundstr÷m, Christer; Enblad, Gunilla; Nilsson, Mats; Zeng, Yixin; Kipling, David; Pan-Hammarstr÷m, Qiang
Identification of translocation products but not K-RAS mutations in memory B cells from patients with multiple myeloma
Background Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells. Design and Methods The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells. Results Cells with the phenotype of tumor plasma cells (CD38++CD19?CD45?/+CD56?/+/++) or memory B cells (CD38?/CD19+/CD27+) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes (FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K-RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K-RAS mutations were identified. Conclusions The increased expression of a specific ōearlyö oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack ōlateö oncogene (K-RAS) mutations but express the ōearlyö oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.
Rasmussen, Thomas; Haaber, Jacob; Dahl, Inger Marie; Knudsen, Lene M.; Kerndrup, Gitte B.; Lodahl, Marianne; Johnsen, Hans E.; Kuehl, Michael
The t(14;18) Chromosome Translocations Involved in B-Cell Neoplasms Result from Mistakes in VDJ Joining
In this study, the joining sequences between chromosomes 14 and 18 on the 14q+ chromosomes of a patient with pre-B-cell leukemia and four patients with follicular lymphoma carrying a t(14;18) chromosome translocation were analyzed. In each case, the involved segment of chromosome 18 has recombined with the immunoglobulin heavy-chain joining segment (JH) on chromosome 14. The sites of the recombination
Yoshihide Tsujimoto; James Gorham; Jeffrey Cossman; Elaine Jaffe; Carlo M. Croce
A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavy-chain genes in B cell tumors
Several methods have been developed for the detection of minimal residual disease (MRD) in B cell tumors. Chromosomal translocations or the rearrangement of the immunoglobulin heavy chain (IgH) and T cell receptor genes are generally employed. We report a novel PCR method to detect MRD using IgH genes. IgH rearranged variable region (VDJ) were amplified from tumor specimens using consensus
C Voena; M Ladetto; M Astolfi; D Provan; JG Gribben; M Boccadoro; A Pileri; P Corradini
A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation.
The biologic and pathologic features of B-cell malignancies bearing a translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 are still poorly described. Herein we report the results of a comprehensive cytogenetic, fluorescence in situ hybridization (FISH), molecular and histopathological survey of a large series of B-cell malignancies with t(14;19) or variant translocations. A total of 56 B-cell malignancies with a FISH-proven BCL3 involvement were identified with the translocation partners being IGH (n=51), IGL (n=2), IGK (n=2) and a non-IG locus (n=1). Hierarchical clustering of chromosomal changes associated with the t(14;19) indicated the presence of two different groups of IG/BCL3-positive lymphatic neoplasias. The first group included 26 B-cell malignancies of various histologic subtypes containing a relatively high number of chromosomal changes and mostly mutated IgVH genes. This cluster displayed three cytogenetic branches, one with rearrangements in 7q, another with deletions in 17p and a third one with rearrangements in 1q and deletions in 6q and 13q. The second group included 19 cases, mostly diagnosed as B-cell chronic lymphocytic leukemia (B-CLL), and characterized by few additional chromosomal changes (e.g. trisomy 12) and unmutated IgVH genes. In conclusion, our study indicates that BCL3 translocations are not restricted to B-CLL but present in a heterogeneous group of B-cell malignancies. PMID:17495977
MartĒn-Subero, J I; Ibbotson, R; Klapper, W; Michaux, L; Callet-Bauchu, E; Berger, F; Calasanz, M J; De Wolf-Peeters, C; Dyer, M J; Felman, P; Gardiner, A; Gascoyne, R D; Gesk, S; Harder, L; Horsman, D E; Kneba, M; K³ppers, R; Majid, A; Parry-Jones, N; Ritgen, M; Salido, M; Solķ, F; Thiel, G; Wacker, H-H; Oscier, D; Wlodarska, I; Siebert, R
Cloning of bcl-6, the Locus Involved in Chromosome Translocations Affecting Band 3q27 in B-Cell Lymphoma1
Chromosomal translocations involving band 3q27 and various chromo somal sites, including the sites of the immunoglobulin (Ig) loci (14q32, 2pl2, 22qll), represent recurrent aberrations in non-Hodgkin's lym- phoma (NHL). In order to identify the putative protooncogene involved in these translocations, we have cloned the breakpoints from two B-cell NHL cases carrying t(3;14)(q27;q32) translocations by screening genomic DNA libraries constructed from
Bihui H. Ye; P. H. Rao; R. S. K. Chaganti; Riccardo Dalla-Favera
Gene Translocations in Musculoskeletal Neoplasms
Establishing the best diagnosis for musculoskeletal neoplasms requires a multidisciplinary approach using clinical, radiographic, and histologic analyses. Despite this rigorous approach, establishing accurate diagnoses and prognoses remains challenging. Improved diagnostic methods are expected as unique molecular signals for specific bone and soft tissue cancers are identified. We performed a systematic review of the best available evidence to explore three major applications of molecular genetics that will best benefit clinical management of musculoskeletal neoplasms: diagnostic, prognostic, and therapeutic applications. The specific questions addressed in this systematic review are: (1) What sets of histopathologic sarcoma subtypes will benefit from molecular evaluation and diagnosis? (2) What molecular methods are best applied to histopathologic sarcomas to distinguish between major subtypes? (3) How do the molecular patterns discovered on genetic diagnosis affect prognosis of certain sarcomas? (4) Which sarcoma translocations can benefit from an improved response and outcome using existing and forthcoming pharmacogenetic approaches targeting molecular events? This review summarizes recent advances in molecular genetics that are available and will soon be available to clinicians to better predict outcomes and subsequently help make future treatment decisions. Level of Evidence: Level IV, diagnostic study. See the Guidelines for Authors for a complete description of levels of evidence.
Krishnan, Balaji; Khanna, Gaurav
Extensive Gene-Specific Translational Reprogramming in a Model of B Cell Differentiation and Abl-Dependent Transformation
To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.
Bates, Jamie G.; Salzman, Julia; May, Damon; Garcia, Patty B.; Hogan, Gregory J.; McIntosh, Martin; Schlissel, Mark S.; Brown, Pat O.
Molecular Cloning of the Chromosomal Breakpoint of B-Cell Lymphomas and Leukemias with the t(11;14) Chromosome Translocation
The chromosomal breakpoint of chronic lymphocytic leukemia (CLL) cells of the B-cell type carrying the translocated long arms of chromosomes 11 and 14 [t(11;14) (q13;q32)] was cloned. The breakpoint was found to be within the joining segment of the human heavy chain locus on the translocated long arm of chromosome 14. A probe that is specific for chromosome 11 and
Yoshihide Tsujimoto; Jorge Yunis; Louise Onorato-Showe; Jan Erikson; Peter C. Nowell; Carlo M. Croce
Patients with diffuse large B-cell lymphoma of germinal center origin with BCL2 translocations have poor outcome, irrespective of MYC status: a report from an International DLBCL rituximab-CHOP Consortium Program Study
Diffuse large B-cell lymphoma can be classified by gene expression profiling into germinal center and activated B-cell subtypes with different prognoses after rituximab-CHOP. The importance of previously recognized prognostic markers, such as Bcl-2 protein expression and BCL2 gene abnormalities, has been questioned in the new therapeutic era. We analyzed Bcl-2 protein expression, and BCL2 and MYC gene abnormalities by interphase fluorescence in situ hybridization in 327 patients with de novo disease treated with rituximab-CHOP. Isolated BCL2 and MYC rearrangements were not predictive of outcome in our patients as a whole, but only in those with the germinal center subtype of lymphoma. The prognostic relevance of isolated MYC rearrangements was weaker than that of BCL2 isolated translocations, but was probably limited by the rarity of the rearrangements. Seven of eight patients with double hit lymphoma had the germinal center subtype with poor outcome. The germinal center subtype patients with isolated BCL2 translocations had significantly worse outcome than the patients without BCL2 rearrangements (P=0.0002), and their outcome was similar to that of patients with the activated B-cell subtype (P=0.30), but not as bad as the outcome of patients with double hit lymphoma (P<0.0001). Bcl-2 protein overexpression was associated with inferior outcome in patients with germinal center subtype lymphoma, but multivariate analysis showed that this was dependent on BCL2 translocations. The gene expression profiling of patients with BCL2 rearrangements was unique, showing activation of pathways that were silent in the negative counterpart. BCL2 translocated germinal center subtype patients have worse prognosis after rituximab-CHOP, irrespective of MYC status, but the presence of combined gene breaks significantly overcomes the prognostic relevance of isolated lesions.
Visco, Carlo; Tzankov, Alexander; Xu-Monette, Zijun Y.; Miranda, Roberto N.; Tai, Yu Chuan; Li, Yan; Liu, Wei-min; d'Amore, Emanuele S. G.; Li, Yong; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Wang, Huan-You; Dunphy, Cherie H.; His, Eric D.; Zhao, X. Frank; Choi, William WL.; Zhao, Xiaoying; van Krieken, J. Han; Huang, Qin; Ai, Weiyun; O'Neill, Stacey; Ponzoni, Maurilio; Ferreri, Andres JM.; Kahl, Brad S.; Winter, Jane N.; Go, Ronald S.; Dirnhofer, Stephan; Piris, Miguel A.; M?ller, Michael B.; Wu, Lin; Medeiros, L. Jeffrey; Young, Ken H.
The pre-B-cell receptor associated protein VpreB3 is a useful diagnostic marker for identifying c-MYC translocated lymphomas
Background During B-cell development, precursor B cells transiently express the pre-B-cell receptor composed of ? heavy chain complexed with VpreB and ?5 surrogate light chain polypeptides. Recent profiling studies unexpectedly revealed abundant transcripts of one member of the VpreB family, VpreB3, in a subset of mature B cells and Burkitt lymphoma. Design and Methods Here we used a novel antibody to investigate the normal expression pattern of VpreB3 protein in human hematopoietic and lymphoid tissues, and to determine whether VpreB3 could serve as a useful diagnostic biomarker for select B-cell lymphomas. Results We found that VpreB3 protein is normally expressed by precursor B cells in bone marrow and by a subset of normal germinal center B cells in secondary lymphoid organs. Among lymphoid malignancies, we found an association between VpreB3 expression and B-cell tumors with c-MYC abnormalities. VpreB3 was highly expressed in all cases of Burkitt lymphoma, whether of endemic or sporadic origin (44/44 cases, 100%), all cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5/5 cases, 100%), and the majority of diffuse large B-cell lymphomas harboring a c-MYC translocation (15/18 cases, 83%). The expression of VpreB3 in diffuse large B-cell lymphomas without a c-MYC translocation was associated with c-MYC polysomy in 25/75 cases (33%) but only rarely observed in diffuse large B-cell lymphomas lacking a c-MYC abnormality (9/98 cases, 9%). Conclusions We conclude that for B-cell tumors with features suggesting a possible c-MYC translocation, such as intermediate to large cell size and high proliferation rate, the presence of VpreB3 should prompt subsequent confirmatory genetic testing, whereas the absence of VpreB3 is virtually always associated with wild-type c-MYC alleles.
Rodig, Scott J.; Kutok, Jeffery L.; Paterson, Jennifer C.; Nitta, Hiroaki; Zhang, Wenjun; Chapuy, Bjoern; Tumwine, Lynette K.; Montes-Moreno, Santiago; Agostinelli, Claudio; Johnson, Nathalie A.; Ben-Neriah, Susana; Farinha, Pedro; Shipp, Margaret A.; Piris, Miguel A.; Grogan, Thomas M.; Pileri, Stefano A.; Gascoyne, Randy D.; Marafioti, Teresa
Expression of the J Chain Gene during B Cell Differentiation is Inversely Correlated with DNA Methylation
During B cell differentiation, transcription of the J chain gene is initiated. To determine the regulatory mechanism involved, we have analyzed the structure of the J chain gene in cell lines representing successive stages in B cell development. Comparison of restriction sites showed that the J chain gene does not require a rearrangement of DNA for expression; cleavage sites present
Mayumi Yagi; Marian Elliott Koshland
Immunoglobulin enhancer and promoter motifs 5' of the B29 B-cell-specific gene.
B29 is a B-cell-specific member of the immunoglobulin gene superfamily that is expressed throughout B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy-chain gene segment rearrangements. We have analyzed the region upstream of the B29 gene to identify DNA sequences involved in transcriptional regulation of this gene. The B29 gene lacks a TATA box and transcription is initiated at multiple sites. The B29 gene sequence 5' of these transcription start sites contains six promoter and enhancer motifs known to control immunoglobulin gene transcription. The most notable is a perfect octamer (5'-ATTTGCAT-3'), which binds the Oct-2 B-cell-specific transcription factor and thereby can account for the tissue-specific expression of this gene. Images
Hermanson, G G; Briskin, M; Sigman, D; Wall, R
The origin of CD95-gene mutations in B-cell lymphoma.
CD95 (Apo-1/Fas) is crucial for the negative selection of B cells within the germinal center (GC). Impairment of CD95-mediated apoptosis results in defective affinity maturation and the persistence of autoreactive B-cell clones. CD95 was defined recently as a tumor-suppressor gene and is silenced in many tumor entities. In contrast to other malignancies, in GC-derived B-cell lymphomas, inactivation of the CD95 gene is often a result of deleterious mutations. Such mutations occur also at a low frequency in normal GC, but not naive, B cells. We propose that CD95 mutations in B-cell lymphomas originate from the GC reaction and are introduced most probably as targeting errors of the somatic hypermutation machinery, which bears--besides its physiological role--an inherent risk of malignant transformation and the persistence of autoreactive B-cell specificities. PMID:11929130
M³schen, Markus; Rajewsky, Klaus; Kr÷nke, Martin; K³ppers, Ralf
Defective B cell tolerance in adenosine deaminase deficiency is corrected by gene therapy
Adenosine deaminase (ADA) gene defects are among the most common causes of SCID. Restoration of purine metabolism and immune functions can be achieved by enzyme replacement therapy, or more effectively by bone marrow transplant or HSC gene therapy (HSC-GT). However, autoimmune complications and autoantibody production, including anti-nuclear antibodies (ANAs), frequently occur in ADA-SCID patients after treatment. To assess whether ADA deficiency affects the establishment of B cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells of ADA-SCID patients before and after HSC-GT. We found that before HSC-GT, new emigrant/transitional and mature naive B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B cell tolerance checkpoints. We further observed impaired B cell receptor (BCR) and TLR functions in B cells after ADA inhibition, which may underlie the defects in B cell tolerance. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance checkpoints, as evidenced by restored removal of developing autoreactive and ANA-expressing B cells. Hence, ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions.
Sauer, Aisha V.; Morbach, Henner; Brigida, Immacolata; Ng, Yen-Shing; Aiuti, Alessandro; Meffre, Eric
The regulation of the B-cell gene expression programme by Pax5
The activity of the transcription factor paired box gene 5 (Pax5) is essential for many aspects of B lymphopoiesis including the initial commitment to the lineage, immunoglobulin rearrangement, pre-B cell receptor signalling and maintaining cell identity in mature B cells. Deregulated or reduced Pax5 activity has also been implicated in B-cell malignancies both in human disease and mouse models. Candidate
Melissa L Holmes; Clare Pridans; Stephen L Nutt
Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia
The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malig- nancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising
Andreas Rosenwald; Ash A. Alizadeh; George Widhopf; Richard Simon; R. Eric Davis; Xin Yu; Liming Yang; Oxana K. Pickeral; Laura Z. Rassenti; John Powell; David Botstein; John C. Byrd; Michael R. Grever; Bruce D. Cheson; Nicholas Chiorazzi; Wyndham H. Wilson; Thomas J. Kipps; Patrick O. Brown; Louis M. Staudt
Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia
The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malig- nancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising
Andreas Rosenwald; Ash A. Alizadeh; George Widhopf; Richard Simon; R. Eric Davis; Xin Yu; Liming Yang; Oxana K. Pickeral; Laura Z. Rassenti; John Powell; David Botstein; John C. Byrd; Michael R. Grever; Bruce D. Cheson; Nicholas Chiorazzi; Wyndham H. Wilson; Thomas J. Kipps; Patrick O. Brown; Louis M. Staudt
Expression of Essential B Cell Development Genes in Horses with Common Variable Immunodeficiency
Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p < 0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p < 0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients.
Tallmadge, R.L.; Such, K.A.; Miller, K.C.; Matychak, M.B.; Felippe, M.J.B.
Expression of essential B cell development genes in horses with common variable immunodeficiency.
Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p<0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p<0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients. PMID:22464097
Tallmadge, R L; Such, K A; Miller, K C; Matychak, M B; Felippe, M J B
In Vivo Ablation of Surface Immunoglobulin on Mature B Cells by Inducible Gene Targeting Results in Rapid Cell Death
Gene targeting experiments have demonstrated that the expression of immunoglobulin heavy chain in the pre-B cell receptor (pBCR) and of heavy and light chains in the B cell antigen receptor (BCR) marks checkpoints in early B cell development that the cells have to pass to survive. To investigate whether the persistence of mature B cells in the peripheral immune system
Kong-Peng Lam; Ralf K³hn; Klaus Rajewsky
LITAF, a BCL6 target gene, regulates autophagy in mature B-cell lymphomas.
We have previously reported that LITAF is silenced by promoter hypermethylation in germinal centre-derived B-cell lymphomas, but beyond these data the regulation and function of lipopolysaccharide-induced tumour necrosis factor (TNF) factor (LITAF) in B cells are unknown. Gene expression and immunohistochemical studies revealed that LITAF and BCL6 show opposite expression in tonsil B-cell subpopulations and B-cell lymphomas, suggesting that BCL6 may regulate LITAF expression. Accordingly, BCL6 silencing increased LITAF expression, and chromatin immunoprecipitation and luciferase reporter assays demonstrated a direct transcriptional repression of LITAF by BCL6. Gain- and loss-of-function experiments in different B-cell lymphoma cell lines revealed that, in contrast to its function in monocytes, LITAF does not induce lipopolysaccharide-mediated TNF secretion in B cells. However, gene expression microarrays defined a LITAF-related transcriptional signature containing genes regulating autophagy, including MAP1LC3B (LC3B). In addition, immunofluorescence analysis co-localized LITAF with autophagosomes, further suggesting a possible role in autophagy modulation. Accordingly, ectopic LITAF expression in B-cell lymphoma cells enhanced autophagy responses to starvation, which were impaired upon LITAF silencing. Our results indicate that the BCL6-mediated transcriptional repression of LITAF may inhibit autophagy in B cells during the germinal centre reaction, and suggest that the constitutive repression of autophagy responses in BCL6-driven lymphomas may contribute to lymphomagenesis. PMID:23795761
Bertolo, Cristina; Roa, Sergio; Sagardoy, Ainara; Mena-Varas, Maria; Robles, Eloy F; Martinez-Ferrandis, Jose I; Sagaert, Xavier; Tousseyn, Thomas; Orta, Alberto; Lossos, Izidore S; Amar, Salomon; Natkunam, Yasodha; Briones, Javier; Melnick, Ari; Malumbres, Raquel; Martinez-Climent, Jose A
Axon growth and guidance genes identify T-dependent germinal centre B cells.
Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms. PMID:17938642
Yu, Di; Cook, Matthew C; Shin, Dong-Mi; Silva, Diego G; Marshall, Jennifer; Toellner, Kai-Michael; Havran, Wendy L; Caroni, Pico; Cooke, Michael P; Morse, Herbert C; MacLennan, Ian C M; Goodnow, Christopher C; Vinuesa, Carola G
The impact of translocations and gene fusions on cancer causation
Chromosome aberrations, in particular translocations and their corresponding gene fusions, have an important role in the initial steps of tumorigenesis; at present, 358 gene fusions involving 337 different genes have been identified. An increasing number of gene fusions are being recognized as important diagnostic and prognostic parameters in malignant haematological disorders and childhood sarcomas. The biological and clinical impact of
Bertil Johansson; Fredrik Mertens; Felix Mitelman
EBF1 acts as a powerful repressor of Blimp-1 gene expression in immature B cells.
The transcription factor, early B cell factor 1 (EBF1) with an atypical zinc-finger and helix-loop-helix motif, is essential for development and differentiation of lymphocytes. In mice, EBF1 is involved in the generation of pre-pro B cells (the first specified progenitors of B cells) from common lymphoid progenitors (CLPs) and transcription regulations of various genes involved in B cell-development, for instance, mb-1 and Pax5. During B lymphopoiesis, interestingly, EBF1 is detected throughout from CLPs to mature B cells. However, in immature B cells, the physiological role of EBF1 remains to be elucidated. Here, by analyzing EBF1-deficient DT40 cells, EBF1(-/-), generated by us, we show that EBF1-deficiency caused significant increases (to ?800%) in both mRNA and protein levels of B lymphocyte-induced maturation protein-1 (Blimp-1), the master gene for plasma cell differentiation. In addition, both transcription and protein synthesis of Blimp-1 were remarkably down-regulated (to ?20%) by re-expression (over-expression) of EBF1. Chromatin immunoprecipitation assay revealed that EBF1 binds to proximal 5'-upstream regions around two putative EBF1 binding motifs of the gene in vivo. These results suggest that EBF1 takes part in transcriptional regulations of the Blimp-1 gene in immature B cells, and may play a key role in B cell differentiation. This is the first report on a novel EBF1 function in immature B cells as a powerful repressor of Blimp-1 gene expression. PMID:22634309
Kikuchi, Hidehiko; Nakayama, Masami; Takami, Yasunari; Kuribayashi, Futoshi; Nakayama, Tatsuo
Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11 ; 14) chromosome translocation
The t(11 ; 14) (ql3 ; q32) chromosome translocation has been reported in diffuse small and large cell lymphomas and in chronic lymphocytic leukaemia (B-CLL)1,2 and multiple myeloma3. Because chromosome band 14q32 is involved in this translocation, as well as in the t(8 ; 14) (q24 ; q32) translocation of the Burkitt tumour4, interruption of the immunoglobulin heavy-chain locus was
Y. Tsujimoto; E. Jaffe; J. Cossman; J. Gorham; P. C. Nowell; C. M. Croce
Targeted gene analysis: increased B-cell lymphoma 6 in preeclamptic placentas.
Preeclampsia is a leading cause for maternal and perinatal mortality and morbidity. Microarray-based transcriptional profiling has been widely used for identifying genes responsible for preeclampsia. These studies deliver multiple pictures of gene signatures, implying the complicated pathophysiology. In the present work, we designed our own gene array containing genes involved in various signaling transduction pathways and analyzed placental samples from patients with preeclampsia and controls. We verify that genes associated with angiogenesis and migration pathways are mostly altered in preeclamptic placentas. Interestingly, several genes including B-cell lymphoma 6 have been identified to be linked to preeclampsia. Increased expression of B-cell lymphoma 6 is correlated with enhanced FLT1 and LEPTIN, the hallmarks of preeclampsia. Moreover, the protein level of B-cell lymphoma 6 is elevated in preeclamptic placentas and is predominantly localized in the nucleus of villous cytotrophoblasts lying directly underneath the syncytial layer, suggestive of an involvement in the function of villous trophoblasts. Altered B-cell lymphoma 6, a key oncogene in B-cell lymphomagenesis, may be involved in the pathogenesis of preeclampsia, and further investigations are required to decipher the molecular mechanisms. PMID:24767250
Louwen, Frank; Muschol-Steinmetz, Cornelia; Friemel, Alexandra; Kõmpf, Anne Kristina; T÷ttel, Eva; Reinhard, Joscha; Yuan, Juping
Prognostic Significance of B-cell Differentiation Genes Encoding Proteins in Diffuse Large B-cell Lymphoma and Follicular Lymphoma Grade 3
Aim To define prognostic significance of B-cell differentiation genes encoding proteins and BCL2 and BCL6 gene abnormalities in diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern. Methods In 53 patients with diffuse large B-cell lymphoma and 20 patients with follicular lymphoma grade 3 with >75% follicular growth pattern the following was performed: 1) determination of protein expression of BCL6, CD10, MUM1/IRF4, CD138, and BCL2 by immunohistochemistry; 2) subclassification into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) groups according to the results of protein expression; 3) detection of t(14;18)(q32;q21)/IgH-BCL2 and BCL6 abnormalities by fluorescent in situ hybridization in diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern as well as in GCB and ABC groups; and 4) assessment of the influence of the analyzed characteristics and clinical prognostic factors on overall survival. Results Isolated BCL6 expression was more frequently found in follicular lymphoma grade 3 with >75% follicular growth pattern than in diffuse large B-cell lymphoma (P?=?0.030). There were no differences in BCL2 and BCL6 gene abnormalities between diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern. Diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern patients were equally distributed in GCB and ABC groups. t(14;18)(q32;q21) was more frequently recorded in GCB group, and t(14;18)(q32;q21) with BCL2 additional signals or only BCL2 and IgH additional signals in ABC group (P?=?0.004). The GCB and ABC groups showed no difference in BCL6 gene abnormalities. There was no overall survival difference between the patients with diffuse large B-cell lymphoma or follicular lymphoma grade 3 with >75% follicular growth pattern, however, GCB group had longer overall survival than ABC group (P?=?0.047). Multivariate analysis showed that BCL6, CD10, and BCL2 expression, BCL2 and BCL6 abnormalities, and International Prognostic Index were not significantly related to overall survival. Conclusion Patients with diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern have very similar characteristics and their prognosis is more influenced by protein expression of B-cell differentiation stage genes than by tumor cells growth pattern, BCL2 and BCL6 abnormalities, and International Prognostic Index.
Borovecki, Ana; Korac, Petra; Nola, Marin; Ivankovic, Davor; Jaksic, Branimir; Dominis, Mara
Identification of Highly Methylated Genes across Various Types of B-Cell Non-Hodgkin Lymphoma
Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n?=?480) when compared to normal B cells (n?=?5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n?=?42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n?=?25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients.
Bethge, Nicole; Honne, Hilde; Hilden, Vera; Tr?en, Gunhild; Eknaes, Mette; Liest?l, Knut; Holte, Harald; Delabie, Jan; Smeland, Erlend B.; Lind, Guro E.
Identification of highly methylated genes across various types of B-cell non-hodgkin lymphoma.
Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n?=?480) when compared to normal B cells (n?=?5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n?=?42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n?=?25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients. PMID:24260260
Bethge, Nicole; Honne, Hilde; Hilden, Vera; Tr°en, Gunhild; Eknµs, Mette; Liest°l, Knut; Holte, Harald; Delabie, Jan; Smeland, Erlend B; Lind, Guro E
BIOMED-2 PCR assays for IGK gene rearrangements are essential for B-cell clonality analysis in follicular lymphoma.
B-cell clonality analysis is commonly performed by polymerase chain reaction (PCR) targeting the IGH genes although a high false-negative rate is recognized for germinal centre/post-germinal centre B-cell malignancies, especially follicular lymphoma. We assessed the diagnostic value of BIOMED-2 IGK assays and investigated the cause of IGH PCR failure in 77 patients with follicular lymphoma. Using the full set of BIOMED-2 reactions, clonal immunoglobulin gene rearrangements were detected in 74 (96%) cases. The clonality detection rate was 86% by two IGK reactions but only 68% by five IGH reactions (P?0Ę001). Sequencing of the clonal PCR products showed significantly fewer somatic mutations in the rearranged IGKV (9/27 cases, 33%, mean mutation rate 0Ę5%) than IGHV (17/17 cases, 100%, rate 11Ę0%) (P?0Ę01). All IGHV-IGHJ PCR failures occurred in cases with at least one mutation at the corresponding IGHV primer binding sites. t(14:18)(q32:q21)/IGH-BCL2 was detected in 50 of 71 (70%) cases and the presence of the translocation was not associated with the poor performance of IGH assays. Our results showed that BIOMED-2 IGK assays are significantly more sensitive than IGH assays in follicular lymphoma due to the fact that the rearranged IGKV is less frequently targeted by somatic hypermutation than IGHV, and therefore, are essential in routine clonality analysis of these lymphomas. PMID:21790530
Payne, Karen; Wright, Penny; Grant, John W; Huang, Yuanxue; Hamoudi, Rifat; Bacon, Chris M; Du, Ming-Qing; Liu, Hongxiang
Gene flow and endangered species translocations: a topic revisited
Understanding the evolutionary role of gene flow is pivotal to the conservation of endangered populations. Gene flow can be enhanced through population translocations that are conducted to maintain genetic variation and combat the negative consequences of inbreeding depression (two of the major concerns in the conservation of subdivided or isolated populations). While researchers have given extensive consideration to the idea
Andrew Storfer
Nuclear positioning, higher-order folding, and gene expression of Mmu15 sequences are refractory to chromosomal translocation
Nuclear localization influences the expression of certain genes. Chromosomal rearrangements can reposition genes in the nucleus and thus could impact the expression of genes far from chromosomal breakpoints. However, the extent to which chromosomal rearrangements influence nuclear organization and gene expression is poorly understood. We examined mouse progenitor B cell lymphomas with a common translocation, der(12)t(12;15), which fuses a gene-rich region of mouse chromosome12 (Mmu12) with a gene-poor region of mouse chromosome15 (Mmu15). We found that sequences 2.3 and 2.7 Mb on either side of the der(12)t(12;15) breakpoint had different nuclear positions measured relative to the nuclear radius. However, their positions were similar to the same loci on unrearranged chromosomes in the same tumor cells, suggesting that changes to nuclear position imposed by the der(12)t(12;15) translocation are constrained within ~2.5 Mb of the breakpoint. In addition, higher-order chromatin folding marked by three-dimensional gene clustering was not significantly altered for the 7 Mb of Mmu15 sequence distal to this translocation breakpoint. Translocation also did not correspond to significant changes in gene expression in this region. These data contrast with those of certain other chromosomal rearrangements and suggest that significant changes to Mmu15 sequence are structurally and functionally tolerated in the tumor cells examined.
Snow, Kathy J.; Wright, Sarah M.; Woo, Yong; Titus, Laura C.; Mills, Kevin D.; Shopland, Lindsay S.
Aldehyde dehydrogenase-1a1 induces oncogene suppressor genes in B cell populations.
The deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cell populations. Aldh1a1 regulated transcription factors during B cell differentiation in a sequential manner: 1) retinoic acid receptor alpha (Rara) in IgG1(+)/CD19(-) and 2) zinc finger protein Zfp423 and peroxisome proliferator-activated receptor gamma (Pparg) in IgG1(+)/CD19(+) splenocytes. In Aldh1a1(-/-) mice, splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma B cell lines also lack Aldh1a1 expression; however, ectopic Aldh1a1 expression rescued Rara and Znf423 expressions in these cells. Our data highlight a mechanism by which an enzyme involved in vitamin A metabolism can improve B cell resistance to oncogenesis. PMID:24080087
Yasmeen, R; Meyers, J M; Alvarez, C E; Thomas, J L; Bonnegarde-Bernard, A; Alder, H; Papenfuss, T L; Benson, D M; Boyaka, P N; Ziouzenkova, O
Fucoidan prevents C{epsilon} germline transcription and NF{kappa}B p52 translocation for IgE production in B cells
Fucoidan, a dietary fiber contained in seaweed, reduces the increase of antigen-specific IgE in mice exposed to ovalbumin. In this study, we investigated the effect of fucoidan on IgE production and intracellular events in B cells in vitro. Fucoidan inhibited the production of IgE and C{epsilon} germline transcription in murine B cells induced by IL-4 (100 ng/ml) and anti-CD40 antibodies (10 {mu}g/ml), whereas it stimulated cell proliferation. A significant effect of fucoidan on IgE production was observed when B cells were stimulated with a higher dose (5 {mu}g/ml) of anti-CD40 antibodies, but not when stimulated with lower doses (1.25, 2.5 {mu}g/ml), regardless of the IL-4 concentrations. Moreover, nuclear translocation of NF{kappa}B p52, but neither that of NF{kappa}B p65, nor the phosphorylation of JAK1 and STAT6 was reduced by fucoidan. These results suggest that fucoidan inhibited IgE production by preventing the NF{kappa}B p52-mediated pathways activated by CD40.
Oomizu, Souichi [Hiroshima Prefectural Institute of Industrial Science and Technology, Higashi-Hiroshima (Japan); Yanase, Yuhki [Hiroshima Prefectural Institute of Industrial Science and Technology, Higashi-Hiroshima (Japan); Department of Dermatology, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima (Japan); Suzuki, Hidenori [Department of Dermatology, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima (Japan); Kameyoshi, Yoshikazu [Department of Dermatology, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima (Japan); Hide, Michihiro [Department of Dermatology, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima (Japan)]. E-mail: firstname.lastname@example.com
Organizational structure and the periphery of the gene regulatory network in B-cell lymphoma
Background The physical periphery of a biological cell is mainly described by signaling pathways which are triggered by transmembrane proteins and receptors that are sentinels to control the whole gene regulatory network of a cell. However, our current knowledge about the gene regulatory mechanisms that are governed by extracellular signals is severely limited. Results The purpose of this paper is three fold. First, we infer a gene regulatory network from a large-scale B-cell lymphoma expression data set using the C3NET algorithm. Second, we provide a functional and structural analysis of the largest connected component of this network, revealing that this network component corresponds to the peripheral region of a cell. Third, we analyze the hierarchical organization of network components of the whole inferred B-cell gene regulatory network by introducing a new approach which exploits the variability within the data as well as the inferential characteristics of C3NET. As a result, we find a functional bisection of the network corresponding to different cellular components. Conclusions Overall, our study allows to highlight the peripheral gene regulatory network of B-cells and shows that it is centered around hub transmembrane proteins located at the physical periphery of the cell. In addition, we identify a variety of novel pathological transmembrane proteins such as ion channel complexes and signaling receptors in B-cell lymphoma.
Role of Egr-1 gene expression in B cell receptor-induced apoptosis in an immature B cell lymphoma.
Ligation of B cell receptor (BCR) on BKS-2, an immature B cell lymphoma by anti-IgM antibodies (Ab) caused apoptosis. Here we report that signaling through B cell receptor in wild type BKS-2 cells down-regulated the expression of Egr-1, a zinc finger-containing transcription factor. A reduction in the level of Egr-1 mRNA could be demonstrated as early as 30 min after the ligation of BCR on BKS-2 cells. Immunocytochemical and Western blot analysis revealed that the expression of EGR-1 protein was also inhibited by anti-IgM treatment. Antisense oligonucleotides to Egr-1 caused growth inhibition and apoptosis in BKS-2 cells, suggesting that expression of Egr-1 is important for the survival of these B lymphoma cells. In contrast to wild type BKS-2 cells, the mutant 1. B5 cell line, which is refractory to B cell receptor-mediated growth-inhibitory signals, showed an increased expression of Egr-1 upon treatment with anti-IgM. These results implicate a role for Egr-1 in blocking B cell receptor-mediated apoptosis in immature B cells. PMID:9346950
Muthukkumar, S; Han, S S; Rangnekar, V M; Bondada, S
Interleukin 4-induced gene 1 is activated in primary mediastinal large B-cell lymphoma.
The molecular markers that distinguish primary mediastinal large B-cell lymphoma (PMBL) from nonmediastinal diffuse large B-cell lymphomas (NM-DLBLs) remain to be identified. Using cDNA representational difference analysis to compare PMBL and NM-DLBL transcripts, we isolated a cDNA fragment homologous to the mouse B-cell interleukin 4 (IL-4)-inducible gene FIG1 (interleukin 4-induced gene 1) transcript. The human FIG1 mRNA encodes a 567 amino acid protein that comprises a signal peptide and a large flavin-binding amino oxidase domain, and shares significant homology with secreted apoptosis-inducing L-amino acid oxidases. Northern blot studies showed that FIG1 mRNA expression is mainly restricted to lymphoid tissues. It is expressed at low levels in thymus, spleen, tonsils, and reactive lymph nodes, and is highly up-regulated in IL-4+CD40-activated tonsillar B cells. Interestingly, in human B-cell lines, FIG1 mRNA expression appeared restricted to the PMBL-derived MedB-1 and Karpas 1106 cell lines. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrated that all but one PMBL (16/17) displayed high FIG1 mRNA levels, whereas most NM-DLBLs (12/18) and all low-grade B-cell lymphomas tested (8/8) exhibited low FIG1 mRNA levels. The difference between PMBLs and NM-DLBLs was statistically significant (Fisher test; P =.0003). Southern blot studies did not show rearrangement of the FIG1 gene. FIG1 gene expression might be due to a constitutive activation of a cytokine signaling pathway in PMBL. PMID:12446450
Copie-Bergman, Christiane; Boulland, Marie-Laure; Dehoulle, Catherine; M÷ller, Peter; Farcet, Jean-Pierre; Dyer, Martin J S; Haioun, Corinne; Romķo, Paul-Henri; Gaulard, Philippe; Leroy, Karen
Immature surface Ig+ B cells can continue to rearrange kappa and lambda L chain gene loci.
Pro and pre B cells possess the long-term capacity to proliferate in vitro on stromal cells and interleukin 7 (IL-7) and can differentiate to surface immunoglobulin (sIg+) cells upon removal of IL-7 from the cultures. A key event in this differentiation is the extensive cell loss due to apoptosis. Because the proto-oncogene bcl-2 can promote cell survival, we established pre-B cell lines from E mu-bcl-2 transgenic mice. These pre-B cells have the same properties as those derived from non-bcl-2 transgenic mice except that they do not die by apoptosis. This allowed us to study the fate of newly formed B cells in vitro for a longer period of time. Here we show that early during the differentiation of pre-B cells, upregulation of RAG-1 and RAG-2 expression go hand in hand with rearrangements of the Ig gene loci. Moreover, the newly formed sIg+ B cells continue to express RAG-1 and RAG-2 and continue to rearrange L chain gene loci, even in the absence of proliferation, in an orderly fashion, so that kappa L+ sIg+ cells can become lambda L+ sIg+ or sIg- cells, whereas lambda L+ sIg+ cells can become sIg-, but not kappa L+ sIg+ cells. Thus, deposition of a complete Ig molecule on the surface of a B cell does not automatically stop the Ig-rearrangement machinery. PMID:8376934
Rolink, A; Grawunder, U; Haasner, D; Strasser, A; Melchers, F
B cell development in mice with a defective lambda 5 gene.
The surrogate light chain encoded by the two pre-B cell-specific genes VpreB and lambda 5 plays a critical role in B cell development of the mouse. It has been shown that targeted disruption of the lambda 5 gene results in a depletion of B220+ CD43- IgM-pre-B cells in bone marrow, and in a delayed appearance both of CD5+ as well as CD5- surface immunoglobulin (sIg)+ B cells in the periphery. In this report we show that DHJH-rearranged B220- and B220+, CD43+, c-kit+, sIgM- pro- and pre-B-I cells with long-term capacity to proliferate in vitro on stromal cells in the presence of interleukin-7 are present in normal numbers in the bone marrow of lambda 5 T/lambda 5 T mice at various ages. They express normal levels of VpreB mRNA but, in contrast to normal pre-B-I cells, do not express surrogate light chain on their surface. Pre-B-I cells from fetal liver and bone marrow of lambda 5 T/lambda 5 T mice differentiate with normal kinetics and in normal numbers to sIg+, mitogen-reactive B cells. These results suggest that the delayed generation of sIg+ B cells in the peripheral, mature compartments of CD5+ and CD5- cells could be accounted for by the daily production of approximately 5 x 10(5) sIg+ B cells from the pre-B-I cell pool in the absence of a normal pool of pre-B-II cells. PMID:7684685
Rolink, A; Karasuyama, H; Grawunder, U; Haasner, D; Kudo, A; Melchers, F
Acute leukemias of different lineages have similar MLL gene fusions encoding related chimeric proteins resulting from chromosomal translocation
The MLL gene, on human chromosome 11q23, undergoes chromosomal translocation in acute leukemias, resulting in gene fusion with AF4 (chromosome 4) and ENL (chromosome 19). The authors report here translocation of MLL with nine different chromosomes and two paracentric chromosome 11 deletions in early B cell, B- or T-cell lineage, or nonlymphocytic acute leukemias. The mRNA translocation junction from 22t(4;11) patients, including six adult leukemias, and nine t(11;19) tumors reveals a remarkable conservation of breakpoints within MLL, AF4, or ENL genes, irrespective of tumor phenotype. Typically, the breakpoints are upstream of the zinc-finger region of MLL, and deletion of this region can accompany translocation, supporting the der(11) chromosome as the important component in leukemogenesis. Partial sequence of a fusion between MLL and the AFX1 gene from chromosome X shows the latter to be rich in Ser/Pro codons, like the ENL mRNA. These data suggest that the heterogeneous 11q23 abnormalities might cause attachment of Ser/Pro-rich segments to the NH[sub 2] terminus of MLL, lacking the zinc-finger region, and that translocation occurs in early hematopoietic cells, before commitment to distinct lineages. 36 refs., 2 figs.
Corral, J.; Forster, A.; Thompson, S.; Rabbitts, T.H. (Medical Research Council Laboratory of Molecular Biology, Cambridge (United Kingdom)); Lampert, F. (Kinderklinik-Universitaet Giessen (Germany)); Kaneko, Y. (Saitama Cancer Centre, Saitama (Japan)); Slater, R.; Kroes, W.G. (Univ. of Amsterdam (Netherlands)); Van Der Schoot, C.E. (Central Laboratory of the Netherlands Red Cross, Amsterdam (Netherlands)); Ludwig, W.D. (Institut fur Humangenetik und Antrhopologie, Heidelberg (Germany)); Karpas, A. (Univ. of Cambridge (United Kingdom)); Pocock, C.; Cotter, F. (Institute of Child Health, London (United Kingdom))
Analysis of the immunoglobulin heavy chain gene variable region of intravascular large B-cell lymphoma
Intravascular large B-cell lymphoma (IVLBL) is a rare neoplasm characterized by proliferation of lymphoma cells within the blood vessels. The cell origin of IVLBL has not yet been determined. We examined cell lineage, with immunohistochemical staining and molecular analysis, using polymerase chain reaction (PCR) of the variable region of the immunoglobulin heavy chain (Ig-VH) gene. We also investigated the cell
M. Kanda; J. Suzumiya; K. Ohshima; S. Haraoka; N. Nakamura; M. Abe; K. Tamura; M. Kikuchi
Conditional inactivation of p53 in mature B cells promotes generation of nongerminal center-derived B-cell lymphomas.
The p53 tumor suppressor exerts a central role in protecting cells from oncogenic transformation. Accordingly, the p53 gene is mutated in a large number of human cancers. In mice, germ-line inactivation of p53 confers strong predisposition to development of different types of malignancies, but the early onset of thymic lymphomas in the majority of the animals prevents detailed studies of tumorigenesis in other tissues. Here, we use the Cre/Lox approach to inactivate p53 in mature B cells in mice (referred to as "CP" B cells) and find that such p53 inactivation results in the routine development of IgM-positive CP peripheral B-cell lymphomas. The CP lymphomas generally appear to arise, even in mice subjected to immunization protocols to activate germinal center reaction, from naive B cells that had not undergone immunoglobulin (Ig) heavy chain gene class switching or somatic hypermutation. In contrast to thymic lymphomas that arise in p53-deficient mice, which generally lack clonal translocations, nearly all analyzed CP B-cell tumors carried clonal translocations. However, in contrast to spontaneous translocations in other mouse B-cell tumor models, CP B-cell tumor translocations were not recurrent and did not involve Ig loci. Therefore, CP tumors might provide models for human lymphomas lacking Ig translocations, such as splenic marginal zone B-cell lymphoma or Waldenstrom macroglobulinemia. Our studies indicate that deletion of p53 is sufficient to trigger transformation of mature B cells and support the notion that p53 deficiency may allow accumulation of oncogenic translocations in B cells. PMID:23382223
Gostissa, Monica; Bianco, Julia M; Malkin, Daniel J; Kutok, Jeffery L; Rodig, Scott J; Morse, Herbert C; Bassing, Craig H; Alt, Frederick W
Conditional inactivation of p53 in mature B cells promotes generation of nongerminal center-derived B-cell lymphomas
The p53 tumor suppressor exerts a central role in protecting cells from oncogenic transformation. Accordingly, the p53 gene is mutated in a large number of human cancers. In mice, germ-line inactivation of p53 confers strong predisposition to development of different types of malignancies, but the early onset of thymic lymphomas in the majority of the animals prevents detailed studies of tumorigenesis in other tissues. Here, we use the Cre/Lox approach to inactivate p53 in mature B cells in mice (referred to as ōCPö B cells) and find that such p53 inactivation results in the routine development of IgM-positive CP peripheral B-cell lymphomas. The CP lymphomas generally appear to arise, even in mice subjected to immunization protocols to activate germinal center reaction, from naive B cells that had not undergone immunoglobulin (Ig) heavy chain gene class switching or somatic hypermutation. In contrast to thymic lymphomas that arise in p53-deficient mice, which generally lack clonal translocations, nearly all analyzed CP B-cell tumors carried clonal translocations. However, in contrast to spontaneous translocations in other mouse B-cell tumor models, CP B-cell tumor translocations were not recurrent and did not involve Ig loci. Therefore, CP tumors might provide models for human lymphomas lacking Ig translocations, such as splenic marginal zone B-cell lymphoma or Waldenstrom macroglobulinemia. Our studies indicate that deletion of p53 is sufficient to trigger transformation of mature B cells and support the notion that p53 deficiency may allow accumulation of oncogenic translocations in B cells.
Gostissa, Monica; Bianco, Julia M.; Malkin, Daniel J.; Kutok, Jeffery L.; Rodig, Scott J.; Morse, Herbert C.; Bassing, Craig H.; Alt, Frederick W.
Harnessing gene conversion in chicken B cells to create a human antibody sequence repertoire.
Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens. PMID:24278246
Schusser, Benjamin; Yi, Henry; Collarini, Ellen J; Izquierdo, Shelley Mettler; Harriman, William D; Etches, Robert J; Leighton, Philip A
Gene transfer of Ig-fusion proteins into B cells prevents and treats autoimmune diseases.
Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PLxSJL F(1) mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7-10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9-23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting. PMID:11971030
Melo, Marco E F; Qian, Jiahua; El-Amine, Moustapha; Agarwal, Rajeev K; Soukhareva, Nadejda; Kang, Yubin; Scott, David W
An AICDA-independent mechanism of secondary VH gene rearrangement in preimmune human B cells Running title: Secondary rearrangement in human B cells
VH replacement is a form of IgH chain receptor editing that is believed to be mediated by recombinase cleavage at cryptic recombination signals (cRSS) embedded in IGHV genes. Whereas there are several reports of IGHV replacement in primary and transformed human B cells and murine models, it remains unclear whether IGHV replacement contributes to the normal human B cell repertoire. We identified VH ? VHDJH compound rearrangements from fetal liver, fetal bone marrow and naive peripheral blood, all of which involved invading and recipient IGHV4 genes that contain a cryptic heptamer, 13 base pair (bp) spacer and nonamer in the 5' portion of framework region (FR) 3. Surprisingly, all pseudohybrid joins lacked molecular processing associated with typical VHDJH recombination or nonhomologous end joining. Although inefficient compared to a canonical RSS, the IGHV4 cRSS was a significantly better substrate for in vitro RAG-mediated cleavage than the IGHV3 cRSS. It has been suggested that activation induced cytidine deamination (AICDA) may contribute to VH replacement. However, we found similar secondary rearrangements utilizing IGHV4 genes in AICDA-deficient human B cells. The data suggest that IGHV4 replacement in preimmune human B cells is mediated by an AICDA-independent mechanism resulting from inefficient but selective RAG activity.
Longo, Nancy S.; Grundy, Gabrielle J.; Lee, Jisoo; Gellert, Martin; Lipsky, Peter E.
Virus-transformed Pre-B Cells Show Ordered Activation but Not Inactivation of Immunoglobulin Gene Rearrangement and Transcription
Summary Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (,u) protein in regulating gene rearrangement
Mark S. Schlissel; Lynn M. Corcoran; David Baltimore
Preferential rearrangement of V kappa 4 gene segments in pre-B cell lines
Examination of the in vitro V kappa gene rearrangements of murine adult bone marrow-derived pre-B cell lines reveals that 21 of 25 (84%) cell lines have rearranged a member of the V kappa 4 family. In contrast, analysis of two V kappa cDNA libraries prepared from LPS-stimulated adult spleen cells indicates that only 17% of the Ig kappa cDNAs contain sequences belonging to the V kappa 4 gene family. Half of the pre-B cell lines examined also share an 8-kbp BamHI reciprocal product (rp). However, these rp do not involve the same V kappa gene, indicating that conserved BamHI sites exist 3' of some V kappa genes. This rp is also readily detected in DNA from normal adult spleen cells, suggesting that the in vitro rearrangements examined in this study are representative of kappa rearrangements that occur in vivo. We suggest that, unlike the diverse V kappa repertoire expressed by mature B cells, the germline V kappa segments involved in initial rearrangements of the Ig kappa locus are highly restricted, and that an initial V kappa 4 rearrangement is probably followed by other, more random recombination events.
Gene expression-based risk score in diffuse large B-cell lymphoma
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma and displays heterogeneous clinical and molecular characteristics. In this study, high throughput gene expression profiling of DLBCL tumor samples was used to design a 12-gene expression¢based risk score (GERS) predictive for patient's overall survival. GERS allowed identifying a high-risk group comprising 46,4% of the DLBCL patients in two independent cohorts (n=414 and n=69). GERS was shown to be an independent predictor of survival when compared to the previously published prognostic factors, including the International Prognostic Index (IPI). GERS displayed a prognostic value in germinal-center B-cell¢like subgroup (GCB) and activated B cell¢like (ABC) molecular subgroups of patients as well as in DLBCL patients treated with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) or Rituximab-CHOP (R-CHOP) regimens. Combination of GERS and IPI lead to a potent prognostic classification of DLBCL patients. Finally, a genomic instability gene signature was highlighted in gene expression profiles of patients belonging to the high-risk GERS-defined group.
Bret, Caroline; Klein, Bernard; Moreaux, Jerome
Gene Expression Profiling of B Cell Chronic Lymphocytic Leukemia Reveals a Homogeneous Phenotype Related to Memory B Cells
B cell-derived chronic lymphocytic leukemia (B-CLL) represents a common malignancy whose cell derivation and pathogenesis are unknown. Recent studies have shown that ? 50% of CLLs display hypermutated immunoglobulin variable region (IgV) sequences and a more fa- vorable prognosis, suggesting that they may represent a distinct subset of CLLs which have transited through germinal centers (GCs), the physiologic site of
Ulf Klein; Yuhai Tu; Gustavo A. Stolovitzky; Michela Mattioli; Giorgio Cattoretti; Hervķ Husson; Arnold Freedman; Giorgio Inghirami; Lilla Cro; Luca Baldini; Antonino Neri; Andrea Califano; Riccardo Dalla-Favera
Role of the translocation partner in protection against AID-dependent chromosomal translocations.
Chromosome translocations between Ig (Ig) and non-Ig genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is c-myc/IgH translocation, which is associated with Burkitt's lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes. c-myc/IgH translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear c-myc/IgH translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCdelta are required for protection against c-myc/IgH translocation, whereas Bcl-XL and BAFF enhance c-myc/IgH translocation. Whether these effects are general or specific to c-myc/IgH translocation and whether AID produces dsDNA breaks in genes other than c-myc and Ig is not known. To examine these questions, we developed an assay for translocation between IgH and Igbeta, both of which are somatically mutated by AID. Igbeta/IgH, like c-myc/IgH translocations, are AID-dependent, and AID is responsible for lesions on IgH and the non-IgH translocation partners. However, ATM, p53, and p19 do not protect against Igbeta/IgH translocations. Instead, B cells are protected against Igbeta/IgH translocations by a BAFF- and PKCdelta-dependent pathway. We conclude that AID-induced double-strand breaks in non-Ig genes other than c-myc lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells. PMID:19966290
Jankovic, Mila; Robbiani, Davide F; Dorsett, Yair; Eisenreich, Thomas; Xu, Yang; Tarakhovsky, Alexander; Nussenzweig, Andre; Nussenzweig, Michel C
PAX5 alteration-associated gene-expression signatures in B-cell acute lymphoblastic leukemia.
The paired box domain gene 5 (PAX5) is frequently altered in both childhood and adult B-cell acute lymphoblastic leukemia, and takes part in leukemogenesis. We analyzed data from the database of Gene Expression Omnibus (accession number: GSE11877) using bioinformatical and statistical methods. The results showed that cases of PAX5 alteration can cluster using unsupervised clustering algorithms, and one gene, zinc and ring finger 1 (ZNRF1), was characterized and validated by quantitative RT-PCR. ZNRF1 may be associated with leukemogenesis of ALL with PAX5 alteration. PMID:23529845
Shang, Zhen; Zhao, Yuechao; Zhou, Kuangguo; Xu, Yanling; Huang, Wei
Gene therapy for immunological tolerance: using 'transgenic' B cells to treat inhibitor formation.
B cells have been shown to function as tolerogenic antigen presenting cells (APCs) both in vivo and in vitro. We have taken advantage of this property, as well as the ability of IgG carriers to be potent 'schleppers' for tolerogenic entities, to develop a gene therapy approach to induce unresponsiveness in a number of systems, including the elimination of haemophilia inhibitors. Thus, peptide-IgG constructs have been engineered into retroviral vectors to create 'transgenic' B cells for tolerance applications. In this paper, we discuss our gene therapy approach mediated by B cells (as well as bone marrow cells) for tolerance acquisition in various mouse models for autoimmune disease and haemophilia A. The mechanisms that are the underpinning of this effort and role of regulatory T cells are discussed herein. Our results indicate that gene therapy strategies can successfully reduce the incidence and or onset of autoimmune diseases and prevent/reverse inhibitor formation in haemophilia A mice. Based on recent success with a model for tolerance with human T cell clones in vitro, plans for future application in patients are discussed. PMID:20536991
Scott, D W
Thymoquinone efficiently inhibits the survival of EBV-infected B cells and alters EBV gene expression.
Epstein--Barr virus (EBV) is a human virus with oncogenic potentials that is implicated in various human diseases and malignancies. In this study, the modulator activity of the potent herbal extract drug thymoquinone on EBV was assessed in vitro. Thymoquinone was tested for cytotoxicity on human cells of lymphoblastoid cells, Raji Burkitt's lymphoma, DG-75 Burkitt's lymphoma, peripheral blood mononuclear cells, and periodontal ligament fibroblast. Apoptosis induction was analyzed via TUNEL assay and activity studies of caspase-3. The effect of thymoquinone on EBV gene expression was determined using real-time polymerase chain reaction. We report here, for the first time, a promising selective inhibitory affect of thymoquinone on EBV-infected B cell lines in vitro, compared with lower activity on EBV negative B cell line and very low toxicity on human peripheral blood mononuclear cells and periodontal ligament fibroblasts. Moreover, the drug was found to efficiently suppress the RNA expression of EBNA2, LMP1, and EBNA1 genes. Specifically, EBNA2 expression levels were the most affected indicating that this gene might have a major contribution to thymoquinone potency against EBV infected cells. Overall, our results suggest that thymoquinone has the potential to suppress the growth of EBV-infected B cells efficiently. PMID:23089554
Zihlif, Malek A; Mahmoud, Ismail S; Ghanim, Majd T; Zreikat, Manar S; Alrabadi, Nasr; Imraish, Amer; Odeh, Fadwa; Abbas, Manal A; Ismail, Said I
Compositions and methods for detecting gene rearrangements and translocations
Disclosed is a series of nucleic acid probes for use in diagnosing and monitoring certain types of leukemia using, e.g., Southern and Northern blot analyses and fluorescence in situ hybridization (FISH). These probes detect rearrangements, such as translocations involving chromosome band 11q23 with other chromosomes bands, including 4q21, 6q27, 9p22, 19p13.3, in both dividing leukemic cells and interphase nuclei. The breakpoints in all such translocations are clustered within an 8.3 kb BamHI genomic region of the MLL gene. A novel 0.7 kb BamH1 cDNA fragment derived from this gene detects rearrangements on Southern blot analysis with a single BamHI restriction digest in all patients with the common 11q23 translocations and in patients with other 11q23 anomalies. Northern blot analyses are presented demonstrating that the MLL gene has multiple transcripts and that transcript size differentiates leukemic cells from normal cells. Also disclosed are MLL fusion proteins, MLL protein domains and anti-MLL antibodies.
Rowley, Janet D. (Chicago, IL); Diaz, Manuel O. (Chicago, IL)
Impaired Immune Responses and B-Cell Proliferation in Mice Lacking the Id3 Gene
B-lymphocyte activation and proliferation induced by the B-cell receptor (BCR) signals are important steps in the initiation of humoral immune responses. How the BCR signals are translated by nuclear transcription factors into cell cycle progression is poorly understood. Id3 is an immediate-early gene responding to growth and mitogenic signals in many cell types including B cells. The primary function of the Id3 protein has been defined as that of inhibitor of basic-helix-loop-helix (bHLH) transcription factors. The interaction between Id3 and bHLH proteins, many of which are essential for cellular differentiation, has been proposed as a key regulatory event leading to cellular proliferation instead of differentiation. To further investigate the role of Id3 in tissue and embryo development and the mechanism of Id3-mediated growth regulation, we generated and analyzed Id3-deficient mice. While these mice display no overt abnormality in tissue and embryo development, their humoral immunity is compromised. The amounts of immunoglobulins produced in Id3-deficient mice immunized with a T-cell-dependent antigen and a type 2 T-cell-independent antigen are attenuated and severely impaired, respectively. Further analysis of lymphocytes isolated from Id3-deficient mice reveals a B-cell defect in their proliferation response to BCR cross-linking but not to lipopolysaccharide or a combination of BCR cross-linking and interleukin-4. Analyses of cultured lymphocytes also suggest involvement of Id3 in cytokine production in T cells and isotype switching in B cells. Finally, the proliferation defect in Id3-deficient B cells can be rescued by ectopic expression of Id1, a homologue of Id3. Taken together, these results define a necessary and specific role for Id3 in mediating signals from BCR to cell cycle progression during humoral immune responses.
Pan, Lihua; Sato, Shinichi; Frederick, Joshua P.; Sun, Xiao-Hong; Zhuang, Yuan
Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains
Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the mu/lambda-expressing cell lines contained both kappa alleles in germline configuration, and synthesis/expression of conventional lambda L chains was directly proven by immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional lambda L chain gene rearrangements without rearranging or deleting either kappa allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface kappa and lambda L chains associated with mu H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both kappa alleles rearranged, and a single lambda rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed kappa and lambda L chains. Multiparameter flow cytometry was used to demonstrate the existence of kappa+/lambda+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface kappa and lambda refutes the widely accepted concept that expression of a single L chain isotype is immutable. The kappa+/lambda+ cells may represent transients undergoing L chain isotype switching.
The CD53 and CEACAM-1 genes are genetic targets for early B cell factor.
Early B cell factor (EBF)-1 is a transcription factor known to be of critical importance for early B lymphocyte development. EBF-1 has been shown to directly interact with and regulate expression of a set of genes involved in the functional formation of the pre-B cell receptor, but the dramatic phenotype observed in the EBF-1-deficient mice suggests that several additional genes are activated by this protein. In order to identify additional target genes for EBF-1, we transduced a hematopoietic progenitor cell line, BaF/3, with an EBF-1-encoding retrovirus and investigated the induced gene expression pattern by micro-arrays. This analysis suggested that among others, the CD53 and the carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-1 genes both were induced by ectopic expression of EBF-1. Identification of the 5' end of the cDNA enabled the identification of promoter elements with functional binding sites for EBF-1 and ability to respond to EBF-1 expression in transient transfection assays. These data suggest that CD53 and CEACAM-1 are direct genetic targets for EBF-1, providing additional information concerning the activity of this crucial transcription factor in hematopoiesis. PMID:17429843
MÕnsson, Robert; Lagergren, Anna; Hansson, Frida; Smith, Emma; Sigvardsson, Mikael
Extensive Gene-Specific Translational Reprogramming in a Model of B Cell Differentiation and Abl-Dependent Transformation
To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib
Jamie G. Bates; Julia Salzman; Damon May; Patty B. Garcia; Gregory J. Hogan; Martin McIntosh; Mark S. Schlissel; Pat O. Brown
Vaccinia Virus Inhibits NF-?B-Dependent Gene Expression Downstream of p65 Translocation
The transcription factor nuclear factor kappa light-chain enhancer of activated B cells (NF-?B) plays a critical role in host defense against viral infection by inducing the production of proinflammatory mediators and type I interferon. Consequently, viruses have evolved many mechanisms to block its activation. The poxvirus vaccinia virus (VACV) encodes numerous inhibitors of NF-?B activation that target multiple points in the signaling pathway. A derivative of VACV strain Copenhagen, called vv811, lacking 55 open reading frames in the left and right terminal regions of the genome was reported to still inhibit NF-?B activation downstream of tumor necrosis factor alpha (TNF-?) and interleukin-1? (IL-1?), suggesting the presence of one or more additional inhibitors. In this study, we constructed a recombinant vv811 lacking the recently described NF-?B inhibitor A49 (vv811?A49), yielding a virus that lacked all currently described inhibitors downstream of TNF-? and IL-1?. Unlike vv811, vv811?A49 no longer inhibited degradation of the phosphorylated inhibitor of ?B? and p65 translocated into the nucleus. However, despite this translocation, vv811?A49 still inhibited TNF-?- and IL-1?-induced NF-?B-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists. This inhibition did not require late viral gene expression. These findings indicate the presence of another inhibitor of NF-?B that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.
Sumner, Rebecca P.; Maluquer de Motes, Carlos; Veyer, David L.
IRF8 Governs Expression of Genes Involved in Innate and Adaptive Immunity in Human and Mouse Germinal Center B Cells
IRF8 (Interferon Regulatory Factor 8) is a transcription factor expressed throughout B cell differentiation except for mature plasma cells. Previous studies showed it is part of the transcriptional network governing B cell specification and commitment in the bone marrow, regulates the distribution of mature B cells into the splenic follicular and marginal zone compartments, and is expressed at highest levels in germinal center (GC) B cells. Here, we investigated the transcriptional programs and signaling pathways affected by IRF8 in human and mouse GC B cells as defined by ChIP-chip analyses and transcriptional profiling. We show that IRF8 binds a large number of genes by targeting two distinct motifs, half of which are also targeted by PU.1. Over 70% of the binding sites localized to proximal and distal promoter regions with ?25% being intragenic. There was significant enrichment among targeted genes for those involved in innate and adaptive immunity with over 30% previously defined as interferon stimulated genes. We also showed that IRF8 target genes contributes to multiple aspects of the biology of mature B cells including critical components of the molecular crosstalk among GC B cells, T follicular helper cells, and follicular dendritic cells.
Morse, Herbert C.
[Expressions of bcl-6, lpp and miR-28 genes in diffuse large B cell lymphoma cell lines].
This study was purposed to explore the expressions of bcl-6, lpp and miR-28 genes in diffuse large B cell lymphoma (DLBCL) cell lines at the levels of gene and protein, and the relationship between them. Northern blot was used to detect bcl-6 and lpp mRNA expression in 8 DLBCL cell lines. Solution hybridization was used to measure miR-28 expression, and Western blot was performed for BCL-6 and LPP protein determinations. The results showed that the expression of bcl-6 mRNA was higher in the cell lines with Ig/BCL-6 translocation (Oc1-ly8, MD903, CTB-1, and MD901), and negative in those without Ig/BCL-6 translocation (HRC57 and K231). The expression of lpp mRNA in CTB-1 cell line was negative. MiR-28 was positive in all cell lines, and the expression levels from high to low were in line as follows: K231, CTB-1, MD903, HRC57, MD901, RCK8, OC1-LY8 and BEVA. BCL-6 protein was also positive in all of cell lines, and the expression levels from high to low were as follows: RCK8, BEVA, MD901, CTB-1, MD903, OC1-LY8, HRC57 and K231. LPP protein was negative in K231 cells, and the expression levels in other cells from high to low were line up as follows: HRC57, OC1-LY8, BEVA, RCK8, CTB-1, MD901 and MD903. The expression levels of bcl-6 and lpp mRNA were not consistent with expression levels of protein. It is concluded that the gene expression levels of bcl-6, lpp and miR-28 are different in various DLBCL cell lines. The expression levels of bcl-6 and lpp mRNA are not parallel with expression levels of protein. The roles of bcl-6, lpp and miR-28 in initiation and development of DLBCL need further investigation. PMID:19236753
Xu, Wei; Li, Jian-Yong; Fan, Lei; Qiao, Chun; Yu, Hui; Shen, Qiu-Dan
Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line
During culture, a chicken B cell line DT40 spontan- eously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytid- ine deaminase (AID)-dependent homologous recom- bination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the
Naoki Kanayama; Kagefumi Todo; Satoko Takahashi; Masaki Magari; Hitoshi Ohmori
MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia
Acute lymphoblastic leukemias carrying a chromosomal translocation involving the mixed-lineage leukemia gene (MLL, ALL1, HRX) have a particularly poor prognosis. Here we show that they have a characteristic, highly distinct gene expression profile that is consistent with an early hematopoietic progenitor expressing select multilineage markers and individual HOX genes. Clustering algorithms reveal that lymphoblastic leukemias with MLL translocations can clearly
Scott A. Armstrong; Jane E. Staunton; Lewis B. Silverman; Rob Pieters; Monique L. den Boer; Mark D. Minden; Stephen E. Sallan; Eric S. Lander; Todd R. Golub; Stanley J. Korsmeyer
Duplication and suppression of chloroplast protein translocation genes in maize.
The HCF106 (high chlorophyll fluorescence) gene of maize encodes a chloroplast membrane protein required for translocation of a subset of proteins across the thylakoid membrane. Mutations in HCF106 caused by the insertion of Robertson's Mutator transposable elements have been mapped to chromosome 2S. Here we show that there is a closely related homolog of HCF106 encoded elsewhere in the maize genome (HCF106c) that can partially compensate for these mutations. This homolog maps on chromosome 10L and is part of the most recent set of segmental duplications in the maize genome. Triple mutants that are disrupted in both the HCF106 and Sec-dependent protein translocation pathways provide evidence that they act independently. The HCF106c gene accounts for a previously reported exception to the correlation between epigenetic suppression of hcf106 and methylation of Mutator transposons. We also demonstrate that insertions of Robertson's Mutator elements into either introns or promoters can lead to mutations whose phenotypes are suppressed in the absence of Mu activity, while alleles with insertions in both positions are not suppressed. The implications of these observations are discussed.
Settles, A M; Baron, A; Barkan, A; Martienssen, R A
Specific 5' and 3' Regions of the mu -chain Gene are Undermethylated at Distinct Stages of B-Cell Differentiation
The mu -chain gene is expressed differently in successive stages of B-lymphocyte development. The heavy chain product appears as a cytoplasmic constituent in pre-B-cells, as part of the IgM receptor in maturing B cells, and as a component in the pentamer IgM antibody synthesized and secreted by the antigen-stimulated cell. We have used the methylation of CpG sequences as an
Marcia A. Blackman; Marian Elliott Koshland
The Regulated Expression of B Lineage Associated Genes during B Cell Differentiation in Bone Marrow and Fetal Liver
Sllmmary The expression of B lineage associated genes during early B cell differentiation stages is not firmly established. Using cell surface markers and multiparameter flow cytometry, bone marrow (BM) cells can be resolved into six fractions, representing sequential stages of development; i.e., pre- Pro-B, early Pro-B, late Pro-B\\/large Pre-B, small Pre-B, immature B, and mature B cells. Here we quantitate
Yue-Sheng Li; Kyoko Hayakawa; Richard R. Hardy
GCN5 is involved in regulation of immunoglobulin heavy chain gene expression in immature B cells.
GCN5 is involved in the acetylation of core histones, which is an important epigenetic event for transcriptional regulation through alterations in the chromatin structure in eukaryotes. To investigate physiological roles of GCN5, we have systematically analyzed phenotypes of homozygous GCN5-deficient DT40 mutants. Here, we report participation of GCN5 in regulation of IgM heavy chain (H-chain) gene expression. GCN5-deficiency down-regulates gene expressions of IgM H-chain (as whole, membrane-bound and secreted forms of its mRNA) but not light chain (L-chain), causing decreases in membrane-bound and secreted forms of IgM proteins. Chromatin immnoprecipitation assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region but not L-chain gene, and acetylate Lys-9 residues of histone H3 within chromatin surrounding the constant region. These results suggest that GCN5 takes part in transcriptional regulation of the IgM H-chain gene via histone acetylation resulting in formation of relaxed chromatin arrangement around its coding region and plays a key role in epigenetic regulation of B cell functions. PMID:24746634
Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo
Deregulated expression of fat and muscle genes in B-cell chronic lymphocytic leukemia with high lipoprotein lipase expression
Lipoprotein lipase (LPL) is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) related to immunoglobulin VH gene (IgVH)mutational status. We determined gene expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high (æLPL+Æ, n=10) or low (æLPL?Æ, n=10) LPL mRNA expression. Selected genes were verified by real-time PCR in an extended patient cohort (n=42).
M Bilban; D Heintel; T Scharl; T Woelfel; M M Auer; E Porpaczy; B Kainz; A Kr÷ber; V J Carey; M Shehata; C Zielinski; W Pickl; S Stilgenbauer; A Gaiger; O Wagner; U Jõger
The DG75 B-cell lymphoma line exhibits biclonal immunoglobulin gene rearrangement
Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement (GR) studies have been successfully employed to investigate the clonality and cell lineage of various lymphoid malignancies. Several lymphoma cell lines, such as BJAB, RAJI, DG75 and Jurkat cell lines, were often used as the positive controls in GR detection assays. Of those, the DG75 B-cell lymphoma line was found to exhibit biclonality [two or more homoduplex and heteroduplex bands in a polymerase chain reaction (PCR) product of clonality assay] in the PCR of GR detection assays. To further explore these characteristics of the biclonal phenomenon, the PCR products were purified and cloned into a pEGM-T clone vector. The sequences were analyzed using DNA analysis software. The results demonstrated that the two bands originated from two forms of GR of DG75 cell lines, i.e., DG75 is a biclonal cell line in Ig GRs, which has not been reported before.
Differential expression of VH gene families in peripheral B cell repertoires of newborn or adult immunoglobulin H chain congenic mice
The pattern of VH gene family expression in the primary B cell repertoire of the mouse is strain dependent. In C57Bl/6 mice, the VH J558 family is expressed by more than 45% of the cells, while the expression of VH 7183, VH Q52, and VH 36-60 families together does not exceed 20%. In BALB/c mice, relative expression of VH J558 is lower than 35%, while the sum of the other three families reaches 25%. To assess which genetic loci control strain-specific VH gene family expression, we studied VH gene family usage in splenic B cell repertoires of different congenic strains of mice. Changes in major histocompatibility complex or immunoglobulin (Ig) K light chain genes did not modify VH gene family expression in adult mice. Differences at the IgH locus, however, modified VH gene family usage. In 1-d-old mice, the strain-specific VH gene family expression pattern is determined by the IgH haplotype. In adult mice, the VH gene family expression pattern of resting B cells is independent of the IgH locus and follows the genetic background of the congenic strain, while it is determined by the IgH haplotype among Ig-secreting spleen cells. In F1(B6 x BALB/c) mice, each of the two spleen B cell populations, sorted on the basis of mu heavy chain allotype expression, shows an independent VH gene family expression pattern, determined by the IgH locus. The implications of these results in the control of VH gene family expression, and in the selection of peripheral B cell repertoires are discussed.
Mutation mismatch repair gene deletions in diffuse large B-cell lymphoma.
To further unravel the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL), we performed high-resolution comparative genomic hybridization on lymph node biopsies from 70 patients. With this strategy, we identified microdeletions of genes involved in the mutation mismatch repair (MMR) pathway in two samples. The first patient presented with a homozygous deletion of MSH2-MSH6 due to duplication of an unbalanced pericentric inversion of chromosome 2. The other case showed a PMS2 heterozygous deletion. PMS2 and MSH2-MSH6 abnormalities, respectively, resulted in a decrease and complete loss of gene expression. However, unlike tumors associated with the hereditary non-polyposis colorectal cancer syndrome or immunodeficiency-related lymphomas, no microsatellite instability was detected. Mutational profiles revealed especially in one patient an aberrant hypermutation without a clear activation-induced cytidine deaminase signature, indicating a breakdown of the high-fidelity repair in favor of the error-prone repair pathway. Our findings suggest that in a rare subset of patients, inactivation of the genes of the MMR pathway is likely an important step in the molecular pathogenesis of DLBCL and does not involve the same molecular mechanisms as other common neoplasms with MMR deficiency. PMID:23066952
Couronnķ, Lucile; Ruminy, Philippe; Waultier-Rascalou, Agathe; Rainville, Vinciane; Cornic, Marie; Picquenot, Jean-Michel; Figeac, Martin; Bastard, Christian; Tilly, Hervķ; Jardin, Fabrice
Cloning of cDNAs of the MLL gene that detect DNA rearrangements and altered RNA transcripts in human leukemic cells with 11q23 translocations.
Recurring chromosomal abnormalities involving translocations at chromosome 11 band q23 are associated with human myeloid and lymphoid leukemia as well as lymphoma. We have identified the gene located at this break-point and have named it MLL (for myeloid-lymphoid, or mixed-lineage, leukemia). The t(4;11), t(6;11), t(9;11), and t(11;19) are among the most common reciprocal translocations in leukemia cells involving this chromosomal band. We now have evidence that the breakpoints in all of these translocations are clustered within a 9-kilobase (kb) BamHI genomic region of the MLL gene. By Southern blot hybridization using a 0.7-kb BamHI cDNA fragment of the MLL gene called MLL 0.7B, we have detected rearrangements of DNA from cell lines and patient material with an 11q23 translocation in this region. Northern blot analyses indicate that this gene has multiple transcripts, some of which appear to be lineage-specific. In normal pre-B cells, four transcripts of 12.5, 12.0, 11.5, and 2.0 kb are detected. These transcripts are also present in monocytoid cell lines with additional hybridization to a 5.0-kb transcript, indicating that expression of different-sized MLL transcripts may be associated with normal hematopoietic lineage development. In a cell line with a t(4;11), the expression of the 12.5-, 12.0-, and 11.5-kb transcripts is reduced, and there is evidence of three other altered transcripts of 11.5, 11.25, and 11.0 kb. Thus, these 11q23 translocations result in rearrangements of the MLL gene and may lead to altered function(s) of MLL and of other gene(s) involved in the translocation. Images
McCabe, N R; Burnett, R C; Gill, H J; Thirman, M J; Mbangkollo, D; Kipiniak, M; van Melle, E; Ziemin-van der Poel, S; Rowley, J D; Diaz, M O
Gene Expression Profiling of the Response to Interferon Beta in Epstein-Barr-Transformed and Primary B Cells of Patients with Multiple Sclerosis
The effects of interferon-beta (IFN-?), one of the key immunotherapies used in multiple sclerosis (MS), on peripheral blood leukocytes and T cells have been extensively studied. B cells are a less abundant leukocyte type, and accordingly less is known about the B cell-specific response to IFN-?. To identify gene expression changes and pathways induced by IFN-? in B cells, we studied the in vitro response of human Epstein Barr-transformed B cells (lymphoblast cell lines-LCLs), and validated our results in primary B cells. LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-? exposure, with over 50 previously unrecognized as IFN-? response genes. Pathways analysis demonstrated that IFN-? affected LCLs in a similar manner to other cell types by activating known IFN-? canonical pathways. Additionally, IFN-? increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Novel response genes identified herein, NEXN, DDX60L, IGFBP4, and HAPLN3, B cell receptor pathway genes, CD79B and SYK, and lymphocyte activation genes, LAG3 and IL27RA, were validated as IFN-? response genes in primary B cells. In this study new IFN-? response genes were identified in B cells, with possible implications to B cell-specific functions. The study's results emphasize the applicability of LCLs for studies of human B cell drug response. The usage of LCLs from patient-based repositories may facilitate future studies of drug response in MS and other immune-mediated disorders with a B cell component.
Khsheibun, Rana; Paperna, Tamar; Volkowich, Anat; Lejbkowicz, Izabella; Avidan, Nili; Miller, Ariel
The X-linked lymphoproliferative syndrome gene product SAP regulates B cell function through the FcgammaRIIB receptor.
X-linked lympho-proliferative (XLP) is an immunodeficiency condition caused by mutation or deletion of the gene encoding the adaptor protein SAP/SH2D1A. Besides defects in T cell and NK cell function and an absence of NKT cells, XLP can also manifest as lymphomas resulting primarily from uncontrolled B cell proliferation upon acute infection by Epstein-Barr virus. While it has been demonstrated that SAP regulates the functions of T cells and NK cells through the SLAM family of immunoreceptors, its role in B cells has not been defined. Here we show that SAP forms a ternary complex with the kinase Lyn and the inhibitory IgG Fc receptor FcgammaRIIB to regulate B cell proliferation and survival. SAP binds directly and simultaneously to the Lyn SH3 domain and an Immuno-receptor Tyrosine-based Inhibitory Motif (ITIM) in FcgammaRIIB, resulting in the activation of the latter. Moreover, SAP associates with FcgammaRIIB in mouse splenic B cells and promotes its tyrosine phosphorylation. Expression of SAP in the A20 B cell line led to a marked reduction in Blnk phosphorylation, a decrease in Akt activation, and a near-complete ablation of phosphorylation of the MAP kinases Erk1/2, p38 and JNK upon colligation of FcgammaRIIB with the B cell receptor (BCR). In contrast, an XLP-causing SAP mutant was much less efficient in eliciting these effects in B cells. Furthermore, compared to A20 cells, SAP transfectants displayed a significantly reduced rate of proliferation and an increased sensitivity to activation-induced cell death. Collectively these data identify an intrinsic function for SAP in inhibitory signaling in B cells and suggests that SAP may play an important role in balancing positive versus negative immune responses. PMID:18662772
Li, Chengjun; Chung, Brian; Tao, Jianping; Iosef, Cristiana; Aoukaty, Ala; Wang, Yefu; Tan, Rusung; Li, Shawn Shun-Cheng
Insights into Gene Expression Changes Impacting B-Cell Transformation: Cross-Species Microarray Analysis of Bovine Leukemia Virus Tax-Responsive Genes in Ovine B Cells
Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor
Pavel Klener; Maud Szynal; Yvette Cleuter; Makram Merimi; Hugues Duvillier; Francoise Lallemand; Claude Bagnis; Philip Griebel; Christos Sotiriou; Arsene Burny; Philippe Martiat; Anne Van den Broeke
A Unified 35Gene Signature for both Subtype Classification and Survival Prediction in Diffuse Large B-Cell Lymphomas
Cancer subtype classification and survival prediction both relate directly to patients' specific treatment plans, making them fundamental medical issues. Although the two factors are interrelated learning problems, most studies tackle each separately. In this paper, expression levels of genes are used for both cancer subtype classification and survival prediction. We considered 350 diffuse large B-cell lymphoma (DLBCL) subjects, taken from
Yu-Dong Cai; Tao Huang; Kai-Yan Feng; Lele Hu; Lu Xie; Vladimir N. Uversky
Global Gene Expression Profiling of Bovine Immature B Cells using Serial Analysis of Gene Expression
The Peyer's patches of the small intestine are important sites of antigen processing. The follicles of the Peyer's patches receive the antigen transported by the M cells at the mucosal surface and then play a major role in development of both protective humoral and mucosal immune responses. Serial analysis of gene expression (SAGE) was employed to derive the global gene
John D. Neill; Julia F. Ridpath; Elizabeth Liebler-Tenorio
Genetic and Physical Interaction of the B-Cell SLE-Associated Genes BANK1 and BLK
Objectives Altered signaling in B-cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signaling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterize the role of BANK1 and BLK in SLE, we performed a genetic interaction analysis hypothesizing that genetic interactions could reveal functional pathways relevant to disease pathogenesis. Methods We Used the method GPAT16 to analyze the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localization, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK. Results Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from Northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, we tested the possibility that BANK1 and BLK could also show a protein-protein interaction. We demonstrated co-immunoprecipitation and co-localization of BLK and BANK1. In a Daudi cell line and primary na've B-cells the endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies. Conclusions Here, we show a genetic interaction between BANK1 and BLK, and demonstrate that these molecules interact physically. Our results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signaling pathway.
Castillejo-Lopez, Casimiro; Delgado-Vega, Angelica M.; Wojcik, Jerome; Kozyrev, Sergey V.; Thavathiru, Elangovan; Wu, Ying-Yu; Sanchez, Elena; Pollmann, David; Lopez-Egido, Juan R.; Fineschi, Serena; Dominguez, Nicolas; Lu, Rufei; James, Judith A.; Merrill, Joan T.; Kelly, Jennifer A.; Kaufman, Kenneth M.; Moser, Kathy; Gilkeson, Gary; Frostegard, Johan; Pons-Estel, Bernardo A.; D'Alfonso, Sandra; Witte, Torsten; Callejas, Jose Luis; Harley, John B.; Gaffney, Patrick; Martin, Javier; Guthridge, Joel M.; Alarcon-Riquelme, Marta E.
Transcription of HLA class II genes in the absence of B-cell-specific octamer-binding factor.
HLA-DR and other human class II histocompatibility genes are expressed by Epstein-Barr virus-transformed B-lymphocyte cell lines but not by most T-cell leukemia lines. We determined by transcriptional run-on experiments that regulation of class II expression in these cells is at the level of gene transcription; nuclei isolated from B-cell lines actively transcribe class II mRNA, whereas nuclei from non-class II-expressing T-cell lines and from the class II transactive factor-deficient B-cell mutant 6.1.6 do not. In searching for DNA-binding proteins which might regulate transcription, we found both a ubiquitous (B1) and a B-cell-specific (B2) factor which bind to the octamer sequence ATTTGCAT 52 base pairs 5' of the cap site in the DR alpha gene. We examined the relationship of these factors to DR alpha transcription. HUT-78, a T-cell line which expresses class II mRNA constitutively, contains only the ubiquitous B1 octamer-binding factor also found in non-class II-expressing T-cell leukemias. Human fibroblast, HeLa, and melanoma cell lines similarly contain only the ubiquitous factor, even when these cells are induced to express class II mRNA by treatment with gamma interferon. Both B1 and B2 binding factors are present in the B-cell mutant 6.1.6, which nevertheless fails to transcribe class II mRNA. Although we have not ruled out the requirement of B-cell-specific octamer-binding factor B2 for class II expression in B cells, it is clear that in other cells substantial DR alpha transcription occurs in the absence of this factor. Images
Stimac, E; Lyons, S; Pious, D
Three distinct signals can induce class II gene expression in a murine pre-B-cell line.
Expression of class II genes of the major histocompatibility complex (MHC) has been studied in an Abelson-murine-leukemia-virus-transformed pre-B-cell line, R8, and its class II molecule (Ia)-negative variant, R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of a trans-acting factor(s) that can regulate class II gene expression. Furthermore, the T-cell-derived lymphokine B-cell-stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes. Images
Polla, B S; Poljak, A; Geier, S G; Nathenson, S G; Ohara, J; Paul, W E; Glimcher, L H
Gene-tagged chromosome translocations in eleven stocks of mice
SummaryĀĀThe cytogenetic problems of correlating linkage groups in the house mouse with their chromosomes, and of establishing the\\u000a chromosomal independence of known linkage groups, call for the use of numerous genetically tagged translocations. Eleven new\\u000a translocations have been induced and tagged; they involve linkage groups I, II, III, V, VIII, IX and XI.
T. G. Cartes; Mary F. Lyon; Rita J. S. Phillips
Gene therapy delivery of myelin oligodendrocyte glycoprotein (MOG) via hematopoietic stem cell transfer induces MOG-specific B cell deletion.
The various mechanisms that have been described for immune tolerance govern our ability to control self-reactivity and minimize autoimmunity. However, the capacity to genetically manipulate the immune system provides a powerful avenue to supplement this natural tolerance in an Ag-specific manner. We have previously shown in the mouse model of experimental autoimmune encephalomyelitis that transfer of bone marrow (BM) transduced with retrovirus encoding myelin oligodendrocyte glycoprotein (MOG) promotes disease resistance and CD4(+) T cell deletion within the thymus. However, the consequence of this strategy on B cell tolerance is not known. Using BM from IgH(MOG) mice that develop MOG-specific B cell receptors, we generated mixed chimeras together with BM-encoding MOG. In these animals, the development of MOG-specific B cells was abrogated, resulting in a lack of MOG-specific B cells in all B cell compartments examined. This finding adds a further dimension to our understanding of the mechanisms of tolerance that are associated with this gene therapy approach to treating autoimmunity and may have important implications for Ab-mediated autoimmune disorders. PMID:24532581
Chung, Jie-Yu; Figgett, William; Fairfax, Kirsten; Bernard, Claude; Chan, James; Toh, Ban-Hock; Mackay, Fabienne; Alderuccio, Frank
Combination of 3? and 5? IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model
To ensure the B cell differentiation stage specificity of the intronic E? element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a VH promoter-GFP reporter gene linked to the 3?LCR region and the E? element. By flow cytometry, GFP+ lymphocytes were observed amongst pro-B cells (B220+CD43+CD117+) and at
Laurence Guglielmi; Marc Le Bert; Isabelle Comte; Marie Laure Dessain; Mireille Drouet; Christiane Ayer-Le Lievre; Michel Cognķ; Yves Denizot
DNA methylation profiles in diffuse large B-cell lymphoma and their relationship to gene expression status
In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2, and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of sixty-seven genes proximal (within 500-bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1, and FLJ21062 expression with increasing methylation suggests that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.
Pike, Brian L.; Greiner, Timothy C.; Wang, Xiaoming; Weisenburger, Dennis D.; Hsu, Ya-Hsuan; Renaud, Gabriel; Wolfsberg, Tyra G.; Kim, Myungjin; Weisenberger, Daniel J.; Siegmund, Kimberly D.; Ye, Wei; Groshen, Susan; Mehrian-Shai, Ruty; Delabie, Jan; Chan, Wing C.; Laird, Peter W.; Hacia, Joseph G.
A unified 35-gene signature for both subtype classification and survival prediction in diffuse large B-cell lymphomas.
Cancer subtype classification and survival prediction both relate directly to patients' specific treatment plans, making them fundamental medical issues. Although the two factors are interrelated learning problems, most studies tackle each separately. In this paper, expression levels of genes are used for both cancer subtype classification and survival prediction. We considered 350 diffuse large B-cell lymphoma (DLBCL) subjects, taken from four groups of patients (activated B-cell-like subtype dead, activated B-cell-like subtype alive, germinal center B-cell-like subtype dead, and germinal center B-cell-like subtype alive). As classification features, we used 11,271 gene expression levels of each subject. The features were first ranked by mRMR (Maximum Relevance Minimum Redundancy) principle and further selected by IFS (Incremental Feature Selection) procedure. Thirty-five gene signatures were selected after the IFS procedure, and the patients were divided into the above mentioned four groups. These four groups were combined in different ways for subtype prediction and survival prediction, specifically, the activated versus the germinal center and the alive versus the dead. Subtype prediction accuracy of the 35-gene signature was 98.6%. We calculated cumulative survival time of high-risk group and low-risk groups by the Kaplan-Meier method. The log-rank test p-value was 5.98e-08. Our methodology provides a way to study subtype classification and survival prediction simultaneously. Our results suggest that for some diseases, especially cancer, subtype classification may be used to predict survival, and, conversely, survival prediction features may shed light on subtype features. PMID:20856936
Cai, Yu-Dong; Huang, Tao; Feng, Kai-Yan; Hu, Lele; Xie, Lu
Proof of the concept to use a malignant B cell line drug screen strategy for identification and weight of melphalan resistance genes in multiple myeloma.
In a conceptual study of drug resistance we have used a preclinical model of malignant B-cell lines by combining drug induced growth inhibition and gene expression profiling. In the current report a melphalan resistance profile of 19 genes were weighted by microarray data from the MRC Myeloma IX trial and time to progression following high dose melphalan, to generate an individual melphalan resistance index. The resistance index was subsequently validated in the HOVON65/GMMG-HD4 trial data set to prove the concept. Biologically, the assigned resistance indices were differentially distributed among translocations and cyclin D expression classes. Clinically, the 25% most melphalan resistant, the intermediate 50% and the 25% most sensitive patients had a median progression free survival of 18, 32 and 28 months, respectively (log-rank P-value ?=?0.05). Furthermore, the median overall survival was 45 months for the resistant group and not reached for the intermediate and sensitive groups (log-rank P-value ?=?0.003) following 38 months median observation. In a multivariate analysis, correcting for age, sex and ISS-staging, we found a high resistance index to be an independent variable associated with inferior progression free survival and overall survival. This study provides clinical proof of concept to use in vitro drug screen for identification of melphalan resistance gene signatures for future functional analysis. PMID:24376673
B°gsted, Martin; Bilgrau, Anders E; Wardell, Christopher P; Bertsch, Uta; Schmitz, Alexander; B°dker, Julie S; Kjeldsen, Malene K; Goldschmidt, Hartmut; Morgan, Gareth J; Dybkaer, Karen; Johnsen, Hans E
Proof of the Concept to Use a Malignant B Cell Line Drug Screen Strategy for Identification and Weight of Melphalan Resistance Genes in Multiple Myeloma
In a conceptual study of drug resistance we have used a preclinical model of malignant B-cell lines by combining drug induced growth inhibition and gene expression profiling. In the current report a melphalan resistance profile of 19 genes were weighted by microarray data from the MRC Myeloma IX trial and time to progression following high dose melphalan, to generate an individual melphalan resistance index. The resistance index was subsequently validated in the HOVON65/GMMG-HD4 trial data set to prove the concept. Biologically, the assigned resistance indices were differentially distributed among translocations and cyclin D expression classes. Clinically, the 25% most melphalan resistant, the intermediate 50% and the 25% most sensitive patients had a median progression free survival of 18, 32 and 28 months, respectively (log-rank P-value ?=?0.05). Furthermore, the median overall survival was 45 months for the resistant group and not reached for the intermediate and sensitive groups (log-rank P-value ?=?0.003) following 38 months median observation. In a multivariate analysis, correcting for age, sex and ISS-staging, we found a high resistance index to be an independent variable associated with inferior progression free survival and overall survival. This study provides clinical proof of concept to use in vitro drug screen for identification of melphalan resistance gene signatures for future functional analysis.
B?gsted, Martin; Bilgrau, Anders E.; Wardell, Christopher P.; Bertsch, Uta; Schmitz, Alexander; B?dker, Julie S.; Kjeldsen, Malene K.; Goldschmidt, Hartmut; Morgan, Gareth J.; Dybkaer, Karen; Johnsen, Hans E.
Regulation of chick early B-cell factor-1 gene expression in feather development.
The chick Ebf1 (early B-cell factor-1) gene is a member of a novel family of helix loop helix transcription factors. The expression profile, regulation and significance of this gene have been extensively studied in lymphatic, nervous, adipose and muscular tissues. However, cEbf1 expression, regulation and function in the feather of chick embryo have not yet been investigated. cEbf1 expression was first detected throughout the mesenchymal core of some few feather placodes (D7-D7.5). After feathers became mature and grew distally (D9 and D10), the mesenchymal expression of cEbf1 became confined to the caudal margin of the proximal half of all formed feather buds. Because this dynamic pattern of expression resembles that of Sonic Hedgehog (Shh) protein and bone morphogenetic protein (Bmp4) plus the crucial role of these two major signals in feather development, we hypothesized that cEbf1 expression in the feather may be regulated by Shh and Bmp4. In a feather explant culture system, Shh signals are necessary to initiate and maintain cEbf1 expression in the posterior half of the feather bud, while Bmp4 is crucial for the initial cEbf1 expression in the anterior half of the feather bud. Inhibition of Shh, not only down-regulates cEbf1, but also changes the morphology of feather buds, which become irregular and fused. This is the first study to demonstrate that cEbf1 expression in the feather bud is under the control of Shh and Bmp4 signals and that expression may play a role in the normal development of feathers. PMID:24365066
El-Magd, Mohammed Abu; Sayed-Ahmed, Ahmed; Awad, Ashraf; Shukry, Mustafa
Correlation between presence of clonal rearrangements of immunoglobulin heavy chain genes and B-cell antigen expression in Hodgkin's disease.
Southern blot analysis of Hodgkin's disease (HD), although often compromised by the small number of abnormal cells present in the tissue, have tended to favor a B-cell derivation of the Hodgkin's and Reed-Sternberg (HRS) cells in cases of nodular sclerosis (NS) and mixed cellularity (MC) Hodgkin's disease. Eighteen frozen and 29 paraffin-embedded sections of lymph node specimens from 29 patients with pretreatment HD (22 NSHD and 7 MCHD) were studied by molecular analysis and immunohistochemistry to determine the phenotype of HRS cells. All cases were reviewed and showed typical morphology and CD45-, CD30+, CD15+, BLA.36+ HRS cells. In 11 of 29 (38%) cases, HRS cells were reactive with at least one B-cell marker (CD20, CD79a, MB2), 7 of 29 (24%) cases showed reactivity with the T-cell marker CD3, and 11 of 29 (38%) cases displayed a "null" phenotype. By using a polymerase chain reaction (PCR) and consensus primers for the V and J regions of the immunoglobulin heavy chain (IgH) gene, the authors were able to detect B-cell clonality in 9 of 18 (50%) frozen samples of HD analyzed. IgH gene rearrangement was present in 8 of 15 (53%) NSHD and in 1 of 3 (33%) MCHD. In five of nine (56%) of these cases, HRS cells were reactive with at least one B-cell marker, whereas one case expressed the T-cell marker CD3. The other three cases with IgH gene rearrangement showed a "null" immunophenotype. IgH gene analysis was negative in all remaining CD3+ cases and in two other cases that expressed B-cell markers by immunohistology. Southern blotting failed to detect rearrangement of the T-cell receptor beta-chain gene and immunoglobulin heavy and light genes in any of these cases. The results show that PCR represents a specific and sensitive technique for the detection of IgH gene rearrangements in cases of Hodgkin's disease. The results also suggest a lymphoid B-cell derivation of HRS cells in a high proportion of the cases. PMID:7572791
Orazi, A; Jiang, B; Lee, C H; English, G W; Cattoretti, G; John, K; Neiman, R S
Protein kinase C? gene expression is oppositely regulated by GCN5 and EBF1 in immature B cells.
In this study, we revealed that GCN5 and early B cell factor 1 (EBF1) participate in regulation of protein kinase C? (PKC?) gene expression in an opposite manner in immature B cells. GCN5-deficiency in DT40 caused drastic down-regulation of transcription of PKC?. In contrast, EBF1-deficiency brought about remarkable up-regulation of that of PKC?, and re-expression of EBF1 dramatically suppressed transcription of PKC?. Chromatin immunoprecipitation assay revealed that GCN5 binds to the 5'-flanking region of the chicken PKC? gene and acetylates histone H3, and EBF1 binds to the 5'-flanking region of the gene surrounding putative EBF1 binding motifs. PMID:24657615
Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo
Helper and killer T cells do not express B cell immunoglobulin joining and constant region gene segments
We have analyzed four kinds of T cells for rearrangement and expression of immunoglobulin genes. These cells include: (a) whole thymus; (b) WEHI-22, a T-cell lymphoma; (c) HT-1, an major histocompatibility complex-restricted T helper line; and (d) CTLLi6, an H-2 alloreactive killer cell line. None of the B-cell joining and constant gene segments are rearranged in the T cells. The monoclonal cells do not express any C kappa, C lambda, Cmu or C alpha RNA species. Small amounts of C kappa, C alpha, and Cmu sequences are present in RNA prepared from the thymus, although the significance of this RNA for T-cell antigen receptor synthesis is uncertain. The data support the hypothesis that expression of B-cell joining and C gene segments is unnecessary for T- cell helper and T-cell killer activity.
T cells from indolent CLL patients prevent apoptosis of leukemic B cells in vitro and have altered gene expression profile.
T cells may have a role in sustaining the leukemic clone in chronic lymphocytic leukemia (CLL). In this study, we have examined the ability of T cells from CLL patients to support the survival of the leukemic B cells in vitro. Additionally, we compared global gene expression of T cells from indolent CLL patients with healthy individuals and multiple myeloma (MM) patients. Apoptosis of purified leukemic B cells was inhibited in vitro when co-cultured with increasing numbers of autologous T cells (p < 0.01) but not autologous B and T cells of normal donors. The anti-apoptotic effect exceeded that of the anti-apoptotic cytokine IL-4 (p = 0.002) and was greater with CD8+ cells (p = 0.02) than with CD4+ cells (p = 0.05). The effect was depended mainly on cell-cell contact although a significant effect was also observed in transwell experiments (p = 0.05). About 356 genes involved in different cellular pathways were deregulated in T cells of CLL patients compared to healthy individuals and MM patients. The results of gene expression profiling were verified for 6 genes (CCL4, CCL5 (RANTES), XCL1, XCL2, KLF6, and TRAF1) using qRT-PCR and immunoblotting. Our results demonstrate that CLL-derived T cells can prevent apoptosis of leukemic B cells and have altered expression of genes that may facilitate the survival of the leukemic clone. PMID:22736254
Kiaii, Shahryar; Kokhaei, Parviz; Mozaffari, Fariba; Rossmann, Eva; Pak, Fatemeh; Moshfegh, Ali; Palma, Marzia; Hansson, Lotta; Mashayekhi, Kaveh; Hojjat-Farsangi, Mohammad; ųsterborg, Anders; Choudhury, Aniruddha; Mellstedt, HÕkan
Transgenic mice bearing the human c-myc gene activated by an immunoglobulin enhancer: A pre-B-cell lymphoma model
Transgenic mice carrying a fusion gene in which the mouse immunoglobulin enhancer has been inserted into an otherwise normal human c-myc gene develop a narrow spectrum of pre-B-cell lymphomas. Tumor occurrence is correlated with expression of the transgene in organs in which large numbers of pre-B cells predominate. These tumors, which arise stochastically, are virtually all lymphoblastic lymphomas of the pre-B-cell type. Evidently the isolated enhancer targets oncogene expression and tumorigenesis to the early B-cell population in preference to more mature B-cell populations. The transgene also confers enhanced in vitro growth properties on nontransformed pre-B cells as observed in bone marrow cultures derived from transgenic animals. These cultured cells represent a population in which the activating function of c-myc can be uncoupled from secondary oncogenic events occurring in vivo.
Schmidt, E.V.; Pattengale, P.K.; Weir, L.; Leder, P. (Harvard Medical School, Boston, MA (USA))
Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma
The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H)- and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a
Robert Kleinfield; Richard R. Hardy; David Tarlinton; Jeffery Dangl; Leonard A. Herzenberg; Martin Weigert
Germline translocations in mice: unique tools for analyzing gene function and long-distance regulatory mechanisms.
Translocations have provided invaluable tools for identifying both cancer-linked genes and loci associated with heritable human diseases, but heritable human translocations are rare and few mouse models exist. Here we report progress on analysis of a collection of heritable translocations generated by treatment of mice with specific chemicals or radiation during late spermatogenic stages. The translocation mutants exhibit a range of visible phenotypes reflecting the disruption of coding sequences or the separation of genes from essential regulatory elements. The breakpoints of both radiation-induced and chemically induced mutations in these mice are remarkably clean, with very short deletions, duplications, or inversions in some cases, and ligation mediated by microhomology, suggesting nonhomologous end joining as the major path of repair. These mutations provide new tools for the discovery of novel genes and regulatory elements linked to human developmental disorders and new clues to the molecular basis of human genetic disease. PMID:18648012
Elso, Colleen; Lu, Xiaochen; Morrison, Stephanie; Tarver, Angela; Thompson, Heather; Thurkow, Hillary; Yamada, N Alice; Stubbs, Lisa
Mechanisms underlying B-cell tolerance induction by antigen-immunoglobulin G gene transfer.
Previous studies on the mechanisms underlying tolerance induction in diabetes have mainly focused on T cells, however B cells also have an important role in diabetes. Based on our previous studies that splenocytes, transduced with glutamic acid decarboxylase (GAD) 65 fused to immunoglobulin (Ig) G carrier, reduced antibody-mediated response in non-obese diabetic (NOD) mice, here we examined the mechanisms underlying B-cell tolerance in this system. We found that GAD-IgG-transduced splenocytes did not reduce CD40 expression on B-cells in NOD mice, but they did downregulate CD40 ligand (CD40L) expression. Furthermore, anti-CD40L injection reduced autoantibody levels in NOD mice and in vitro experiments demonstrated that CD40L blockade reduced the antigenpresenting capability of B-cells. In conclusion, the results of this study suggest that downregulation of CD40L may be one mechanism underlying the induction of B-cell tolerance in GAD-IgG-treated NOD mice. PMID:18034991
Wang, R; Wang, J; Han, G; Song, L; Chen, G; Xu, R; Yu, M; Qian, J; Shen, B; Li, Y
A global DNA methylation and gene expression analysis of early human B-cell development reveals a demethylation signature and transcription factor network
The epigenetic changes during B-cell development relevant to both normal function and hematologic malignancy are incompletely understood. We examined DNA methylation and RNA expression status during early B-cell development by sorting multiple replicates of four separate stages of pre-B cells derived from normal human fetal bone marrow and applied high-dimension DNA methylation scanning and expression arrays. Features of promoter and gene body DNA methylation were strongly correlated with RNA expression in multipotent progenitors (MPPs) both in a static state and throughout differentiation. As MPPs commit to pre-B cells, a predominantly demethylating phenotype ensues, with 79% of the 2966 differentially methylated regions observed involving demethylation. Demethylation events were more often gene body associated rather than promoter associated; predominantly located outside of CpG islands; and closely associated with EBF1, E2F, PAX5 and other functional transcription factor (TF) sites related to B-cell development. Such demethylation events were accompanied by TF occupancy. After commitment, DNA methylation changes appeared to play a smaller role in B-cell development. We identified a distinct development-dependent demethylation signature which has gene expression regulatory properties for pre-B cells, and provide a catalog reference for the epigenetic changes that occur in pre-B-cell leukemia and other B-cell-related diseases.
Lee, Seung-Tae; Xiao, Yuanyuan; Muench, Marcus O.; Xiao, Jianqiao; Fomin, Marina E.; Wiencke, John K.; Zheng, Shichun; Dou, Xiaoqin; de Smith, Adam; Chokkalingam, Anand; Buffler, Patricia; Ma, Xiaomei; Wiemels, Joseph L.
High Frequency of Monoallelic Retinoblastoma Gene Deletion in B-Cell Chronic Lymphoid Leukemia Shown by Interphase Cytogenetics
Inactivation of the retinoblastoma tumor-suppressor gene (RB-I ) has been associated with tumorigenicity in various human malignancies. In chronic lymphoid leukemias of B- cell origin (B-CLL) an involvement of RB-1 has been sug- gested based on cytogenetic data. We examined RB-1 and its chromosomal locus 13ql4 in 35 cases of B-CLL by dual- color in situ hybridization to interphase nuclei
Stephan Stilgenbauer; Hartmut Dohner; Mehtap Bulgay-Morschel; Sandra Weitz; Martin Bentz; Peter Lichter
The X-linked lymphoproliferative syndrome gene product SAP regulates B cell function through the Fc?RIIB receptor
X-linked lympho-proliferative (XLP) is an immunodeficiency condition caused by mutation or deletion of the gene encoding the adaptor protein SAP\\/SH2D1A. Besides defects in T cell and NK cell function and an absence of NKT cells, XLP can also manifest as lymphomas resulting primarily from uncontrolled B cell proliferation upon acute infection by Epstein¢Barr virus. While it has been demonstrated that
Chengjun Li; Brian Chung; Jianping Tao; Cristiana Iosef; Ala Aoukaty; Yefu Wang; Rusung Tan; Shawn Shun-Cheng Li
Molecular mechanisms and selective influences that shape the kappa gene repertoire of IgM+ B cells.
To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified Vkappa Jkappa rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive Vkappa Jkappa rearrangements were sequenced. Nearly every functional Vkappa gene segment was used in rearrangements, although six Vkappa gene segments, A27, L2, L6, L12a, A17, and O12/O2 were used preferentially. Of these, A27, L2, L6, and L12a showed evidence of positive selection based on the variable region and not CDR3, whereas A17 was overrepresented because of a rearrangement bias based on molecular mechanisms. Utilization of Jkappa segments was also nonrandom, with Jkappa1 and Jkappa2 being overrepresented and Jkappa3 and Jkappa5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two Vkappa Jkappa rearrangements, marked differences were noted in the Vkappa segments used for the initial and subsequent rearrangements, whereas Jkappa segments were used comparably. Junctional diversity was generated by n-nucleotide addition in 60% and by exonuclease trimming in 75% of the Vkappa Jkappa rearrangements analyzed. Despite this large degree of diversity, a strict CDR3 length was maintained in both productive and nonproductive rearrangements. More than 23% of the productive rearrangements, but only 7% of the nonproductive rearrangements contained somatic hypermutations. Mutations were significantly more frequent in Vkappa sequences derived from CD5- as compared with CD5+ B cells. These results document that the gene segment utilization within the Vkappa repertoire is biased by both intrinsic molecular processes as well as selection after light chain expression. Moreover, IgM+ memory cells with highly mutated kappa genes reside within the CD5- but not the CD5+ B cell compartment.
Foster, S J; Brezinschek, H P; Brezinschek, R I; Lipsky, P E
AID-induced remodeling of immunoglobulin genes and B cell fate
Survival and phenotype of normal and malignant B lymphocytes are critically dependent on constitutive signals by the B cell receptor (BCR) for antigen. In addition, either antigen ligation of the BCR or various mitogenic stimuli result in B cell activation and induction of activation-induced deaminase (AID). AID activity can in turn mediate somatic hypermutation (SHM) of immunoglobulin (Ig) V regions and also deeply remodel the Ig heavy chain locus through class switch recombination (CSR) or locus suicide recombination (LSR). In addition to changes linked to affinity for antigen, modifying the class/isotype (i.e. the structure and function) of the BCR or suddenly deleting BCR expression also modulates the fate of antigen-experienced B cells.
Laffleur, Brice; Denis-Lagache, Nicolas; Peron, Sophie; Sirac, Christophe; Moreau, Jeanne; Cogne, Michel
AID-induced remodeling of immunoglobulin genes and B cell fate.
Survival and phenotype of normal and malignant B lymphocytes are critically dependent on constitutive signals by the B cell receptor (BCR) for antigen. In addition, either antigen ligation of the BCR or various mitogenic stimuli result in B cell activation and induction of activation-induced deaminase (AID). AID activity can in turn mediate somatic hypermutation (SHM) of immunoglobulin (Ig) V regions and also deeply remodel the Ig heavy chain locus through class switch recombination (CSR) or locus suicide recombination (LSR). In addition to changes linked to affinity for antigen, modifying the class/isotype (i.e. the structure and function) of the BCR or suddenly deleting BCR expression also modulates the fate of antigen-experienced B cells. PMID:24851241
Laffleur, Brice; Denis-Lagache, Nicolas; Pķron, Sophie; Sirac, Christophe; Moreau, Jeanne; Cognķ, Michel
CD10-MUM1+ follicular lymphoma lacks BCL2 gene translocation and shows characteristic biologic and clinical features.
CD10 and MUM1 are representative B cell differentiation markers. Follicular lymphoma (FL) is usually positive for CD10 and negative for MUM1. In this study, however, we compared 22 FLs with peculiar phenotype CD10-MUM1+ with 119 typical CD10+MUM1- FLs. All CD10-MUM1+ FL patients exhibited follicular structure with follicular dendritic meshwork, and a high rate of somatic hypermutation and ongoing mutation, similar to typical FL. However, CD10-MUM1+ FLs were encountered frequently in the elderly compared with CD10+MUM1- typical FLs (67.0 versus 58.7 years, P < .01), showed high grade (grade 3A or 3B) morphology (91% versus 17%, P < .001), diffuse proliferation (59% vs 19%, P < .001), and lacked BCL2/IGH translocation (5% versus 92.5%, P < .001), which is the most characteristic aberration in FL, and 88% showed BCL6 gene abnormalities (translocation or amplification). Our results indicate that CD10-MUM1+ FL is different from typical FL with respect to biologic and clinical features. PMID:17138820
Karube, Kennosuke; Guo, Ying; Suzumiya, Junji; Sugita, Yasuo; Nomura, Yuko; Yamamoto, Kohei; Shimizu, Kei; Yoshida, Shirou; Komatani, Hideki; Takeshita, Morishige; Kikuchi, Masahiro; Nakamura, Naoya; Takasu, Osamu; Arakawa, Fumiko; Tagawa, Hiroyuki; Seto, Masao; Ohshima, Koichi
Immunization and Infection Change the Number of Recombination Activating Gene (RAG)-expressing B Cells in the Periphery by Altering Immature Lymphocyte Production
Recombination activating gene ( RAG ) expression in peripheral B cells increases after immuni- zation with (4-hydroxy-3-nitrophenyl) acetyl coupled to chicken gamma globulin (NP-CGG) in alum. This increase could result from reinduction of RAG expression or, alternatively, from accumulation of RAG -expressing immature B cells in the periphery. We have used mice that carry a green fluorescent protein (GFP) RAG
Hitoshi Nagaoka; Gloria Gonzalez-Aseguinolaza; Moriya Tsuji; Michel C. Nussenzweig
Immune tolerance induction to factor IX through B cell gene transfer: TLR9 signaling delineates between tolerogenic and immunogenic B cells.
A subset of patients with severe hemophilia B, the X-linked bleeding disorder resulting from absence of coagulation factor IX (FIX), develop pathogenic antibody responses during replacement therapy. These inhibitors block standard therapy and are often associated with anaphylactic reactions to FIX. Established clinical immune tolerance induction protocols often fail for FIX inhibitors. In a murine model of this immune complication, retrovirally transduced primary B cells expressing FIX antigen fused with immunoglobulin-G heavy chain prevented antibody formation to FIX and was also highly effective in desensitizing animals with preexisting response. In contrast, transplant of B cells that received the identical expression cassette via nucleofection of plasmid vector substantially heightened antibody formation against FIX, a response that could be blocked by toll-like receptor 9 (TLR9) inhibition. While innate responses to TLR4 activation or to retrovirus were minimal in B cells, plasmid DNA activated TLR9, resulting in CpG-dependent NF-?B activation/IL-6 expression and adaptor protein 3 dependent, CpG-independent induction of IFN-I. Neither response was seen in TLR9-deficient B cells. Therefore, TLR9 signaling in B cells, in particular in response to plasmid vector, is highly immunogenic and has to be avoided in design of tolerance protocols. PMID:24609143
Wang, Xiaomei; Moghimi, Babak; Zolotukhin, Irene; Morel, Laurence M; Cao, Ou; Herzog, Roland W
PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells
Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylationin-dependent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.
Bougel, Stephanie; Renaud, Stephanie; Braunschweig, Richard; Loukinov, Dmitri; Morse, Herbert C; Bosman, Fred T.; Lobanenkov, Victor; Benhattar, Jean
Diffuse large B-cell lymphoma outcome prediction by gene- expression profiling and supervised machine learning
Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy in adults, is curable in less than 50% of patients. Prognostic models based on pre-treatment characteristics, such as the International Prognostic Index (IPI), are currently used to predict outcome in DLBCL. However, clinical outcome models identify neither the molecular basis of clinical heterogeneity, nor specific therapeutic targets. We analyzed the
An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion
The Ig heavy (H) chain plays a pivotal role in the regulation of primary B cell development through its association with a variety of other proteins including Iga and Igb, the surrogate light chain components and bona fide L chains, to form transmembrane signaling complexes. Little is known about how alterations in the structure of the H chain variable region
Lynn Heltemes-Harris; Xiaohe Liu; Tim Manser
Profiling of Hodgkin's Lymphoma Cell Line L1236 and Germinal Center B Cells: Identification of Hodgkin's Lymphoma-specific Genes
The malignant cells of classical HodgkinÆs lymphoma (cHL), Hodgkin and Reed-Sternberg (HRS) cells, appear to be derived from germinal center (GC) B cells in most cases of the disease. Apart from recent findings of constitutive activation of some transcription factors and autocrine stimulation by cytokine receptors, the mechanisms of malignant transformation in cHL still remain poorly understood. We performed a large scale gene expression study using serial analysis of gene expression (SAGE), comparing the cHL cell line L1236 and human GC B cells. Semiquantitative RT-PCR was used to confirm results from the SAGE and to analyze gene expression in 3 additional cHL cell lines. To investigate expression of some genes in cHL cases, we applied RT-PCR on microdissected HRS cells. In total, 464 genes showed a change in expression level of 5-fold or higher. For 12 genes (out of 177) identified as upregulated in L1236 cells, RT-PCR confirmed the SAGE results and also showed elevated expression in 3 other cHL cell lines. For 3 of the upregulated genes, expression by HRS cells in the tissue also was confirmed. Several of the differentially expressed genes may play a role in the pathogenesis of cHL because they represent potential oncogenes, such as rhoC, L-myc, and PTP4A, or transcription factors, such as ATF-5, ATBF1, and p21SNFT. The genes that showed significantly deregulated expression in HRS cells should be helpful not only for the identification of genes involved in the pathogenesis of cHL but also for discovering potential prognostic markers or therapeutic targets.
Schwering, Ines; Brauninger, Andreas; Distler, Verena; Jesdinsky, Julia; Diehl, Volker; Hansmann, Martin-Leo; Rajewsky, Klaus; Kuppers, Ralf
Distinct gene expression signature in Btk-defective T1 B-cells
BrutonÆs tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. Mutations in Btk give rise to the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (Xid) in mice. Recent studies have subdivided the mouse immature, or transitional, B-cells into two distinct subsets according to their respective surface markers. Transitional type 1 (T1) and transitional
Jessica M. Lindvall; K. Emelie M. Blomberg; Anna Bergl÷f; C. I. Edvard Smith
p53 Gene Mutation in B-Cell Chronic Lymphocytic Leukemia Is Associated With Drug Resistance and Is Independent of MDRl\\/MDR3 Gene Expression
We studied 53 patients with B-cell chronic lymphocytic leukemia (B-CLL) and found mutations of the p53 gene in 15%. Patients with p53 gene mutations were found to have an aggressive form of B-CLL disease characterized by advanced Rai stage, rapid lymphocyte doubling time (LDT), and resistance to chemotherapy. While 27 of 29 treated patients (93%) without p53 mutations achieved a
Soumaya El Rouby; Anju Thomas; Dan Costin; Carl R. Rosenberg; Milan Potmesil; Robert Silber; Elizabeth W. Newcomb
Coordinated Expression of MicroRNA-155 and Predicted Target Genes in Diffuse Large B-cell Lymphoma
MicroRNAs (miRNAs) attenuate gene expression by pairing to the 3?UTR of target transcripts inducing RNA cleavage or translational inhibition. Overexpression of microRNA-155 (miR-155), measured either at the primary (BIC gene) or mature transcript level, was recently described in diffuse large B-cell lymphomas (DLBCL). However, these studies have been limited in size and have not attempt to link miR-155 expression to that of putative target genes. To start to address these issues we examined a collection of 22 well-characterized DLBCL cell lines. The expression of miR-155 is heterogeneous in these cell lines and associates with NF-?B activity. Importantly, we found that the expression of the primary miR-155 transcript reliably reflects that of the functional mature miR-155. Since many gene array platforms include probe sets for the primary miR-155 sequences, these findings allowed us to confidently examine large array-based expression datasets of primary DLBCLs in the context of miR-155 levels. Our investigation revealed that that miR-155 expression segregates with specific molecular subgroups of DLBCL and it is highest in the Activated B-cell (ABC)-type lymphomas. These findings were particularly relevant because these tumors are characterized by constitutive activation of NF-?B signals supporting the data derived from our cell lines. More importantly, using supervised learning algorithms, we identified a robust gene signature driven by the differential expression of miR-155. These profiles contained several gene markers, including predicted targets, consistently downregulated in tumors expressing the high levels of miR-155. Our data start to unveil the genome wide effects of miR-155 expression in DLBCL and indicate the utility of this strategy in the identification and validation of miRNA target genes.
Rai, Deepak; Karanti, Shailaja; Jung, Inkyung; Dahia, Patricia L.M.; Aguiar, Ricardo C.T.
Sucking genes into pores: insight into driven translocation.
Flexible polymers such as long DNA, RNA molecules, and proteins, can pass through a narrow pore whose size is comparable to their molecular thickness. We highlight the richness and complexity involved in the dynamics of this unique mode of molecular transport, called translocation, actively driven by external forces. In particular, the process takes place in the condition far from equilibrium accompanying of large conformational distortion in line with the propagation of the tensile force along the chain backbone. A general framework is proposed, which captures such essential features, whereby can account for reported various experimental data from a unified viewpoint. PMID:20481745
Sakaue, Takahiro
Epstein - Barr Virus Transforming Protein LMP-1 Alters B Cells Gene Expression by Promoting Accumulation of the Oncoprotein ?Np73?
Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ?Np73?, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ?Np73? promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ?Np73? mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ?Np73? accumulation. The recruitment of p73 to the ?Np73? promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ?Np73? expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ?Np73? down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.
Accardi, Rosita; Fathallah, Ikbal; Gruffat, Henri; Mariggio, Giuseppe; Le Calvez-Kelm, Florence; Voegele, Catherine; Bartosch, Birke; Hernandez-Vargas, Hector; McKay, James; Sylla, Bakary S.; Manet, Evelyne; Tommasino, Massimo
Translocations at 8q24 juxtapose MYC with genes that harbor superenhancers resulting in overexpression and poor prognosis in myeloma patients.
Secondary MYC translocations in myeloma have been shown to be important in the pathogenesis and progression of disease. Here, we have used a DNA capture and massively parallel sequencing approach to identify the partner chromosomes in 104 presentation myeloma samples. 8q24 breakpoints were identified in 21 (20%) samples with partner loci including IGH, IGK and IGL, which juxtapose the immunoglobulin (Ig) enhancers next to MYC in 8/23 samples. The remaining samples had partner loci including XBP1, FAM46C, CCND1 and KRAS, which are important in B-cell maturation or myeloma pathogenesis. Analysis of the region surrounding the breakpoints indicated the presence of superenhancers on the partner chromosomes and gene expression analysis showed increased expression of MYC in these samples. Patients with MYC translocations had a decreased progression-free and overall survival. We postulate that translocation breakpoints near MYC result in colocalization of the gene with superenhancers from loci, which are important in the development of the cell type in which they occur. In the case of myeloma these are the Ig loci and those important for plasma cell development and myeloma pathogenesis, resulting in increased expression of MYC and an aggressive disease phenotype. PMID:24632883
Walker, B A; Wardell, C P; Brioli, A; Boyle, E; Kaiser, M F; Begum, D B; Dahir, N B; Johnson, D C; Ross, F M; Davies, F E; Morgan, G J
Construction of a microarray specific to the chicken immune system: profiling gene expression in B cells after lipopolysaccharide stimulation
The objective of this study was to profile gene expression in cells of the chicken immune system. A low-density immune-specific microarray was constructed that contained genes with known functions in the chicken immune system, in addition to chicken-expressed sequence tags (ESTs) homologous with mammalian immune system genes, which were systematically characterized by bioinformatic analyses. Genes and ESTs that met the annotation criteria were amplified and placed on a microarray. The microarray contained 84 immune system gene elements. As a means of calibration, the microarray was then used to examine gene expression in chicken B cells after lipopolysaccharide stimulation. Differential gene expression was observed at 6, 12, and 24 h but not at 48 h after stimulation. The results were validated by semiquantitative polymerase chain reaction. The microarray showed a high degree of reproducibility, as demonstrated by intra- and interassay correlation coefficients of 0.97 and 0.95, respectively. Thus, the low-density microarray developed in this study may be used as a tool for monitoring gene expression in the chicken immune system.
Sarson, Aimie J.; Read, Leah R.; Haghighi, Hamid R.; Lambourne, Melissa D.; Brisbin, Jennifer T.; Zhou, Huaijun; Sharif, Shayan
Conserved upstream sequences of human class II major histocompatibility genes enhance expression of class II genes in wild-type but not mutant B-cell lines.
Class II major histocompatibility genes contain a conserved upstream sequence (CUS) that is important in the expression of these genes. This region has been divided into two major elements, the X box and the Y box. The ability of these elements to mediate transcription of a heterologous promoter was assayed upon transfection into a B-cell line (Raji), a class II-specific trans-acting factor-deficient B-cell line (RJ2.2.5 cells), and a T-cell line (Jurkat). The results showed that the X box element was responsible for directing tissue-specific expression when Raji cells were compared to Jurkat cells. The X box could not direct expression of the heterologous promoter in the trans-acting factor-deficient cell line, indicating that the X box is an ultimate target of the missing or defective factor in the RJ2.2.5 cell line. The Y box directed an equal but extremely low level of transcription in this system in both the mutant and wild-type B-cell lines, suggesting that this element is not involved in B-cell expression or as a target of the mutant factor. Images
Sloan, J H; Boss, J M
Conserved upstream sequences of human class II major histocompatibility genes enhance expression of class II genes in wild-type but not mutant B-cell lines
Class II major histocompatibility genes contain a conserved upstream sequence (CUS) that is important in the expression of these genes. This region has been divided into two major elements, the X box and the Y box. The ability of these elements to mediate transcription of a heterologous promoter was assayed upon transfection into a B-cell line (Raji), a class II-specific trans-acting factor-deficient B-cell line (RJ2.2.5 cells), and a T-cell line (Jurkat). The results showed that the X box element was responsible for directing tissue-specific expression when Raji cells were compared to Jurkat cells. The X box could not direct expression of the heterologous promoter in the trans-acting factor-deficient cell line, indicating that the X box is an ultimate target of the missing or defective factor in the RJ2.2.5 cell line. The Y box directed an equal but extremely low level of transcription in this system in both the mutant and wild-type B-cell lines, suggesting that this element is not involved in B-cell expression or as a target of the mutant factor.
Sloan, J.H.; Boss, J.M. (Emory Univ., Atlanta, GA (USA))
Fusion of the NUP98 gene with the LEDGF/p52 gene defines a recurrent acute myeloid leukemia translocation
Background The NUP98 gene is involved in multiple rearrangements in haematological malignancy. The leukemic cells in an acute myeloid leukemia (AML) patient with a t(9;11)(p22;p15) were recently shown to have a fusion between the NUP98 gene and the LEDGF gene but it was not demonstrated that this fusion was recurrent in other leukaemia patients with the same translocation. Results We used RT-PCR to analyse the leukemic cells from an AML patient who presented with a cytogenetically identical translocation as the sole chromosomal abnormality. A NUP98-LEDGF fusion transcript was observed and confirmed by sequencing. The reciprocal transcript was also observed. The fusion transcript was not detectable during remission and recurred at relapse. The breakpoints in the NUP98 and LEDGF genes were different to those previously reported. The NUP98 breakpoint occurs in the intron between exons 8 and 9. It is the most 5' breakpoint reported in a translocation involving the NUP98 gene. All of the LEDGF gene is included in the fusion except for exon 1 which codes for the first 24 amino terminal amino acids. Conclusions Our results show that fusion of the NUP98 and LEDGF genes is a new recurrent translocation in AML.
Hussey, Damian J; Moore, Sarah; Nicola, Mario; Dobrovic, Alexander
Induction of sterile transcription from the kappa L chain gene locus in V(D)J recombinase-deficient progenitor B cells.
B cell development in RAG-2-deficient (RAG-2T) mice is impeded at an early stage, due to the inability of these animals to rearrange their endogenous ig gene loci. Expression of an E mu-bcl-2 transgene in these mice did not change this phenotype. However, stromal cell/IL-7-reactive B cell progenitors (pro-B cells) were found in fetal live and bone marrow of RAG-2T and RAG-2T/E mu-bcl-2 transgenic mice in numbers comparable to normal mice. Like cells from normal mice they are c-kit+, surrogate L chain+ and CD25-, and can proliferate in vitro for long periods of time. Upon IL-7 deprivation, they can be induced to differentiate into c-kit-, surrogate L chain- and CD25+ cells that are no longer clonable on stromal cells and IL-7. Furthermore, sterile transcription from the kappa L chain gene loci is induced. The latter was also observed with pro-B cells directly isolated ex vivo from the bone marrow of RAG-2-deficient animals. The results suggest that progenitor B cell differentiation can occur in cells from V(D)J recombinase-deficient mice to the stage where kL chain gene rearrangements would normally be initiated. It further indicates that some molecular programs of early B cell differentiation can take place in the absence of Ig gene rearrangements. PMID:8746561
Grawunder, U; Rolink, A; Melchers, F
Silencing and Nuclear Repositioning of the ?5 Gene Locus at the Pre-B Cell Stage Requires Aiolos and OBF-1
The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes ?5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the ?5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.
Karnowski, Alexander; Cao, Chun; Matthias, Gabriele; Carotta, Sebastian; Corcoran, Lynn M.; Martensson, Inga-Lill; Skok, Jane A.; Matthias, Patrick
Rearrangement and expression of kappa light chain genes can occur without mu heavy chain expression during differentiation of pre-B cells.
The kinetics of kappa light (kappa L) chain gene rearrangement and expression on mRNA and protein level has been studied with four stromal cell/IL-7 reactive, long-term in vitro proliferating pre-B cell lines and clones, two from fetal liver of normal mice and two from fetal liver of E microH-bcl-2 transgenic (bcl-2-tg) mice. These pre-B cell lines and clones are DJH-rearranged on both H chain alleles. Two of the clones harbor H chain rearrangements which do not allow the expression of VHDJH rearranged H chain genes as microH chain proteins. Upon removal of IL-7 from the pre-B cell cultures all four cell lines rearrange VH-DJH and VL-JL gene segments, loose the surface expression of c-kit, CD43, and surrogate light chain, as well as the capacity to be clonable on stromal cells in the presence of IL-7. Pre-B cells from normal mice die by apoptosis during differentiation, while those from bcl-2-tg mice do not. All four lines and clones express comparable levels of mRNA for microH and kappa L chains with the same time kinetics during 3 days of differentiation. However, only two of the four pre-B cell lines and clones express microH chain protein, whereas all four pre-B cell lines and clones express kappa L chain protein at comparable levels between 2 x 10(5) and 1.4 x 10(6) kappa L chain molecules per cell. These results suggest that microH chain expression is not mandatory for rearrangement and normal expression of kappa L chain genes when pre-B cells differentiate to B cells. PMID:8312230
Grawunder, U; Haasner, D; Melchers, F; Rolink, A
Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma
Although the identification and characterization of translocations have rapidly increased, little is known about the mechanisms of how translocations occur in vivo. We used anaplastic large cell lymphoma (ALCL) with and without the characteristic t(2;5)(p23;q35) translocation to study the mechanisms of formation of translocations and of ALCL transformation. We report deregulation of several genes located near the ALCL translocation breakpoint, regardless of whether the tumor contains the t(2;5). The affected genes include the oncogenic transcription factor Fra2 (located on 2p23), the HLH protein Id2 (2p25), and the oncogenic tyrosine kinase CSF1-receptor (5q33.1). Their up-regulation promotes cell survival and repression of T cell-specific gene expression programs that are characteristic for ALCL. The deregulated genes are in spatial proximity within the nuclear space of t(2;5)-negative ALCL cells, facilitating their translocation on induction of double-strand breaks. These data suggest that deregulation of breakpoint-proximal genes occurs before the formation of translocations, and that aberrant transcriptional activity of genomic regions is linked to their propensity to undergo chromosomal translocations. Also, our data demonstrate that deregulation of breakpoint-proximal genes has a key role in ALCL.
Mathas, Stephan; Kreher, Stephan; Meaburn, Karen J.; Johrens, Korinna; Lamprecht, Bjorn; Assaf, Chalid; Sterry, Wolfram; Kadin, Marshall E.; Daibata, Masanori; Joos, Stefan; Hummel, Michael; Stein, Harald; Janz, Martin; Anagnostopoulos, Ioannis; Schrock, Evelin; Misteli, Tom; Dorken, Bernd
An update of preimplantation genetic diagnosis in gene diseases, chromosomal translocation, and aneuploidy screening
Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.
Chang, Li-Jung; Chen, Shee-Uan; Tsai, Yi-Yi; Hung, Chia-Cheng; Fang, Mei-Ya
Variant B Cell Receptor Isotype Functions Differ in Hairy Cell Leukemia with Mutated BRAF and IGHV Genes
A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD?ve mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL.
Weston-Bell, Nicola J.; Forconi, Francesco; Kluin-Nelemans, Hanneke C.; Sahota, Surinder S.
Comparative RNA-Seq and Microarray Analysis of Gene Expression Changes in B-Cell Lymphomas of Canis familiaris
Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq appeared more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.
Monks, Noel; Eugster, Emily; Cherba, David; Berlinski, Pamela; Kamerling, Steve; Marotti, Keith; Simpson, Heather; Rusk, Tony; Tembe, Waibhav; Legendre, Christophe; Benson, Hollie; Liang, Winnie; Webb, Craig Paul
In multiple myeloma, 14q32 translocations are nonrandom chromosomal fusions driving high expression levels of the respective partner genes.
In studies of patients with multiple myeloma (MM), gene expression profiling (GEP) of myeloma cells demonstrates substantially higher expression of MMSET, FGFR3, CCND3, CCND1, MAF, and MAFB--the partner genes of 14q32 translocations--than GEP of plasma cells from healthy individuals. Interphase fluorescent in situ hybridization (FISH) was used to discriminate between chromosomal translocations involving different regions of the immunoglobulin heavy chain (IGH) genes at 14q32. With special probes designed for the constant region (IGHC) and the variable region (IGHV), IGH translocations were shown to be definite, nonrandom chromosomal fusions of IGHC with the loci of FGFR3, CCND1, CCND3, MAF, and MAFB genes; and IGHV with the locus of MMSET gene. When correlated with GEP results, the IGH translocations were found to drive expression levels of the partner genes to significantly higher levels (spikes) than copy-number variations. Hence, 42% of IGH translocations were identified among newly diagnosed MM patients (448/1,060). As GEP has become essential for assessing cancer risk, this novel approach is highly consistent with the cytogenetic features of the chromosomal translocations to effectively stratify molecular subgroups of MM on the basis of gene expression profiles of the IGH translocation partner genes in myeloma cells. ® 2014 Wiley Periodicals, Inc. PMID:24638926
Tian, Erming; Sawyer, Jeffrey R; Heuck, Christoph J; Zhang, Qing; van Rhee, Frits; Barlogie, Bart; Epstein, Joshua
Pax5: a master regulator of B cell development and leukemogenesis.
The B cell lineage of the hematopoietic system is responsible for the generation of high-affinity antibodies, which provide humoral immunity for protection against foreign pathogens. B cell commitment and development depend on many transcription factors including Pax5. Here, we review the different functions of Pax5 in regulating various aspects of B lymphopoiesis. At B cell commitment, Pax5 restricts the developmental potential of lymphoid progenitors to the B cell pathway by repressing B-lineage-inappropriate genes, while it simultaneously promotes B cell development by activating B-lymphoid-specific genes. Pax5 thereby controls gene transcription by recruiting chromatin-remodeling, histone-modifying, and basal transcription factor complexes to its target genes. Moreover, Pax5 contributes to the diversity of the antibody repertoire by controlling V(H)-DJ(H) recombination by inducing contraction of the immunoglobulin heavy-chain locus in pro-B cells, which is likely mediated by PAIR elements in the 5' region of the V(H) gene cluster. Importantly, all mature B cell types depend on Pax5 for their differentiation and function. Pax5 thus controls the identity of B lymphocytes throughout B cell development. Consequently, conditional loss of Pax5 allows mature B cells from peripheral lymphoid organs to develop into functional T cells in the thymus via dedifferentiation to uncommitted progenitors in the bone marrow. Pax5 has also been implicated in human B cell malignancies because it can function as a haploinsufficient tumor suppressor or oncogenic translocation fusion protein in B cell precursor acute lymphoblastic leukemia. PMID:21970955
Medvedovic, Jasna; Ebert, Anja; Tagoh, Hiromi; Busslinger, Meinrad
Gene targeting of the KI¢KII sequence elements in a model pre-B cell line: effects on germline transcription and rearrangement of the ? locus
To study the role of individual sequence elements in the coordinate regulation of rearrangement and germline transcription of the ? locus, we have developed a gene targeting system with a mouse model pre-B cell line, 38B9. This line can be induced to initiate ? germline transcription and V¢J rearrangement. Importantly, the effects of gene disruption in the cell line can
Xiangdong Liu; Brian Van Ness
Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1.
The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact.
Bogdan, J A; Adams-Burton, C; Pedicord, D L; Sukovich, D A; Benfield, P A; Corjay, M H; Stoltenborg, J K; Dicker, I B
Anti-vascular endothelial growth factor receptor (VEGFR) 2 autoantibody identification in glioblastoma patient using single B cell-based antibody gene cloning.
Antibody direct cloning from single B cells is simple and efficient and has been successful in antibody identification of infectious diseases. However, although a recent whole-exome sequencing revealed abundant heterogeneic mutation accumulation in cancers, identification and synthesis of autoantibodies against specific cancer-associated antigens is still difficult in cancer patients owing to the very small number of B cells producing autoantibodies. In the present study, to identify autoantibodies targeting tumor antigens, we measured the titer of autoantibodies in high-grade glioma patients' plasma and identified two patients with elevated autoantibodies to a few transmembrane proteins. Specific B cells producing autoantibody against vascular endothelial growth factor receptor (VEGFR) 2 were immunostained with labeled protein and anti-human IgG antibody, and then collected by a single cell sorter. Finally, 22 antibody genes were successfully identified using direct IgG cloning from single B cell mRNA, and two antibody clones were found to have significant VEGFR2-specific binding affinity. The current direct human IgG gene cloning technique for identifying human antibodies derived from IgG-memory B cells avoids time-consuming procedures such as phage display-based antibody-library screening, and therefore may be applicable to identifying human autoantibodies in a variety of disorders including cancers even when antibody elevation is not detected because of a very small number of memory B cells. PMID:24534640
Iizuka, Akira; Komiyama, Masaru; Oshita, Chie; Kume, Akiko; Ashizawa, Tadashi; Mitsuya, Koichi; Hayashi, Nakamasa; Nakasu, Yoko; Yamaguchi, Ken; Akiyama, Yasuto
Application of HSVtk suicide gene to X-SCID gene therapy: Ganciclovir treatment offsets gene corrected X-SCID B cells
Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human {gamma}c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the {gamma}c chain, the cells were treated with ganciclovir (GCV). The {gamma}c chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the {gamma}c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.
Uchiyama, Toru [Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575 (Japan); Kumaki, Satoru [Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575 (Japan)]. E-mail: firstname.lastname@example.com; Ishikawa, Yoshinori [Department of Microbiology and Immunity, Graduate School of Medicine, Tohoku University, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575 (Japan); Onodera, Masafumi [Major of Medical Sciences, Graduate School of Comprehensive Human Sciences, Tsukuba University, Tennodai 1-1-1, Tsukuba 305-8575 (Japan); Sato, Miki [Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575 (Japan); Du, Wei [Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575 (Japan); Sasahara, Yoji [Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575 (Japan); Tanaka, Nobuyuki [Department of Microbiology and Immunity, Graduate School of Medicine, Tohoku University, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575 (Japan); Sugamura, Kazuo [Department of Microbiology and Immunity, Graduate School of Medicine, Tohoku University, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575 (Japan); Tsuchiya, Shigeru [Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575 (Japan)
ZBTB32 is an early repressor of the class II transactivator and MHC class II gene expression during B cell differentiation to plasma cells1
The MHC class II transactivator (CIITA) and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when Blimp-1, the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. ShRNA depletion of ZBTB32 in a plasma cell line resulted in reexpression of CIITA and I-A. Compared to conditional Blimp-1 knock out and wild-type B cells, B cells from ZBTB32/ROG-knock out mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.
Yoon, Hyesuk; Scharer, Christopher D.; Majumder, Parimal; Davis, Carl W.; Butler, Royce; Zinzow-Kramer, Wendy; Skountzou, Ioanna; Koutsonanos, Dimitrios G.; Ahmed, Rafi; Boss, Jeremy M.
Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes.
The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes. Images
Ohara, N; Matsuo, K; Yamaguchi, R; Yamazaki, A; Tasaka, H; Yamada, T
The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion
Background Epstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth. Results We investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-?B p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation. Conclusions Our data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts.
The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion.
BACKGROUND: Epstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth. RESULTS: We investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-?B p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation. CONCLUSIONS: Our data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts. PMID:23320978
Hernando, Henar; Shannon-Lowe, Claire; Islam, Abul B; Al-Shahrour, Fatima; RodrĒguez-Ubreva, Javier; RodrĒguez-Cortez, Virginia C; Javierre, Biola M; Mangas, Cristina; Fernßndez, AgustĒn F; Parra, Maribel; Delecluse, Henri-Jacques; Esteller, Manel; L¾pez-Granados, Eduardo; Fraga, Mario F; L¾pez-Bigas, Nuria; Ballestar, Esteban
Germline Variation in Complement Genes and Event-Free Survival in Follicular and Diffuse Large B-Cell Lymphoma
The complement pathway plays a central role in innate immunity, and also functions as a regulator of the overall immune response. We evaluated whether polymorphisms in complement genes are associated with event-free survival (EFS) in follicular (FL) and diffuse large B-cell (DLBCL) lymphoma. We genotyped 167 single nucleotide polymorphisms (SNPs) from 30 complement pathway genes in a prospective cohort study of newly diagnosed FL (N=107) and DLBCL (N=82) patients enrolled at the Mayo Clinic from 2002¢2005. Cox regression was used to estimate Hazard Ratios (HRs) for individual SNPs with EFS, adjusting for FLIPI or IPI and treatment. For gene-level analyses, we used a principal components based gene-level test. In gene-level analyses for FL EFS, CFH (p=0.009), CD55 (p=0.006), CFHR5 (p=0.01), C9 (p=0.02), CFHR1 (p=0.03), and CD46 (p=0.03) were significant at p<0.05, and these genes remained noteworthy after accounting for multiple testing (q<0.15). SNPs in CFH, CFHR1, and CFHR5 showed stronger associations among patients receiving any rituximab, while SNPs from CD55 and CD46 showed stronger associations among patients who were observed. For DLBCL, only CLU (p=0.001) and C7 (p=0.03) were associated with EFS, but did not remain noteworthy after accounting for multiple testing (q>0.15). Genes from the Regulators of Complement Activation (CFH, CD55, CFHR1, CFHR5, CD46) at 1q32-q32.1, along with C9, were associated with FL EFS after adjusting for clinical variables, and if replicated, these findings add further support for the role of host innate immunity in FL prognosis.
Charbonneau, Bridget; Maurer, Matthew J.; Fredericksen, Zachary S.; Zent, Clive S.; Link, Brian K.; Novak, Anne J.; Ansell, Stephen M.; Weiner, George J.; Wang, Alice H.; Witzig, Thomas E.; Dogan, Ahmet; Slager, Susan L.; Habermann, Thomas M.; Cerhan, James R.
Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement.
Two waves of immunoglobulin gene rearrangements, first of the heavy, then of the light chain chain gene loci form functional immunoglobulin genes during B cell development. In mouse bone marrow the differential surface expression of B220 (CD45R), c-kit, CD25, and surrogate light chain as well as the cell cycle status allows FACS separation of the cells in which these two waves of rearrangements occur. The gene products of two recombination activating genes, RAG1 and RAG2 are crucial for this rearrangement process. Here, we show that the expression of the RAG genes is twice up- and down-regulated, at the transcriptional level for RAG1 and RAG2, and at the postranscriptional level for RAG2 protein. Expression levels are high in D-->JH and VH-->DJH rearranging proB and preB-I cells, low in preB cells expressing the preB cell receptor on the cell surface, and high again in VL-->JL rearranging small preB-II cells. In immature B cells expressing on the cell surface RAG1 and RAG2 mRNA is down-regulated, whereas RAG2 protein levels are maintained. Down-regulation of RAG1 and RAG2 gene expression after productive rearrangement at one heavy chain allele might be part of the mechanisms that prevent further rearrangements at the other allele. PMID:7584150
Grawunder, U; Leu, T M; Schatz, D G; Werner, A; Rolink, A G; Melchers, F; Winkler, T H
Recent advances in genomics and proteomics, combined with the facilitated generation and analysis of transgenic and gene-knockout animals, have revealed new complexities in classical biological systems, including the B-cell compartment. Studies on an 'old', but poorly characterized, B-cell subset Ś the naive, marginal-zone (MZ) B-cell subset Ś over the past two years have spawned an avalanche of data that encompass
Flavius Martin; John F. Kearney
B-cell-specific enhancer activity of conserved upstream elements of the class II major histocompatibility complex DQB gene
A 95-base-pair immediate upstream sequence of the human class II major histocompatibility complex DQB gene containing the conserved X and Y elements showed enhancer activity in a transient expression assay. An enhancer test plasmid harboring the bacterial chloramphenicol acetyltransferase gene under the control of a truncated simian virus 40 enhancerless early promoter was employed. The DQB sequence inserted into this plasmid was active as an enhancer in Raji cells (human Burkitt lymphoma cells) but not active in Jurkat cells (human T-cell leukemia cells) or in HeLa cells (human cervical carcinoma cells). This cell-type specificity suggests that this enhancer activity may be involved in the tissue specificity of the DQB gene that is normally expressed only in mature B cells, macrophages, and thymic epithelial cells. Deletion analysis showed that both X and Y box sequences are essential for the full activity of the enhancer sequence and that these two sequences may function in a cooperative manner as cis-acting elements. Further deletions were used to define the 5' border of the X element. These results suggest that previously characterized protein factors that bind to X and Y include transcription factors involved in the cell-type specificity of this enhancer activity.
Sakurai, M.; Strominger, J.L.
Profound obesity associated with a balanced translocation that disrupts the SIM1 gene
Studies of mice and humans have revealed a number of genes that when mutated result in severe obesity. We have studied a unique girl with early-onset obesity and a de novo balanced translocation between chromo- somes 1p22.1 and 6q16.2. Her weight gain is most likely due to excessive food intake, since measured energy expenditure was normal. We cloned and sequenced
J. L. Jr; Nancy F. Butte; Andrew R. Zinn
Comparison of Gene Expression Profiles in Chromate Transformed BEAS-2B Cells
BackgroundHexavalent chromium [Cr(VI)] is a potent human carcinogen. Occupational exposure has been associated with increased risk of respiratory cancer. Multiple mechanisms have been shown to contribute to Cr(VI) induced carcinogenesis, including DNA damage, genomic instability, and epigenetic modulation, however, the molecular mechanism and downstream genes mediating chromium's carcinogenicity remain to be elucidated.Methods\\/ResultsWe established chromate transformed cell lines by chronic exposure
Hong Sun; Harriet A. Clancy; Thomas Kluz; Jiri Zavadil; Max Costa
A 57-gene expression signature in B-cell chronic lymphocytic leukemia.
B-CLL is the most frequent type of leukemia in the Western countries. The disease, common among the elderly, follows a variable course in terms of survival time and symptoms. There is evidence that the accumulation of lymphocytes in peripheral blood and bone marrow is due to a cell resistance to apoptosis rather than to highly proliferative cells. Genetic mechanisms that lead to the development and progression of disease are mainly unknown, although a number of prognostically and diagnostically important genetic markers have been identified. The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered. IMP dehydrogenase, adenine phosphoribosyltransferase, adenylosuccinate lyase, adenylate kinase, ADORA1, G-protein-coupled receptor kinase 6, Bcl-2-like 1 isoform 2, caspase 6, and 8 were found underexpressed; while ADORA3, Gars-Airs-Gart, adenylate kinase 3, adenylate deaminase, NMN adenylyltransferase, CD26, CD38, interleukins 18 and 4 were found overexpressed. The microarray technique is a powerful method for identification of potential important diagnostic and prognostic markers, besides giving prominence to genes candidate for further studies. PMID:19278812
Carlucci, F; Marinello, E; Tommassini, V; Pisano, B; Rosi, F; Tabucchi, A
Next-Generation Sequencing of Apoptotic DNA Breakpoints Reveals Association with Actively Transcribed Genes and Gene Translocations
DNA fragmentation is a well-recognized hallmark of apoptosis. However, the precise DNA sequences cleaved during apoptosis triggered by distinct mechanisms remain unclear. We used next-generation sequencing of DNA fragments generated in Actinomycin D-treated human HL-60 leukemic cells to generate a high-throughput, global map of apoptotic DNA breakpoints. These data highlighted that DNA breaks are non-random and show a significant association with active genes and open chromatin regions. We noted that transcription factor binding sites were also enriched within a fraction of the apoptotic breakpoints. Interestingly, extensive apoptotic cleavage was noted within genes that are frequently translocated in human cancers. We speculate that the non-random fragmentation of DNA during apoptosis may contribute to gene translocations and the development of human cancers.
Fullwood, Melissa J.; Lee, Joanne; Lin, Lifang; Li, Guoliang; Huss, Mikael; Ng, Patrick; Sung, Wing-Kin; Shenolikar, Shirish
Cell-type-specific regulation of the human tumor necrosis factor alpha gene in B cells and T cells by NFATp and ATF-2/JUN.
The human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest genes transcribed after the stimulation of a B cell through its antigen receptor or via the CD-40 pathway. In both cases, induction of TNF-alpha gene transcription can be blocked by the immunosuppressants cyclosporin A and FK506, which suggested a role for the NFAT family of proteins in the regulation of the gene in B cells. Furthermore, in T cells, two molecules of NFATp bind to the TNF-alpha promoter element kappa 3 in association with ATF-2 and Jun proteins bound to an immediately adjacent cyclic AMP response element (CRE) site. Here, using the murine B-cell lymphoma cell line A20, we show that the TNF-alpha gene is regulated in a cell-type-specific manner. In A20 B cells, the TNF-alpha gene is not regulated by NFATp bound to the kappa 3 element. Instead, ATF-2 and Jun proteins bind to the composite kappa 3/CRE site and NFATp binds to a newly identified second NFAT site centered at -76 nucleotides relative to the TNF-alpha transcription start site. This new site plays a critical role in the calcium-mediated, cyclosporin A-sensitive induction of TNF-alpha in both A20 B cells and Ar-5 cells. Consistent with these results, quantitative DNase footprinting of the TNF-alpha promoter using increasing amounts of recombinant NFATp demonstrated that the -76 site binds to NFATp with a higher affinity than the kappa 3 site. Two other previously unrecognized NFATp-binding sites in the proximal TNF-alpha promoter were also identified by this analysis. Thus, through the differential use of the same promoter element, the composite kappa 3/CRE site, the TNF-alpha gene is regulated in a cell-type-specific manner in response to the same extracellular signal.
Tsai, E Y; Yie, J; Thanos, D; Goldfeld, A E
Differences in gene expression between B-cell chronic lymphocytic leukemia and normal B cells: a meta-analysis of three microarray studies
Motivation: A major focus of current cancer research is to identify genes that can be used as markers for prognosis and diagnosis, and as targets for therapy. Microarray technology has been applied extensively for this purpose, even though it has been reported that the agreement between microarray platforms is poor. A critical question is: how can we best com- bine
Jing Wang; Kevin R. Coombes; W. Edward Highsmith; M. J. Keating; Lynne V. Abruzzo
Variant translocation of the bcl-2 gene to immunoglobulin. lambda. light chain gene in chronic lymphocytic leukemia
The bcl-2 gene has been identified as a gene directly involved in the consistent chromosome translocation t(14;18), which is found in {approx} 90% of human follicular lymphoma cases, and is a prime candidate for the oncogene playing a crucial role in follicular lymphomagenesis. In this paper, the authors describe a case of chronic lymphocytic leukemia showing the juxtaposition of the bcl-2 gene on chromosome 18 to immunoglobulin {lambda} light chain (Ig{lambda}) gene on chromosome 22 in a head-to-head configuration. Sequencing analysis of the joining site of the bcl-2 gene and Ig{lambda} gene has shown that the breakpoint is within the 5{prime} flanking region of the bcl-2 gene and about 2.2 kilobases 5{prime} to the joining segment of Ig{lambda} locus in a germ-line configuration. The extranucleotide, commonly appearing at the joining site of the t(14;18) translocation involving the IgH locus, is absent from the joining site of bcl-2 and Ig{lambda}. The lack of extranucleotide suggests that the juxtaposition of the bcl-2 and Ig{lambda} genes occurred during physiological rearrangement of the Ig{lambda} gene since it has been shown that the rearrangement of the Ig{lambda} locus is not accompanied by extranucleotides.
Adachi, M.; Cossman, J.; Longo, D.; Croce, C.M.; Tsujimoto, Y. (Wistar Institute of Anatomy and Biology, Philadelphia, PA (USA))
Expressed sequences as candidates for a novel tumor suppressor gene at band 13q14 in B-cell chronic lymphocytic leukemia and mantle cell lymphoma
Deletions affecting the interval between the RB1 gene and marker D13S25 at band 13q14 are the most frequent genetic abnormalities of B-cell chronic lymphocytic leukemia (B-CLL) and indicate the presence of a novel tumor suppressor gene in this region. In the current study, a high resolution physical map of fragments spanning one megabasepair (Mb) of genomic DNA at the critical
Stephan Stilgenbauer; Jeremy Nickolenko; Jens Wilhelm; Stephan Wolf; Sandra Weitz; Konstanze D÷hner; Thomas Boehm; Hartmut D÷hner; Peter Lichter
Colorimetric in situ hybridization identifies MYC gene signal clusters correlating with increased copy number, mRNA, and protein in diffuse large B-cell lymphoma.
Abnormalities of the MYC oncogene on chromosome 8 are characteristic of Burkitt lymphoma and other aggressive B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL). We recently described a colorimetric in situ hybridization (CISH) method for detecting extra copies of the MYC gene in DLBCL and the frequent occurrence of excess copies of discrete MYC signals in the context of diploidy or polyploidy of chromosome 8, which correlated with increased mRNA signals. We further observed enlarged MYC signals, which were counted as a single gene copy but, by their dimension and unusual shape, likely consisted of "clusters" of MYC genes. In this study, we sought to further characterize these clusters of MYC signals by determining whether the presence of these correlated with other genetic features, mRNA levels, protein, and overall survival. We found that MYC clusters correlated with an abnormal MYC locus and with increased mRNA. MYC mRNA correlated with protein levels, and both increased mRNA and protein correlated with poorer overall survival. MYC clusters were seen in both the germinal center and activated B-cell subtypes of DLBCL. Clusters of MYC signals may be an underappreciated, but clinically important, feature of aggressive B-cell lymphomas with potential prognostic and therapeutic relevance. PMID:23355209
Valentino, Carlo; Kendrick, Samantha; Johnson, Nathalie; Gascoyne, Randy; Chan, Wing C; Weisenburger, Dennis; Braziel, Rita; Cook, James R; Tubbs, Raymond; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Jaffe, Elaine; Zhang, Wenjun; Brunhoeber, Patrick; Nitta, Hiro; Grogan, Tom; Rimsza, Lisa
Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling
Engagement of the B-cell antigen receptor (BCR) leads to activation of the Raf¢MEK¢ERK pathway and Raf kinases play an important role in the modulation of ERK activity. B lymphocytes express two Raf isoforms, Raf-1 and B-Raf. Using an inducible deletion system in DT40 cells, the contribution of Raf-1 and B-Raf to BCR signalling was dissected. Loss of Raf-1 has no effect on BCR-mediated ERK activation, whereas B-Raf-deficient DT40 cells display a reduced basal ERK activity as well as a shortened BCR-mediated ERK activation. The Raf-1/B-Raf double deficient DT40 cells show an almost complete block both in ERK activation and in the induction of the immediate early gene products c-Fos and Egr-1. In contrast, BCR-mediated activation of nuclear factor of activated T cells (NFAT) relies predominantly on B-Raf. Furthermore, complementation of Raf-1/B-Raf double deficient cells with various Raf mutants demonstrates a requirement for Ras-GTP binding in BCR-mediated activation of both Raf isoforms and also reveals the important role of the S259 residue for the regulation of Raf-1. Our study shows that BCR-mediated ERK activation involves a cooperation of both B-Raf and Raf-1, which are activated specifically in a temporally distinct manner.
Brummer, Tilman; Shaw, Peter E.; Reth, Michael; Misawa, Yukiko
Ten-Eleven Translocation-2 gene mutations: A potential new molecular marker in malignant gliomas (Review)
Alterations of the Ten-Eleven Translocation-2 (TET2) gene in myeloid malignancies and isocitrate dehydrogenase (IDH) gene mutations in gliomas and myeloid malignancies have recently been identified using molecular, comparative genomic hybridization and single nucleotide polymorphism array techniques. The mutations of the TET2 gene have been shown to be mutually exclusive with IDH1/2 mutations in acute myeloid leukemia (AML) and evidence has been found to provide a biochemical basis for the mutual exclusivity of IDH1/2 and TET2 gene mutations. Based on mounting evidence, we aimed to investigate whether TET2 mutations may be identified as novel mutations in malignant gliomas without IDH1/2 mutations, and indicate their possible significance in gliomas.
Biallelic somatic inactivation of the NF1 gene through chromosomal translocations in a sporadic neurofibroma.
Neurofibroma is a benign tumor originating from Schwann cells in peripheral nerve sheaths and may occur as a sporadic tumor or as part of the dominantly inherited tumor syndrome NF1. NF1 is caused by constitutional mutations in the NF1 gene, located in chromosome band 17q11. Whereas the involvement of the NF1 gene in neurofibroma development in NF1 patients has been fairly well characterized, the significance of inactivation of this gene in sporadic neurofibromas remains less well investigated. Inactivation of both copies of NF1 has been described in a few neurofibromas from NF1 patients, and LOH at the same locus has been reported in additional cases. In the present study, we report the cytogenetic and molecular cytogenetic findings in a sporadic neurofibroma that at G-banding analysis showed a translocation between one chromosome 2 and the long arms of both copies of chromosome 17. FISH analysis using a set of 3 BAC clones covering the entire coding region of NF1 revealed the complete loss of one allele and the deletion of the 5' portion of the second allele as a result of 2 translocation events. To the best of our knowledge, this represents the first demonstration of a somatic biallelic inactivation of the NF1 gene in neurofibroma, providing further evidence for the importance of NF1 inactivation also in sporadic neurofibromas. PMID:15986446
Storlazzi, Clelia Tiziana; Von Steyern, Fredrik Vult; Domanski, Henryk A; Mandahl, Nils; Mertens, Fredrik
The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats.
Vale, AM; Foote, JB; Granato, A; Zhuang, Y; Pereira, RMS; Lopes, UG; Bellio, M; Burrows, PD; Schroeder, HW; Nobrega, A
Microarray gene expression analysis of fixed archival tissue permits molecular classification and identification of potential therapeutic targets in diffuse large B-cell lymphoma.
Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) has a poor prognosis. Novel drugs targeting the constitutively activated NF-?B pathway characteristic of ABC-DLBCL are promising, but evaluation depends on accurate activated B cell-like (ABC)/germinal center B cell-like (GCB) molecular classification. This is traditionally performed on gene microarray expression profiles of fresh biopsies, which are not routinely collected, or by immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue, which lacks reproducibility and classification accuracy. We explored the possibility of using routine archival FFPE tissue for gene microarray applications. We examined Affymetrix HG U133 Plus 2.0 gene expression profiles from paired archival FFPE and fresh-frozen tissues of 40 ABC/GCB-classified DLBCL cases to compare classification accuracy and test the potential for this approach to aid the discovery of therapeutic targets and disease classifiers in DLBCL. Unsupervised hierarchical clustering of unselected present probe sets distinguished ABC/GCB in FFPE with remarkable accuracy, and a Bayesian classifier correctly assigned 32 of 36 cases with >90% probability. Enrichment for NF-?B genes was appropriately seen in ABC-DLBCL FFPE tissues. The top discriminatory genes expressed in FFPE separated cases with high statistical significance and contained novel biology with potential therapeutic insights, warranting further investigation. These results support a growing understanding that archival FFPE tissues can be used in microarray experiments aimed at molecular classification, prognostic biomarker discovery, and molecular exploration of rare diseases. PMID:22446084
Linton, Kim; Howarth, Christopher; Wappett, Mark; Newton, Gillian; Lachel, Cynthia; Iqbal, Javeed; Pepper, Stuart; Byers, Richard; Chan, Wing John; Radford, John
Tyrosine kinase chromosomal translocations mediate distinct and overlapping gene regulation events
Background Leukemia is a heterogeneous disease commonly associated with recurrent chromosomal translocations that involve tyrosine kinases including BCR-ABL, TEL-PDGFRB and TEL-JAK2. Most studies on the activated tyrosine kinases have focused on proximal signaling events, but little is known about gene transcription regulated by these fusions. Methods Oligonucleotide microarray was performed to compare mRNA changes attributable to BCR-ABL, TEL-PDGFRB and TEL-JAK2 after 1 week of activation of each fusion in Ba/F3 cell lines. Imatinib was used to control the activation of BCR-ABL and TEL-PDGFRB, and TEL-JAK2-mediated gene expression was examined 1 week after Ba/F3-TEL-JAK2 cells were switched to factor-independent conditions. Results Microarray analysis revealed between 800 to 2000 genes induced or suppressed by two-fold or greater by each tyrosine kinase, with a subset of these genes commonly induced or suppressed among the three fusions. Validation by Quantitative PCR confirmed that eight genes (Dok2, Mrvi1, Isg20, Id1, gp49b, Cxcl10, Scinderin, and collagen V?1(Col5a1)) displayed an overlapping regulation among the three tested fusion proteins. Stat1 and Gbp1 were induced uniquely by TEL-PDGFRB. Conclusions Our results suggest that BCR-ABL, TEL-PDGFRB and TEL-JAK2 regulate distinct and overlapping gene transcription profiles. Many of the genes identified are known to be involved in processes associated with leukemogenesis, including cell migration, proliferation and differentiation. This study offers the basis for further work that could lead to an understanding of the specificity of diseases caused by these three chromosomal translocations.
Familial cryptic translocation resulting in Angelman syndrome: Implications for imprinting or location of the Angelman gene?
Angelman syndrome (AS) is associated with a loss of maternal genetic information, which typically occurs as a result of a deletion at 15q11-q13 or paternal uniparental disomy of chromosome 15. We report a patient with AS as a result of an unbalanced cryptic translocation whose breakpoint, at 15q11.2, falls within this region. The proband was diagnosed clinically as having Angelman syndrome, but without a detectable cytogenetic deletion, by using high-resolution G-banding. FISH detected a deletion of D15S11 (IR4-3R), with an intact GABRB3 locus. Subsequent studies of the proband`s mother and sister detected a cryptic reciprocal translocation between chromosomes 14 and 15 with the breakpoint being between SNRPN and D15S10. The proband was found to have inherited an unbalanced form, being monosomic from 15pter through SNRPN and trisomic for 14pter to 14q11.2. DNA methylation studies showed that the proband had a paternal-only DNA methylation pattern at SNRPN, D15S63 (PW71), and ZNF127. The mother and unaffected sister, both having the balanced translocation, demonstrated normal DNA methylation patterns at all three loci. These data suggest that the gene for AS most likely lies proximal to D15S10, in contrast to the previously published position, although a less likely possibility is that the maternally inherited imprinting center acts in trans in the unaffected balanced translocation carrier sister. 27 refs., 6 figs.
Burke, L.W.; Wiley, J.E.; Smith, A.J.W.; Kushnick, T. [East Carolina Univ. School of Medicine, Greenville, NC (United States)] [and others
Early gene expression changes by Epstein-Barr virus infection of B-cells indicate CDKs and survivin as therapeutic targets for post-transplant lymphoproliferative diseases.
Lymphoproliferative diseases (LPDs) associated with Epstein-Barr virus (EBV) infection cause significant morbidity and mortality in bone marrow and solid organ transplant recipients. To gain insight into LPD pathogenesis and to identify potential effective therapeutic approaches, we investigated early molecular events leading to B-cell transformation by gene expression profiling of EBV-infected B-cells from tonsils by Affymetrix microarray 72 hr postinfection when the B-cells hyperproliferation phase starts. Cell cycle and apoptosis were the most significantly affected pathways and enriched gene sets. In particular, we found significantly increased expression of cyclin-dependent kinase (CDK)1 and CCNB1 (cyclin B1) and of one of their downstream targets BIRC5 (survivin). Importantly, the strong upregulation of the antiapoptotic protein survivin was confirmed in lymphoblastoid cell lines (LCLs) and 71% of EBV-positive post-transplant EBV-LPD lesions scored positive for survivin. The validity of early transforming events for the identification of therapeutic targets for EBV-LPD was confirmed by the marked antiproliferative effect of the CDK inhibitor flavopiridol on LCLs and by the strong induction of apoptosis by survivin inhibition with YM155 or terameprocol. Our results suggest that targeting of CDKs and/or survivin in post-transplant EBV-LPD by specific inhibitors might be an important approach to control and eliminate EBV-transformed B-cells that should be further considered. PMID:23640782
Bernasconi, Michele; Ueda, Seigo; Krukowski, Patricia; Bornhauser, Beat C; Ladell, Kristin; Dorner, Marcus; Sigrist, Juerg A; Campidelli, Cristina; Aslandogmus, Roberta; Alessi, Davide; Berger, Christoph; Pileri, Stefano A; Speck, Roberto F; Nadal, David
A Novel Translocation Breakpoint within the BPTF Gene Is Associated with a Pre-Malignant Phenotype
Partial gain of chromosome arm 17q is an abundant aberrancy in various cancer types such as lung and prostate cancer with a prominent occurrence and prognostic significance in neuroblastoma ¢ one of the most common embryonic tumors. The specific genetic element/s in 17q responsible for the cancer-promoting effect of these aberrancies is yet to be defined although many genes located in 17q have been proposed to play a role in malignancy. We report here the characterization of a naturally-occurring, non-reciprocal translocation der(X)t(X;17) in human lung embryonal-derived cells following continuous culturing. This aberrancy was strongly correlated with an increased proliferative capacity and with an acquired ability to form colonies in vitro. The breakpoint region was mapped by fluorescence in situ hybridization (FISH) to the 17q24.3 locus. Further characterization by a custom-made comparative genome hybridization array (CGH) localized the breakpoint within the Bromodomain PHD finger Transcription Factor gene (BPTF), a gene involved in transcriptional regulation and chromatin remodeling. Interestingly, this translocation led to elevation in the mRNA levels of the endogenous BPTF. Knock-down of BPTF restricted proliferation suggesting a role for BPTF in promoting cellular growth. Furthermore, the BPTF chromosomal region was found to be amplified in various human tumors, especially in neuroblastomas and lung cancers in which 55% and 27% of the samples showed gain of 17q24.3, respectively. Additionally, 42% percent of the cancer cell lines comprising the NCI-60 had an abnormal BPTF locus copy number. We suggest that deregulation of BPTF resulting from the translocation may confer the cells with the observed cancer-promoting phenotype and that our cellular model can serve to establish causality between 17q aberrations and carcinogenesis.
Lipson, Doron; Milyavsky, Michael; Polak-Charcon, Sylvie; Mardoukh, Corine; Solomon, Hilla; Kalo, Eyal; Madar, Shalom; Brosh, Ran; Perelman, Marina; Navon, Roy; Goldfinger, Naomi; Barshack, Iris; Yakhini, Zohar; Rotter, Varda
A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene
OF the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains1. Recent data suggest that pre-B cells express mu chains on the membrane together with the
Daisuke Kitamura; J³rgen Roes; Ralf K³hn; Klaus Rajewsky
Hidden gene amplifications in aggressive B-cell non-Hodgkin lymphomas detected by microarray-based comparative genomic hybridization
DNA amplifications are important mechanisms for proto-oncogene activation. Comparative genomic hybridization (CGH) to metaphase chromosome preparations has revealed amplifications in 10¢20% of B-cell lymphomas (B-NHL). We analysed a series of 16 aggressive non-Hodgkin lymphomas by the new approach termed Matrix-CGH (M-CGH) using genomic DNA microarrays as hybridization target. For M-CGH, a dedicated B-cell lymphoma chip was constructed containing 496 genomic
Swen Wessendorf; Carsten Schwaenen; Holger Kohlhammer; Dirk Kienle; Gunnar Wrobel; Thomas FE Barth; Michelle Nessling; Peter M÷ller; Hartmut D÷hner; Peter Lichter; Martin Bentz
Gene expression profiling of plasma cells and plasmablasts: toward a better understanding of the late stages of B-cell differentiation.
Plasma cells (PCs), the end point of B-cell differentiation, are a heterogeneous cell compartment comprising several cell subsets from short-lived highly proliferative plasmablasts to long-lived nondividing fully mature PCs. Whereas the major transcription factors driving the differentiation of B cells to PCs were recently identified, the subtle genetic changes that underlie the transition from plasmablasts to mature PCs are poorly understood. We recently described an in vitro model making it possible to obtain a large number of cells with the morphologic, phenotypic, and functional characteristics of normal polyclonal plasmablastic cells (PPCs). Using Affymetrix microarrays we compared the gene expression profiles of these PPCs with those of mature PCs isolated from tonsils (TPCs) and bone marrow (BMPCs), and with those of B cells purified from peripheral blood (PBB cells) and tonsils (TBCs). Unsupervised principal component analysis clearly distinguished the 5 cell populations on the basis of their differentiation and proliferation status. Detailed statistical analysis allowed the identification of 85 PC genes and 40 B-cell genes, overexpressed, respectively, in the 3 PC subsets or in the 2 B-cell subsets. In addition, several signaling molecules and antiapoptotic proteins were found to be induced in BMPCs compared with PPCs and could be involved in the accumulation and prolonged survival of BMPCs in close contact with specialized stromal microenvironment. These data should help to better understand the molecular events that regulate commitment to a PC fate, mediate PC maintenance in survival niches, and could facilitate PC immortalization in plasma cell dyscrasias. PMID:12663452
Tarte, Karin; Zhan, Fenghuang; De Vos, John; Klein, Bernard; Shaughnessy, John
Light-chain gene expression before heavy-chain gene rearrangement in pre-B cells transformed by Epstein-Barr virus.
Epstein-Barr virus transformation of B-cell-depleted bone marrow cells from human fetuses allowed us to identify novel cell types characterized by the expression of immunoglobulin kappa or lambda light chain without heavy chains. Four kappa-only clones with normal karyotype were obtained and examined for their immunoglobulin gene configurations and expression. All four clones had kappa-chain gene rearrangements at either one or both alleles, but the heavy-chain gene loci in these clones either were in germ-line context or had undergone only D-JH rearrangements (D and JH represent diversity and joining gene segments). All clones contained kappa mRNA of normal size at levels consistent with the protein level, except for one clone that no longer produced kappa protein. No mu mRNA or immunoglobulin heavy-chain molecules were detected in any of the kappa+ clones. The results suggest that the mu heavy-chain protein is not an obligatory prerequisite for light-chain gene rearrangements. Images
Kubagawa, H; Cooper, M D; Carroll, A J; Burrows, P D
Long-range oncogenic activation of Igh-c-myc translocations by the Igh 3' regulatory region.
B-cell malignancies, such as human Burkitt's lymphoma, often contain translocations that link c-myc or other proto-oncogenes to the immunoglobulin heavy chain locus (IgH, encoded by Igh). The nature of elements that activate oncogenes within such translocations has been a long-standing question. Translocations within Igh involve DNA double-strand breaks initiated either by the RAG1/2 endonuclease during variable, diversity and joining gene segment (V(D)J) recombination, or by activation-induced cytidine deaminase (AID, also known as AICDA) during class switch recombination (CSR). V(D)J recombination in progenitor B (pro-B) cells assembles Igh variable region exons upstream of mu constant region (Cmu) exons, which are the first of several sets of C(H) exons ('C(H) genes') within a C(H) locus that span several hundred kilobases (kb). In mature B cells, CSR deletes Cmu and replaces it with a downstream C(H) gene. An intronic enhancer (iEmu) between the variable region exons and Cmu promotes V(D)J recombination in developing B cells. Furthermore, the Igh 3' regulatory region (Igh3'RR) lies downstream of the C(H) locus and modulates CSR by long-range transcriptional enhancement of C(H) genes. Transgenic mice bearing iEmu or Igh3'RR sequences fused to c-myc are predisposed to B lymphomas, demonstrating that such elements can confer oncogenic c-myc expression. However, in many B-cell lymphomas, Igh-c-myc translocations delete iEmu and place c-myc up to 200 kb upstream of the Igh3'RR. Here we address the oncogenic role of the Igh3'RR by inactivating it in two distinct mouse models for B-cell lymphoma with Igh-c-myc translocations. We show that the Igh3'RR is dispensable for pro-B-cell lymphomas with V(D)J recombination-initiated translocations, but is required for peripheral B-cell lymphomas with CSR-associated translocations. As the Igh3'RR is not required for CSR-associated Igh breaks or Igh-c-myc translocations in peripheral B-cell lymphoma progenitors, we conclude that this regulatory region confers oncogenic activity by long-range and developmental stage-specific activation of translocated c-myc genes. PMID:20010689
Gostissa, Monica; Yan, Catherine T; Bianco, Julia M; Cognķ, Michel; Pinaud, Eric; Alt, Frederick W
Rgs13 Constrains Early B Cell Responses and Limits Germinal Center Sizes
Germinal centers (GCs) are microanatomic structures that develop in secondary lymphoid organs in response to antigenic stimulation. Within GCs B cells clonally expand and their immunoglobulin genes undergo class switch recombination and somatic hypermutation. Transcriptional profiling has identified a number of genes that are prominently expressed in GC B cells. Among them is Rgs13, which encodes an RGS protein with a dual function. Its canonical function is to accelerate the intrinsic GTPase activity of heterotrimeric G-protein ? subunits at the plasma membrane, thereby limiting heterotrimeric G-protein signaling. A unique, non-canonical function of RGS13 occurs following translocation to the nucleus, where it represses CREB transcriptional activity. The functional role of RGS13 in GC B cells is unknown. To create a surrogate marker for Rgs13 expression and a loss of function mutation, we inserted a GFP coding region into the Rgs13 genomic locus. Following immunization GFP expression rapidly increased in activated B cells, persisted in GC B cells, but declined in newly generated memory B and plasma cells. Intravital microscopy of the inguinal lymph node (LN) of immunized mice revealed the rapid appearance of GFP+ cells at LN interfollicular regions and along the T/B cell borders, and eventually within GCs. Analysis of WT, knock-in, and mixed chimeric mice indicated that RGS13 constrains extra-follicular plasma cell generation, GC size, and GC B cell numbers. Analysis of select cell cycle and GC specific genes disclosed an aberrant gene expression profile in the Rgs13 deficient GC B cells. These results indicate that RGS13, likely acting at cell membranes and in nuclei, helps coordinate key decision points during the expansion and differentiation of naive B cells.
Hwang, Il-Young; Hwang, Kyung-Sun; Park, Chung; Harrison, Kathleen A.; Kehrl, John H.
Potential tumor suppressive function of miR-196b in B-cell lineage acute lymphoblastic leukemia.
Keeping in view the fact that genes coding microRNAs (miRNAs) have been found to be localized in chromosomal regions susceptible to genetic translocations, this study was addressed to identify and characterize the miRNAs that are present near/within the regions involved in genetic translocations characteristic of B-cell acute lymphoblastic leukemia (B-cell ALL). Out of six such identified miRNAs miR-196b was not only found to be significantly down-regulated in both EB-3 cell line as well as B-cell ALL patients as compared to that found in the corresponding controls, but also had the inherent capacity to down-regulate the highly expressed c-myc gene, a consequence of genetic translocation characteristic of EB-3 cells at both transcriptional and translational level. This phenomenon was in conformity with the observed reciprocal relationship between the expressed genes coding for miR-196b and c-myc in B-cells derived from ALL patients as well as c-myc gene was found to be a putative target of miR-196b as predicted by bioinformatic algorithms. Also down-regulation of c-myc gene was accompanied by decreased expressions of c-myc effector genes coding for hTERT, Bcl-2, and AATF. Based upon these results, we propose for the first time that miR-196b has the inherent capacity to down-regulate the overamplified c-myc gene recognized as a common pathognomonic feature leading to cancer in general and B-cell ALL in particular. Hence miR-196b can be assigned with the tumor suppressor function and can be of therapeutic importance in paving the way toward the treatment of B-cell ALL. PMID:20549547
Bhatia, Suman; Kaul, Deepak; Varma, Neelam
Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti-transferrin receptor antibodies and an immunoglobulin promoter
Background We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, due to widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of dual targeted lentiviral-mediated gene therapy approaches to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter. Methods Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored by an MTS assay. Results We found that EGFP was specifically expressed in a panel of human malignant B cells, but not in human T cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC. Conclusions These studies demonstrate that gene expression can be restricted to malignant B cells and suggest that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches.
Leoh, Lai Sum; Morizono, Kouki; Kershaw, Kathleen M.; Chen, Irvin S. Y.; Penichet, Manuel L.; Daniels-Wells, Tracy R.
Treatment of diabetes in NOD mice by gene transfer of Ig-fusion proteins into B cells: role of T regulatory cells.
We previously reported that retrovirally mediated gene expression of Ig fusion proteins leads to specific immunologic tolerance and successful treatment of autoimmune conditions. Thus, a single dose of GAD65-IgG- or (Pro) Insulin-IgG-transduced B cells delays the onset and decreases the incidence of diabetes in young (7-12 weeks old) NOD female mice. Herein, we tested the role of regulatory T cells by in vivo treatment with anti-CD25 before B-cell gene therapy or by in vitro ablation of CD25+ cells from tolerized hosts in an adoptive transfer model. Our results demonstrate that anti-CD25 treatment, like cyclophosphamide, partially blocks the efficacy of gene therapy for tolerance. Moreover, B-cell therapy is effective at preventing diabetes transfer by female T cells (from older diabetic mice) into intact male recipients with normal islets, but failed to do so in NOD-scid recipients. This is due in part to homeostatic proliferation but also to the absence of CD25+ T cells in the latter hosts. Tolerance induced in younger NOD females can be stably transferred to NOD-scid recipients. However, physical removal of CD25+ cells abrogates the transfer of tolerance. Therefore, we conclude that CD4+, CD25+ regulatory T cells are required for the induction as well as maintenance of tolerance in this gene therapy model. The phenotype of these induced regulatory T cells is under investigation. PMID:16860296
Soukhareva, Nadejda; Jiang, Yufei; Scott, David W
PAX5 mutations occur frequently in adult B-cell progenitor acute lymphoblastic leukemia and PAX5 haploinsufficiency is associated with BCR-ABL1 and TCF3-PBX1 fusion genes: a GRAALL study.
Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count. PMID:19587702
Familiades, J; Bousquet, M; Lafage-Pochitaloff, M; Bķnķ, M-C; Beldjord, K; De Vos, J; Dastugue, N; Coyaud, E; Struski, S; Quelen, C; Prade-Houdellier, N; Dobbelstein, S; Cayuela, J-M; Soulier, J; Grardel, N; Preudhomme, C; Cavķ, H; Blanchet, O; Lhķritier, V; Delannoy, A; Chalandon, Y; Ifrah, N; Pigneux, A; Brousset, P; Macintyre, E A; Huguet, F; Dombret, H; Broccardo, C; Delabesse, E
A novel heterozygous point mutation in the p63 gene in a patient with ectodermal dysplasia associated with B-cell leukemia.
We report a 7-year-old boy with a past medical history of B-cell leukemia with dysmorphic features, including cleft palate, hypotrichosis with trichorrhexis nodosa, hypohidrosis, oligodontia, and ridging of nails. A heterozygous germline mutation, Ala111Thr, in the p63 gene was detected in the boy and in his mother, who had no clinical expression. This case emphasizes the spectrum of different phenotypical manifestations of mutations in the p63 gene and underlines the possible role of this gene as a tumor suppressor. PMID:21906144
Cabanillas, Miguel; Torrelo, Antonio; Monteagudo, Benigno; Sußrez-Amor, Oscar; RamĒrez-Santos, Aquilina; Gonzßlez-Vilas, Daniel; de las Heras, Cristina
MDR1 (ABCB1) gene polymorphism C3435T is associated with P-glycoprotein activity in B-cell chronic lymphocytic leukemia
Functional single nucleotide polymorphism (SNP) C3435T in exon 26 of the MDR1 (ABCB1) gene encoding the xenobiotic transporter P-glycoprotein (P-gp, MDR1, ABCB1) may influence susceptibility to several diseases as well as clinical outcome of treatment with P-gp substrates. Exposure to environmental chemicals is thought to be involved in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) and P-gp-transported drugs are
Krzysztof Jamroziak; Ewa Balcerczak; Piotr Smolewski; Robert W. Robey; Barbara Cebula; Mariusz Panczyk; Monika Kowalczyk; Anna Szmigielska; Marek Mirowski; Susan E. Bates; Tadeusz Robak
Detailed mapping of chromosome 17p deletions reveals HIC1 as a novel tumor suppressor gene candidate telomeric to TP53 in diffuse large B-cell lymphoma
Deletions in the short arm of chromosome 17 (17p) involving the tumor suppressor TP53 occur in up to 20% of diffuse large B-cell lymphomas (DLBCLs). Although inactivation of both alleles of a tumor suppressor gene is usually required for tumor development, the overlap between TP53 deletions and mutations is poorly understood in DLBCLs, suggesting the possible existence of additional tumor
H St÷cklein; J Smardova; J Macak; T Katzenberger; S H÷ller; S Wessendorf; G Hutter; M Dreyling; E Haralambieva; U Mõder; H K M³ller-Hermelink; A Rosenwald; G Ott; J Kalla
Activation of the Early B-Cell-Specific mb-1 (IgĀ) Gene by Pax5 Is Dependent on an Unmethylated Ets Binding Site
Methylation of cytosine in CpG dinucleotides promotes transcriptional repression in mammals by blocking transcription factor binding and recruiting methyl-binding proteins that initiate chromatin remodeling. Here, we use a novel cell-based system to show that retrovirally expressed Pax-5 protein activates endogenous early B-cell-specific mb-1 genes in plasmacytoma cells, but only when the promoter is hypomethylated. CpG meth- ylation does not directly
Holly Maier; Jeff Colbert; Daniel Fitzsimmons; Dawn R. Clark; James Hagman
64. Rescue of B Cell Development in an Animal Model of X-Linked Agammaglobulinemia (XLA) Via B Lineage-Specific Lentiviral Gene Therapy
X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton's tyrosine kinase (Btk); and characterized by a block in pre B-cell development leading to absence of serum immunoglobulin and recurrent bacterial infections. Using Btk and Tec double deficient (Btk\\/Tec?\\/?) mice as a model for XLA, we previously showed that onco-retroviral-mediated Btk gene transfer into hematopoietic stem cells (HSC)
Wenying Zhang; Stephanie Humblet-Baron; Kevin Kipp; Socheath Khim; Jordan Jarjour; Karen Sommer; Brigid Stirling; Lia Pernell; David J. Rawlings
Identification of Bach2 as a B-cell-specific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3' enhancer.
Maf family transcription factors are important regulators in various differentiation systems. Putative Maf recognition elements (MAREs) are found in the 3' enhancer region of the immunoglobulin heavy chain (IgH) gene. These elements are bound in B-cell extracts by a heterodimeric protein complex containing both Bach2 and a small Maf protein. Analysis of normal hematopoietic cells revealed that Bach2 is specifically expressed in B cells. Bach2 is abundantly expressed in the early stages of B-cell differentiation and turned off in terminally differentiated cells. Bach2 acts together with MafK as a negative effector of the IgH 3' enhancer and binds to the co-repressor SMRT (silencing mediator of retinoid and thyroid receptor). Hence the Bach2-small-Maf heterodimer may represent the first example of a B-cell lineage, and of a developmental stage-restricted negative effector of the MARE in the IgH 3' enhancer region.
Muto, A; Hoshino, H; Madisen, L; Yanai, N; Obinata, M; Karasuyama, H; Hayashi, N; Nakauchi, H; Yamamoto, M; Groudine, M; Igarashi, K
A targeted kappa immunoglobulin gene containing a deletion of the nuclear matrix association region exhibits spontaneous hyper-recombination in pre-B cells.
Previous studies employing ectopic integration of reporter genes have shown that the nuclear matrix association region (MAR) adjacent to the intronic enhancer of the mouse kappa immunoglobulin (Ig) gene is required for high level transcription of rearranged genes, demethylation, reduction of position effects and maximal somatic hypermutation in B cells. To test for the function of this MAR in its natural chromosomal environment, we pursued the 'HIT-and-RUN' procedure with the mouse pre-B cell line 103 to create a targeted MAR deletion. We observed a 'HIT' targeting frequency of 1/684 but 0/2100 'RUN' clones maintained the MAR-deleted germline locus because of an unexpected hyper-recombination for Vkappa-Jkappa joining, specifically to the MAR-deleted allele, and primarily at Jkappa4 and Jkappa5. This hyper-recombination was correlated with undermethylation of the Jkappa-Ckappa region but not with the level of local transcription. These results are consistent with the possibility that the MAR and/or DNA methylation negatively regulate(s) Vkappa-Jkappa joining during the pre-B cell stage of development. PMID:9823759
Hale, M A; Garrard, W T
VH gene analysis of spontaneously activated B cells in adult MRL-lpr/lpr mice. J558 bias is not limited to classic lupus autoantibodies.
To determine the genetic origins of lupus auto-antibodies, we analyzed the relationship between VH gene usage and auto-Ag-binding properties of 352 B cell hybridomas derived from MRL-lpr/lpr mice. The hybridomas were derived from neonatal, 1-month-old, 3-month-old, and 6-month-old mice. The experimental strategy provided that the hybridomas were monoclonal at initial evaluation, so the Ag binding and V gene frequencies of the entire population could be determined. Initially, 1032 Ig-producing hybridomas were evaluated for binding to six Ag; VH gene family use was determined in 119 anti-DNA and anti-rabbit thymus extract (RTE) antibodies (autoantibodies) and in 233 age-matched Ig that did not bind to any of the six Ag (nonbinders). Neonatal B cells, including cross-reactive IgM autoantibodies and nonbinder IgM, used relatively 3' VH genes. The majority of B cells in adult mice used VH genes of the J558 family. Although J558 use was significantly higher among the autoantibodies (anti-DNA and anti-RTE) than among the nonbinder Ig, this difference was due to a higher frequency of J558 use by 1-month-old mice. At 3 months, J558 use by the nonbinder Ig increased to the same frequency of J558 use as in the autoantibody population. J558 use in both groups of antibodies exceeded a previously reported estimation of J558 expression in the functional B cell repertoire of young adult MRL-lpr/lpr mice. Several subgroups of antibodies that share properties with pathogenic Ig, including IgG, cross-reactive Ig, and anti-dsDNA autoantibodies, demonstrated a marked preferential expression of the J558 family. These results suggest that there is an age-related bias in the activation of B cells using J558 VH genes in MRL-lpr/lpr mice that is under the influence of a selective force distinct from, or in addition to, an ssDNA or RTE auto-Ag-driven response. PMID:1908876
Foster, M H; MacDonald, M; Barrett, K J; Madaio, M P
Morphologic and Functional Effects of Gamma Secretase Inhibition on Splenic Marginal Zone B Cells
The ?-secretase complex is a promising target in Alzheimer's disease because of its role in the amyloidogenic processing of ?-amyloid precursor protein. This enzyme also catalyzes the cleavage of Notch receptor, resulting in the nuclear translocation of intracellular Notch where it modulates gene transcription. Notch signaling is essential in cell fate decisions during embryogenesis, neuronal differentiation, hematopoiesis, and development of T and B cells, including splenic marginal zone (MZ) B cells. This B cell compartment participates in the early phases of the immune response to blood-borne bacteria and viruses. Chronic treatment with the oral ?-secretase inhibitor RO4929097 resulted in dose-dependent decreased cellularity (atrophy) of the MZ of rats and mice. Significant decreases in relative MZ B-cell numbers of RO4929097-treated animals were confirmed by flow cytometry. Numbers of MZ B cells reverted to normal after a sufficient RO4929097-free recovery period. Functional characterization of the immune response in relation to RO4929097-related MZ B cell decrease was assessed in mice vaccinated with inactivated vesicular stomatitis virus (VSV). Compared with the immunosuppressant cyclosporin A, RO4929097 caused only mild and reversible delayed early neutralizing IgM and IgG responses to VSV. Thus, the functional consequence of MZ B cell decrease on host defense is comparatively mild.
de Vera Mudry, Maria Cristina; Regenass-Lechner, Franziska; Ozmen, Laurence; Altmann, Bernd; Festag, Matthias; Singer, Thomas; Muller, Lutz; Jacobsen, Helmut; Flohr, Alexander
In many B-cell lymphomas, chromosomal translocations are biologic and diagnostic hallmarks of disease. An intriguing subset is formed by the so-called double- hit (DH) lymphomas that are defined by a chromosomal breakpoint affecting the MYC/8q24 locus in combination with another recurrent breakpoint, mainly a t(14;18)(q32;q21) involving BCL2. Recently, these lymphomas have received increased attention, which contributed to the introduction of a novel category of lymphomas in the 2008 WHO classification, "B cell lymphoma unclassifiable with features intermediate between DLBCL and BL." In this review we explore the existing literature for the most recurrent types of DH B-cell lymphomas and the involved genes with their functions, as well as their pathology and clinical aspects including therapy and prognosis. The incidence of aggressive B-cell lymphomas other than Burkitt lymphoma with a MYC breakpoint and in particular a double hit is difficult to assess, because screening by methods like FISH has not been applied on large, unselected series, and the published cytogenetic data may be biased to specific categories of lymphomas. DH lymphomas have been classified heterogeneously but mostly as DLBCL, the majority having a germinal center phenotype and expression of BCL2. Patients with DH lymphomas often present with poor prognostic parameters, including elevated LDH, bone marrow and CNS involvement, and a high IPI score. All studies on larger series of patients suggest a poor prognosis, also if treated with RCHOP or high-intensity treatment modalities. Importantly, this poor outcome cannot be accounted for by the mere presence of a MYC/8q24 breakpoint. Likely, the combination of MYC and BCL2 expression and/or a related high genomic complexity are more important. Compared to these DH lymphomas, BCL6(+)/MYC(+) DH lymphomas are far less common, and in fact most of these cases represent BCL2(+)/BCL6(+)/MYC(+) triple-hit lymphomas with involvement of BCL2 as well. CCND1(+)/MYC(+) DH lymphomas with involvement of 11q13 may also be relatively frequent, the great majority being classified as aggressive variants of mantle cell lymphoma. This suggests that activation of MYC might be an important progression pathway in mantle cell lymphoma as well. Based on clinical significance and the fact that no other solid diagnostic tools are available to identify DH lymphomas, it seems advisable to test all diffuse large B-cell and related lymphomas for MYC and other breakpoints. PMID:21119107
Aukema, Sietse M; Siebert, Reiner; Schuuring, Ed; van Imhoff, Gustaaf W; Kluin-Nelemans, Hanneke C; Boerma, Evert-Jan; Kluin, Philip M
Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning
Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy in adults, is curable in less than 50% of patients. Prognostic models based on pre-treatment characteristics, such as the International Prognostic Index (IPI), are currently used to predict outcome in DLBCL. However, clinical outcome models identify neither the molecular basis of clinical heterogeneity, nor specific therapeutic targets. We analyzed the
Ken N. Ross; Pablo Tamayo; Andrew P. Weng; Jeffery L. Kutok; Ricardo C. T. Aguiar; Michelle Gaasenbeek; Michael Angelo; Michael Reich; Geraldine S. Pinkus; Tane S. Ray; Margaret A. Koval; Kim W. Last; Andrew Norton; T. Andrew Lister; Jill Mesirov; Donna S. Neuberg; Eric S. Lander; Jon C. Aster; Margaret A. Shipp; Todd R. Golub
Hu-ets-1 and Hu-ets-2 Genes are Transposed in Acute Leukemias with (4;11) and (8;21) Translocations
Human probes identifying the cellular homologs of the v-ets gene, Hu-ets-1 and Hu-ets-2, and two panels of rodent-human cell hybrids were used to study specific translocations occurring in acute leukemias. The human ets-1 gene was found to translocate from chromosome 11 to 4 in the t(4;11)(q21;23), a translocation characteristic of a subtype of leukemia that represents the expansion of a
Nicoletta Sacchi; Dennis K. Watson; Ad H. M. Guerts van Kessel; Anne Hagemeijer; John Kersey; Harry D. Drabkin; David Patterson; Takis S. Papas
RFP2, c13ORF1, and FAM10A4 are the most likely tumor suppressor gene candidates for B-cell chronic lymphocytic leukemia
Occurrence of 13q14 deletions between D13S273 and D13S25 in B-cell chronic lymphocytic leukemia (B-CLL) suggests that the region contains a tumor suppressor gene. We constructed a PAC\\/cosmid contig largely corresponding to a 380-kb 13q14 YAC insert that we found deleted in a high proportion of B-CLL patients. We found seven genes by exon trapping, cDNA screening and analysis\\/cDNA extension of
W. J van Everdink; A Baranova; C Lummen; T Tyazhelova; M. W. G Looman; D Ivanov; E Verlind; A Pestova; H Faber; A. Y van der Veen; N Yankovsky; E Vellenga; C. H. C. M Buys
Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(-)/IgM+ B cells.
To analyze the immunoglobulin repertoire of human IgM+ B cells and the CD5(+) and CD5(-) subsets, individual CD19(+)/ IgM+/CD5(+) or CD5(-) B cells were sorted and non-productive as well as productive VH gene rearrangements were amplified from genomic DNA and sequenced. In both subsets, the VH3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members. In the CD5(+) B cell subset, all other VH families were found at a frequency expected from random usage, whereas in the CD5(-) population, VH4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the VH1 family was significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used VH gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells. Evidence for selection based on the D segment and the JH gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity. Somatically hypermutated VHDJH rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM+ B cells, indicating that IgM+ memory B cells were more frequent in the CD5(-) B cell population. Of note, the frequency of specific VH genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulation imposed additional biases on the repertoire of IgM+ B cells. These results indicate that the expressed repertoire of IgM+ B cell subsets is shaped by recombinational bias, as well as selection before and after antigen exposure. Moreover, the influences on the repertoires of CD5(+) and CD5(-) B cells are significantly different, suggesting that human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, respectively.
Brezinschek, H P; Foster, S J; Brezinschek, R I; Dorner, T; Domiati-Saad, R; Lipsky, P E
Long-range Oncogenic Activation of IgH/c-myc Translocations by the IgH 3' Regulatory Region
B cell malignancies, such as human BurkittÆs lymphoma (BL), often harbor translocations that link c-myc or other proto-oncogenes to the immunoglobulin heavy chain locus (IgH)1. The nature of elements that activate oncogenes within such translocations has been a longstanding question. Translocations within IgH involve DNA double strand breaks (DSBs) initiated either by the RAG1/2 endonuclease during V(D)J recombination or by activation induced cytidine deaminase (AID) during class switch recombination (CSR)2-4. V(D)J recombination in progenitor B (pro-B) cells assembles IgH variable region exons upstream of ? constant region (C?) exons, which are the first of several sets of CH exons (ōCH genesö) within a CH locus that spans several hundred kilobases5,6. In mature B cells, CSR deletes C? and replaces it with a downstream CH gene6. An enhancer (iE?) between the variable region exons and C? promotes V(D)J recombination in developing B cells7. In addition, the IgH 3Æ regulatory region (IgH3ÆRR) lies downstream of the CH locus and modulates CSR by long-range transcriptional enhancement of CH genes8-10. Transgenic mice bearing iE? or IgH3ÆRR sequences fused to c-myc are predisposed to B lymphomas, demonstrating such elements can confer oncogenic c-myc expression11-16. However, in many B cell lymphomas, IgH/c-myc translocations delete iE? and place c-myc up to 200kb upstream of the IgH3ÆRR1. We now address the oncogenic role of the IgH3ÆRR by inactivating it in two distinct mouse models for B cell lymphoma with IgH/c-myc translocations. The IgH3ÆRR is dispensable for pro-B lymphomas with V(D)J recombination-initiated translocations, but required for peripheral B cell lymphomas with CSR-associated translocations. As the IgH3ÆRR is not required for CSR-associated IgH breaks or IgH/c-myc translocations in peripheral B cell lymphoma progenitors, we conclude this regulatory region confers oncogenic activity via long-range and developmental stage-specific activation of translocated c-myc genes.
Gostissa, Monica; Yan, Catherine T.; Bianco, Julia M.; Cogne, Michel; Pinaud, Eric; Alt, Frederick W.
LHFP,a Novel Translocation Partner Gene of HMGICin a Lipoma, Is a Member of a New Family of LHFP-like Genes
A major cytogenetic subgroup among human lipomas is characterized by translocations involving theHMGICgene at 12q15. In the context of an ongoing research program aiming at the elucidation of the functional consequences ofHMGICtranslocations in the etiology of lipomas, we have isolated a novel human gene,LHFP(lipomaHMGICfusion partner), that acts as a translocation partner ofHMGICin a lipoma with t(12;13). TheLHFPgene was mapped to
Marleen M. R. Petit; Eric F. P. M. Schoenmakers; Christel Huysmans; Jan M. W. Geurts; Nils Mandahl
[Role of T-cell leukemia translocation-associated gene (TCTA) protein in human osteoclastogenesis].
Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-kappaB ligand (RANKL), TNF-alpha, IL-6, IL-17, and IFN-gamma. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibit human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported, including our report published in 2009. In the current review paper, we summarized papers on TCTA protein before 2009 and our recent findings. PMID:20046013
Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki
Methylenetetrahydrofolate reductase gene polymorphisms association with the risk of diffuse large B cell lymphoma: a meta-analysis.
To date, case-control studies on the association between methylenetetrahydrofolate reductase (MTHFR) and diffuse large B cell lymphoma (DLBCL) have provided either controversial or inconclusive results. To clarify the effect of MTHFR on the risk of diffuse large B cell lymphoma, a meta-analysis of all case-control observational studies was performed. The fixed effects and random effects model showed that the C677T polymorphism was associated with a risk of DLBCL among East Asian populations, and A1298C polymorphism was not associated with a risk of DLBCL among Caucasian and East Asian populations. Our pooled data suggest evidence for a major role of MTHFR C677T polymorphism in the carcinogenesis of DLBCL among East Asian populations. PMID:23812728
Sun, Yun-Yu; An, Li; Xie, Yu-Lan; Xu, Jing-Yan; Wang, Jing
Fine mapping of the EDA gene: A translocation breakpoint is associated with a CpG island that is transcribed
In order to identify the gene for human X-linked anhidrotic ectodermal dysplasia (EDA), a translocation breakpoint in a female with t(X;1)(q13.1;p36.3) and EDA (patient AK) was finely mapped. The EDA region contains five groups of rare-cutter restriction sites that define CpG islands. The two more centromeric of these islands are associated with transcripts of 3.5 kb and 1.8 kb. The third CpG island maps within <1 kb of the translocation breakpoint in patient AK, as indicated by a genomic rearrangement, and {approximately}100 kb centromeric from another previously mapped translocation breakpoint (patient AnLy). Northern analysis with a probe from this CpG island detected an {approximately}6-kb mRNA in several fetal tissues tested. An extended YAC contig of 1,200 kb with an average of fivefold coverage was constructed. The two most telomeric CpG islands map 350 kb telomeric of the two translocations. Taken together, the results suggest that the CpG island just proximal of the AK translocation breakpoint lies at the 5{prime} end of a candidate gene for EDA. 26 refs., 4 figs., 1 tab.
Srivastava, A.K.; Schlessinger, D. [Washington Univ. School of Medicine, St. Louis, MO (United States); Montonen, O. [Univ. of Helsinki (Finland)] [and others
E?/miR-125b transgenic mice develop lethal B-cell malignancies.
MicroRNA-125b-1 (miR-125b-1) is a target of a chromosomal translocation t(11;14)(q24;q32) recurrently found in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation results in overexpression of miR-125b controlled by immunoglobulin heavy chain gene (IGH) regulatory elements. In addition, we found that six out of twenty-one BCP-ALL patients without t(11;14)(q24;q32) showed overexpression of miR-125b. Interestingly, four out of nine patients with BCR/ABL-positive BCP-ALL and one patient with B-cell lymphoid crisis that had progressed from chronic myelogenous leukemia overexpressed miR-125b. To examine the role of the deregulated expression of miR-125b in the development of B-cell tumor in vivo, we generated transgenic mice mimicking the t(11;14)(q24;q32) (E?/miR-125b-TG mice). E?/miR-125b-TG mice overexpressed miR-125b driven by IGH enhancer and promoter and developed IgM-negative or IgM-positive lethal B-cell malignancies with clonal proliferation. B cells obtained from the E?/miR-125b-TG mice were resistant to apoptosis induced by serum starvation. We identified Trp53inp1, a pro-apoptotic gene induced by cell stress, as a novel target gene of miR-125b in hematopoietic cells in vitro and in vivo. Our results provide direct evidence that miR-125b has important roles in the tumorigenesis of precursor B cells. PMID:21738213
Enomoto, Y; Kitaura, J; Hatakeyama, K; Watanuki, J; Akasaka, T; Kato, N; Shimanuki, M; Nishimura, K; Takahashi, M; Taniwaki, M; Haferlach, C; Siebert, R; Dyer, M J S; Asou, N; Aburatani, H; Nakakuma, H; Kitamura, T; Sonoki, T
Tet2 facilitates the de-repression of myeloid target genes during C/EBPa induced transdifferentiation of pre-B cells
SUMMARY The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is C/EBPa induced transdifferentiation of pre-B cells into macrophages. Here we found that C/EBPa binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps C/EBPa rapidly de-repress myeloid genes during the conversion of pre-B cells into macrophages.
Kallin, Eric M.; Rodriguez-Ubreva, Javier; Christensen, J esper; Cimmino, Luisa; Aifantis, Iannis; Helin, Kristian; Ballestar, Esteban; Graf, Thomas
Comparative Gene Expression Profiling Identifies Common Molecular Signatures of NF-?B Activation in Canine and Human Diffuse Large B Cell Lymphoma (DLBCL)
We present the first comparison of global transcriptional changes in canine and human diffuse large B-cell lymphoma (DLBCL), with particular reference to the nuclear factor-kappa B (NF-?B) pathway. Microarray data generated from canine DLBCL and normal lymph nodes were used for differential expression, co-expression and pathway analyses, and compared with analysis of microarray data from human healthy and DLBCL lymph nodes. The comparisons at gene level were performed by mapping the probesets in canine microarrays to orthologous genes in humans and vice versa. A considerable number of differentially expressed genes between canine lymphoma and healthy lymph node samples were also found differentially expressed between human DLBCL and healthy lymph node samples. Principal component analysis using a literature-derived NF-?B target gene set mapped to orthologous canine array probesets and human array probesets clearly separated the healthy and cancer samples in both datasets. The analysis demonstrated that for both human and canine DLBCL there is activation of the NF-?B/p65 canonical pathway, indicating that canine lymphoma could be used as a model to study NF-?B-targeted therapeutics for human lymphoma. To validate this, tissue arrays were generated for canine and human NHL and immunohistochemistry was employed to assess NF-?B activation status. In addition, human and canine B-cell lymphoma lines were assessed for NF-?B activity and the effects of NF-?B inhibition.
Mudaliar, Manikhandan A. V.; Haggart, Ross D.; Miele, Gino; Sellar, Grant; Tan, Karen A. L.; Goodlad, John R.; Milne, Elspeth; Vail, David M.; Kurzman, Ilene
Molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth.
Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, although its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. PMID:7607676
Ishikawa, J; Kaisho, T; Tomizawa, H; Lee, B O; Kobune, Y; Inazawa, J; Oritani, K; Itoh, M; Ochi, T; Ishihara, K
Modulation of B-cell tolerance by Murine Gammaherpesvirus 68 infection: requirement for Orf73 viral gene expression and follicular helper T cells
Viruses such as Epstein¢Barr virus (EBV) have been linked to mechanisms that support autoantibody production in diseases such as systemic lupus erythematosus. However, the mechanisms by which viruses contribute to autoantibody production remain poorly defined. This stems in part, from the high level of seropositivity for EBV (> 95%) and the exquisite species specificity of EBV. In this study we overcame these problems by using murine gammaherpesvirus 68 (MHV68), a virus genetically and biologically related to EBV. We first showed that MHV68 drives autoantibody production by promoting a loss of B-cell anergy. We next showed that MHV68 infection resulted in the expansion of follicular helper T (Tfh) cells in vivo, and that these Tfh cells supported autoantibody production and a loss of B-cell anergy. Finally, we showed that the expansion of Tfh cells and autoantibody production was dependent on the establishment of viral latency and expression of a functional viral gene called Orf73. Collectively, our studies highlighted an unexpected role for viral latency in the development of autoantibodies following MHV68 infection and suggest that virus-induced expansion of Tfh cells probably plays a key role in the loss of B-cell anergy.
Gauld, Stephen B; Santis, Jessica L; Kulinski, Joseph M; McGraw, Jennifer A; Leonardo, Steven M; Ruder, Elizabeth A; Maier, Weston; Tarakanova, Vera L
Structural profiles of TP53 gene mutations predict clinical outcome in diffuse large B-cell lymphoma: an international collaborative study
The purpose of this study is to correlate the presence of TP53 gene mutations with the clinical outcome of a cohort of patients with diffuse large B-cell lymphoma (DLBCL) assembled from 12 medical centers. TP53 mutations were identified in 102 of 477 patients, and the overall survival (OS) of patients with TP53 mutations was significantly worse than those with wild-type TP53 (P < .001). However, subsets of TP53 mutations were found to have different effects on OS. Mutations in the TP53 DNA-binding domains were the strongest predictors of poor OS (P < .001). Mutations in the Loop-Sheet-Helix and Loop-L3 were associated with significantly decreased OS (P = .002), but OS was not significantly affected by mutations in Loop-L2. A subset of missense mutations (His158, His175, Ser245, Gln248, His273, Arg280, and Arg282) in the DNA-binding domains had the worst prognosis. Multivariate analysis confirmed that the International Prognostic Index and mutations in the DNA-binding domains were independent predictors of OS. TP53 mutations also stratified patients with germinal center B cell¢like DLBCL, but not nongerminal center B cell¢like DLBCL, into molecularly distinct subsets with different survivals. This study shows the prognostic importance of mutations in the TP53 DNA-binding domains in patients with DLBCL.
Leroy, Karen; M?ller, Michael B.; Colleoni, Gisele W. B.; Sanchez-Beato, Margarita; Kerbauy, Fabio R.; Haioun, Corinne; Eickhoff, Jens C.; Young, Allen H.; Gaulard, Philippe; Piris, Miguel A.; Oberley, Terry D.; Rehrauer, William M.; Kahl, Brad S.; Malter, James S.; Campo, Elias; Delabie, Jan; Gascoyne, Randy D.; Rosenwald, Andreas; Rimsza, Lisa; Huang, James; Braziel, Rita M.; Jaffe, Elaine S.; Wilson, Wyndham H.; Staudt, Louis M.; Vose, Julie M.; Chan, Wing C.; Weisenburger, Dennis D.; Greiner, Timothy C.
Increased AICD generation does not result in increased nuclear translocation or activation of target gene transcription
A sequence of amyloid precursor protein (APP) cleavages culminates in the sequential release of the APP intracellular domain (AICD) and the amyloid {beta} peptide (A{beta}) and/or p3 fragment. One of the environmental factors favouring the accumulation of AICD appears to be a rise in intracellular pH. Here we further identified the metabolism and subcellular localization of artificially expressed constructs under such conditions. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely cleaved from C83. While the AICD surplus was unable to further activate transcription of a luciferase reporter via a Gal4-DNA-binding domain, it failed entirely via the endogenous promoter regions of proposed target genes, APP and KAI1. The lack of a specific transactivation potential was also demonstrated by the unchanged levels of target gene mRNA. However, rather than translocating to the nucleus, the AICD surplus remains membrane tethered or free in the cytosol where it interacts with Fe65. Therefore we provide strong evidence that an increase in AICD generation does not directly promote gene activation of previously proposed target 0011gen.
Waldron, Elaine; Isbert, Simone [Institute of Physiological Chemistry and Pathobiochemistry, Molecular Neurodegeneration, Johannes Gutenberg-University Mainz, 55099 Mainz (Germany); Kern, Andreas [Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University Mainz, 55099 Mainz (Germany); Jaeger, Sebastian; Martin, Anne M. [Institute of Physiological Chemistry and Pathobiochemistry, Molecular Neurodegeneration, Johannes Gutenberg-University Mainz, 55099 Mainz (Germany); Hebert, Sebastien S. [Department for Molecular and Developmental Genetics, VIB, Leuven (Belgium); Center for Human Genetics, KULeuven, Herestraat 49, Leuven 3000 (Belgium); Behl, Christian [Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University Mainz, 55099 Mainz (Germany); Weggen, Sascha [Institute of Neuropathology, Heinrich-Heine-University Duesseldorf, 40225 Duesseldorf (Germany); De Strooper, Bart [Department for Molecular and Developmental Genetics, VIB, Leuven (Belgium); Center for Human Genetics, KULeuven, Herestraat 49, Leuven 3000 (Belgium); Pietrzik, Claus U. [Institute of Physiological Chemistry and Pathobiochemistry, Molecular Neurodegeneration, Johannes Gutenberg-University Mainz, 55099 Mainz (Germany)], E-mail: firstname.lastname@example.com
Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias
Recurring chromosomal translocations involving chromosome 11, band q23, have been observed in acute lymphoid leukemias and especially in acute myeloid leukemias. The authors recently showed that breakpoints in four 11q23 translocations, t(4,11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13.3), were contained within a yeast artificial chromosome clone bearing the CD3D and CD3G gene loci. They have identified within the CD3 yeast artificial chromosome
S. Ziemin-van der Poel; N. R. McCabe; H. J. Gill; R. Espinosa; Y. Patel; A. Harden; P. Rubinelli; S. D. Smith; M. M. LeBeau; J. D. Rowley; M. O. Diaz
The Pontin series of recombinant alien translocations in bread wheat: single translocations integrating combinations of Bdv2, Lr19 and Sr25 disease-resistance genes from Thinopyrum intermedium and Th. ponticum.
Two bread wheat lines each with a translocation on chromosome 7DL from either Thinopyrum intermedium (TC5 and TC14) or Thinopyrum ponticum (T4m), were hybridized in a ph1b mutant background to enhance recombination between the two translocated chromosomal segments. The frequency of recombinants was high in lines derived from the larger and similar-sized translocations (TC5/T4m), but much lower when derived from different-sized translocations (TC14/T4m). Recombinant translocations contained combinations of resistance genes Bdv2, Lr19 and Sr25 conferring resistance to Barley yellow dwarf virus (BYDV), leaf rust and stem rust, respectively. Their genetic composition was identified using bioassays and molecular markers specific for the two progenitor Thinopyrum species. This set of 7DL Th. ponticum/intermedium recombinant translocations was termed the Pontin series. In addition to Thinopyrum markers, the size of the translocation was estimated with the aid of wheat markers mapped on each of the 7DL deletion bins. Bioassays for BYDV, leaf rust and stem rust were performed under greenhouse and field conditions. Once separated from ph1b background, the Pontin recombinant translocations were stable and showed normal inheritance in successive backcrosses. The reported Pontin translocations integrate important resistance genes in a single linkage block which will allow simultaneous selection of disease resistance. Combinations of Bdv2Ā+ĀLr19 or Lr19Ā+ĀSr25 in both long and short translocations, are available to date. The smaller Pontins, comprising only 20Ā% of the distal portion of 7DL, will be most attractive to breeders. PMID:23807636
Ayala-Navarrete, L I; Mechanicos, A A; Gibson, J M; Singh, D; Bariana, H S; Fletcher, J; Shorter, S; Larkin, Philip J
A single recessive gene controls cadmium translocation in the cadmium hyperaccumulating rice cultivar Cho-Ko-Koku
The heavy metal cadmium (Cd) is highly toxic to humans and can enter food chains from contaminated crop fields. Understanding\\u000a the molecular mechanisms of Cd accumulation in crop species will aid production of safe Cd-free food. Here, we identified\\u000a a single recessive gene that allowed higher Cd translocation in rice, and also determined the chromosomal location of the\\u000a gene. The
Kouichi Tezuka; Hidenori Miyadate; Kazunao Katou; Ikuko Kodama; Shinichi Matsumoto; Tomohiko Kawamoto; Satoshi Masaki; Hideki Satoh; Masayuki Yamaguchi; Kenji Sakurai; Hidekazu Takahashi; Namiko Satoh-Nagasawa; Akio Watanabe; Tatsuhito Fujimura; Hiromori Akagi
An epigenetic chromatin remodeling role for NFATc1 in transcriptional regulation of growth and survival genes in diffuse large B-cell lymphomas
The nuclear factor of activated T cells (NFAT) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway in the pathogenesis of various malignancies, including diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanism(s) underlying NFATc1 regulation of genes controlling lymphoma cell growth and survival is still unclear. In this study, we demonstrate that the transcription factor NFATc1 regulates gene expression in DLBCL cells through a chromatin remodeling mechanism that involves recruitment of the SWItch/Sucrose NonFermentable chromatin remodeling complex ATPase enzyme SMARCA4 (also known as Brahma-related gene 1) to NFATc1 targeted gene promoters. The NFATc1/Brahma-related gene 1 complex induces promoter DNase I hypersensitive sites and recruits other transcription factors to the active chromatin site to regulate gene transcription. Targeting NFATc1 with specific small hairpin RNA inhibits DNase I hypersensitive site formation and down-regulates target gene expression. Our data support a novel epigenetic control mechanism for the transcriptional regulation of growth and survival genes by NFATc1 in the pathophysiology of DLBCL and suggests that targeting NFATc1 could potentially have therapeutic value.
Tamayo, Archito T.; Li, Changping; Bueso-Ramos, Carlos; Ford, Richard J.
The BCL6 transcriptional program features repression of multiple oncogenes in primary B cells and is deregulated in DLBCL
The BCL6 transcriptional repressor is required for development of germinal center (GC) B cells and when expressed constitutively causes diffuse large B-cell lymphomas (DLBCLs). We examined genome-wide BCL6 promoter binding in GC B cells versus DLBCLs to better understand its function in these settings. BCL6 bound to both distinct and common sets of functionally related gene in normal GC cells versus DLBCL cells. Certain BCL6 target genes were preferentially repressed in GC B cells, but not DLBCL cells. Several such genes have prominent oncogenic functions, such as BCL2, MYC, BMI1, EIF4E, JUNB, and CCND1. BCL6 and BCL2 expression was negatively correlated in primary DLBCLs except in the presence of BCL2 translocations. The specific BCL6 inhibitor retro-inverso BCL6 peptidomimetic inhibitor-induced expression of BCL2 and other oncogenes, consistent with direct repression effects by BCL6. These data are consistent with a model whereby BCL6 can directly silence oncogenes in GC B cells and counterbalance its own tumorigenic potential. Finally, a BCL6 consensus sequence and binding sites for other physiologically relevant transcription factors were highly enriched among target genes and distributed in a pathway-dependent manner, suggesting that BCL6 forms specific regulatory circuits with other B-cell transcriptional factors.
Ci, Weimin; Polo, Jose M.; Cerchietti, Leandro; Shaknovich, Rita; Wang, Ling; Yang, Shao Ning; Ye, Kenny; Farinha, Pedro; Horsman, Douglas E.; Gascoyne, Randy D.
Rearrangements of MYC gene facilitate risk stratification in diffuse large B-cell lymphoma patients treated with rituximab-CHOP.
In order to address the debatable prognostic role of MYC rearrangements in diffuse large B-cell lymphoma patients treated with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, we evaluated MYC rearrangements by fluorescence in situ hybridization in 563 cases using break-apart probes and IGH/MYC dual-fusion probes. Concurrent BCL2 and BCL6 aberrations were also assessed. Data were correlated with clinicopathological variables and prognostic parameters. MYC rearrangements were observed in 39/432 evaluable cases (9%), including 4 rearrangements detectable only with the dual-fusion probes, 15 detectable only with the break-apart probes and 20 detectable with both dual-fusion probes and break-apart probes. MYC rearrangements correlated with germinal center B-cell origin (P=0.02), MYC protein expression (P=0.032), and larger tumor mass size (P=0.0003). Patients with MYC rearrangements were more likely to be treatment resistant (P<0.0001). All types of MYC rearrangements were associated with poorer disease-specific survival, that is, 20/39 dead, median disease-specific survival 42 months, compared with 98/393 dead among the non-rearranged cases, median disease-specific survival not reached (P=0.0002). Cases with MYC rearrangements that overexpressed MYC protein were at risk with respect to disease-specific survival independent of the International Prognostic Index (P=0.046 and P<0.001, respectively). Presence of concurrent BCL2 aberrations but not of BCL6 aberrations was prognostically additive. Radiotherapy seemed to diminish the prognostic effects of MYC rearrangements in diffuse large B-cell lymphoma patients since only 2/10 irradiated patients with MYC rearrangements died of/with disease, compared with 16/28 non-irradiated patients with MYC rearrangements. We conclude that MYC rearrangements add prognostic information for individual risk estimation and such cases might represent a distinct, biologically determined disease subgroup. PMID:24336156
Tzankov, Alexandar; Xu-Monette, Zijun Y; Gerhard, Marc; Visco, Carlo; Dirnhofer, Stephan; Gisin, Nora; Dybkaer, Karen; Orazi, Attilio; Bhagat, Govind; Richards, Kristy L; Hsi, Eric D; Choi, William Wl; van Krieken, J Han; Ponzoni, Maurilio; Ferreri, Andrķs Jm; Ye, Qing; Winter, Jane N; Farnen, John P; Piris, Miguel A; M°ller, Michael B; You, M James; McDonnell, Timothy; Medeiros, L Jeffrey; Young, Ken H
Bortezomib induces nuclear translocation of I?B? resulting in gene-specific suppression of NF-?B--dependent transcription and induction of apoptosis in CTCL.
Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor ?B (NF-?B), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-?B activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-?B activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-?B activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of I?B? (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-?B p65 and p50 in the nucleus and inhibits NF-?B DNA binding activity. Surprisingly, however, while expression of NF-?B-dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-?B p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-?B-dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-?B dimers recruited to NF-?B-responsive promoters. PMID:21224428
Juvekar, Ashish; Manna, Subrata; Ramaswami, Sitharam; Chang, Tzu-Pei; Vu, Hai-Yen; Ghosh, Chandra C; Celiker, Mahmut Y; Vancurova, Ivana
Clinical features and outcome of MLL gene rearranged acute lymphoblastic leukemia in infants with additional chromosomal abnormalities other than 11q23 translocation
The treatment outcome for infant acute lymphoblastic leukemia (ALL) with positive MLL gene rearrangements remains poor. We analyzed whether additional chromosomal abnormalities (ACA) other than 11q23 translocation could affect the disease behavior and its prognosis.Eighteen of seventy-four patients with infant acute lymphoblastic leukemia showed ACA, including three-way translocations in four, other novel translocations in four, and complex structural chromosomal changes
Hisamichi Tauchi; Daisuke Tomizawa; Mariko Eguchi; Minenori Eguchi-Ishimae; Katsuyoshi Koh; Masahiro Hirayama; Noriko Miyamura; Naoko Kinukawa; Yasuhide Hayashi; Keizo Horibe; Eiichi Ishii
The Transcriptional Co-Repressor Myeloid Translocation Gene 16 Inhibits Glycolysis and Stimulates Mitochondrial Respiration
The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor¢containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline¢dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia¢stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein¢protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen¢activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti¢tumor effect.
Kumar, Parveen; Sharoyko, Vladimir V.; Spegel, Peter; Gullberg, Urban; Mulder, Hindrik; Olsson, Inge; Ajore, Ram
Nuclear translocation uncovers the amyloid Peptide a?42 as a regulator of gene transcription.
Although soluble species of the amyloid-? peptide A?42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-? (A?). Here, we show that A? peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear A?42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled A? in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of A? in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for A?42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that A?42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic A?42 wild-type peptide. Shorter peptides (A?38 or A?40) and other longer peptides (nontoxic A?42 G33A substitution or A?43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of A?42 impacts gene regulation, and deleterious effects of A?42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes. PMID:24878959
Barucker, Christian; Harmeier, Anja; Weiske, Joerg; Fauler, Beatrix; Albring, Kai Frederik; Prokop, Stefan; Hildebrand, Peter; Lurz, Rudi; Heppner, Frank L; Huber, Otmar; Multhaup, Gerhard
B-cell neoplasia associated gene with multiple splicing (BCMS): the candidate B-CLL gene on 13q14 comprises more than 560 kb covering all critical regions
Deletions in chromosomal band 13q14.3 occur in >50% of B-cell chronic lymphocytic leukemias (B-CLL) and mantle cell lymphoma, indicating the localiza- tion of a tumor suppressor gene involved in the pathomechanism of these diseases. Within a 400 kb recurrently deleted segment at least two minimally deleted subregions had been reported. For the two genes residing in the proximal subregion, initially
Stephan Wolf; Claudia Schaffner; Christian Korz; Hartmut D÷hner; Stephan Stilgenbauer; Peter Lichter
Action of glucagon-like peptide 1 and glucose levels on corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells.
Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic-pituitary-adrenal (HPA) axis. Glucagon-like peptide-1 (GLP-1), an important regulator of metabolism or energy homeostasis, is implicated in the regulation of the HPA axis in response to stress and may act directly on CRF and AVP neurons. To elucidate the direct regulation of CRF and AVP genes by GLP-1 in the hypothalamus, we examined the effect of GLP-1 in hypothalamic 4B cells, which show the characteristics of hypothalamic paraventricular nucleus neurons. The mRNA of GLP-1 receptor was detected in 4B cells by RT-PCR. GLP-1 significantly stimulated both CRF and AVP mRNA levels. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. GLP-1 directly stimulated the activities of both CRF and AVP promoters in hypothalamic 4B cells. Basal promoter activities of both CRF and AVP were increased in higher glucose medium. In addition, CRF and AVP promoter activities were increased by GLP-1 in standard or low glucose medium but not in higher glucose medium. An equimolar concentration of metabolically inactive l-glucose failed to mimic the effect of d-glucose, indicating that the event was caused by changes in glucose levels and not by hyperosmolality. Together, these data suggest that GLP-1 would contribute to stress responses through activation of CRF and AVP genes in the hypothalamic cells. Hyperglycemia may be one of the stressors enhancing the syntheses of CRF and AVP in the hypothalamus. PMID:22801106
Kageyama, Kazunori; Yamagata, Satoshi; Akimoto, Kanako; Sugiyama, Aya; Murasawa, Shingo; Suda, Toshihiro
Over half of breakpoints in gene pairs involved in cancer-specific recurrent translocations are mapped to human chromosomal fragile sites
Background Gene rearrangements such as chromosomal translocations have been shown to contribute to cancer development. Human chromosomal fragile sites are regions of the genome especially prone to breakage, and have been implicated in various chromosome abnormalities found in cancer. However, there has been no comprehensive and quantitative examination of the location of fragile sites in relation to all chromosomal aberrations. Results Using up-to-date databases containing all cancer-specific recurrent translocations, we have examined 444 unique pairs of genes involved in these translocations to determine the correlation of translocation breakpoints and fragile sites in the gene pairs. We found that over half (52%) of translocation breakpoints in at least one gene of these gene pairs are mapped to fragile sites. Among these, we examined the DNA sequences within and flanking three randomly selected pairs of translocation-prone genes, and found that they exhibit characteristic features of fragile DNA, with frequent AT-rich flexibility islands and the potential of forming highly stable secondary structures. Conclusion Our study is the first to examine gene pairs involved in all recurrent chromosomal translocations observed in tumor cells, and to correlate the location of more than half of breakpoints to positions of known fragile sites. These results provide strong evidence to support a causative role for fragile sites in the generation of cancer-specific chromosomal rearrangements.
Burrow, Allison A; Williams, Laura E; Pierce, Levi CT; Wang, Yuh-Hwa
Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation.
Dietary polyunsaturated fatty acids (PUFAs) are potent inhibitors of hepatic glycolysis and lipogenesis. Recently, carbohydrate-responsive element-binding protein (ChREBP) was implicated in the regulation by glucose of glycolytic and lipogenic genes, including those encoding L-pyruvate kinase (L-PK) and fatty acid synthase (FAS). The aim of our study was to assess the role of ChREBP in the control of L-PK and FAS gene expression by PUFAs. We demonstrated in mice, both in vivo and in vitro, that PUFAs [linoleate (C18:2), eicosapentanoic acid (C20:5), and docosahexaenoic acid (C22:6)] suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translocation from the cytosol to the nucleus, independently of an activation of the AMP-activated protein kinase, previously shown to regulate ChREBP activity. In contrast, saturated [stearate (C18)] and monounsaturated fatty acids [oleate (C18:1)] had no effect. Since glucose metabolism via the pentose phosphate pathway is determinant for ChREBP nuclear translocation, the decrease in xylulose 5-phosphate concentrations caused by a PUFA diet favors a PUFA-mediated inhibition of ChREBP translocation. In addition, overexpression of a constitutive nuclear ChREBP isoform in cultured hepatocytes significantly reduced the PUFA inhibition of both L-PK and FAS gene expression. Our results demonstrate that the suppressive effect of PUFAs on these genes is primarily caused by an alteration of ChREBP nuclear translocation. In conclusion, we describe a novel mechanism to explain the inhibitory effect of PUFAs on the genes encoding L-PK and FAS and demonstrate that ChREBP is a pivotal transcription factor responsible for coordinating the PUFA suppression of glycolytic and lipogenic genes. PMID:16184193
Dentin, Renaud; Benhamed, Fadila; Pķgorier, Jean-Paul; Foufelle, Fabienne; Viollet, Benoit; Vaulont, Sophie; Girard, Jean; Postic, Catherine
Molecular portraits of B cell lineage commitment.
In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B cells (PBCs) by using serial analysis of gene expression. From more than 100,000 serial analysis of gene expression tags collected from human CD34(+) HSCs and CD10(+) CD19(+) PBCs, 42,399 unique transcripts were identified in HSCs but only 16,786 in PBCs, suggesting that more than 60% of transcripts expressed in HSCs were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptor (pre-BCR)-associated genes are virtually missing in HSCs but account for more than 10% of the transcriptome of PBCs, which also show increased expression of apoptosis-related genes. Both concentration of the transcriptional repertoire on pre-BCR-related genes together with marked up-regulation of apoptosis mediators in PBC might reflect selection for the expression of a functional pre-BCR within the bone marrow. Besides known regulator genes of early B cell development such as PAX5, E2A, and EBF, the most abundantly expressed genes in PBCs include ATM, PDGFRA, SIAH1, PIM2, C/EBPB, WNT16, and TCL1, the role of which has not been established yet in early B cell development. PMID:12119411
M³schen, Markus; Lee, Sanggyu; Zhou, Guolin; Feldhahn, Niklas; Barath, Varun Singh; Chen, Jianjun; Moers, Cordula; Kr÷nke, Martin; Rowley, Janet D; Wang, San Ming
Amplified RPS6KB1 and CDC2 genes are potential biomarkers for aggressive HIV+/EBV+ diffuse large B-cell lymphomas
RPS6KB1 encodes p70S6K/p85S6K, which plays a role in the PI3K/Akt/mTOR signal transduction pathway. CDC2 gene encodes cdc2, which is critical for G2/M cell cycle progression. We had previously shown that amplified RPS6KB1 and CDC2 are commonly detected in the EBV+ diffuse large B-cell lymphoma (DLBCL) in HIV patients. In current study, we further evaluated the amplified RPS6KB1 and CDC2 genes in 12 HIV-related aggressive B-cell lymphomas and 10 non-HIV-related DLBCL using real time quantitative PCR. The cases were divided into 4 groups: 1) HIV-/EBV-; 2) HIV-/EBV+; 3) HIV+/EBV-; and 4) HIV+/EBV+. Receiver operating characteristic (ROC) curve and the area under the curve (AUC) was used to assess the ability of each gene to distinguish non-HIV+/EBV+ cases from HIV+/EBV+ cases. The AUC was estimated to be 0.76 for RPS6KB1 and 0.74 for CDC2 by using the Mann-Whitney statistic. Amplified RPS6KB1 and CDC2 genes were more frequently detected in common variants of DLBCL associated with HIV infection. Taken together, amplified RPS6KB1 and CDC2 are potential biomarkers for the aggressive DLBCL, particularly in HIV+/EBV+ patients. This study also suggests that the HIV+/EBV+ aggressive DLBCL could be potentially treated by targeting RPS6KB1 and CDC2 genes.
Zhao, Xianfeng F; Zhao, Merry Y; Chai, Ling; Kukuruga, Debra; Tan, Ming; Stass, Sanford A
Coffee constituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression.
Oxidative cellular stress initiates Nrf2 translocation into the nucleus, thus inducing antioxidant response element (ARE)-mediated expression of Phase II enzymes involved in detoxification and antioxidant defence. We investigated whether coffee extracts (CEs) of different proveniences and selected constituents have an impact on the Nrf2/ARE pathway in human colon carcinoma cells (HT29). Assessed as increased nuclear Nrf2 protein, Nrf2 nuclear translocation was modulated by different CEs as observed by Western blot analysis. In addition to the known Nrf2 activator 5-O-caffeoylquinic acid (CGA), pyridinium derivatives like the N-methylpyridinium ion (NMP) were identified as potent activators of Nrf2 nuclear translocation and ARE-dependent gene expression of selected antioxidative Phase II enzymes in HT29. Thereby, the substitution pattern at the pyridinium core structure determined the impact on Nrf2-signalling. In contrast, trigonelline was found to interfere with Nrf2 activation, effectively suppressing the NMP-mediated induction of Nrf2/ARE-dependent gene expression. In conclusion, several coffee constituents, partly already present in the raw material as well as those generated during the roasting process, contribute to the Nrf2-translocating properties of consumer-relevant coffee. A fine tuning in the degradation/formation of activating and deactivating constituents of the Nrf2/ARE pathway during the roasting process appears to be critical for the chemopreventive properties of the final coffee product. PMID:20655719
Boettler, Ute; Sommerfeld, Katharina; Volz, Nadine; Pahlke, Gudrun; Teller, Nicole; Somoza, Veronika; Lang, Roman; Hofmann, Thomas; Marko, Doris
Translocation of the c-myc Gene into the Immunoglobulin Heavy Chain Locus in Human Burkitt Lymphoma and Murine Plasmacytoma Cells
The consistent appearance of specific chromosomal translocations in human Burkitt lymphomas and murine plasmacytomas has suggested that these translocations might play a role in malignant transformation. Here we show that transformation of these cells is frequently accompanied by the somatic rearrangement of a cellular analogue of an avian retrovirus transforming gene, c-myc. Moreover, we map c-myc to human chromosome 8
R. Taub; I. Kirsch; C. Morton; G. Lenoir; D. Swan; S. Tronick; S. Aaronson; P. Leder
Structural analysis of the mouse chromosomal gene encoding interleukin 4 which expresses B cell, T cell and mast cell stimulating activities.
Based on homology with the mouse interleukin 4 (IL-4) cDNA that expresses B cell, T cell, and mast cell stimulating activities (Lee, F. et al., (1986) Proc. Natl. Acad. Sci. USA 83, 2061), we have isolated from a Balb/c mouse liver DNA library the mouse chromosomal gene and analyzed its overall structure. The gene occurs as a single copy in the haploid genome and contains four exons and three introns. The exon sequences almost completely match the cloned cDNA sequence. Interestingly, there is a fairly high degree of homology between mouse IL-4 and mouse IL-2 genes extending more than 200 bp upstream of a "TATA" like sequence located 20 bp upstream of the transcription initiation site. These sequences may play an important role in the regulated expression of this gene in concanavalin A or antigen-stimulated T lymphocytes. The supernatant of COS7 cells transfected with plasmid DNA containing the entire gene exhibited both T cell growth factor and mast cell growth factor activities. Images
Otsuka, T; Villaret, D; Yokota, T; Takebe, Y; Lee, F; Arai, N; Arai, K
Inactivation of the DCC tumor suppressor gene in a B-cell lymphoma cell line with the alteration of chromosome 18.
A B-cell lymphoma cell line, designated KML-1, was established from pleural effusion of a patient with non-Hodgkin's lymphoma of large-cell type. The lymphoma arose in the pelvis and ran an aggressive clinical course. Chromosome analysis of the cell line exhibited a complex karyotype including the loss of chromosome 18. To evaluate the molecular events in the cell line that may be associated with the development of the lymphoma, we investigated the expression and/or alterations of several classes of human genes, including oncogenes, tumor suppressor genes, and cytokine genes. The expression of the DCC (deleted in colorectal cancer) gene, located on the chromosome 18q21, was extremely reduced in KML-1 cell line, as compared with that in a normal spleen tissue and other 4 lymphoma cell lines by the reverse transcription-polymerase-chain-reaction (RT-PCR) method. This finding suggests that inactivation of the DCC gene might play a role in the pathogenesis of the case of lymphoma. PMID:7572991
Ikezoe, T; Miyagi, T; Kubota, T; Taguchi, T; Ohtsuki, Y; Miyake, K; Inokuchi, K; Nomura, T; Koeffler, H P; Miyoshi, I
Clinicopathological study of gene rearrangement and microRNA expression of primary central nervous system diffuse large B-cell lymphomas.
We studied the clinicopathological and imaging characteristics of primary central nervous system diffuse large B-cell lymphomas (PCNS-DLBCL). Imaging, pathologic histology, and immunohistochemical staining characteristics were analyzed, and the immunoglobulin heavy and light chain gene rearrangement of 25 PCNS-DLBCL cases was examined. MicroRNA was extracted from 10 cases each of PCNS-DLBCL, extracerebral germinal center DLBCL (GC-DLBCL), and extracerebral non-GC-DLBCL (NGC-DLBCL); we conducted chip hybridization and comparatively analyzed the difference among the three. PCNS-DLBCLs typically involved no less than two cerebral lobes (10/25); the frontal lobe was affected most often (6/25). Target-shaped structures were observed in all PCNS-DLBCLs due to the proliferation of centroblast-like large lymphocytes surrounding the vessels. There was strong and diffuse immunostaining for CD20 and CD79a, and negative immunostaining for CD3, CD5, CD23, and cyclin D1 for all PCNS-DLBCLs. The percentage of cells with nuclear positivity for anti-Ki67 antibody ranged 50-90% (mean, 80%). Three, 19, and 22 PCNS-DLBCLs were CD10-, Bcl-6-, and melanoma ubiquitous mutated 1-positive, respectively. Twenty-four PCNS-DLBCLs were B-cell monoclonal. MicroRNA hybridization showed that 788 PCNS-DLBCL microRNAs/segments increased to at least twice that of NGC-DLBCLs, and 401 PCNS-DLBCL microRNAs/segments declined to less than half of that of NGC-DLBCLs. Six hundred and eleven PCNS-DLBCL microRNAs/segments increased to at least twice that of GC-DLBCLs, and 229 PCNS-DLBCL microRNAs/segments declined to less than half of that in GC-DLBCLs. PCNS-DLBCL typically affected multiple sites, tended to occur in older men, arose from activated B cells, had high B-cell monoclonality; its microRNA expression differed from that of NGC-DLBCL and GC-DLBCL. PMID:24133582
Zheng, Jinfeng; Xu, Jiagang; Ma, Shufang; Sun, Xiyan; Geng, Ming; Wang, Lin
Suppression of IgE B cells and IgE binding to Fc?RI by gene therapy with single chain anti-IgE1
This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org. Immunoglobulin E (IgE) plays a pivotal role in allergic reactions and asthma through its ability to bind to the mast cell Fc receptor for IgE (Fc?RI). Current therapies to suppress such reactions include passive treatment with neutralizing antibodies to IgE that block its binding to Fc?RI. In theory, induction of immune tolerance in the B lymphocytes that carry IgE antigen receptors and give rise to IgE secreting cells should provide longer term efficacy. However, recent data have suggested that such memory cells may lack cell surface IgE. Using a gene therapy approach, we show that a recombinant single-chain neutralizing anti-IgE could not only neutralize circulating IgE, but also reduce IgE+ B cell numbers and H-chain transcripts. Therapeutic anti-IgE stimulated a calcium response in primary B cells or in a B cell line expressing membrane IgE and suppressed IgE secretion in vitro suggesting that active signaling through membrane IgE likely promoted tolerance. Interestingly, upon subsequent challenge of anti-IgE treated mice with an IgE crosslinking reagent capable of inducing activation of IgE-decorated mast cells, an anaphylaxis reaction was induced, apparently via a Fc?RIII pathway involving recognition of anti-IgE antibody itself. These studies have important implications for the optimal design of safe and effective anti-IgE therapies and suggest that the IgE memory B cells may be targeted by such genetic antibody therapies.
Ota, Takayuki; Aoki-Ota, Miyo; Duong, Bao Hoa; Nemazee, David
Systemic autoimmunity such as systemic lupus erythematosus (SLE) is associated with the loss of B-cell tolerance, B-cell dysregulation and autoantibody production. While some autoantibodies may contribute to the pathology seen with SLE, numerous studies have shown that dysregulation of T-cell function is another critical aspect driving disease. The positive results obtained in clinical trials using T-cell- or B-cell-specific treatments have
Joanna Groom; Fabienne Mackay
Molecular cloning and characterization of genes for antibodies generated by orbital tissue-infiltrating B-cells in Graves` ophthalmopathy
Graves` ophthalmopathy is a distressing autoimmune disease of unknown etiology. Analysis of the genes for antibodies secreted by orbital tissue-infiltrating plasma cells might provide insight into the pathogenesis of this disease. The authors, therefore, constructed an immunoglobulin heavy (H) chain and an immunoglobulin k light (L) chain cDNA library from the orbital tissue of a patient with active Graves` ophthalmopathy. Analysis of 15 H (IgG1) and 15 L (k) chains revealed a restricted spectrum of variable region genes. Fourteen of 15 variable k genes were about 94% homologous to the closest known germline gene, KL012. Thirteen of 15 H chain genes were 91% and 90% homologous to the closest germline genes, DP10 and hv1263, respectively. Remarkably, these germline genes also code for other autoantibodies to striated muscle (KL012) and thyroid peridase (KL012 and hv1263). These studies raise the possibility that particular germline genes may be associated with autoimmunity in humans. Further, the present study opens the way to identifying ocular autoantigens that may be the target of an humoral immune response. 29 refs., 4 figs., 1 tab.
Jaume, J.C.; Portolano, S.; Prummel, M.F.; McLachlan, S.M.; Rapoport, B. [Univ. of California, San Francisco, CA (United States)] [Univ. of California, San Francisco, CA (United States)
Analysis of Chromosomal Translocations
\\u000a Chromosomal translocations were the first target for the specific detection of residual tumor cells in bone marrow and peripheral\\u000a blood. Some types of leukemia are regularly or generally associated with translocations. In chronic myelogenous leukemia (CML)\\u000a and a proportion of patients with acute lymphoblastic leukemia (ALL), the t(9;22)(q34;q11) translocation leads to the fusion\\u000a of the ABL gene with part of
Andreas Hochhaus
CD19 is a major B cell receptor-independent activator of MYC-driven B-lymphomagenesis
PAX5, a B cell¢specific transcription factor, is overexpressed through chromosomal translocations in a subset of B cell lymphomas. Previously, we had shown that activation of immunoreceptor tyrosine-based activation motif (ITAM) proteins and B cell receptor (BCR) signaling by PAX5 contributes to B-lymphomagenesis. However, the effect of PAX5 on other oncogenic transcription factor-controlled pathways is unknown. Using a MYC-induced murine lymphoma model as well as MYC-transformed human B cell lines, we found that PAX5 controls c-MYC protein stability and steady-state levels. This promoter-independent, posttranslational mechanism of c-MYC regulation was independent of ITAM/BCR activity. Instead it was controlled by another PAX5 target, CD19, through the PI3K-AKT-GSK3? axis. Consequently, MYC levels in B cells from CD19-deficient mice were sharply reduced. Conversely, reexpression of CD19 in murine lymphomas with spontaneous silencing of PAX5 boosted MYC levels, expression of its key target genes, cell proliferation in vitro, and overall tumor growth in vivo. In human B-lymphomas, CD19 mRNA levels were found to correlate with those of MYC-activated genes. They also negatively correlated with the overall survival of patients with lymphoma in the same way that MYC levels do. Thus, CD19 is a major BCR-independent regulator of MYC-driven neoplastic growth in B cell neoplasms.
Chung, Elaine Y.; Psathas, James N.; Yu, Duonan; Li, Yimei; Weiss, Mitchell J.; Thomas-Tikhonenko, Andrei
A Constitutional Translocation t(1;17)(p36.2;q11.2) in a Neuroblastoma Patient Disrupts the Human NBPF1 and ACCN1 Genes
The human 1p36 region is deleted in many different types of tumors, and so it probably harbors one or more tumor suppressor genes. In a Belgian neuroblastoma patient, a constitutional balanced translocation t(1;17)(p36.2;q11.2) may have led to the development of the tumor by disrupting or activating a gene. Here, we report the cloning of both translocation breakpoints and the identification
Karl Vandepoele; Vanessa Andries; Nadine van Roy; Katrien Staes; Jo Vandesompele; GeneviĶve Laureys; Els de Smet; Geert Berx; Frank Speleman; Frans van Roy; Anja-Katrin Bielinsky
Characterization of a heavy metal translocating P-type ATPase gene from an environmental heavy metal resistance Enterobacter sp. isolate.
Heavy metals are common contaminants found in polluted areas. We have identified a heavy metal translocating P-type ATPase gene (hmtp) via fosmid library and in vitro transposon mutagenesis from an Enterobacter sp. isolate. This gene is believed to participate in the bacterium's heavy metal resistance traits. The complete gene was identified, cloned, and expressed in a suitable Escherichia coli host cell. E. coli W3110, RW3110 (zntA::Km), GG48 (?zitB::Cm zntA::Km), and GG51 (?zitB::Cm) were used to study the possible effects of this gene for heavy metal (cadmium and zinc in particular) resistance. Among the E. coli strains tested, RW3110 and GG48 showed more sensitivity to cadmium and zinc compared to the wild-type E. coli W3110 and strain GG51. Therefore, strains RW3110 and GG48 were chosen for the reference hosts for further evaluation of the gene's effect. The results showed that expression of this heavy metal translocating P-type ATPase gene could increase the ability for zinc and cadmium resistance in the tested microorganisms. PMID:23344939
Chien, Chih-Ching; Huang, Chia-Hsuan; Lin, Yi-Wei
Early B-Cell Factor (O/E-1) Is a Promoter of Adipogenesis and Involved in Control of Genes Important for Terminal Adipocyte Differentiation
Olf-1/early B-cell factor (O/E-1) is a transcription factor important for B-lymphocyte and neuronal gene regulation. Here we report that all three known O/E genes (O/E-1, -2, and -3) are expressed in mouse adipose tissue and are upregulated during adipocyte differentiation. Forced expression of O/E-1 in either the preadipocyte cell line 3T3-L1 or mouse embryonic fibroblasts augmented adipogenesis, and constitutive expression of O/E-1 in uncommitted NIH 3T3 fibroblasts led to initiation of adipocyte differentiation. Furthermore, a dominant negative form of O/E-1 partially suppressed 3T3-L1 adipogenesis, indicating that expression from endogenous O/E target genes is required for 3T3-L1 terminal differentiation. Thus, our data point to the importance of O/E target genes for adipocyte differentiation and suggest a novel role for O/E-1 as an initiator and stimulator of adipogenesis.
Akerblad, Peter; Lind, Ulrika; Liberg, David; Bamberg, Krister; Sigvardsson, Mikael
Genetic variation in chromosomal translocation breakpoint and immune function genes and risk of non-Hodgkin lymphoma
BackgroundĀĀTumor necrosis factor (TNF) and interleukin 10 (IL10) are promising candidate susceptibility genes for non-Hodgkin lymphoma (NHL). Chromosomal translocation breakpoint genes\\u000a are of interest, given their documented involvement in lymphoma progression.\\u000a \\u000a \\u000a \\u000a \\u000a MethodsĀĀWe analyzed 11 polymorphisms in BCL2, CCND1, MYC, TNF, and IL10 in a large, population-based, Danish-Swedish case¢control study including 2,449 NHL cases and 1,980 controls. Relative risk\\u000a of NHL
Pia Fernberg; Ellen T. Chang; Kristina Duvefelt; Henrik Hjalgrim; Sandra Eloranta; Karina Meden S°rensen; Anna Porwit; Keith Humphreys; Mads Melbye; Karin Ekstr÷m Smedby
The human PD1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors
We report the complete cDNA sequence and the genomic structure of the human PD-1 homologue. An analysis of the expression pattern of the human PD-1 gene (hPD-1) and the murine PD-1 gene (mPD-1) in developing bone marrow B-lineage cells was also undertaken. The full length hPD-1 cDNA is 2106 nucleotides long and encodes a predicted protein of 288 amino acid
Lawrence R Finger; Jaiyu Pu; Robert Wasserman; Rajeev Vibhakar; Elaine Louie; Richard R Hardy; Peter D Burrows; Linda G Billips
Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation.
Sequence analysis of rearranged VHDJH genes of B lineage cells from various stages of ontogeny indicates that short sequence homologies at the breakpoints of recombination contribute to V region gene assembly. Such homologies are regularly seen at DJH junctions of neonatal pre-B cells, most of which do not contain N sequences. In the same cells, but not at later developmental stages, preferential usage of the JH1 element is observed. After birth, N sequence insertion increases with time and is always more prominent at the VHD border than the DJH border. In pre-B cells from adult animals and in mature B cells, in cases where N sequences were not detectable, sequence homologies at the DJH border were found in only half of the instances. This lower incidence could be due to N sequence addition to one of the recombining DNA ends and/or cellular selection. Inspection of VHDJH junctions for N sequence insertion, sequence homologies at the DJH border and JH1 usage allows the estimation of the timepoint in ontogeny at which particular B cell subsets are seeded into the immune system. Specifically, the present data show that the cells of the Ly1 B cell subset are generated not only neonatally but also beyond the first weeks of life. However, the DJH junctions of the progenitors of chronic B cell leukemias which originate from the same B cell subset resemble those of neonatal pre-B cells, suggesting that these cells have already undergone a transforming event at this early developmental stage.
Gu, H; Forster, I; Rajewsky, K
B-cell Lymphoma Lymphomas are cancers that arise from lymphoid cells, which are part of the immune system. The World Health Organization currently recognizes about 70 different types of lymphoma and divides them into four major groups: mature B-cell neoplasms,
Activation of EVI1 Gene Expression in Human Acute Myelogenous Leukemias by Translocations Spanning 300-400 Kilobases on Chromosome Band 3q26
Retroviral activation of Evi-1 gene expression is one of the most common transforming events in murine myeloid leukemias. To evaluate the role of the EVI1 gene in human acute myelogenous leukemia (AML), leukemic blasts or cell lines from 116 patients were examined. In eight patients the EVI1 gene was expressed and all but one had cytogenetically detectable translocations of chromosome
Kazuhiro Morishita; Evan Parganas; Cheryl L. Willman; Michael H. Whittaker; Harry Drabkin; John Oval; Raymond Taetle; Marcus B. Valentine; James N. Ihle
B-cell receptor, clinical course and prognosis in chronic lymphocytic leukaemia: the growing saga of the IGHV3 subgroup gene usage.
The immunoglobulin heavy chain variable gene (IGHV) mutational status has been recognized as an important predictor of prognosis in chronic lymphocytic leukaemia (CLL) since 1999. More recently, other features of the B-cell receptor, such as stereotypy, have been identified as capable of refining the prognostic potential of IGHV status in the clinical assessment of CLL patients. In this context, different genes belonging to the IGHV3 subgroup, the most frequently used subgroup in CLL, have been shown to denote disease subsets that either display a bad prognosis (i.e. IGHV3-21, IGHV3-23) or are associated with particularly good clinical outcomes, including a highly stable/indolent clinical course, even prone to spontaneous regression (i.e. IGHV3-72, IGHV3-30). The present review focuses on the molecular and biological features of CLL-expressing specific genes belonging to the IGHV3 subgroup that are known to mark disease subsets with completely different clinical courses, and may be possibly related to CLL pathogenesis via antigen and/or superantigen involvement. PMID:21303354
Dal-Bo, Michele; Del Giudice, Ilaria; Bomben, Riccardo; Capello, Daniela; Bertoni, Francesco; Forconi, Francesco; Laurenti, Luca; Rossi, Davide; Zucchetto, Antonella; Pozzato, Gabriele; Marasca, Roberto; Efremov, Dimitar G; Guarini, Anna; Del Poeta, Giovanni; FoÓ, Robin; Gaidano, Gianluca; Gattei, Valter
Analysis of Epstein-Barr Virus-Regulated Host Gene Expression Changes through Primary B-Cell Outgrowth Reveals Delayed Kinetics of Latent Membrane Protein 1-Mediated NF-?B Activation
Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-?B activity. It was previously thought that the major viral protein responsible for NF-?B activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-?B activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-?B activation during the first week after infection increased the transformation efficiency, while early NF-?B inhibition had no effect on transformation. Rather, inhibition of NF-?B was not toxic to EBV-infected cells until LMP1 levels and NF-?B activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.
Price, Alexander M.; Tourigny, Jason P.; Forte, Eleonora; Salinas, Raul E.; Dave, Sandeep S.
Site-Specific Expression of Polycomb-Group Genes Encoding the HPC-HPH\\/PRC1 Complex in Clinically Defined Primary Nodal and Cutaneous Large B-Cell Lymphomas
Polycomb-group (PcG) genes preserve cell identity by gene silencing, and contribute to regulation of lymphopoiesis and malignant transformation. We show that primary nodal large B-cell lymphomas (LBCLs), and secondary cutaneous deposits from such lymphomas, abnormally express the BMI-1, RING1, and HPH1 PcG genes in cycling neoplastic cells. By contrast, tumor cells in primary cutaneous LBCLs lacked BMI-1 expression, whereas RING1
Frank M. Raaphorst; Maarten Vermeer; Elly Fieret; Tjasso Blokzijl; Danny Dukers; Richard G. A. B. Sewalt; Arie P. Otte; Rein Willemze; Chris J. L. M. Meijer
Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88.
Human TLR10 is an orphan member of the TLR family. Genomic studies indicate that TLR10 is in a locus that also contains TLR1 and TLR6, two receptors known to function as coreceptors for TLR2. We have shown that TLR10 was not only able to homodimerize but also heterodimerized with TLRs 1 and 2. In addition, unlike TLR1 and TLR6, TLR10 was expressed in a highly restricted fashion as a highly N-glycosylated protein, which we detected in B cell lines, B cells from peripheral blood, and plasmacytoid dendritic cells from tonsil. We were also able to detect TLR10 in a CD1a(+) DC subset derived from CD34(+) progenitor cells which resemble Langerhans cells in the epidermis. Although we were unable to identify a specific ligand for TLR10, by using a recombinant CD4TLR10 molecule we also demonstrated that TLR10 directly associates with MyD88, the common Toll IL-1 receptor domain adapter. Additionally, we have characterized regions in the Toll IL-1 receptor domain of TLR10 that are essential in the activation of promoters from certain inflammatory cytokines. Even though TLR10 expression has not been detected in mice, we have identified a partial genomic sequence of the TLR10 gene that was present but nonfunctional and disrupted by a retroviral insertion in all mouse strains tested. However, a complete TLR10 sequence could be detected in the rat genome, indicating that a functional copy may be preserved in this species. PMID:15728506
Hasan, Uzma; Chaffois, Claire; Gaillard, Claude; Saulnier, Virginie; Merck, Estelle; Tancredi, Sandra; Guiet, Chantal; BriĶre, Francine; Vlach, Jaromir; Lebecque, Serge; Trinchieri, Giorgio; Bates, Elizabeth E M
Most peripheral B cells in mice are ligand selected
Using amplified cDNA and genomic libraries, we have analyzed the VH gene repertoire of pre-B cells and various B cell subsets of conventional mice at the level of VH genes belonging to the J558 VH gene family. The sequence data were evaluated on the basis of a newly established list of 67 J558 VH genes that comprise approximately two- thirds of the J558 VH genes of the murine IgHb haplotype. The results of the analysis demonstrate that VH gene utilization in pre-B cells, although biased to some extent by B cell autonomous VH gene selection, scatters over the whole range of J558 VH genes present in the germline. In contrast, in mature, peripheral B cells comprising long-lived mu + delta high B cells as well as Ly-1 B cells, small overlapping sets of germline VH genes are dominantly expressed. The data indicate that the recruitment of newly generated B cells into the long-lived peripheral B cell pool is mediated through positive selection by internal and/or external antigens. Because of the absence of immunoglobulin class switching and somatic hypermutation, this process is different from the selection of memory B cells in T cell-dependent immune responses.
MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program
Diffuse large B-cell lymphoma (DLBCL) is stratified into prognostically favorable germinal center B-cell (GCB)¢like and unfavorable activated B-cell (ABC)¢like subtypes based on gene expression signatures. In this study, we analyzed 893 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We show that MYC/BCL2 protein coexpression occurred significantly more commonly in the ABC subtype. Patients with the ABC or GCB subtype of DLBCL had similar prognoses with MYC/BCL2 coexpression and without MYC/BCL2 coexpression. Consistent with the notion that the prognostic difference between the 2 subtypes is attributable to MYC/BCL2 coexpression, there is no difference in gene expression signatures between the 2 subtypes in the absence of MYC/BCL2 coexpression. DLBCL with MYC/BCL2 coexpression demonstrated a signature of marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. We conclude that MYC/BCL2 coexpression in DLBCL is associated with an aggressive clinical course, is more common in the ABC subtype, and contributes to the overall inferior prognosis of patients with ABC-DLBCL. In conclusion, the data suggest that MYC/BCL2 coexpression, rather than cell-of-origin classification, is a better predictor of prognosis in patients with DLBCL treated with R-CHOP.
Hu, Shimin; Xu-Monette, Zijun Y.; Tzankov, Alexander; Green, Tina; Wu, Lin; Balasubramanyam, Aarthi; Liu, Wei-min; Visco, Carlo; Li, Yong; Miranda, Roberto N.; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W. L.; Zhao, Xiaoying; van Krieken, J. Han; Huang, Qin; Huh, Jooryung; Ai, Weiyun; Ponzoni, Maurilio; Ferreri, Andres J. M.; Zhou, Fan; Slack, Graham W.; Gascoyne, Randy D.; Tu, Meifeng; Variakojis, Daina; Chen, Weina; Go, Ronald S.; Piris, Miguel A.; M?ller, Michael B.; Medeiros, L. Jeffrey; Young, Ken H.
Polymerase chain reaction (PCR) detection of B cell clonality in Sj÷gren's syndrome patients: a diagnostic tool of clonal expansion.
Sj÷gren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell-activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non-Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non-specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi-nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR-enzyme-linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl-2 oncogene coupled with a variable segment of the IgH to assess the Bcl-2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P<0.01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients. PMID:20408860
Guzmßn, L M; Castillo, D; Aguilera, S O
Effects of B-Cell Lymphoma 2 Gene Transfer to Myoblast Cells on Skeletal Muscle Tissue Formation Using Magnetic Force-Based Tissue Engineering
Tissue-engineered skeletal muscle should possess a high cell-dense structure with unidirectional cell alignment. However, limited nutrient and/or oxygen supply within the artificial tissue constructs might restrict cell viability and muscular functions. In this study, we genetically modified myoblast cells with the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene and evaluated their function in artificial skeletal muscle tissue constructs. Magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of skeletal muscle bundles by applying a magnetic force. Bcl-2-overexpressing muscle bundles formed highly cell-dense and viable tissue constructs, while muscle bundles without Bcl-2 overexpression exhibited substantial necrosis/apoptosis at the central region of the bundle. Bcl-2-overexpressing muscle bundles contracted in response to electrical pulses and generated a significantly higher physical force. These findings indicate that the incorporation of anti-apoptotic gene-transduced myoblast cells into tissue constructs significantly enhances skeletal muscle formation and function.
Sato, Masanori; Ito, Akira; Akiyama, Hirokazu; Kawabe, Yoshinori
Influence of polymorphisms in genes encoding immunoregulatory proteins and metabolizing enzymes on susceptibility and outcome in patients with diffuse large B-cell lymphoma treated with rituximab.
We analyzed the allelic distribution of 12 candidate polymorphisms in a large retrospective study of 486 patients with diffuse large B-cell lymphoma (DLBCL) treated at Oslo University Hospital with 1056 blood donors serving as controls. Variants in TNF? (rs1800629) (GG vs. AG/AA, p < 0.001) and LTA (rs909253) (AA vs. AG/GG, p = 0.02) and deletions in GSTM1 and GSTT1 (undeleted vs. deleted, p = 0.01 and p = 0.01, respectively) were associated with increased susceptibility of developing DLBCL. IL-10 (rs1800896) variants (GG vs. AG/AA, p = 0.03) were associated with decreased susceptibility. In line with several previous reports, patients carrying the TNF? (rs1800629) A allele treated in the pre-rituximab era had inferior outcome compared to patients carrying the homozygous GG genotype (p = 0.004, n = 33). However, patients receiving at least one dose of rituximab had equal outcome regardless of their TNF? genotype (HR = 0.94, p = 0.79, n = 307). Deletion in GSTM1 was associated with inferior outcome for patients with low International Prognostic Index (IPI) score (p = 0.04). Our findings support the suggestions that polymorphisms in genes encoding immunoregulatory proteins and enzymes that metabolize carcinogens and chemotherapeutic drugs influence DLBCL susceptibility and possibly treatment outcome. The influence of polymorphisms in immunoregulatory genes on outcome in DLBCL should be reevaluated in the rituximab era. PMID:23391141
Yri, Olav E; Ekstr°m, Per Olaf; Hilden, Vera; Gaudernack, Gustav; Liest°l, Knut; Smeland, Erlend B; Holte, Harald
Phenotypic markers and BCL-1 gene rearrangements in B-cell chronic lymphocytic leukemia: a Cancer and Leukemia Group B study.
The markers, CD11b, CD11c, CD14, CD21, CD23, CD25, CD38, and FMC7 were correlated with morphologic and other laboratory and clinical characteristics of 127 patients with untreated CD5+ chronic lymphocytic leukemia (CLL). Only CD38 and CD21 were significantly associated with atypical CLL morphology. The integrin associated markers CD11b and CD11c were associated with lower leukocyte count (white blood cell count [WBC]) and lower Rai stage. By contrast, the activation antigen CD23 was associated with a higher WBC, higher Rai stage, younger age group, and the presence of lymphadenopathy. Therefore, we conclude that CD23 positivity may reflect a more aggressive form of CLL, and CD11b and CD11c positivity a less aggressive form. The BCL-1 gene rearrangement was present in 5 of 84 (6%) CLL cases examined and was associated with atypical morphology and surface expression of CD11b. Patients with a BCL-1 gene rearrangement may represent a CLL subset or possibly a different B-cell disease. PMID:8102560
Newman, R A; Peterson, B; Davey, F R; Brabyn, C; Collins, H; Brunetto, V L; Duggan, D B; Weiss, R B; Royston, I; Millard, F E
Exposure of B cell chronic lymphocytic leukemia (B-CLL) cells to nutlin-3 induces a characteristic gene expression profile, which correlates with nutlin-3-mediated cytotoxicity.
By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 B-CLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type B-CLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing. PMID:19519319
Zauli, G; di Iasio, M G; Secchiero, P; Dal Bo, M; Marconi, D; Bomben, R; Del Poeta, G; Gattei, V
Gene expression profiling of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly reveals alterations of characteristic oncogenetic pathways.
Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly (EBV[+]DLBCL-E) is classified as a subtype of DLBCL. Until now, its molecular pathogenesis has remained unknown. To identify pathways characteristic of EBV(+)DLBCL-E, gene expression profiling of five EBV(+)DLBCL-E and seven EBV-negative DLBCL (EBV[-]DLBCL) cases was undertaken using human oligonucleotide microarray analysis. Gene set enrichment analysis and gene ontology analysis showed that gene sets of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and nuclear factor kappa B (NF-?B) pathways were enriched in EBV(+)DLBCL-E cases. To confirm the results of the expression profiles, in vitro analysis was performed. Expression profiling analysis showed that high activation of the JAK-STAT and NF-?B pathways was induced by EBV infection into DLBCL cell lines. Activation of the NF-?B pathway was confirmed in EBV-infected cell lines using an electrophoretic mobility shift assay. Western blot analysis revealed an increased protein expression level of phosphorylated signal transducer and activator of transcription 3 (STAT3) in an EBV-infected cell line. Protein expression of phosphorylated STAT3 was frequently observed in lymphoma cells of EBV(+)DLBCL-E clinical samples using immunohistochemistry (EBV[+]DLBCL-E: 80.0% [n = 20/25] versus EBV[-]DLBCL: 38.9% [n = 14/36]; P = 0.001). The results of the present study suggest that activation of the JAK-STAT and NF-?B pathways was characteristic of EBV(+)DLBCL-E, which may reflect the nature of EBV-positive tumor cells. Targeting these pathways as therapies might improve clinical outcomes of EBV(+)DLBCL-E. PMID:24581222
Kato, Harumi; Karube, Kennosuke; Yamamoto, Kazuhito; Takizawa, Jun; Tsuzuki, Shinobu; Yatabe, Yasushi; Kanda, Teru; Katayama, Miyuki; Ozawa, Yukiyasu; Ishitsuka, Kenji; Okamoto, Masataka; Kinoshita, Tomohiro; Ohshima, Koichi; Nakamura, Shigeo; Morishima, Yasuo; Seto, Masao
NSDL National Science Digital Library
This Flash animation shows intracellular and extracellular interactions that illustrate the maturation stages of B cells in the bone marrow. It uses sound and mouse-over identification to help students learn more and retain the information.
American Society For Microbiology;
CD30 expression defines a novel subgroup of diffuse large B-cell lymphoma with favorable prognosis and distinct gene expression signature: a report from the International DLBCL Rituximab-CHOP Consortium Program Study.
CD30, originally identified as a cell-surface marker of Reed-Sternberg and Hodgkin cells of classical Hodgkin lymphoma, is also expressed by several types of non-Hodgkin lymphoma, including a subset of diffuse large B-cell lymphoma (DLBCL). However, the prognostic and biological importance of CD30 expression in DLBCL is unknown. Here we report that CD30 expression is a favorable prognostic factor in a cohort of 903 de novo DLBCL patients. CD30 was expressed in ?14% of DLBCL patients. Patients with CD30(+) DLBCL had superior 5-year overall survival (CD30(+), 79% vs CD30(-), 59%; P = .001) and progression-free survival (P = .003). The favorable outcome of CD30 expression was maintained in both the germinal center B-cell and activated B-cell subtypes. Gene expression profiling revealed the upregulation of genes encoding negative regulators of nuclear factor ?B activation and lymphocyte survival, and downregulation of genes encoding B-cell receptor signaling and proliferation, as well as prominent cytokine and stromal signatures in CD30(+) DLBCL patients, suggesting a distinct molecular basis for its favorable outcome. Given the superior prognostic value, unique gene expression signature, and significant value of CD30 as a therapeutic target for brentuximab vedotin in ongoing successful clinical trials, it seems appropriate to consider CD30(+) DLBCL as a distinct subgroup of DLBCL. PMID:23343832
Hu, Shimin; Xu-Monette, Zijun Y; Balasubramanyam, Aarthi; Manyam, Ganiraju C; Visco, Carlo; Tzankov, Alexander; Liu, Wei-min; Miranda, Roberto N; Zhang, Li; Montes-Moreno, Santiago; Dybkµr, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L; Hsi, Eric D; Choi, William W L; Han van Krieken, J; Huang, Qin; Huh, Jooryung; Ai, Weiyun; Ponzoni, Maurilio; Ferreri, Andrķs J M; Zhao, Xiaoying; Winter, Jane N; Zhang, Mingzhi; Li, Ling; M°ller, Michael B; Piris, Miguel A; Li, Yong; Go, Ronald S; Wu, Lin; Medeiros, L Jeffrey; Young, Ken H
CD30 expression defines a novel subgroup of diffuse large B-cell lymphoma with favorable prognosis and distinct gene expression signature: a report from the International DLBCL Rituximab-CHOP Consortium Program Study
CD30, originally identified as a cell-surface marker of Reed-Sternberg and Hodgkin cells of classical Hodgkin lymphoma, is also expressed by several types of non-Hodgkin lymphoma, including a subset of diffuse large B-cell lymphoma (DLBCL). However, the prognostic and biological importance of CD30 expression in DLBCL is unknown. Here we report that CD30 expression is a favorable prognostic factor in a cohort of 903 de novo DLBCL patients. CD30 was expressed in ?14% of DLBCL patients. Patients with CD30+ DLBCL had superior 5-year overall survival (CD30+, 79% vs CD30¢, 59%; P = .001) and progression-free survival (P = .003). The favorable outcome of CD30 expression was maintained in both the germinal center B-cell and activated B-cell subtypes. Gene expression profiling revealed the upregulation of genes encoding negative regulators of nuclear factor ?B activation and lymphocyte survival, and downregulation of genes encoding B-cell receptor signaling and proliferation, as well as prominent cytokine and stromal signatures in CD30+ DLBCL patients, suggesting a distinct molecular basis for its favorable outcome. Given the superior prognostic value, unique gene expression signature, and significant value of CD30 as a therapeutic target for brentuximab vedotin in ongoing successful clinical trials, it seems appropriate to consider CD30+ DLBCL as a distinct subgroup of DLBCL.
Hu, Shimin; Xu-Monette, Zijun Y.; Balasubramanyam, Aarthi; Manyam, Ganiraju C.; Visco, Carlo; Tzankov, Alexander; Liu, Wei-min; Miranda, Roberto N.; Zhang, Li; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W. L.; Han van Krieken, J.; Huang, Qin; Huh, Jooryung; Ai, Weiyun; Ponzoni, Maurilio; Ferreri, Andres J. M.; Zhao, Xiaoying; Winter, Jane N.; Zhang, Mingzhi; Li, Ling; M?ller, Michael B.; Piris, Miguel A.; Li, Yong; Go, Ronald S.; Wu, Lin; Medeiros, L. Jeffrey; Young, Ken H.
Rapid enhancement of. beta. /sub 2/-interferon/B-cell differentiation factor BSF-2 gene expression in human fibroblasts by diacylglycerols and the calcium ionophore A23187
The expression in human fibroblasts of the ..beta../sub 2/-interferon (IFN-..beta../sub 2/) gene, which is now recognized to be identical to the gene encoding B-cell differentiation factor BSF-2, is enhanced by several cytokines that affect cell growth (tumor necrosis factor, interleukin 1, platelet-derived growth factor, and ..beta../sub 1/-interferon). The authors have examined the possibility that IFN-..beta../sub 2/ gene expressions is regulated through activation, by diacylglycerol of the protein kinase C pathway. The synthetic diacylglycerols 1,2-dioctanoylglycerol (diC/sub 8/) and 1-oleoyl-2-acetylglycerol strongly enhanced IFN-..beta../sub 2/, but not IFN-..beta../sub 1/, gene expression in human fibroblasts (FS-4 strain). An increase in IFN-..beta../sub 2/ mRNA level was detected within 15 min after addition of diC/sub 8/ (290 ..mu..M) to FS-4 cells and was maximal approximately 20 hr later. An increase in IFN-..beta../sub 2/ gene transcription was detected within 5 min of addition of diC/sub 8/, and the rate of transcription was near-maximal by 15-30 min. The enhancement of IFN-..beta../sub 2/ gene expression by diC/sub 8/, interleukin 1, or tumor necrosis factor was not prevented by H8, a preferential inhibitor of cAMP- and cGMP-dependent protein kinases, but was blocked by H7, an inhibitor of protein kinases C as well as of cyclic nucleotide-dependent protein kinases. The calcium ionophore A23187 (1-10 ..mu..M) also elicited an increase in the level of IFN-..beta../sub 2/ mRNA in FS-4 fibroblasts; appropriate combinations of A23187 and diC/sub 8/ had at least an additive effect in enhancing IFN-..beta../sub 2/ mRNA levels. These results show that protein kinase C-activating or (Ca/sup 2 +/)-elevating agents rapidly increase the expression of the IFN-..beta../sub 2/ gene in human fibroblasts.
Sehgal, P.B.; Walther, Z.; Tamm, I.
Rapid enhancement of beta 2-interferon/B-cell differentiation factor BSF-2 gene expression in human fibroblasts by diacylglycerols and the calcium ionophore A23187.
The expression in human fibroblasts of the beta 2-interferon (IFN-beta 2) gene, which is now recognized to be identical to the gene encoding B-cell differentiation factor BSF-2, is enhanced by several cytokines that affect cell growth (tumor necrosis factor, interleukin 1, platelet-derived growth factor, and beta 1-interferon). We have examined the possibility that IFN-beta 2 gene expression is regulated through activation, by diacylglycerol, of the protein kinase C pathway. The synthetic diacylglycerols 1,2-dioctanoylglycerol (diC8) and 1-oleoyl-2-acetylglycerol strongly enhanced IFN-beta 2, but not IFN-beta 1, gene expression in human fibroblasts (FS-4 strain). An increase in IFN-beta 2 mRNA level was detected within 15 min after addition of diC8 (290 microM) to FS-4 cells and was maximal approximately 20 hr later. An increase in IFN-beta 2 gene transcription was detected within 5 min of addition of diC8, and the rate of transcription was near-maximal by 15-30 min. The enhancement of IFN-beta 2 gene expression by diC8, interleukin 1, or tumor necrosis factor was not prevented by H8, a preferential inhibitor of cAMP- and cGMP-dependent protein kinases, but was blocked by H7, an inhibitor of protein kinase C as well as of cyclic nucleotide-dependent protein kinases. diC8 was found to protect FS-4 cells from the cytopathic effect of vesicular stomatitis virus; this protection was blocked by polyclonal or monoclonal antibodies that neutralize IFN-beta, suggesting that the antiviral effect was due to the secretion of IFN-beta 2 by the diC8-treated fibroblasts. The calcium ionophore A23187 (1-10 microM) also elicited an increase in the level of IFN-beta 2 mRNA in FS-4 fibroblasts; appropriate combinations of A23187 and diC8 had at least an additive effect in enhancing IFN-beta 2 mRNA levels. These results show that protein kinase C-activating or [Ca2+]-elevating agents rapidly increase the expression of the IFN-beta 2 gene in human fibroblasts. Images
Sehgal, P B; Walther, Z; Tamm, I
Cyclin D2 dysregulation by chromosomal translocations to TCR loci in T-cell acute lymphoblastic leukemias
Strong expression of at least one of the three D-type cyclins is common in human cancers. While the cyclin D1 and D3 genes (CCND1 and CCND3) are recurrently involved in genomic rearrangements, especially in B-cell lymphoid neoplasias, no clear involvement of the cyclin D2 gene (CCND2) has been reported to date. Here, we identified chromosomal translocations targeting the CCND2 locus
E Clappier; W Cuccuini; J-M Cayuela; D Vecchione; A Baruchel; H Dombret; F Sigaux; J Soulier
Evidence that Yaa-induced loss of marginal zone B cells is a result of dendritic cell-mediated enhanced activation
The development of systemic lupus is accelerated by the Yaa (Y-linked autoimmune acceleration) mutation, which is the consequence of a translocation of the telomeric end containing the Tlr7 gene from the X chromosome onto the Y chromosome. However, the loss of marginal zone (MZ) B cells, one of the Yaa-linked cellular abnormalities, has previously been shown to be unrelated to
Marie-Laure Santiago-Raber; Hirofumi Amano; Eri Amano; Liliane Fossati-Jimack; Lee Kim Swee; Antonio Rolink; Shozo Izui
MicroRNA 28 controls cell proliferation and is down-regulated in B-cell lymphomas.
Burkitt lymphoma (BL) is a highly aggressive B-cell non-Hodgkin lymphoma (B-NHL), which originates from germinal center (GC) B cells and harbors translocations deregulating v-myc avian myelocytomatosis viral oncogene homolog (MYC). A comparative analysis of microRNAs expressed in normal and malignant GC B cells identified microRNA 28 (miR-28) as significantly down-regulated in BL, as well as in other GC-derived B-NHL. We show that reexpression of miR-28 impairs cell proliferation and clonogenic properties of BL cells by modulating several targets including MAD2 mitotic arrest deficient-like 1, MAD2L1, a component of the spindle checkpoint whose down-regulation is essential in mediating miR-28-induced proliferation arrest, and BCL2-associated athanogene, BAG1, an activator of the ERK pathway. We identify the oncogene MYC as a negative regulator of miR-28 expression, suggesting that its deregulation by chromosomal translocation in BL leads to miR-28 suppression. In addition, we show that miR-28 can inhibit MYC-induced transformation by directly targeting genes up-regulated by MYC. Overall, our data suggest that miR-28 acts as a tumor suppressor in BL and that its repression by MYC contributes to B-cell lymphomagenesis. PMID:24843176
Schneider, Christof; Setty, Manu; Holmes, Antony B; Maute, Roy L; Leslie, Christina S; Mussolin, Lara; Rosolen, Angelo; Dalla-Favera, Riccardo; Basso, Katia
Evaluation of the NOD/SCID xenograft model for glucocorticoid-regulated gene expression in childhood B-cell precursor acute lymphoblastic leukemia
Background Glucocorticoids such as prednisolone and dexamethasone are critical drugs used in multi-agent chemotherapy protocols used to treat acute lymphoblastic leukemia (ALL), and response to glucocorticoids is highly predictive of outcome. The NOD/SCID xenograft mouse model of ALL is a clinically relevant model in which the mice develop a systemic leukemia which retains the fundamental biological characteristics of the original disease. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Cells from a glucocorticoid-sensitive xenograft derived from a child with B-cell precursor ALL were inoculated into NOD/SCID mice. When highly engrafted the mice were randomized into groups of 4 to receive dexamethasone 15 mg/kg by intraperitoneal injection or vehicle control. Leukemia cells were harvested from mice spleens at 0, 8, 24 or 48 hours thereafter, and gene expression analyzed on Illumina WG-6_V3 chips, comparing all groups to time 0 hours. Results The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes, with fewer observed at the 24 and 48 hour timepoints, and with minimal changes seen across the time-matched controls. When compared to publicly available datasets of glucocorticoid-induced gene expression from an in vitro cell line study and from an in vivo study of patients with ALL, at the level of pathways, expression changes in the 8 hour xenograft samples showed a similar response to patients treated with glucocorticoids. Replicate analysis revealed that at the 8 hour timepoint, a dataset with high signal and differential expression, using data from 3 replicates instead of 4 resulted in excellent recovery scores of > 0.9. However at other timepoints with less signal very poor recovery scores were obtained with 3 replicates. Conclusions The NOD/SCID xenograft mouse model provides a reproducible experimental system in which to investigate clinically-relevant mechanisms of drug-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates.
Structural organization of the bcr gene and its role in the Ph' translocation
The Philadelphia (Ph') chromosome, an abnormal chromosome 22 (ref. 1), is one of the best-known examples of a specific human chromosomal abnormality strongly associated with one form of human leukaemia, chronic myelocytic leukaemia (CML). The finding2 that a small region of chromosome 9 which includes the c-abl oncogene is translocated to chromosome 22 prompted studies to elucidate the molecular mechanisms
Nora Heisterkamp; Kees Stam; John Groffen; Annelies de Klein; Gerard Grosveld
Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours
EWING'S sarcoma and related subtypes of primitive neuroectodermal tumours share a recurrent and specific t(ll;22) (q24;q12) chromosome translocation1-8, the breakpoints of which have recently been cloned9. Phylogenetically conserved restriction fragments in the vicinity of EWSR1 and EWSR2, the genomic regions where the breakpoints of chromosome 22 and chromosome 11 are, respectively, have allowed identification of transcribed sequences from these regions
Olivier Delattre; Jessica Zucman; Bķatrice Plougastel; Chantal Desmaze; Thomas Melot; Martine Peter; Heinrich Kovar; Isabelle Joubert; Pieter de Jong; Guy Rouleau; Alain Aurias; Gilles Thomas
An unbalanced translocation unmasks a recessive mutation in the follicle-stimulating hormone receptor (FSHR) gene and causes FSH resistance
Follicle-stimulating hormone (FSH) mediated by its receptor (FSHR) is pivotal for normal gametogenesis. Inactivating FSHR mutations are known to cause hypergonadotropic hypogonadism with disturbed follicular maturation in females. So far, only very few recessive point mutations have been described. We report on a 17-year-old female with primary amenorrhea, hypergonadotropic hypogonadism and disturbed folliculogenesis. Chromosome analysis detected a seemingly balanced translocation 46,XX,t(2;8)(p16.3or21;p23.1)mat. FSHR sequence analysis revealed a novel non-synonymous point mutation in exon 10 (c.1760C>A, p.Pro587His), but no wild-type allele. The mutation was also found in the father, but not in the mother. Furthermore, molecular-cytogenetic analyses of the breakpoint region on chromosome 2 showed the translocation to be unbalanced, containing a deletion with one breakpoint within the FSHR gene. The deletion size was narrowed down by array analysis to approximately 163?kb, involving exons 9 and 10 of the FSHR gene. Functional studies of the mutation revealed the complete lack of signal transduction presumably caused by a changed conformational structure of transmembrane helix 6. To our knowledge, this is the first description of a compound heterozygosity of an inactivating FSHR point mutation unmasked by a partial deletion. This coincidence of two rare changes caused clinical signs consistent with FSH resistance.
Kuechler, Amla; Hauffa, Berthold P; Koninger, Angela; Kleinau, Gunnar; Albrecht, Beate; Horsthemke, Bernhard; Gromoll, Jorg
A constitutional translocation t(1;17)(p36.2;q11.2) in a neuroblastoma patient disrupts the human NBPF1 and ACCN1 genes.
The human 1p36 region is deleted in many different types of tumors, and so it probably harbors one or more tumor suppressor genes. In a Belgian neuroblastoma patient, a constitutional balanced translocation t(1;17)(p36.2;q11.2) may have led to the development of the tumor by disrupting or activating a gene. Here, we report the cloning of both translocation breakpoints and the identification of a novel gene that is disrupted by this translocation. This gene, named NBPF1 for Neuroblastoma BreakPoint Family member 1, belongs to a recently described gene family encoding highly similar proteins, the functions of which are unknown. The translocation truncates NBPF1 and gives rise to two chimeric transcripts of NBPF1 sequences fused to sequences derived from chromosome 17. On chromosome 17, the translocation disrupts one of the isoforms of ACCN1, a potential glioma tumor suppressor gene. Expression of the NBPF family in neuroblastoma cell lines is highly variable, but it is decreased in cell lines that have a deletion of chromosome 1p. More importantly, expression profiling of the NBPF1 gene showed that its expression is significantly lower in cell lines with heterozygous NBPF1 loss than in cell lines with a normal 1p chromosome. Meta-analysis of the expression of NBPF and ACCN1 in neuroblastoma tumors indicates a role for the NBPF genes and for ACCN1 in tumor aggressiveness. Additionally, DLD1 cells with inducible NBPF1 expression showed a marked decrease of clonal growth in a soft agar assay. The disruption of both NBPF1 and ACCN1 genes in this neuroblastoma patient indicates that these genes might suppress development of neuroblastoma and possibly other tumor types. PMID:18493581
Vandepoele, Karl; Andries, Vanessa; Van Roy, Nadine; Staes, Katrien; Vandesompele, Jo; Laureys, GeneviĶve; De Smet, Els; Berx, Geert; Speleman, Frank; van Roy, Frans
The role of T-cell leukemia translocation-associated gene protein in human tumorigenesis and osteoclastogenesis.
Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-?B ligand (RANKL), TNF-?, IL-6, IL-17, and IFN-?. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibit human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported. Genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene. In the current paper, we reviewed the roles of TCTA protein in lung cancer cell lines and human osteoclastogenesis. PMID:22174563
Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki
The Role of T-Cell Leukemia Translocation-Associated Gene Protein in Human Tumorigenesis and Osteoclastogenesis
Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-?B ligand (RANKL), TNF-?, IL-6, IL-17, and IFN-?. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibit human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported. Genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene. In the current paper, we reviewed the roles of TCTA protein in lung cancer cell lines and human osteoclastogenesis.
Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki
MicroRNAs in B cell development and malignancy
MicroRNAs are small RNA molecules that regulate gene expression and play critical roles in B cell development and malignancy. miRNA expression is important globally, as B cell specific knockouts of Dicer show profound defects in B cell development; and is also critical at the level of specific miRNAs. In this review, we discuss miRNAs that are involved in normal B cell development in the bone marrow and during B cell activation and terminal differentiation in the periphery. Next, we turn to miRNAs that are dysregulated during diseases of B cells, including malignant diseases and autoimmunity. Further study of miRNAs and their targets will lead to a better understanding of B cell development, and should also lead to the development of novel therapeutic strategies against B cell diseases.
Islands of non-essential genes, including a DNA translocation operon, in the genome of bacteriophage 0305?8-36
We investigate genes of lytic, Bacillus thuringiensis bacteriophage 0305?8-36 that are non-essential for laboratory propagation, but might have a function in the wild. We isolate deletion mutants to identify these genes. The non-permutation of the genome (218.948 Kb, with a 6.479 Kb terminal repeat and 247 identified orfs) simplifies isolation of deletion mutants. We find two islands of non-essential genes. The first island (3.01% of the genomic DNA) has an informatically identified DNA translocation operon. Deletion causes no detectable growth defect during propagation in a dilute agarose overlay. Identification of the DNA translocation operon begins with a DNA relaxase and continues with a translocase and membrane-binding anchor proteins. The relaxase is in a family, first identified here, with homologs in other bacteriophages. The second deleted island (3.71% of the genome) has genes for two metallo-protein chaperonins and two tRNAs. Deletion causes a significant growth defect. In addition, (1) we find by ōin situö (in-plaque) single-particle fluorescence microscopy that adsorption to the host occurs at the tip of the 486 nm long tail, (2) we develop a procedure of 0305?8-36 purification that does not cause tail contraction, and (3) we then find by electron microscopy that 0305?8-36 undergoes tail tip-tail tip dimerization that potentially blocks adsorption to host cells, presumably with effectiveness that increases as the bacteriophage particle concentration increases. These observations provide an explanation of the previous observation that 0305?8-36 does not lyse liquid cultures, even though 0305?8-36 is genomically lytic.
Pathria, Saurav; Rolando, Mandy; Lieman, Karen; Hayes, Shirley; Hardies, Stephen; Serwer, Philip
Activation-induced Cytidine Deaminase in B Cell Immunity and Cancers
Activation-induced cytidine deaminase (AID) is an enzyme that is predominantly expressed in germinal center B cells and plays a pivotal role in immunoglobulin class switch recombination and somatic hypermutation for antibody (Ab) maturation. These two genetic processes endow Abs with protective functions against a multitude of antigens (pathogens) during humoral immune responses. In B cells, AID expression is regulated at the level of either transcriptional activation on AID gene loci or post-transcriptional suppression of AID mRNA. Furthermore, AID stabilization and targeting are determined by post-translational modifications and interactions with other cellular/nuclear factors. On the other hand, aberrant expression of AID causes B cell leukemias and lymphomas, including Burkitt's lymphoma caused by c-myc/IgH translocation. AID is also ectopically expressed in T cells and non-immune cells, and triggers point mutations in relevant DNA loci, resulting in tumorigenesis. Here, I review the recent literatures on the function of AID, regulation of AID expression, stability and targeting in B cells, and AID-related tumor formation.
Bruton's tyrosine kinase links the B cell receptor to nuclear factor kappaB activation.
The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation. Bruton's tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases, is essential for the full repertoire of IgM signals to be transduced. We examined the ability of BTK to regulate the nuclear factor (NF)-kappaB/Rel family of transcription factors, as the activation of these factors is required for a B cell response to mIgM. We found greatly diminished IgM- but not CD40-mediated NF-kappaB/Rel nuclear translocation and DNA binding in B cells from X-linked immunodeficient (xid) mice that harbor an R28C mutation in btk, a mutation that produces a functionally inactive kinase. The defect was due, in part, to a failure to fully degrade the inhibitory protein of NF-kappaB, IkappaBalpha. Using a BTK-deficient variant of DT40 chicken B cells, we found that expression of wild-type or gain-of-function mutant BTK, but not the R28C mutant, reconstituted NF-kappaB activity. Thus, BTK is essential for activation of NF-kappaB via the B cell receptor. PMID:10811866
Bajpai, U D; Zhang, K; Teutsch, M; Sen, R; Wortis, H H
Bruton's Tyrosine Kinase Links the B Cell Receptor to Nuclear Factor ?b Activation
The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation. Bruton's tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases, is essential for the full repertoire of IgM signals to be transduced. We examined the ability of BTK to regulate the nuclear factor (NF)-?B/Rel family of transcription factors, as the activation of these factors is required for a B cell response to mIgM. We found greatly diminished IgM- but not CD40-mediated NF-?B/Rel nuclear translocation and DNA binding in B cells from X-linked immunodeficient (xid) mice that harbor an R28C mutation in btk, a mutation that produces a functionally inactive kinase. The defect was due, in part, to a failure to fully degrade the inhibitory protein of NF-?B, I?B?. Using a BTK-deficient variant of DT40 chicken B cells, we found that expression of wild-type or gain-of-function mutant BTK, but not the R28C mutant, reconstituted NF-?B activity. Thus, BTK is essential for activation of NF-?B via the B cell receptor.
Bajpai, Urmila D.; Zhang, Keming; Teutsch, Mark; Sen, Ranjan; Wortis, Henry H.
Relationship between B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas.
B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase III▀ (topoIII▀) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIII▀, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA-1 (p= 0.003), H2AX (p=0.024) and topoIII▀ (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas. PMID:22936454
da Silveira, Gi¾rgia Gobbi; Oliveira-Costa, JoŃo Paulo; Soave, Danilo Figueiredo; Zanetti, Juliana Silva; Soares, Fernando Augusto; Ribeiro-Silva, Alfredo
Analysis of translocations that involve the NUP98 gene in patients with 11p15 chromosomal rearrangements.
The NUP98 gene has been reported to be fused with at least 15 partner genes in leukemias with 11p15 translocations. We report the results of screening of cases with cytogenetically documented rearrangements of 11p15 and the subsequent identification of involvement of NUP98 and its partner genes. We identified 49 samples from 46 hematology patients with 11p15 (including a few with 11p14) abnormalities, and using fluorescence in situ hybridization (FISH), we found that NUP98 was disrupted in 7 cases. With the use of gene-specific FISH probes, in 6 cases, we identified the partner genes, which were PRRX1 (PMX1; in 2 cases), HOXD13, RAP1GDS1, HOXC13, and TOP1. In the 3 cases for which RNA was available, RT-PCR was performed, which confirmed the FISH results and identified the location of the breakpoints in patient cDNA. Our data confirm the previous findings that NUP98 is a recurrent target in various types of leukemia. PMID:15390187
Kobzev, Yuri N; Martinez-Climent, Jose; Lee, Sanggyu; Chen, Jianjun; Rowley, Janet D
Gene expression profiling of plasma cells and plasmablasts: toward a better understanding of the late stages of B-cell differentiation
Plasma cells (PCs), the end point of B-cell differentiation, are a heterogeneous cell compartment comprising several cell sub- sets from short-lived highly proliferative plasmablasts to long-lived nondividing fully mature PCs. Whereas the major tran- scription factors driving the differentia- tion of B cells to PCs were recently identi- fied, the subtle genetic changes that underlie the transition from plasmablasts to
Karin Tarte; Fenghuang Zhan; John De Vos; Bernard Klein; John Shaughnessy Jr
Effect of the E? IgH enhancer on expression of a GFP reporter gene in transfected B cells and transgenic mice
Transgenic mice were generated to identify the first B cell maturation stage showing expression of an immunoglobulin transcriptional enhancer element (E?)-green fluorescent protein (GFP) transgene, and to check the ability of the E? element to behave as a locus control region. Flow cytometry experiments indicated that stably transfected 18¢81 cells (a murine pre-B cell line) and A20 cells (a murine
Laurence Guglielmi; Marc Le Bert; Michel Cognķ; Yves Denizot
B-cell tolerance checkpoints in health and autoimmunity.
The enormous diversity of the antibody repertoire is generated by two mechanisms: recombination of immunoglobulin (Ig) gene variable (V), diversity (D), and joining (J) gene segments during the early stages of B-cell development in the bone marrow and somatic hypermutation (SHM) of functional Ig genes from antigen-activated B cells within secondary lymphoid organs. Diversity by V(D)J recombination and SHM not only provides protective humoral immunity but also generates potentially harmful clones expressing autoantibodies. Under normal circumstances, several mechanisms regulate the removal of autoreactive B cells and defects in central and peripheral B cell tolerance checkpoints are associated with the development of autoimmunity in humans. PMID:18848883
Meffre, Eric; Wardemann, Hedda
The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in upregulation of PLAG1 gene expression under control of the LIFR promoter
We have previously shown that the PLAG1 gene on chromosome 8q12 is consistently rearranged in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between the PLAG1 gene, which encodes a novel zinc finger protein, and the constitutively expressed gene for ?-catenin (CTNNB1), a protein with roles in cell-cell adhesion and the WG\\/WNT signalling
Marianne L Voz; Anna-Karin ┼str÷m; Koen Kas; Joachim Mark; G÷ran Stenman; Wim JM Van de Ven; WJM Van de Ven
The transient expression of pre-B cell receptors governs B cell development.
Only a subpopulation of relatively large pre-B cells express pre-B cell receptors (preBCR) that can be seen with very sensitive immunofluorescence methods. Inefficient assembly of the multicomponent preBCR coupled with their ligand-induced endocytosis may account for the remarkably low in vivo levels of preBCR expression. Signaling initiated via the preBCR promotes cellular proliferation and RAG-1 and RAG-2 downregulation to interrupt the immunoglobulin V(D)J gene rearrangement process. Silencing of the surrogate light chain genes, VpreB and lambda5, then terminates preBCR expression to permit cell cycle exit, recombinase gene upregulation, and VJ(L) rearrangement by small pre-B cells destined to become B cells. PMID:12220935
Burrows, Peter D; Stephan, Robert P; Wang, Yui-Hsi; Lassoued, Ka'ss; Zhang, Zhixin; Cooper, Max D
Molecular analysis of rheumatoid factor (RF)-negative B cell hybridomas from rheumatoid synovial tissue: evidence for an antigen-induced stimulation with selection of high mutated IgVH and low mutated IgVL/lambda genes.
The mutational pattern of IgVH and IgVL genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgVH genes of the B cell hybridomas belonged to the VH3 family (DP42; DP47, n = 2; DP53), the VH1 family (DP75), the VH4 family (DP71) and the VH5 family (DP73); 7/7 IgVH genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgVH genes and the mean R/S ratio of all IgVH genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgVL/lambda genes belonged to the Vlambda1 family (DPL2, DPL5, DPL8nf), the Vlambda2 family (DPL11, n = 2) and to the Vlambda6 family (IGLV6S1); 6/6 IgVL genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgVL genes and the mean R/S ratio of all IgVL was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4+ follicular dendritic cells and Ki-67+ proliferating cells and a dominance of IgA+ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgVH and IgVL/lambda genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA. PMID:9933438
Krenn, V; K÷nig, A; Hensel, F; Berek, C; Souto Carneiro, M M; Haedicke, W; Wang, Y; Vollmers, H; M³ller-Hermelink, H K
Molecular analysis of rheumatoid factor (RF)-negative B cell hybridomas from rheumatoid synovial tissue: evidence for an antigen-induced stimulation with selection of high mutated IgVH and low mutated IgVL/? genes
The mutational pattern of IgVH and IgVL genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgVH genes of the B cell hybridomas belonged to the VH3 family (DP42; DP47, n = 2; DP53), the VH1 family (DP75), the VH4 family (DP71) and the VH5 family (DP73); 7/7 IgVH genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgVH genes and the mean R/S ratio of all IgVH genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgVL/? genes belonged to the V?1 family (DPL2, DPL5, DPL8nf), the V?2 family (DPL11, n = 2) and to the V?6 family (IGLV6S1); 6/6 IgVL genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgVL genes and the mean R/S ratio of all IgVL was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4+ follicular dendritic cells and Ki-67+ proliferating cells and a dominance of IgA+ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgVH and IgVL/? genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA.
Krenn, V; KOnig, A; Hensel, F; Berek, C; Souto Carneiro, M M; Haedicke, W; Wang, YK; Vollmers, H-P; MUller-Hermelink, H K
Immunohistochemical Detection of MYC-driven Diffuse Large B-Cell Lymphomas
Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. A small subset of DLBCLs has translocations involving the MYC locus and an additional group has a molecular signature resembling Burkitt lymphoma (mBL). Presently, identification of such cases by morphology is unreliable and relies on cytogenetic or complex molecular methods such as gene transcriptional profiling. Herein, we describe an immunohistochemical (IHC) method for identifying DLBCLs with increased MYC protein expression. We tested 77 cases of DLBCL and identified 15 cases with high MYC protein expression (nuclear staining in >50% of tumor cells). All MYC translocation positive cases had increased MYC protein expression by this IHC assay. In addition, gene set enrichment analysis (GSEA) of the DLBCL transcriptional profiles revealed that tumors with increased MYC protein expression (regardless of underlying MYC translocation status) had coordinate upregulation of MYC target genes, providing molecular confirmation of the IHC results. We then generated a molecular classifier derived from the MYC IHC results in our cases and employed it to successfully classify mBLs from two previously reported independent case series, providing additional confirmation that the MYC IHC results identify clinically important subsets of DLBCLs. Lastly, we found that DLBCLs with high MYC protein expression had inferior overall survival when treated with R-CHOP. In conclusion, the IHC method described herein can be used to readily identify the biologically and clinically distinct cases of MYC-driven DLBCL, which represent a clinically significant subset of DLBCL cases due to their inferior overall survival.
Kluk, Michael J.; Chapuy, Bjoern; Sinha, Papiya; Roy, Alyssa; Cin, Paola Dal; Neuberg, Donna S.; Monti, Stefano; Pinkus, Geraldine S.; Shipp, Margaret A.; Rodig, Scott J.
B cell therapies for rheumatoid arthritis: beyond B cell depletion.
Initially suggested by the presence of rheumatoid factor autoantibodies, multiple pathogenic roles for B cells (both antibody-mediated and antibody-independent) in rheumatoid arthritis (RA) now are supported by a growing body of experimental observations and human studies. The pathogenic significance of B cells in this disease also has been established conclusively by the proven benefit of Rituximab-induced B cell depletion in RA patients refractory to tumor necrosis factor (TNF) blockade. This article reviews the rationale for the use of B cell-targeting therapies in RA and discusses the caveats and limitations of indiscriminate B cell depletion as currently applied, ncluding incomplete depletion of pathogenic B cells and elimination of protective B cells. Finally, it presents alternative therapeutic strategies that exploit current knowledge of B cell activation, survival, and differentiation to provide more selective B cell and plasma cell targeting. PMID:20510237
Calero, Ismael; Nieto, Jose Antonio; Sanz, I±aki
Integrative Gene Expression Profiling Reveals G6PD-Mediated Resistance to RNA-Directed Nucleoside Analogues in B-Cell Neoplasms
The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including G6PD which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce de novo resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of G6PD expression may be particularly efficacious.
McBrayer, Samuel K.; Yarrington, Michael; Qian, Jun; Feng, Gang; Shanmugam, Mala; Gandhi, Varsha; Krett, Nancy L.; Rosen, Steven T.
Genomic structure of the EWS gene and its relationship to EWSR1, a site of tumor-associated chromosome translocation
The EWS gene has been identified based on its location at the chromosome 22 breakpoint of the t(11;22)(q24;q12) translocation that characterizes Ewing sarcoma and related neuroectodermal tumors. The EWS gene spans about 40 kb of DNA and is encoded by 17 exons. The nucleotide sequence of the exons is identical to that of the previously described cDNA. The first 7 exons encode the N-terminal domain of EWS, which consists of a repeated degenerated polypeptide of 7 to 12 residues rich in tyrosine, serine, threonine, glycine, and glutamine. Exons 11, 12, and 13 encode the putative RNA binding domain. The three glycine- and arginine-rich motifs of the gene are mainly encoded by exons 8-9, 14, and 16. The DNA sequence in the 5[prime] region of the gene has features of a CpG-rich island and lacks canonical promoter elements, such as TATA and CCAAT consensus sequences. Positions of the chromosome 22 breakpoints were determined for 19 Ewing tumors. They were localized in introns 7 or 8 in 18 cases and in intron 10 in 1 case. 26 refs., 5 figs.
Plougastel, B.; Zucman, J.; Peter, M.; Thomas, G.; Delattre, O. (Laboratoire de Genetique des Tumeurs, Paris (France))
The tal gene undergoes chromosome translocation in T cell leukemia and potentially encodes a helix-loop-helix protein.
We have analyzed t(1;14)(p32;q11) chromosome translocations from two patients with T cell acute lymphocytic leukemia. The chromosome 1 breakpoints of these patients lie within a kilobasepair of each other, and thus define a genetic locus (designated tal) involved in T cell oncogenesis. Moreover, we have identified sequences within tal that potentially encode an amphipathic helix-loop-helix motif, a DNA-binding domain found in a variety of proteins that control cell growth and differentiation. The homology domain of tal is especially related to that of lyl-1, a gene on chromosome 19 that has also been implicated in T cell oncogenesis. Hence, tal and lyl-1 encode a distinct family of helix-loop-helix proteins involved in the malignant development of lymphocytes. Images Fig. 1. Fig. 3. Fig. 6.
Chen, Q; Cheng, J T; Tasi, L H; Schneider, N; Buchanan, G; Carroll, A; Crist, W; Ozanne, B; Siciliano, M J; Baer, R
Estradiol modulates uterine 18 kDa translocator protein gene expression in uterus and kidney of rats.
We examined the effect of ovariectomy, with and without estradiol treatment, on 18 kDa translocator protein (TSPO) gene expression and its binding density in the uterus and kidney of rats. Ovariectomy causes a significant decrease in uterine, but not renal TSPO binding density, while estradiol treatment of ovariectomized rats restored TSPO binding density in the uterus. These TSPO density levels did not correlate with steady state or new RNA transcription. Our in vivo study suggests that estradiol is responsible for the maintenance of uterine TSPO density via transcriptional mechanisms. Our in vivo study also suggests that in the kidney estradiol appears to operate via post-transcriptional mechanisms to maintain TSPO density. PMID:19524125
Mazurika, Caroline; Veenman, Leo; Weizman, Ronit; Bidder, Miri; Leschiner, Svetlana; Golani, Idit; Spanier, Ilana; Weisinger, Gary; Gavish, Moshe
Immunoglobulin heavy chain expression shapes the B cell receptor repertoire in human B cell development
Developing B cells must pass a series of checkpoints that are regulated by membrane-bound Ig? through the Ig?-Ig? signal transducers. To determine how Ig? expression affects B cell development and Ab selection in humans we analyzed Ig gene rearrangements in pro-B cells from two patients who are unable to produce Ig? proteins. We find that Ig? expression does not affect VH, D, or JH segment usage and is not required for human Ig? and Ig? recombination or expression. However, the heavy and light chains found in pro-B cells differed from those in peripheral B cells in that they showed unusually long CDR3s. In addition, the Ig? repertoire in Ig?-deficient pro-B cells was skewed to downstream J?s and upstream V?s, consistent with persistent secondary V(D)J rearrangements. Thus, Ig? expression is not required for secondary V(D)J recombination in pro-B cells. However, B cell receptor expression shapes the Ab repertoire in humans and is essential for selection against AbÆs with long CDR3s.
Meffre, Eric; Milili, Michele; Blanco-Betancourt, Carla; Antunes, Henedina; Nussenzweig, Michel C.; Schiff, Claudine
Genetic polymorphisms in oxidative stress-related genes are associated with outcomes following treatment for aggressive B-cell non-Hodgkin lymphoma.
Variable survival outcomes are seen following treatment for aggressive non-Hodgkin lymphoma (NHL). This study examined whether outcomes for aggressive B-cell NHL are associated with single nucleotide polymorphisms (SNPs) in oxidative stress-related genes, which can alter drug metabolism and immune responses. Genotypes for 53 SNPs in 29 genes were determined for 337 patients given anthracycline-based therapies. Their associations with progression-free survival (PFS) and overall survival (OS) were estimated by Cox proportional hazard regression; associations with hematologic toxicity were estimated by logistic regression. To validate the findings, the top three SNPs were tested in an independent cohort of 572 DLBCL patients. The top SNPs associated with PFS in the discovery cohort were the rare homozygotes for MPO rs2243828 (hazard ratio [HR]?=?1.87, 95% confidence interval [CI]?=?1.14-3.06, P?=?0.013), AKR1C3 rs10508293 (HR?=?2.09, 95% CI?=?1.28-3.41, P?=?0.0032) and NCF4 rs1883112 (HR?=?0.66, 95% CI?=?0.43-1.02, P?=?0.06). The association of the NCF4 SNP with PFS was replicated in the validation dataset (HR?=?0.66, 95% CI?=?0.44-1.01, P?=?0.05) and the meta-analysis was significant (HR?=?0.66, 95% CI?=?0.49-0.89, P?0.01). The association of the MPO SNP was attenuated in the validation dataset, while the meta-analysis remained significant (HR?=?1.64, 95% CI?=?1.12-2.41). These two SNPs showed similar trends with OS in the meta-analysis (for NCF4, HR?=?0.72, 95% CI?=?0.51-1.02, P?=?0.07 and for MPO, HR?=?2.06, 95% CI?=?1.36-3.12, P?0.01). In addition, patients with the rare homozygote of the NCF4 SNP had an increased risk of hematologic toxicity. We concluded that genetic variations in NCF4 may contribute to treatment outcomes for patients with aggressive NHL. Am. J. Hematol. 89:639-645, 2014. ® 2014 Wiley Periodicals, Inc. PMID:24633940
Gustafson, Heather L; Yao, Song; Goldman, Bryan H; Lee, Kristy; Spier, Catherine M; LeBlanc, Michael L; Rimsza, Lisa M; Cerhan, James R; Habermann, Thomas M; Link, Brian K; Maurer, Matthew J; Slager, Susan L; Persky, Daniel O; Miller, Thomas P; Fisher, Richard I; Ambrosone, Christine B; Briehl, Margaret M
Frequent allelic losses of 9p21 markers and low incidence of mutations at p16(CDKN2) gene in non-Hodgkin lymphomas of B-cell lineage
We present an allelotype analysis of 35 cases of non-Hodgkin lymphomas and normal pairs using four microsatellite markers that flank the region occupied by the CDKN2 gene locus at 9p21. Frequent allelic losses (LOH) were detected in B-cell lineage NHLs, including Burkitt lymphoma (33.3% of total, if we only consider high grade tumors). In five of these tumors LOH did
Josķ Fernßndez-Piqueras; Javier Santos; Ignacio Pķrez de Castro; Bßrbara Melķndez; BeatrĒz MartĒnez; Mercedes Robledo; Carmen Rivas; Javier BenĒtez
11p15 translocations involving the NUP98 gene in childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome.
In a survey of childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS) in Japan, we found 11p15 translocations in 5 (6%) of 81 children with t-AML/MDS. t(11;17)(p15;q21), t(11;12)(p15;q13), t(7;11)(p15;p15), inv(11)(p15q22), and add(11)(p15) were each found in one patient. Southern blotting and/or RT-PCR analyses revealed rearrangements of the NUP98 gene in tumor samples of all five patients. Rearrangements of DDX10 were detected in t-AML/MDS cells with inv(11), and rearrangements of HOXA9 were detected in t-AML cells with t(7;11). The 17q21 breakpoint of t(11;17) and the 12q13 breakpoint of t(11;12)(p15;q13) coincided with the loci of the HOXB and HOXC gene families, respectively. Therefore, it is reasonable to speculate that one of the HOXB genes and one of the HOXC genes were fused to NUP98 by t(11;17) and t(11;12), respectively, in t-AML/MDS cells. We propose that NUP98 may be a target gene for t-AML/MDS, and that t-AML/MDS with a fusion of NUP98 and HOX or DDX10 genes may be more frequent in children than in patients of other age groups. PMID:10502319
Nishiyama, M; Arai, Y; Tsunematsu, Y; Kobayashi, H; Asami, K; Yabe, M; Kato, S; Oda, M; Eguchi, H; Ohki, M; Kaneko, Y
Identification of COL3A1 and RAB2A as novel translocation partner genes of PLAG1 in lipoblastoma.
Lipoblastoma is a rapidly growing, benign neoplasm in children. Surgical excision is usually curative, with a recurrence rate of about 20%. Because the histology of lipoblastoma is heterogeneous and overlaps with other lipomatous tumors, some lipoblastoma cases have been difficult to diagnose. The detection of PLAG1 gene rearrangement is useful for the diagnosis of lipoblastoma. Three fusion partner genes are known in relation to PLAG1 in lipoblastoma HAS2 at 8q24.1, COL1A2 at 7q22, and RAD51L1 at 14q24. Herein, we describe another two novel fusion genes in lipoblastoma tumor specimens. We checked six tumors for the presence of two known fusion genes, HAS2-PLAG1 and COL1A2-PLAG1. Only HAS2-PLAG1 was found in one of the cases. Next, we attempted to identify potential PLAG1 fusion partners using 5'RACE. Sequence analysis revealed two novel fusion genes, COL3A1-PLAG1 in three cases and RAB2A-PLAG1 in one case, respectively. As a result of the translocations, the constitutively active promoter of the partner gene drives the ectopic expression of PLAG1. We also evaluated whether a high level of PLAG1 expression can be used to help differentiate lipomatous tumors. PLAG1 expression was evaluated by real-time PCR in five lipoblastoma tumor specimens. The expressions were 70-150 times higher in lipoblastomas than in human adipocytes. However, PLAG1 expression was low in one case of lipoma. These results demonstrate that PLAG1 overexpression is a potential marker of lipoblastoma. Our findings, in agreement with previous studies, show that lipoblastoma is a group of lipomatous tumors with PLAG1 rearrangement and overexpression. ® 2014 Wiley Periodicals, Inc. PMID:24700772
Yoshida, Hideki; Miyachi, Mitsuru; Ouchi, Kazutaka; Kuwahara, Yasumichi; Tsuchiya, Kunihiko; Iehara, Tomoko; Konishi, Eiichi; Yanagisawa, Akio; Hosoi, Hajime
Immunoglobulin. gamma. light-chain-related genes 14. 1 and 16. 1 are expressed in pre-B cells and may encode the human immunoglobulin. omega. light-chain protein
Human pre-B cells, which produce immunoglobulin heavy chain but do not produce immunoglobulin light chain, are shown to contain a 1-kilobase transcript homologous to immunoglobulin {lambda} light-chain genes. Detailed analysis of RNA and cDNA clones derived from these transcripts reveals that they originate from the distinct immunoglobulin {lambda}-like genes 14.1/16.1. Sequence analysis of these clones reveals a long open reading frame, beginning with an ATG, capable of encoding a protein of 214 amino acids with an unprocessed molecular weight of 22,944. The C-terminal half of this predicted protein is highly homologous to immunoglobulin {lambda} light-chain joining and constant region protein sequence, while the amino-terminal end does not share homology with variable regions. Antisera raised against a peptide whose sequence was predicted from the 14.1 cDNA sequence identifies a 22-kDa protein in human pre-B cells. Immunoprecipitation of immunoglobulin {mu}-chain from these pre-B cells with anti-immunoglobulin {mu} antibody coprecipitates a 22-kDa protein, which is a candidate for the human immunoglobulin {omega} light-chain protein and may be the protein product of the 14.1/16.1 genes.
Hollis, G.F.; Evans, R.J.; Stafford-Hollis, J.M.; Korsmeyer, S.J.; McKearn, J.P. (Monsanto Co., St. Louis, MO (USA))
Disruption of the ATE1 and SLC12A1 Genes by Balanced Translocation in a Boy with Non-Syndromic Hearing Loss
We report on a boy with non-syndromic hearing loss and an apparently balanced translocation t(10;15)(q26.13;q21.1). The same translocation was found in the normally hearing brother, father and paternal grandfather; however, this does not exclude its involvement in disease pathogenesis, for example, by unmasking a second mutation. Breakpoint analysis via FISH with BAC clones and long-range PCR products revealed a disruption of the arginyltransferase 1 (ATE1) gene on translocation chromosome 10 and the solute carrier family 12, member 1 gene (SLC12A1) on translocation chromosome 15. SNP array analysis revealed neither loss nor gain of chromosomal regions in the affected child, and a targeted gene enrichment panel consisting of 130 known deafness genes was negative for pathogenic mutations. The expression patterns in zebrafish and humans did not provide evidence for ear-specific functions of the ATE1 and SLC12A1 genes. Sanger sequencing of the 2 genes in the boy and 180 GJB2 mutation-negative hearing-impaired individuals did not detect homozygous or compound heterozygous pathogenic mutations. Our study demonstrates the many difficulties in unraveling the molecular causes of a heterogeneous phenotype. We cannot directly implicate disruption of ATE1 and/or SLC12A1 to the abnormal hearing phenotype; however, mutations in these genes may have a role in polygenic or multifactorial forms of hearing impairment. On the other hand, it is conceivable that our patient carries a disease-causing mutation in a so far unidentified deafness gene. Evidently, disruption of ATE1 and/or SLC12A1 gene function alone does not have adverse effects.
Vona, B.; Neuner, C.; El Hajj, N.; Schneider, E.; Farcas, R.; Beyer, V.; Zechner, U.; Keilmann, A.; Poot, M.; Bartsch, O.; Nanda, I.; Haaf, T.
Disruption of the ATE1 and SLC12A1 Genes by Balanced Translocation in a Boy with Non-Syndromic Hearing Loss.
We report on a boy with non-syndromic hearing loss and an apparently balanced translocation t(10;15)(q26.13;q21.1). The same translocation was found in the normally hearing brother, father and paternal grandfather; however, this does not exclude its involvement in disease pathogenesis, for example, by unmasking a second mutation. Breakpoint analysis via FISH with BAC clones and long-range PCR products revealed a disruption of the arginyltransferase 1 (ATE1) gene on translocation chromosome 10 and the solute carrier family 12, member 1 gene (SLC12A1) on translocation chromosome 15. SNP array analysis revealed neither loss nor gain of chromosomal regions in the affected child, and a targeted gene enrichment panel consisting of 130 known deafness genes was negative for pathogenic mutations. The expression patterns in zebrafish and humans did not provide evidence for ear-specific functions of the ATE1 and SLC12A1 genes. Sanger sequencing of the 2 genes in the boy and 180 GJB2 mutation-negative hearing-impaired individuals did not detect homozygous or compound heterozygous pathogenic mutations. Our study demonstrates the many difficulties in unraveling the molecular causes of a heterogeneous phenotype. We cannot directly implicate disruption of ATE1 and/or SLC12A1 to the abnormal hearing phenotype; however, mutations in these genes may have a role in polygenic or multifactorial forms of hearing impairment. On the other hand, it is conceivable that our patient carries a disease-causing mutation in a so far unidentified deafness gene. Evidently, disruption of ATE1 and/or SLC12A1 gene function alone does not have adverse effects. PMID:24550759
Vona, B; Neuner, C; El Hajj, N; Schneider, E; Farcas, R; Beyer, V; Zechner, U; Keilmann, A; Poot, M; Bartsch, O; Nanda, I; Haaf, T
B cell receptor signal strength determines B cell fate
B cell receptor (BCR)-mediated antigen recognition is thought to regulate B cell differentiation. BCR signal strength may also influence B cell fate decisions. Here, we used the Epstein-Barr virus protein LMP2A as a constitutively active BCR surrogate to study the contribution of BCR signal strength in B cell differentiation. Mice carrying a targeted replacement of Igh by LMP2A leading to
Kevin L Otipoby; Marat Alimzhanov; Sibille Humme; Nathalie Uyttersprot; Jeffery L Kutok; Michael C Carroll; Stefano Casola; Klaus Rajewsky
Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia.
Expression of Hoxa7 and Hoxa9 is activated by proviral integration in BXH2 murine myeloid leukaemias. This result, combined with the mapping of the HOXA locus to human chromosome 7p15, suggested that one of the HOXA genes might be involved in the t(7;11)(p15;p15) translocation found in some human myeloid leukaemia patients. Here we show that in three patients with t(7;11), the chromosome rearrangement creates a genomic fusion between the HOXA9 gene and the nucleoporin gene NUP98 on chromosome 11p15. The translocation produces an invariant chimaeric NUP98/HOXA9 transcript containing the amino terminal half of NUP98 fused in frame to HOXA9. These studies identify HOXA9 as an important human myeloid leukaemia gene and suggest an important role for nucleoporins in human myeloid leukaemia given that a second nucleoporin, NUP214, has also been implicated in human myeloid leukaemia. PMID:8563753
Nakamura, T; Largaespada, D A; Lee, M P; Johnson, L A; Ohyashiki, K; Toyama, K; Chen, S J; Willman, C L; Chen, I M; Feinberg, A P; Jenkins, N A; Copeland, N G; Shaughnessy, J D
A recurrent 11q aberration pattern characterizes a subset of MYC-negative high-grade B-cell lymphomas resembling Burkitt lymphoma.
The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q. PMID:24398325
Salaverria, Itziar; Martin-Guerrero, Idoia; Wagener, Rabea; Kreuz, Markus; Kohler, Christian W; Richter, Julia; Pienkowska-Grela, Barbara; Adam, Patrick; Burkhardt, Birgit; Claviez, Alexander; Damm-Welk, Christine; Drexler, Hans G; Hummel, Michael; Jaffe, Elaine S; K³ppers, Ralf; Lefebvre, Christine; Lisfeld, Jasmin; L÷ffler, Markus; Macleod, Roderick A F; Nagel, Inga; Oschlies, Ilske; Rosolowski, Maciej; Russell, Robert B; Rymkiewicz, Grzegorz; Schindler, Detlev; Schlesner, Matthias; Scholtysik, Renķ; Schwaenen, Carsten; Spang, Rainer; Szczepanowski, Monika; Tr³mper, Lorenz; Vater, Inga; Wessendorf, Swen; Klapper, Wolfram; Siebert, Reiner
Glucocorticoids stimulate inflammatory 5-lipoxygenase gene expression and protein translocation in the brain.
In the brain, the expression of 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of inflammatory leukotrienes, increases during aging. Antiinflammatory drugs are currently being evaluated for the treatment of aging-associated neurodegenerative diseases such as Alzheimer's disease. Although generally considered antiinflammatory, glucocorticoids, whose production also increases during aging, are not particularly effective in this disease. In human monocytes, 5-LO mRNA content increases on exposure to the synthetic glucocorticoid dexamethasone, which prompted us to hypothesize that glucocorticoids might increase 5-LO expression in the brain as well. We treated rats for 10 days either with corticosterone (implanted subcutaneously) or with dexamethasone (injected daily); they were killed on day 10 after pellet implantation or 24 h after the 10th dexamethasone injection. We found increased levels of 5-LO mRNA and protein in hippocampus and cerebellum of glucocorticoid-treated rats; 5-LO-activating protein (FLAP) mRNA content was not affected. Using western immunobloting, we also observed the concurrent translocation of 5-LO protein from cytosol to membrane, an indication of its activation. Thus, glucocorticoid-mediated up-regulation of the neuronal 5-LO pathway may contribute to rendering an aging brain vulnerable to degeneration. PMID:10428066
Uz, T; Dwivedi, Y; Savani, P D; Impagnatiello, F; Pandey, G; Manev, H
T cell CD40LG gene expression and the production of IgG by autologous B cells in systemic lupus erythematosus
CD40 ligand (CD40LG), encoded on the X chromosome, has been reported to be overexpressed on lupus Tcells. Herein, we investigated the effect of DNA demethylation on Tcell CD40LG expression and the production of IgG by autologous B cells in lupus. We found normal human T cells transfected with CD40LG induced autologous B cell activation and plasma cell differentiation. Both female lupus CD4+ T cells and demethylating agents treated CD4+ T cells overexpressed CD40LG mRNA. Further, lupus T cells from both genders or demethylated CD4+ T cells from healthy women overstimulated autologous B cells, and this could be reversed with anti-CD40LG Ab in only females. We demonstrated that female lupus CD4+ T cells and demethylated CD4+ T cells express high level of CD40LG and overstimulate B cells to produce IgG. This is due to DNA demethylation and thereby reactivation of the inactive X chromosome in female.
Zhou, Ying; Yuan, Jun; Pan, Yujun; Fei, Yiping; Qiu, Xiangning; Hu, Nan; Luo, Yongqi; Lei, Wenzhi; Li, Yaping; Long, Hai; Sawalha, Amr H; Richardson, Bruce; Lu, Qianjin
A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia.
A novel class of conserved transcription factors has been identified from the molecular cloning of AF10, the gene involved in the t(10;11)(p12;q23) translocation of acute myeloid leukemias. AF10 encodes a 109-kD protein of 1,027 amino acids and contains an N-terminal zinc finger region and a C-terminal leucine zipper. These structures have been found to be conserved in sequence and position in three other proteins, AF17, BR140, and a previously unrecognized Caenorhabditis elegans gene, provisionally named CEZF. The overall structure, level of sequence conservation, and expression pattern suggest that these genes encode a new class of transcription factors, some of which are targets for chromosomal translocation in acute leukemia. PMID:7888665
Chaplin, T; Ayton, P; Bernard, O A; Saha, V; Della Valle, V; Hillion, J; Gregorini, A; Lillington, D; Berger, R; Young, B D
t(4;11)(q21;p15) translocation involving NUP98 and RAP1GDS1 genes: characterization of a new subset of T acute lymphoblastic leukaemia.
Two cases of T acute lymphoblastic leukaemia (T-ALL) with an identical t(4;11)(q21;p15) translocation were identified within a prospective study on the biological and clinical features of adult ALL patients enrolled into the therapeutic protocol ALL0496 of the GIMEMA Italian Group. In both cases, the molecular characterization showed an involvement of the NUP98 gene on 11p15 which rearranges with the RAP1GDS1 gene on 4q21. The morphological and immunological features of the leukaemic cells, as well as the clinical behaviour and response to induction therapy, were the same in both patients. Based on the available data, the t(4;11)(q21;p15) translocation involving the NUP98-RAP1GDS1 fusion gene emerges as a new highly specific genetic abnormality that characterizes a subset of T-ALL. PMID:10929031
Mecucci, C; La Starza, R; Negrini, M; Sabbioni, S; Crescenzi, B; Leoni, P; Di Raimondo, F; Krampera, M; Cimino, G; Tafuri, A; Cuneo, A; Vitale, A; FoÓ, R
Human aryl hydrocarbon receptor nuclear translocator gene (ARNT) D/N511 polymorphism.
We found a novel A-->C change in codon 511 of the ARNT gene, which predicted the substitution of Asn (AAC) for Asp (GAC) at this position. Amplification using mismatched primers allowed the ARNT D/N511 polymorphism to be detected by digestion with endonuclease Tth111I. The frequency of the ARNT N511 allele was 0.019 in Caucasians and 0.026 in Africans. Because of the importance of the ARNT gene product in the metabolism of xenobiotics, this polymorphism may be useful in the study of associations with metabolic phenotypes and in pharmacogenetic studies. PMID:10721670
Cao, H; Hegele, R A
Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes.
Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1-AHCY and RIF1-PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer. PMID:18084325
Howarth, K D; Blood, K A; Ng, B L; Beavis, J C; Chua, Y; Cooke, S L; Raby, S; Ichimura, K; Collins, V P; Carter, N P; Edwards, P A W
Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes
Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1Mb resolution on BAC arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Secondly, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300, and FOXP4. Two gene fusions were demonstrated, TAX1BP1-AHCY and RIF1-PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.
Howarth, KD; Blood, KA; Ng, BL; Beavis, JC; Chua, Y; Cooke, SL; Raby, S; Ichimura, K; Collins, VP; Carter, NP; Edwards, PAW
MYC protein expression and genetic alterations have prognostic impact in patients with diffuse large B-cell lymphoma treated with immunochemotherapy
MYC alterations influence the survival of patients with diffuse large B-cell lymphoma. Most studies have focused on MYC translocations but there is little information regarding the impact of numerical alterations and protein expression. We analyzed the genetic alterations and protein expression of MYC, BCL2, BCL6, and MALT1 in 219 cases of diffuse large B-cell lymphoma. MYC rearrangement occurred as the sole abnormality (MYC single-hit) in 3% of cases, MYC and concurrent BCL2 and/or BCL6 rearrangements (MYC double/triple-hit) in 4%, MYC amplifications in 2% and MYC gains in 19%. MYC single-hit, MYC double/triple-hit and MYC amplifications, but not MYC gains or other gene rearrangements, were associated with unfavorable progression-free survival and overall survival. MYC protein expression, evaluated using computerized image analysis, captured the unfavorable prognosis of MYC translocations/amplifications and identified an additional subset of patients without gene alterations but with similar poor prognosis. Patients with tumors expressing both MYC/BCL2 had the worst prognosis, whereas those with double-negative tumors had the best outcome. High MYC expression was associated with shorter overall survival irrespectively of the International Prognostic Index and BCL2 expression. In conclusion, MYC protein expression identifies a subset of diffuse large B-cell lymphoma with very poor prognosis independently of gene alterations and other prognostic parameters.
Valera, Alexandra; Lopez-Guillermo, Armando; Cardesa-Salzmann, Teresa; Climent, Fina; Gonzalez-Barca, Eva; Mercadal, Santiago; Espinosa, Inigo; Novelli, Silvana; Briones, Javier; Mate, Jose L.; Salamero, Olga; Sancho, Juan M.; Arenillas, Leonor; Serrano, Sergi; Erill, Nadina; Martinez, Daniel; Castillo, Paola; Rovira, Jordina; Martinez, Antonio; Campo, Elias; Colomo, Luis
Functional characterization of the human translocator protein (18kDa) gene promoter in human breast cancer cell lines.
The translocator protein (18kDa; TSPO) is a mitochondrial drug- and cholesterol-binding protein that has been implicated in several processes, including steroidogenesis, cell proliferation, and apoptosis. Expression of the human TSPO gene is elevated in several cancers. To understand the molecular mechanisms that regulate TSPO expression in human breast cancer cells, the TSPO promoter was identified, cloned, and functionally characterized in poor-in-TSPO hormone-dependent, non-aggressive MCF-7 cells and rich-in-TSPO hormone-independent, aggressive, and metastatic MDA-MB-231 breast cancer cells. RNA ligase-mediated 5'-rapid amplification of cDNA ends analysis indicated transcription initiated at multiple sites downstream of a GC-rich promoter that lacks functional TATA and CCAAT boxes. Deletion analysis indicated that the region from -121 to +66, which contains five putative regulatory sites known as GC boxes, was sufficient to induce reporter activity up to 24-fold in MCF-7 and nearly 120-fold in MDA-MB-231 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1, Sp3 and Sp4 bind to these GC boxes in vitro and to the endogenous TSPO promoter. Silencing of Sp1, Sp3 and Sp4 gene expression reduced TSPO levels. In addition, TSPO expression was epigenetically regulated at one or more of the identified GC boxes. Disruption of the sequence downstream of the main start site of TSPO differentially regulated TSPO promoter activity in MCF-7 and MDA-MB-231 cells, indicating that essential elements contribute to its differential expression in these cells. Taken together, these experiments constitute the first in-depth functional analysis of the human TSPO gene promoter and its transcriptional regulation. PMID:21958735
Batarseh, Amani; Barlow, Keith D; Martinez-Arguelles, Daniel B; Papadopoulos, Vassilios
Promiscuous genes involved in recurrent chromosomal translocations in soft tissue tumours.
Soft tissue tumours represent a heterogeneous group of mesenchymal lesions and their classification continues to evolve as a result of incorporating advances in cytogenetic and molecular techniques. In the last decade, traditional diagnostic approaches were supplemented with a significant number of reliable molecular diagnostic tools, detecting tumour type specific genetic alterations. Additionally, the successful application of some of these techniques to formalin fixed, paraffin embedded tissue enabled a broader range of clinical material to be subjected to molecular analysis. However, despite all these remarkable advances, the realisation that some of the genetic abnormalities are not fully histotype specific and that certain gene aberrations can be shared among different sarcoma types, otherwise completely unrelated clinically or immunophenotypically, has introduced some drawbacks in surgical pathology practice. One such common example is the presence of EWSR1 gene rearrangements by fluorescence in situ hybridisation (FISH), a test now preferred over the elaborate RT-PCR testing, in a variety of benign and highly malignant soft tissue tumours, in addition to a subset of carcinomas. Furthermore, the presence of identical gene fusions in completely different sarcoma types (i.e., EWSR1-ATF1, EWSR1-CREB1) or in non-mesenchymal malignancies (epithelial or haematological) has raised skepticism as to their diagnostic utility, and their lack of specificity has been compared to the limitations of other ancillary techniques, in particular immunohistochemistry. This review catalogues the main groups of genes that behave in a promiscuous manner within recurrent fusion events in soft tissue tumours. Although we acknowledge that the present molecular classification of soft tissue tumours is much more complex than two decades ago, when EWSR1 gene rearrangements had been described as the hallmark of Ewing sarcoma, we make the strong argument that with very few exceptions, the prevalence of fusion transcripts in most sarcomas is such that they come to define these entities and can be used as highly specific molecular diagnostic markers in the right clinical and pathological context. PMID:24378390
Antonescu, Cristina R; Dal Cin, Paola
Comparative Gene Expression Profiling Identifies Common Molecular Signatures of NF-kB Activation in Canine and Human Diffuse Large B Cell Lymphoma (DLBCL)
We present the first comparison of global transcriptional changes in canine and human diffuse large B-cell lymphoma (DLBCL), with particular reference to the nuclear factor-kappa B (NF-kB) pathway. Microarray data generated from canine DLBCL and normal lymph nodes were used for differential expression, co-expression and pathway analyses, and compared with analysis of microarray data from human healthy and DLBCL lymph
Manikhandan A. V. Mudaliar; Ross D. Haggart; Gino Miele; Grant Sellar; Karen A. L. Tan; John R. Goodlad; Elspeth Milne; David M. Vail; Ilene Kurzman; Daniel Crowther; David J. Argyle; Easter Bush
MHC class I bound peptides of a colon carcinoma cell line, a Ki-ras gene-targeted progeny cell line and a B cell line
MHC class I associated peptides on cancer cells represent potential targets for CD8+ cytotoxic T cell activity against tumor cells. We eluted the naturally bound MHC class I peptides of a colon carcinoma cell line and compared them to peptides isolated from a B cell line and a slow-growing activated Ki-ras-disrupted colon cancer cell line. While we failed to detect
Christopher J Savoie; Nobuhiro Kamikawaji; Tohru Sudo; Masanori Furuse; Senji Shirasawa; Takeshi Tana; Takehiko Sasazuki
FCGR3A gene polymorphisms may correlate with response to frontline R-CHOP therapy for diffuse large B-cell lymphoma
The precise mechanism of rituximab plus cyclophosphamide\\/doxorubicin\\/vincris- tine\\/prednisone (R-CHOP) therapy in dif- fuse large B-cell lymphoma (DLBCL) is not fully elucidated. Besides overcoming bcl-2-mediated chemoresistance, anti- body-dependent cellular cytotoxicity (ADCC), which is activated by effector cells via immunoglobulin G (IgG) frag- ment C receptors (FcRs), was also pro- posed as a mechanism of rituximab. The current study evaluated the impact
Dong Hwan Kim; Hee Du Jung; Jong Gwang Kim; Je-Jung Lee; Deok-Hwan Yang; Yeon Hee Park; Ho Jin Shin; Min Kyoung Kim; Myung Soo Hyun; Sang Kyun Sohn
The Majority of Human Memory B Cells Recognizing RhD and Tetanus Resides in IgM+ B Cells
B cell memory to T cell¢dependent (TD) Ags are considered to largely reside in class-switched CD27+ cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D+ erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid¢specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27? B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27+IgG+ B cells was observed. Next, B cells were enriched with D+ erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27?IgM+ and CD27+IgM+ B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27?IgM+, CD27+IgM+, and CD27+IgG+ B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM+ B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27+ B cells from Ag-experienced precursors.
Della Valle, Luciana; Dohmen, Serge E.; Verhagen, Onno J. H. M.; Berkowska, Magdalena A.; Vidarsson, Gestur
Targeted Deletion of the Gene Encoding the La Autoantigen (Sj?gren's Syndrome Antigen B) in B Cells or the Frontal Brain Causes Extensive Tissue Loss
La antigen (Sj÷gren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sj÷gren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1Cre La-deleted mice produce no B cells. Consistent with ?CamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ?5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.
Gaidamakov, Sergei; Maximova, Olga A.; Chon, Hyongi; Blewett, Nathan H.; Wang, Hongsheng; Crawford, Amanda K.; Day, Amanda; Tulchin, Natalie; Crouch, Robert J.; Morse, Herbert C.; Blitzer, Robert D.
Lon protease inactivation, or translocation of the lon gene, potentiate bacterial evolution to antibiotic resistance.
Previous work demonstrated that selection for Escherichia coli mutants with low antibiotic resistance frequently resulted in co-selection of lon mutations and that lon(-) mutants evolved higher-level resistance faster than a lon(+) strain. Here we show that lon mutation causes a very low multidrug resistance by inducing the AcrAB-TolC pump via stabilization of the acrAB transcriptional activators MarA and SoxS, which are substrates of the Lon protease. Fast evolution of lon(-) mutants towards higher resistance involves selection of frequent next-step mutations consisting of large duplications including acrAB and the mutated lon gene. Resistance results from the combined effects of acrAB duplication and lon mutation increasing dosage of efflux pump. In contrast, when acrAB duplication occurs as the first step mutation, increased Lon activity caused by lon(+) co-duplication mitigates the effect of acrAB duplication on resistance, and faster evolution towards higher resistance is not observed. As predicted, when the functional lon gene is relocated far from acrAB to prevent their co-duplication, first-step acrAB duplication confers higher resistance, which then allows selection of frequent next-step mutations and results in faster evolution towards higher resistance. Our results demonstrate how order of appearance of mutations and gene location can influence the rate of resistance evolution. PMID:24325250
Nicoloff, Hervķ; Andersson, Dan I
Differential programming of B cells in AID deficient mice.
The Aicda locus encodes the activation induced cytidine deaminase (AID) and is highly expressed in germinal center (GC) B cells to initiate somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. Besides these Ig specific activities in B cells, AID has been implicated in active DNA demethylation in non-B cell systems. We here determined a potential role of AID as an epigenetic eraser and transcriptional regulator in B cells. RNA-Seq on different B cell subsets revealed that Aicda(-/-) B cells are developmentally affected. However as shown by RNA-Seq, MethylCap-Seq, and SNP analysis these transcriptome alterations may not relate to AID, but alternatively to a CBA mouse strain derived region around the targeted Aicda locus. These unexpected confounding parameters provide alternative, AID-independent interpretations on genotype-phenotype correlations previously reported in numerous studies on AID using the Aicda(-/-) mouse strain. PMID:23922811
Hogenbirk, Marc A; Heideman, Marinus R; Velds, Arno; van den Berk, Paul C M; Kerkhoven, Ron M; van Steensel, Bas; Jacobs, Heinz
Ibrutinib for B cell malignancies
Research over the role of BrutonÆs agammaglobulinemia tyrosine kinase (BTK) in B-lymphocyte development, differentiation, signaling and survival has led to better understanding of the pathogenesis of B-cell malignancies. Down-regulation of BTK activity is an attractive novel strategy for treating patients with B-cell malignancies. Ibrutinib (PCI-32765), a potent inhibitor of BTK induces impressive responses in B-cell malignancies through irreversible bond with cysteine-481 in the active site of BTK (TH/SH1 domain) and inhibits BTK phosphorylation on Tyr223. This review discussed in details the role of BTK in B-cell signaling, molecular interactions between B cell lymphoma/leukemia cells and their microenvironment. Clinical trials of the novel BTK inhibitor, ibrutinib (PCI-32765), in B cell malignancies were summarized.
Ibrutinib for B cell malignancies.
Research over the role of Bruton's agammaglobulinemia tyrosine kinase (BTK) in B-lymphocyte development, differentiation, signaling and survival has led to better understanding of the pathogenesis of B-cell malignancies. Down-regulation of BTK activity is an attractive novel strategy for treating patients with B-cell malignancies. Ibrutinib (PCI-32765), a potent inhibitor of BTK induces impressive responses in B-cell malignancies through irreversible bond with cysteine-481 in the active site of BTK (TH/SH1 domain) and inhibits BTK phosphorylation on Tyr223. This review discussed in details the role of BTK in B-cell signaling, molecular interactions between B cell lymphoma/leukemia cells and their microenvironment. Clinical trials of the novel BTK inhibitor, ibrutinib (PCI-32765), in B cell malignancies were summarized. PMID:24472371
Novero, Aileen; Ravella, Pavan M; Chen, Yamei; Dous, George; Liu, Delong
B cell modulation in rheumatology
Summary of recent advances While evidence of dysregulation of the B-cell compartment was first demonstrated with the identification of autoantibodies, other functional roles of B lymphocytes in autoimmune pathogenesis have generally been underappreciated or completely overlooked. With the recent approval of the first B-cell targeting agent in rheumatoid arthritis, new strategies are being developed to target B cells through a range of membrane-associated lineage-specific molecules, and also by interfering with B-cell specific pro-survival signals. B-cell directed agents are therefore providing an effective new mechanistic approach to treatment, and also enabling new perspectives from the dissection of the contributions of B cells in physiologic and pathologic immune responses.
Silverman, Gregg J.; Khanna, Sahil
Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome
BACKGROUND: Prader-Willi syndrome (MIM #176270; PWS) is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon
Birgitt Sch³le; Mohammed Albalwi; Emma Northrop; David I Francis; Margaret Rowell; Howard R Slater; RJ McKinlay Gardner; Uta Francke
B-cell differentiation in EBV-positive Burkitt lymphoma is impaired at posttranscriptional level by miRNA-altered expression.
Endemic, sporadic and HIV-associated Burkitt lymphoma (BL) all have a B-cell phenotype and a MYC translocation, but a variable association with the Epstein-Barr virus (EBV). However, there is still no satisfactory explanation of how EBV participates in the pathogenesis of BL. A recent investigation suggested that EBV-positive and EBV-negative BL have different cells of origin. In particular, according to immunoglobulin gene mutation analysis, EBV-negative BLs may originate from early centroblasts, whereas EBV-positive BLs seem to arise from postgerminal center B cells or memory B cells. The appearance of a germinal center phenotype in EBV-positive cells might thus derive from a block in B-cell differentiation. The exit from the germinal center involves a complex series of events, which require the activation of BLIMP-1, and the consequent downregulation of several target genes. Here, we investigated the expression of specific miRNAs predicted to be involved in B-cell differentiation and found that hsa-miR-127 is differentially expressed between EBV-positive and EBV-negative BLs. In particular, it was strongly upregulated only in EBV-positive BL samples, whereas EBV-negative cases showed levels of expression similar to normal controls, including microdissected germinal centers (GC) cells. In addition, we found evidence that hsa-miR-127 is involved in B-cell differentiation process through posttranscriptional regulation of BLIMP1 and XBP1. The overexpression of this miRNA may thus represent a key event in the lymphomagenesis of EBV positive BL, by blocking the B-cell differentiation process. PMID:19530237
Leucci, Eleonora; Onnis, Anna; Cocco, Mario; De Falco, Giulia; Imperatore, Francesco; Giuseppina, Antonicelli; Costanzo, Valentina; Cerino, Giovanna; Mannucci, Susanna; Cantisani, Rocco; Nyagol, Joshua; Mwanda, Walter; Iriso, Robert; Owang, Martin; Schurfeld, Karin; Bellan, Cristiana; Lazzi, Stefano; Leoncini, Lorenzo
Mantle cell lymphoma harboring Burkitt's-like translocations presents differential expression of aurora kinase genes compared with others 8q abnormalities.
We compared the levels of AURKA and AURKB in 24 (mantle cell lymphoma) MCL patients harboring 8q abnormalities and its relationship with MYCC gene status. Two distinct subgroups were observed, in terms of MYCC expression. Except for the patients with Burkitt's-like translocation, none of the patients harboring 8q abnormalities, including balanced translocations or duplications of MYCC band, identified both by G-banding and SKY, showed differential expression levels of MYCC. These previous findings also reflected in the differential expression of AURKA and AURKB genes. We found that AURKA and AURKB mRNA were expressed at significantly higher levels in MCL patients harboring Burkitt's-like translocation, when compared to patients with 8q rearrangements. The high expression of aurora kinase genes is reported to be associated with some parameters of clinical oncologic aggressiveness, such as high histological grade, invasion and increased rates of metastasis in several types of cancers. It is possible that in MCL patients expressing abnormal levels of MYCC together with a high expression of AURKA might offer some resistant to the conventional therapy purposes. Thus, aurora kinase inhibitors may also be considered for this specific subgroup on MCL, whose aggressive clinical course resembles high-grade lymphoma. PMID:24683001
de Oliveira, Fßbio Morato; Rodrigues-Alves, Ana Paula Nunes; Lucena-Ara·jo, Ant¶nio Roberto; de Paula Silva, Ferdinando; da Silva, Fernanda Borges; FalcŃo, Roberto Passetto
Analysis of the Ten-Eleven Translocation 2 (TET2) Gene Mutation in Myeloproliferative Neoplasms.
Loss-of-function mutations in the putative tumor suppressor gene, Ten-Eleven Ttranslocation 2(TET2), have been identified recently in myeloproliferative neoplasms (MPNs). The present study analyzed the TET2 gene in 99 MPNs patients. The overall TET2 mutational frequency was 12.1% (22.2% in polycythemia vera (PV), 9.7% in essential thrombocythemia (ET), 18.2% in primary myelofibrosis (PMF,) and 0% in unclassified MPNs), and 11 mutations (p.Lys95Asnfs*18, p.Gln967Asnfs*40, p.Lys1022Glufs*4, p.Asp1314Metfs*49, p.Gln1534Alafs*43, p.Tyr1618Leufs*4, p.Leu1609Glufs*45, p.Gly1735*, Q599R, c.3409+1G>T, c.4044+2insT) were identified. All the patients with TET2 mutation were accompanied by the JAK2 V617F mutation. The existence of the TET2 mutation was not related to the patient's age, hematologic indices, JAK2 V617F allele burden, frequencies of organomegaly, marrow fibrosis, or thrombotic/hemorrhagic complications in entire MPN patients. However, tendencies toward higher JAK2 V617F allele burdens (88.0▒4.3% vs. 19.1▒28.7%, P=0.034) and higher Hct (47.4▒5.4% vs. 25.5▒6.2%, P=0.037) were detected in PMF patients harboring TET2 mutations. Moreover, a significantly higher frequency of organomegaly was identified in ET patients harboring the TET2 mutation (50% vs. 19.6%, P=0.018). The TET2 mutation most likely contributes to clinical phenotypes and shows a high accompanying rate with JAK2 V617F; larger scale studies involving more MPN patients are needed. PMID:24795056
Ha, Jung-Sook; Jeon, Dong-Seok; Kim, Jae-Ryong; Ryoo, Nam-Hee; Suh, Jang-Soo
Comparative analyses of B cell populations in trout kidney and mouse bone marrow; establishing "B cell signatures"
This study aimed to identify the frequency and distribution of developing B cell populations in the kidney of the rainbow trout, using four molecular B cell markers that are highly conserved between species, including two transcription factors, Pax5 and EBF1, recombination activating gene RAG1, and the immunoglobulin heavy chain mu. Three distinct B cell stages were defined: early developing B cells (CLP, pro-B, and early pre-B cells), late developing B cell (late pre-B, immature B, and mature B cells), and IgM-secreting cells. Developmental stage-specific, combinatorial expression of Pax5, EBF1, RAG1 and immunoglobulin mu was determined in trout anterior kidney cells by flow cytometry. Trout staining patterns were compared to a well-defined primary immune tissue, mouse bone marrow, and using mouse surface markers B220 and CD43. A remarkable level of similarity was uncovered between the primary immune tissues of both species. Subsequent analysis of the entire trout kidney, divided into five contiguous segments K1-K5, revealed a complex pattern of early developing, late developing, and IgM-secreting B cells. Patterns in anterior kidney segment K1 were most similar to those of mouse bone marrow, while the most posterior part of the kidney, K5, had many IgM-secreting cells, but lacked early developing B cells. A potential second B lymphopoiesis site was uncovered in segment K4 of the kidney. The B cell patterns, or ōB cell signaturesö described here provide information on the relative abundance of distinct developing B cell populations in the trout kidney, and can be used in future studies on B cell development in other vertebrate species.
Zwollo, Patty; Mott, Katrina; Barr, Maggie
Attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice.
Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made. PMID:17942549
Cooper, David; Wright, Kevin J; Calderon, Priscilla C; Guo, Min; Nasar, Farooq; Johnson, J Erik; Coleman, John W; Lee, Margaret; Kotash, Cheryl; Yurgelonis, Irene; Natuk, Robert J; Hendry, R Michael; Udem, Stephen A; Clarke, David K
Attenuation of Recombinant Vesicular Stomatitis Virus-Human Immunodeficiency Virus Type 1 Vaccine Vectors by Gene Translocations and G Gene Truncation Reduces Neurovirulence and Enhances Immunogenicity in Mice?
Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.
Cooper, David; Wright, Kevin J.; Calderon, Priscilla C.; Guo, Min; Nasar, Farooq; Johnson, J. Erik; Coleman, John W.; Lee, Margaret; Kotash, Cheryl; Yurgelonis, Irene; Natuk, Robert J.; Hendry, R. Michael; Udem, Stephen A.; Clarke, David K.
Translocated effectors of Yersinia
Summary Currently, all known translocated effectors of Yersinia are delivered into host cells by type III secretion systems (T3SSs). Pathogenic Yersinia maintain the plasmid-encoded Ysc T3SS for the specific delivery of the well-studied Yop effectors. New horizons for effector biology have opened with the discovery of the Ysps of Y. enterocolitica Biovar 1B, which are translocated into host cells by the chromosome-endoded Ysa T3SS. The reported arsenal of effectors is likely to expand since genomic analysis has revealed gene-clusters in some Yersinia that code for other T3SSs. These efforts also revealed possible type VI secretion (T6S) systems, which may indicate translocation of effectors occurs by multiple mechanisms.
Matsumoto, Hiroyuki; Young, Glenn M.
Immunoglobulin heavy chain gene analysis in bone marrow biopsies and corresponding lymph node specimens: dependency on pre-treatment, histological subtype and extension of B-cell lymphoma.
Bone marrow biopsies (BMB) are the conventional staging method for assessing marrow involvement by lymphoma. Morphological criteria provide basic data determining their dignity, but concerning microfocal infiltrates, these criteria are rather inaccurate. Here, by examination of immunoglobulin H (IgH) receptor rearrangement and comparison of medullar and nodular lymphoma sites, diagnostic reliability was improved. Employing non-nested IgH rearrangement analysis with FR3A and JHa consensus primers, B-cell clonality was assessed on glutardialdehyde fixed, decalcified BMB with and without lymphoma infiltration and on the corresponding lymph node specimens. Malignancy was confirmed when polymerase chain reaction (PCR) generated no more than two peaks and was observed in 60% of the medullar B-cell lymphoma. Comparison of lymph node tissues and BMB revealed an identical pattern in 50% of the probes. In 25% of the cases a single clonal peak derived from the lymph node tissues was also observed in the BMB but was surrounded by additional peaks. Here, direct comparison of the data permitted determination of lymphoma in the BMB. Therefore, IgH FR3 PCR analysis is a suitable tool to examine small lymphoid infiltrates in BMB, and direct comparison with corresponding nodal lymphoma can further facilitate estimation of their dignity. PMID:18425348
Odenthal, Margarete; Siebolts, Udo; Ernestus, Karen; Disse, Daniel; Dienes, Hans Peter; Wickenhauser, Claudia
Translocations in epithelial cancers
Genomic translocations leading to the expression of chimeric transcripts characterize several hematologic, mesenchymal and epithelial malignancies. While several gene fusions have been linked to essential molecular events in hematologic malignancies, the identification and characterization of recurrent chimeric transcripts in epithelial cancers has been limited. However, the recent discovery of the recurrent gene fusions in prostate cancer has sparked a revitalization of the quest to identify novel rearrangements in epithelial malignancies. Here, the molecular mechanisms of gene fusions that drive several epithelial cancers and the recent technological advances that increase the speed and reliability of recurrent gene fusion discovery are explored.
Chad Brenner, J.; Chinnaiyan, Arul M.
The inv(11)(p15q22) chromosome translocation of de novo and therapy-related myeloid malignancies results in fusion of the nucleoporin gene, NUP98, with the putative RNA helicase gene, DDX10.
The inv(11)(p15q22) is a recurrent chromosomal abnormality associated with de novo and therapy-related myeloid malignancies. Here we report the molecular definition of this chromosomal aberration in four patients. Positional cloning showed the consistent rearrangement of the DDX10 gene on chromosome 11q22, which encodes a putative RNA helicase. The translocation targets the NUP98 gene on 11p15, a member of the FG peptide repeat nucleoporin family. In DDX10 and NUP98, the inv(11) breakpoints occurred within two introns of each gene and the two genes merged in-frame to produce the chimeric transcripts characteristic of this translocation. Although two reciprocal chimeric products, NUP98-DDX10 and DDX10-NUP98, were predicted, only NUP98-DDX10 appears to be implicated in tumorigenesis. DDX10 is predicted to be involved in ribosome assembly. NUP98 has been identified as a nuclear pore complex protein and a target of chromosomal translocation in acute myeloid leukemia through the t(7;11)(p15;p15) translocation. The predicted NUP98-DDX10 fusion protein may promote leukemogenesis through aberrant nucleoplasmic transport of mRNA or alterations in ribosome assembly. PMID:9166830
Arai, Y; Hosoda, F; Kobayashi, H; Arai, K; Hayashi, Y; Kamada, N; Kaneko, Y; Ohki, M
Anaplastic Lymphoma Kinase-Positive Large B-Cell Lymphoma: An Underrecognized Aggressive Lymphoma
Anaplastic lymphoma kinase-(ALK-) positive large B-cell lymphoma (ALK+ LBCL) is a rare, aggressive tumor characterized by an immunoblastic or plasmablastic morphologic appearance, expression of ALK, CD138, CD45, EMA, and often IgA by immunohistochemistry, and characteristic chromosomal translocations or rearrangements involving the ALK locus. The morphologic and immunophenotypic overlap of this tumor with other hematologic and nonhematologic malignancies may result in misdiagnosis. The tumor has been identified in both pediatric and adult populations and demonstrates a male predominance. Presentation is most often nodal, particularly cervical. No association with immunocompromise or geographic location has been recognized. The most common gene rearrangement is between clathrin and ALK (t(2;17)(p23;q23)), resulting in the CLTC-ALK chimeric protein, although other fusions have been described. Response to conventional chemotherapy is poor. The recent introduction of the small molecule ALK inhibitor, crizotinib, may provide a potential new therapeutic option for patients with this disease.
Morgan, Elizabeth A.; Nascimento, Alessandra F.
Entropic effects in formation of chromosome territories: towards understanding of radiation-induced gene translocation frequency
NASA Astrophysics Data System (ADS)
A detailed understanding of structural organization of biological target, such as geometry of an inter-phase chromosome, is an essential prerequisite for gaining deeper insight into relationship between radiation track structure and radiation-induced biological damage [1]. In particular, coupling of biophysical models aimed to describe architecture of chromosomes and their positioning in a cell nucleus [2-4] with models of local distribution of ionizations caused by passing projectiles, are expected to result in more accurate estimates of aberration induction caused by radiation. There is abundant experimental evidence indicating that arrangements of chromosomes in eukaryotic cell nucleus is non-random and has been evolutionary conserved in specific cell types. Moreover, the radial position of a given chromosome territory (CT) within the cell nucleus has been shown to correlate with its size and gene density. Usually it is assumed that chromosomal geometry and positioning result from the action of specific forces acting locally, such as hydrogen bonds, electrostatic, Van der Waals or hydrophobic interactions operating between nucleosomes and within their interiors. However, it is both desirable and instructive to learn to what extend organization of inter-phase chromosomes is affected by nonspecific entropic forces. In this study we report results of a coarse-grained analysis of a chromatin structure modeled by two distinct approaches. In the first method, we adhere to purely statistical analysis of chromatin packing within a chromosome territory. On the basis of the polymer theory, the chromatin fiber of diameter 30nm is approximated by a chain of spheres, each corresponding to about 30 kbp. Random positioning of the center of the domain is repeated for 1000 spherical nuclei. Configuration of the domain is determined by a random packing of a polymer (a string of identical beads) in estimated fraction of space occupied by a chromosome of a given length and mass. The degree of condensation of the chromatin fiber is modeled by changing length of the string: e.g. loosening of the structure is achieved by distributing the chromosome mass into a higher number of smaller beads and tighter configuration corresponds to a lower number of fragments (balls) with a bigger radius. Additionally, for each configuration, a degree of possible overlapping between domains is assumed. This procedure effectively intensifies loosening/tightening of the chromosome structure by changing the radial dimension of the domain while keeping a constant volume of the polymer chain. Such a positioning model is confronted with a minimalistic molecular dynamics model [5] on a similar structure, in which a chain of beads becomes connected by entropic spring energy and subjected to thermal fluctuations. Comparison of both Monte Carlo models allows to discuss variability of possible configurations as observed in static and dynamic models of chromosome territories along with the effect of compaction and relative arrangements of territorial polymer structures. Acknowledgements: Project is operated within the Foundation for Polish Science International Ph.D. Projects Programme co-financed by the European Regional Development Fund covering, under the agreement no. MPD/2009/6, the Jagiellonian University International Ph.D. Studies in Physics of Complex Systems. References: [1] F. Ballarini, M. Biaggi, and A. Ottolenghi, Radiation Protection Dosimetry 99, 175 (2002). [2] M. Nicodemi and A. Prisco, Biophysical Journal 96, 2168 (2009). [3] P. Cook and D. Marenduzzo, Journal of Cell Biology 186, 825 (2009). [4] M. Tark-Dame, R. van Driel, and D. Heermann, Journal of Cell Science 124, 839 (2011). [5] W. Swope, H. Andersen, P. Berens, and K. Wilson, J. Chem. Phys. 76, 637 (1982).
Gudowska-Nowak, Ewa; Ritter, Sylvia; Durante, Marco; Deperas-Standylo, Joanna; Ciesla, Michal
The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene.
The gene on chromosome 10 at band p12 (AF10), involved in the t(10;11) translocation in acute myeloid leukemia, has been identified and shown to contain conserved zinc finger and leucine zipper domains. These regions are highly homologous to the equivalent regions on AF17, the gene involved in the t(11;17) translocations. A series of adult, childhood, and infant leukemias with either simple or complex versions of the t(10;11) has been examined by Southern analysis and shown to involve rearrangement to the HRX locus. Reverse transcriptase-polymerase chain reaction from either bone marrow or peripheral blood cells showed that HRX sequence was fused to AF10 sequence in all 8 cases and subsequent sequence analysis showed an in-frame fusion between the HRX and AF10 sequence. A consistent feature of these fusions was the juxtaposition of the leucine dimerization motif of AF10 onto the NH2-terminal region of HRX. The published data suggest that a similar conclusion can be drawn about the t(11;17) translocation, implying a critical role for this motif in the chimaeric HRX protein. PMID:7662954
Chaplin, T; Bernard, O; Beverloo, H B; Saha, V; Hagemeijer, A; Berger, R; Young, B D
Smith-Lemli-Opitz syndrome in a female with a de novo, balanced translocation involving 7q32: Probable disruption of an SLOS gene
A 3-month-old infant girl had manifestations of the Smith-Lemli-Opitz syndrome (SLOS) including typical positional anomalies of the limbs, apparent Hirschsprung disease, cataracts, ptosis, anteverted nares, cleft of the posterior palate, small tongue, broad maxillary alveolar ridges, and abnormally low serum cholesterol levels. Chromosomal analysis showed a de novo balanced translocation interpreted as 46,XX,t(7;20)(q32.1;q13.2). We hypothesize that the translocation breakpoint in this case interrupts one SLOS allele and that the other allele at the same locus has a more subtle mutation that was inherited from the other parent. This case, as well as cytogenetic observations in other SLOS cases, suggests that SLOS could be due to autosomal recessive mutation at a gene in 7q32. 33 refs., 3 figs., 1 tab.
Wallace, M.; Zori, R.T.; Alley, T.; Whidden, E.; Gray, B.A.; Williams, C.A. [Univ. of Florida College of Medicine, Gainesville, FL (United States)
Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders
Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes.
Henriques, Ana; Rodriguez-Caballero, Arancha; Criado, Ignacio; Langerak, Anton W.; Nieto, Wendy G.; Lecrevisse, Quentin; Gonzalez, Marcos; Cortesao, Emilia; Paiva, Artur; Almeida, Julia; Orfao, Alberto
Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders.
Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes. PMID:24488564
Henriques, Ana; RodrĒguez-Caballero, Arancha; Criado, Ignacio; Langerak, Anton W; Nieto, Wendy G; Lķcrevisse, Quentin; Gonzßlez, Marcos; CortesŃo, EmĒlia; Paiva, Artur; Almeida, Julia; Orfao, Alberto
Fusion of the ZC3H7B and BCOR genes in endometrial stromal sarcomas carrying an X;22-translocation.
Endometrial stromal sarcomas (ESS) are genetically heterogeneous uterine tumors in which a JAZF1-SUZ12 chimeric gene resulting from the chromosomal translocation t(7;17)(p15;q21) as well as PHF1 rearrangements (in chromosomal band 6p21) with formation of JAZF1-PHF1, EPC1-PHF1, and MEAF6-PHF1 chimeras have been described. Here, we investigated two ESS characterized cytogenetically by the presence of a der(22)t(X;22)(p11;q13). Whole transcriptome sequencing one of the tumors identified a ZC3H7-BCOR chimeric transcript. Reverse transciptase-PCR with the ZC3H7B forward and BCOR reverse primer combinations confirmed the presence of a ZC3H7-BCOR chimeric transcript in both ESS carrying a der(22)t(X;22) but not in a control ESS with t(1;6) and the MEAF6-PHF1 fusion. Sequencing of the amplified cDNA fragments showed that in both cases ESS exon 10 of ZC3H7B (from 22q13; accession number NM_017590 version 4) was fused to exon 8 of BCOR (from Xp11; accession number NM_001123385 version 1). Reciprocal multiple BCOR-ZC3H7B cDNA fragments were amplified in only one case suggesting that ZC3H7B-BCOR, on the der(22)t(X;22), is the pathogenetically important fusion gene. The putative ZC3H7B-BCOR protein would contain the tetratricopeptide repeats and LD motif from ZC3H7B and the AF9 binding site (1093-1233aa), the 3 ankyrin repeats (1410-1509 aa), and the NSPC1 binding site of BCOR. Although the presence of these motifs suggests various functions of the chimeric protein, it is possible that its most important role may be in epigenetic regulation. Whether or not the (patho)genetic subsets JAZF1-SUZ12, PHF1 rearrangements, and ZC3H7B-BCOR correspond to any phenotypic, let alone clinically important, differences in ESS remain unknown. PMID:23580382
Panagopoulos, Ioannis; Thorsen, Jim; Gorunova, Ludmila; Haugom, Lisbeth; Bjerkehagen, Bodil; Davidson, Ben; Heim, Sverre; Micci, Francesca
Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.
Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. A small subset of DLBCLs has translocations involving the MYC locus and an additional group has a molecular signature resembling Burkitt lymphoma (mBL). Presently, identification of such cases by morphology is unreliable and relies on cytogenetic or complex molecular methods such as gene transcriptional profiling. Herein, we describe an immunohistochemical (IHC) method for identifying DLBCLs with increased MYC protein expression. We tested 77 cases of DLBCL and identified 15 cases with high MYC protein expression (nuclear staining in >50% of tumor cells). All MYC translocation positive cases had increased MYC protein expression by this IHC assay. In addition, gene set enrichment analysis (GSEA) of the DLBCL transcriptional profiles revealed that tumors with increased MYC protein expression (regardless of underlying MYC translocation status) had coordinate upregulation of MYC target genes, providing molecular confirmation of the IHC results. We then generated a molecular classifier derived from the MYC IHC results in our cases and employed it to successfully classify mBLs from two previously reported independent case series, providing additional confirmation that the MYC IHC results identify clinically important subsets of DLBCLs. Lastly, we found that DLBCLs with high MYC protein expression had inferior overall survival when treated with R-CHOP. In conclusion, the IHC method described herein can be used to readily identify the biologically and clinically distinct cases of MYC-driven DLBCL, which represent a clinically significant subset of DLBCL cases due to their inferior overall survival. PMID:22511926
Kluk, Michael J; Chapuy, Bjoern; Sinha, Papiya; Roy, Alyssa; Dal Cin, Paola; Neuberg, Donna S; Monti, Stefano; Pinkus, Geraldine S; Shipp, Margaret A; Rodig, Scott J
Synergistic Attenuation of Vesicular Stomatitis Virus by Combination of Specific G Gene Truncations and N Gene Translocations?
A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their ōwild-typeö progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD50) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10- to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD50 of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.
Clarke, David K.; Nasar, Farooq; Lee, Margaret; Johnson, J. Erik; Wright, Kevin; Calderon, Priscilla; Guo, Min; Natuk, Robert; Cooper, David; Hendry, R. Michael; Udem, Stephen A.
B Cell Subsets in Atherosclerosis
Atherosclerosis, the underlying cause of heart attacks and strokes, is a chronic inflammatory disease of the artery wall. Immune cells, including lymphocytes modulate atherosclerotic lesion development through interconnected mechanisms. Elegant studies over the past decades have begun to unravel a role for B cells in atherosclerosis. Recent findings provide evidence that B cell effects on atherosclerosis may be subset-dependent. B-1a B cells have been reported to protect from atherosclerosis by secretion of natural IgM antibodies. Conventional B-2 B cells can promote atherosclerosis through less clearly defined mechanism that may involve CD4 T cells. Yet, there may be other populations of B cells within these subsets with different phenotypes altering their impact on atherosclerosis. Additionally, the role of B cell subsets in atherosclerosis may depend on their environmental niche and/or the stage of atherogenesis. This review will highlight key findings in the evolving field of B cells and atherosclerosis and touch on the potential and importance of translating these findings to human disease.
Perry, Heather M.; Bender, Timothy P.; McNamara, Coleen A.
Computational and Functional Analysis of Growth Hormone (GH)-Regulated Genes Identifies the Transcriptional Repressor B-Cell Lymphoma 6 (Bc16) as a Participant in GH-Regulated Transcription
For insight into transcriptional mechanisms mediating physiological responses to GH, data mining was performed on a profile of GH-regulated genes induced or inhibited at different times in highly responsive 3T3-F442A adipocytes. Gene set enrichment analysis indicated that GH-regulated genes are enriched in pathways including phosphoinositide and insulin signaling and suggested that suppressor of cytokine signaling 2 (SOCS2) and phosphoinositide 3? kinase regulatory subunit p85? (Pik3r1) are important targets. Model-based Chinese restaurant clustering identified a group of genes highly regulated by GH at times consistent with its key physiological actions. This cluster included IGF-I, phosphoinositide 3? kinase p85?, SOCS2, and cytokine-inducible SH2-containing protein. It also contains the most strongly repressed gene in the profile, B cell lymphoma 6 (Bcl6), a transcriptional repressor. Quantitative real-time PCR verified the strong decrease in Bcl6 mRNA after GH treatment and induction of the other genes in the cluster. Transcriptional network analysis of the genes implicated signal transducer and activator of transcription (Stat) 5 as hub regulating the most responsive genes, Igf1, Socs2, Cish, and Bcl6. Transcriptional activation analysis demonstrated that Bcl6 inhibits SOCS2-luciferase and blunts its stimulation by GH. Occupancy of endogenous Bcl6 on SOCS2 DNA decreased after GH treatment, whereas occupancy of Stat5 increased concomitantly. Thus, GH-mediated inhibition of Bcl6 expression may reverse the repression of SOCS2 and facilitate SOCS2 activation by GH. Together these analyses identify Bcl6 as a participant in GH-regulated gene expression and suggest an interplay between the repressor Bcl6 and the activator Stat5 in regulating genes, which contribute to GH responses.
Chen, Yili; Lin, Grace; Huo, Jeffrey S.; Barney, Deborah; Wang, Zhenni; Livshiz, Tamara; States, David J.; Qin, Zhaohui S.; Schwartz, Jessica
Single nucleotide polymorphisms at erythropoietin, superoxide dismutase 1, splicing factor, arginine/serin-rich 15 and plasmacytoma variant translocation genes association with diabetic nephropathy.
A number of genes have been identified in diabetic nephropathy. Association between diabetes-associated nephropathy and polymorphisms in the erythropoietin (EPO) gene, variants in the superoxide dismutase 1 (SOD1) gene and plasmacytoma variant translocation 1 (PVT1) gene have been identified. The EPO, SOD1:SFRS15 and PVT1 genes were genotyped using the single nucleotide polymorphism (SNP) technique in 38 diabetic nephropathy patients (Group 1) compared with 64 diabetic type 2 subjects without nephropathy (Group 2) at the Mubarak Alkabeer Hospital, Kuwait. The frequency of the risk allele T of the EPO (rs1617640) gene was high in both groups (0.96 in Group 1 and 0.92 in Group 2). Similarly, SNPs of the PVT1 (rs2720709) gene showed a higher frequency of the risk allele G in both groups (0.70 in the Group 1 and 0.68 in Group 2). Although the frequency of the risk allele A was higher than the frequency of the non-risk allele C of the SOD1:SFRS15 gene in both groups, the lowest probability value was observed in those gene SNPs (P = 0.05). We observed that the A allele of the SOD1:SFRS15 gene (rs17880135) was more frequently present in Group 1 (0.75) compared with Group 2 (0.62). Susceptibility to diabetes-associated nephropathy is partially mediated by genetic predisposition, and screening tests may open the gate for new therapeutic approaches. PMID:24821155
Alwohhaib, Maisaa; Alwaheeb, Salah; Alyatama, Nour; Dashti, Ali A; Abdelghani, Amal; Hussain, Naser
Expression of HOXB genes is significantly different in acute myeloid leukemia with a partial tandem duplication of MLL vs. a MLL translocation: a cross-laboratory study.
In acute myeloid leukemia (AML), the mixed lineage leukemia (MLL) gene may be rearranged to generate a partia