{'fraction_non_alphanumeric': 0.03698098308876752, 'fraction_numerical': 0.023294071198262815, 'mean_word_length': 5.002369668246446, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Strain‐dependency of Chromosomal Abnormalities in Lymphomas Developed in Eμ‐myc Transgenic Mice

We previously showed that B and T cell lymphoma development in Eμ (immunoglobulin heavy chain enhancer)‐mye transgenic mice is dependent on the mouse strain. To determine whether any non‐random chromosomal abnormality that was present was caused by variations in the lymphoma cell type or by a different genetic background, we crossed C3H transgenic mice with other inbred strains of mice, C57BL/6 or BALB/c. Cytogenetic analysis showed a high frequency of non‐random chromosomal aberrations, namely, duplication or amplification of part of chromosome 5 containing the transgene and trisomy of chromosome 1, 6, or 12 in the genetic background of C3HXC57BL/6 mouse and C3HxBALB/c mouse, respectively, regardless of cell type of lymphoma. These results suggest that non‐random chromosomal abnormalities in lymphoma cells are dependent on the genetic background of mouse, not on the tumor cell type in Eμ‐myc transgenic

Cytogenetic studies have revealed many kinds of chromosomal abnormalities in human malignancies. Initially, most researchers preferred to regard these as secondary features of the established tumor itself, features having little to do with the tumor's origin. However, with the advent of technical improvements, non-random chromosomal abnormalities have been identified in experimental tumors of animal models as well as in human malignant hematological diseases, for instance, chromosomal translocation t (8;14) in Burkitt's lymphomas, 1) t (14;18) in follicular lymphomas, 2) t(9;22) in chronic myelogenous leukemias (CML), 3) t (8;21) in acute myeloid leukemia (M2) 4) and t (15;17) in acute promyelocytic leukemias (M3). 5) In recent years, several oncogenes were shown to be localized at the breakpoint and included in the exchanged segments of two chromosomes leading to the production of a fused transcript. These results clearly suggest that chromosomal abnormalities are directly involved in the malignant process.
On the other hand, it is known that a wide variety of tumors arise spontaneously or experimentally in inbred strains of mouse, and that certain types of tumors appear in high frequencies in some inbred strains. These findings suggest that there are genetic factors responsible for tumor susceptibility. The role of genetic factors in tumor susceptibility was originally recognized in rare instances of familial occurrence of tumors of a certain type such as retinoblastoma. Thus, it seems likely that the genetic factor is involved in the generation of non-random chromosomal abnormalities leading to tumor development in some cases. For example, the generation of mineral oil-or pristane-induced plasmacytomas is restricted to a few selected strains of mice, such as BALB/c and NZB, and most of these plasmacytomas have been shown to contain non-random chromosomal translocations, t(6;15) or t(12;15). 6,7) We previously produced transgenic mice by introducing Eµ (immunoglobulin heavy chain enhancer)-myc gene into two inbred strains of mice, C57BL/6J (B6) and C3H/HeJ (C3H). 8) By analyses of these transgenic mice we found that most B6 transgenic mice developed B cell lymphomas while T cell lymphomas developed in most C3H transgenic mice. Furthermore, the average age of onset in inbred strains of mice is much earlier than in outbred strains. 9) Subsequent analysis revealed the abnormality of chromosome 6 in all cases of T cell lymphoma that developed in C3H transgenic mice. To examine whether this chromosomal abnormality is related to the development of T cell lymphoma or to the genetic background of this mouse strain, we crossed C3H transgenic mice with other inbred strains of mice, B6 or BALB/c. We show here that the generation of specific chromosomal abnormality is dependent on the genetic background in mice.

MATERIALS AND METHODS
Production of transgenic mice Eµ-myc transgenic mice have previously been described. 8,9) The C3H Eµ-myc 24 line was chosen because offspring were obtained only from this line. The F 1 hybrid Eµ-myc mice were obtained by crossing C3H/HeJ transgenic mice with either B6 or BALB/c mice (Clea Japan, Inc., Tokyo). Offspring were screened for the presence of the transgene by Southern blot analysis and PCR as previously described. 9,10) Flow cytometry analysis The monoclonal antibodies used were FITC-anti-IgM (Zymed Laboratories, Inc., South San Francisco, CA), FITC-anti-Thy1.2, PE-anti-L3T4, FITC-anti-Lyt2 (Becton Dickinson Co., Franklin Lakes, NJ), anti-B220 (6B2 rat g2a, k), and FITC-anti-RAT K. Single cell suspensions were prepared from lymph node in phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS) and 0.1% NaN 3 . These cell suspensions were incubated with antibodies for 20 min at 4°C. The stained cells were examined and analyzed by FACScan equipped with Consort 30 program (Becton Dickinson Co.). Chromosome analysis Chromosomal analysis was performed in C3H Eµ-myc mouse lines, 6, 24, and 34. Lymphoma cells were washed with PBS solution, treated with 0.075 M KCl including Colcemid (0.02 µg/ml) for 15 min, fixed with ethanol/acetic acid (1:1), spread onto slides, and dried with air. G-banding analysis was performed as described. 6) Well-spread metaphase plates were photographed for karyotype analysis. Identification of mouse chromosomes and characterization of structurally changed chromosomes were carried out according to the standardized system for mice. 11) Probe Eµ-myc gene inserted into pBR322 was labeled by nick translation with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. The labeled probe was ethanol-precipitated with 625 µg/ml sonicated salmon sperm DNA and tRNA, denatured for 10 min at 75°C in 100% formamide and kept at 4°C. Chromosome in situ hybridization Chromosome in situ hybridization was performed according to the method of Takahashi et al. 12) and Matsuda et al. 13) After destaining in ethanol/acetic acid (1:1), the chromosomal slides were heated to 65°C for 3 h in the oven. They were denatured in 70% formamide, 2× standard saline citrate (SSC) pH 7.0, at 70°C for 2 min, and dehydrated in 70%, 85%, 100% ethanol series. The hybridization solution was mixed with an equal volume of denatured probe in formamide to make a final concentration of 50% formamide, 2× SSC, 10% dextran sulfate and 2 mg/ml bovine serum albumin (BSA). The biotin-labeled DNA was mixed at a final concentration of 5 µg/ml and used at 100 ng per slide. After incubation in a humid chamber at 37°C overnight, the slides were washed for 20 min each in 50% formamide, 2× SSC at 37°C, 2× SSC and then 1× SSC at room temperature. The slides were incubated for 45 min with 3 µg/ ml fluoresceinated avidin in 4× SSC containing 1% BSA at 37°C, then rinsed by shaking in 4× SSC, 4× SSC with 0.05% Triton X, and 4× SSC for 10 min each at room temperature. They were stained with 1.25 µg/ml propidium iodide in fluorescence antifade solution (1% diazabicyclooctane in glycerol with 10% PBS) and observed under a fluorescence microscope. The metaphase cells were photographed with Ektachrome ASA400 film.

RESULTS
C3H Eµ µ µ µ-myc mice Most C3H Eµ-myc mice developed T cell lymphomas. We analyzed the karyotypes of eight T cell lymphomas and one B cell lymphoma that developed in C3H Eµ-myc mice. All of them had structural abnormalities between the B3 and G regions of chromosome 6 ( Fig. 1). To examine whether these abnormalities were a direct consequence of transgene integration, we determined the integration site using chromosome in situ hybridization analysis. The hybridization signal was detected in the A area of chromosome 5 (Fig. 4), suggest-ing that integration of transgenes is not directly involved in the chromosome 6 abnormality. To determine whether this abnormality is related to T cell lymphoma, we also analyzed the karyotypes of spleen cells of normal C3H mouse. A similar abnormality of chromosome 6 was observed in most, but not all, these cells of a normal C3H mouse purchased from Clea Japan. Interestingly, this abnormality was never observed in spleen cells of a C3H mouse purchased from Shizuoka Jikken Doubutsu Laboratory. This suggests that abnormality of chromosome 6 is a constitutional change accidentally introduced into the colony at Clea Japan. Other chromosomal changes, such as Only those chromosomes in which aberration was found are listed. loss of chromosome X or Y, were found in one of eight T cell lymphomas or one B cell lymphoma, respectively. None of these were consistent chromosomal abnormalities (Table I).
One interesting finding was the appearance of chromosome 5 abnormality in hybrid transgenic mice. Six of 10 and 5 of 6 lymphomas showed duplication at area A of chromosome 5 in (C3H×B6)F 1 and (F 1 ×B6)F 2 mice, respectively ( Fig. 2 and Table I). Other chromosomal abnormalities were observed at a low frequency (Table I).
It is of interest that the abnormality of chromosome 5 was never observed in lymphoma cells which developed in C3H, B6, or (C3H×BALB/c)F 1 transgenic mice, suggesting that this abnormality is related to the genetic background of the C3H×B6 hybrid strain. As described before, transgenes were integrated into area A of chromosome 5. Therefore, this amplification of area A of chromosome 5 may be related to the transgene integration. In fact, Eµmyc transgene was amplified concomitantly with the amplification of area A of chromosome 5 (Fig. 4). (C3H× × × ×BALB/c)F 1 Eµ µ µ µ-myc mice To examine whether Eµ-myc gene expression can induce plasmacytomas and non-random chromosomal changes in BALB/c background, we mated C3H Eµ-myc mice with BALB/c mice and obtained six (C3H×BALB/c)F 1 mice (Table II). None of these mice developed plasmacytoma. Two of the six developed T cell lymphomas, one with trisomy of chromosome 12, the other with trisomy of both chromosomes 6 and 12 (Fig. 3). Two others developed B cell lymphomas, one with trisomy of chromosome 1 and the other with trisomy of both chromosomes 1 and 6. In the other two mice, cell surface analysis was not performed, but karyotype analysis revealed that both had trisomy of chromosome 1, 6, and 12. These results suggest an association Only those chromosomes in which aberration was found are listed. between trisomy of chromosomes 1, 6, or 12 and the C3H×BALB/c genetic background.

DISCUSSION
In this report, we show that specific chromosomal abnormality is linked to the genetic background of a mouse strain. The amplification of the area of chromosome 5 containing the transgenes, and trisomy of chromosome 1, 6, or 12 were observed in the genetic background of C3H×C57BL/6 mouse and C3H×BALB/c mouse, respectively. Although more analyses are required to reach a final conclusion, non-random chromosomal abnormalities are associated with the genetic background of the mouse strain, but not with the tumor cell type in Eµ-myc transgenic mice so far examined.
Several lines of evidence have demonstrated the preferential involvement of specific chromosomal anomalies in a certain types of tumors in a certain genetic background. For example, trisomy of chromosome 15 was found at high frequency in spontaneous lymphomas as well as in lymphomas induced by carcinogens, X-rays, RadLV or Moloney MuLV in AKR/J mice. 14) In addition, a preferential duplication of chromosome 15 derived from the AKR mouse was demonstrated in T cell lymphomas in (AKR×B6)F 1 mice. 15) Ohno et al. demonstrated that mineral oil-or pristane-induced plasmacytomas contain nonrandom chromosomal translocations, t(6;15) or t(12;15), 6,7) and Suematsu et al. were able to generate monoclonal transplantable plasmacytomas with the chromosomal translocation t(12;15) in interleukin 6 transgenic mice of BALB/c background, but not in those of C57BL/6 background. 16,17) We also observed trisomy of chromosome 6 and/or 12 in lymphoma cells developed in the BALB/c background. These results clearly indicate that non-random chromosomal aberrations in the development of some tumors are related to the genetic background.
Accumulation of chromosomal aberrations would result in loss of tumor-suppressor genes or activation of oncogenes, leading to excessive cell proliferation, transformation and metastasis. In fact, amplification of Eµ-myc transgenes in lymphoma cells was detected by FISH and tended to be seen in more progressive lymphoma cells (Fig. 4). A similar finding was observed in the case of Nmyc gene, whose copy number is related to the clinical stage of malignant neuroblastoma. 18) Mai et al. recently demonstrated that c-myc is involved in gene amplification and locus-specific genomic instability. 19) The molecular mechanisms are unknown, though genetic instability is one of the characteristics of cancer. 20) Recent studies have revealed that genetic instability occurs in at least two different forms in cancer. In a small number of cancers, defect of mismatch repair causes microsatellite instability (MSI). Such microsatellite instability has been found to be associated with hereditary nonpolyposis colorectal cancer (HNPCC), and is implicated in the mutation of mismatch repair genes such as hMSH2, hPMS1, 2 and hMLH1. [21][22][23][24] In contrast, abnormalities of chromosome number (aneuploidy) due to a chromosomal instability have been observed in many tumors. The human homologue of the yeast BUB1 gene, which controls mitotic checkpoints and chromosome segregation in yeast, was shown to be involved in chromosomal instability in human colorectal cancer. 25) Furthermore, a human homologue of yeast mitotic checkpoint MAD1 protein was identified as the cellular target of the HTLV-1 oncoprotein Tax. This provides a molecular explanation for HTLV-1 associated chromosomal abnormalities in adult T-cell leukemia (ATL) cells. 26) Evidence on the causes and effects of genetic instability continues to accumulate. A mutation in the tumor suppressor gene encoding p53 has been shown to eliminate a cell cycle check point and thus to enhance genomic instability, demonstrating that cell cycle checkpoint genes are involved in maintaining genetic stability. 27,28) Some autosomal recessive disorders, such as Bloom syndrome, Fanconi anemia, ataxia telangiectasia and Nijmegen breakage syndrome reveal the phenotype of chromosomal instability. These diseases also predispose to cancer, and the genes involved have been identified recently. [29][30][31][32] Even though the genes related to genetic instability are being identified, the molecular mechanisms are compli-cated and remain elusive. At present, it is difficult to explain the relationship between specific chromosomal abnormality and genetic background.
Studies on transgenic mice carrying viral oncogenes (SV40-T antigen), viral genome (papillomavirus type 1) or c-myc oncogene (MMTV-myc) also revealed that specific chromosomal abnormalities were associated with tumor progression. [33][34][35] Transgenic or knockout mouse models of human carcinogenesis, together with advanced techniques of FISH analysis, such as spectral karyotyping (SKY), 36) may prove to be attractive tools for the study of genetic instability.