PMC3556036_01
Severe skin reaction of a Dubowitz Syndrome patient to radiation therapy.. Shown is the severe skin injury on the anal-rectal region after 10 fractions of 2 Gy radiation-therapy for anal cancer. The patient died of the metastatic anal cancer later.
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Over the ensuing several weeks the patient severe moist desquamation gradually healed, with significant residual induration and fibrosis in the region (Figure 1).
Primary culture of human fibroblasts.. Fibroblast cell lines, designated CoDa, were established from the autopsy skin tissues. Panel a shows the representative images of a normal fibroblast control line (FBCL) and CoDa cells in in vitro culture. Images were taken under bright field microscopy with various confluences. Panel b shows the cell cycle distribution of fibroblasts in log-phase of in vitro culture, based on flow cytometry analysis of the DNA content. Panels c and d show population doubling time (panel c) and plating efficiency (panel d) of CoDa and control fibroblasts in vitro. Shown are averages of 2-3 independent experiments with CoDa-33 and FBCL cells. Error bars are standard errors.
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The CoDa cells displayed typical fibroblast morphology in primary culture, and have comparable growth properties and cell cycle distribution as normal skin fibroblasts at log-phase of growth (Figs. 2a and 2b). As shown in Figure 2c, both cells have population doubling time of ~30 hours. As shown in Figure 2d, the plating efficiency (PE) for both cells is around 30-34% at early passages, and gradually decreases as the passage numbers increase. Collectively, Figure 2 suggests that the CoDa cells have similar growth properties as the normal control cell line.
CoDa cells are hyper-sensitive to ionizing radiation and have defects in DNA double-strand break repair.. (a) Cellular sensitivity of human fibroblasts to Cs-137 gamma-radiation measured by colony formation assay. Shown are the averages of 3-6 experiments with three CoDa clones (CoDa-2, 21, and 33), and 3-7 experiments with three independent control fibroblasts cell lines (FBCL, HSF-42, and HSF-43). Error bars are standard errors. (b) The numbers of gammaH2AX foci per cell in a normal fibroblast control line (FBCL) and CoDa cells. At different time points after the cells were treated with gamma-radiation, cells were fixed and stained by immunoflurescent techniques with an anti-gammaH2AX antibody. The number of gammaH2AX foci was counted in >400 cells per time point in each experiment. Shown are averages and standard deviations of three experiments. (c) The representative images of gammaH2AX nuclear foci at 4 and 24 hours after IR treatment. DAPI staining shows the location of nuclear. (d) The total gammaH2AX protein level measured by anti-gammaH2AX western blot after irradiation (2 Gy) in FBCL and CoDa cells. beta-actin was used as a loading control.
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As shown in Figure 3a, the CoDa cells are ~30 fold more sensitive to ionizing radiation than normal fibroblasts at the dose of 2 Gy (0.99% survival of CoDa cells vs. 29.7% survival for normal fibroblasts). The number of foci per cell increased to a similar level in CoDa and control cells shortly after 2 Gy irradiation (Fig. 3b, c), suggesting that irradiation induced a similar level of initial amount of DNA damage in these cells. The same extend of initial activation but delayed clearance of gammaH2AX was confirmed by western blot after 2 Gy of irradiation (Fig. 3d). It is clear that, similar as gammaH2AX (Fig. 3), the CoDa cells have normal initial 53BP1 focus formation in response to ionizing radiation but a slower removal of the 53BP1 foci after IR (Fig. 4a). CoDa cells. Collectively, Figures 3 and 4 suggest a delayed repair of DSBs in CoDa cells but the DNA damage signaling to activate gammaH2AX, and 53BP1 recruitment are unlikely affected in CoDa cells. This is consistent with the delay of DSB repair in CoDa cells (Figs. 3 and 4). CoDa cells maintained a higher ATM phosphorylation level up to 24 hours compared to less than 8 hours in control cells, likely due to a high level of unrepaired DNA damage. Collectively, these data (Figures 3, 4, 5, 6) support a scenario that CoDa cells possess normal initial DNA damage response signaling, and cell cycle checkpoint activations but have an impaired repair of DSB.
CoDa cells have normal IR-induced formations of 53BP1 nuclear foci, but displayed clearance of 53BP1 foci.. The number of nuclear 53BP1 foci was scored at various time points after 2 Gy of gamma-irradiation in two control fibroblasts (FBCL and HSF42) and three CoDa populations (CoDa-2, CoDa-21, and CoDa-33). (a) The average number of 53BP1 foci from control fibroblasts (Delta) and their average (blue line), three CoDa populations ( ) and their average (pink line). Shown are the averages of two experiments for each cell line. (b) The representative images of 53BP1 foci in the CoDa and FBCL cells at 8 and 24 hours after 2 Gy radiation treatment. DAPI staining shows the location of nuclear. In addition, double staining with 53BP1 and gammaH2AX antibodies was performed in FBCL and CoDa cells at various time points after 2 Gy of gamma-irradiation. (c) The representative images of 53BP1 and gammaH2AX foci in FBCL and CoDa cells at 30 min, 8 and 24 hours after 2 Gy of radiation treatment. DAPI staining shows the location of nuclear.
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Figure 4a shows the average number of 53BP1 nuclear foci among three CoDa cell population (CoDa-2, CoDa-21, and CoDa-33) and three normal fibroblasts (FBCL, HSF42, and HSF43). Figure 4b shows representative images of 53BP1 foci. Figure 4c shows the representative images for double staining of 53BP1 and gammaH2AX foci. CoDa cells. Collectively, Figures 3 and 4 suggest a delayed repair of DSBs in CoDa cells but the DNA damage signaling to activate gammaH2AX, and 53BP1 recruitment are unlikely affected in CoDa cells. This is consistent with the delay of DSB repair in CoDa cells (Figs. 3 and 4). CoDa cells maintained a higher ATM phosphorylation level up to 24 hours compared to less than 8 hours in control cells, likely due to a high level of unrepaired DNA damage. Collectively, these data (Figures 3, 4, 5, 6) support a scenario that CoDa cells possess normal initial DNA damage response signaling, and cell cycle checkpoint activations but have an impaired repair of DSB.
Normal G1/S and G2/M checkpoint activation and impaired recovery from G2 arrest in CoDa cells after irradiation.. To assess G1/S checkpoint activation (panel a), cells were treated with or without 6 Gy gamma-rays and nocodazole was added into medium right after irradiation. Cells were collected for DNA content analysis at 0, 8, 16 and 32 hours after nocodazole added. Shown in panel-a are the histographs of DNA content measured from fibroblasts with 0 Gy and 6 Gy radiation treatment. To measure G2/M checkpoint activation (panel b), cells were treated with or without 6 Gy gamma-rays and nocodazole was added into medium right after radiation. Cells were fixed at 0, 6, 12, 24 and 36 hours after adding nocodazole. Then immunofluorescent staining was performed with anti-phosphorylated histone-3 (Phospho-H3 at Ser10) antibody. Percentage of mitotic cells (Phospho-H3 positive) was counted by flow cytomertry and the experiment was repeated once. Error bars are standard deviation.
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As shown in Figure 5a (top of the panel), without irradiation, CoDa and FBCL have similar cell cycle profiles during the course of G1 phase cell population shift to G2/M phases. After 6 Gy of radiation, the there was a similar delay of the G1 shift in both CoDa and FBCL as shown in Figure 5a (bottom of the panel), indicating the irradiation induced the activation of G1/S checkpoint in both cell lines. As shown in Figure 5b, there is a similar delay of mitotic index initially after 6 Gy-irradiation in both control and CoDa cells. CoDa cells maintained a higher ATM phosphorylation level up to 24 hours compared to less than 8 hours in control cells, likely due to a high level of unrepaired DNA damage. Collectively, these data (Figures 3, 4, 5, 6) support a scenario that CoDa cells possess normal initial DNA damage response signaling, and cell cycle checkpoint activations but have an impaired repair of DSB.
Normal activation but with delayed recovery of ATM in response to irradiation but with delayed recovery.. FBCL (Control) and CoDa cells were treated with 2 Gy gamma-rays and harvested at 0.5, 2, 4, 8 and 24 hours after IR for whole cell extract preparation. The protein samples were subjected to SDS-PAGE, then the membrane was blotted with antibodies specified in the figure, including ATM and phosphorylated ATM at Ser-1981, and alpha-tubulin was used as loading control.
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ATM phosphorylation at Ser-1981 is an early event after DNA damage induced by radiation. As shown in Figure 6, ATM phosphorylation was detected as early as half an hour after radiation in both control and CoDa cells. CoDa cells maintained a higher ATM phosphorylation level up to 24 hours compared to less than 8 hours in control cells, likely due to a high level of unrepaired DNA damage. Collectively, these data (Figures 3, 4, 5, 6) support a scenario that CoDa cells possess normal initial DNA damage response signaling, and cell cycle checkpoint activations but have an impaired repair of DSB.
Cellular sensitivity of CoDa cells to various DNA damaging agents.. The cellular sensitivity of CoDa cells was assessed using growth inhibition assay with continuous drug treatment (see Materials and Methods for details), including bleomycin (b), doxorubicin (c), mitomycin-C (d), cisplatin (e), hydroxyperoxide(f) and camptothecin(g). Panels a and h show the sensitivities with ionizing radiation and ultra violet treatment using the same growth inhibition assay as drug treatments. Shown are the averages form 3-5 independent experiments. Error bars are standard deviations.
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As shown in Figure 7, CoDa cells are more sensitive to bleomycin and doxorubicin than the control cells (Fig. 7b and c), but are not to topoisomerase I poison (camptothecin) and DNA cross-link agents (cisplatin and mitomycin-C) (Fig. 7d, e and g). The sensitivity to UV and H2O2 are not significantly different between CoDa and controls (Fig. 7f and h). HR defects cause hypersensitivity to DNA cross-links but the CoDa cells are not sensitive to mitomycin-C and cisplatin (Fig. 7d and 7e), and we did not observe defective RAD51 recruitment to DNA damage at early stage after irradiation (Fig.
Mutations of LIG4 identified in CoDa genomic DNA.. Shown are sequencing results from control fibroblast FBCL (Wild-type) and CoDa cells (mixture of wild-type and mutated alleles). (a) The 2440C>T (R814X) mutation. Single nucleotide mutation (C>T) at position of 2440 of coding region changes Arg-814 to stop codon, which results in premature truncation of the DNA ligase IV protein. (b) The 613DeltaT mutation. Single nucleotide deletion at nucleotide position 613 of coding region leads to a frame shift at L205 (L205FS) and causes a premature stop codon 18 aa downstream. Mutated amino acids are labeled with numerical number in protein sequence and shown in red. Pink arrows indicate the location of mutated nucleotides. (c) The schematic structure of DNA Ligase IV protein (Adapted from Marchetti et al.). LIG4 comprises three domains: DNA-binding domain (DBD), adenylation domain (AdD) and oligo-binding domain (OBD). AdD and OBD form the catalytic domain. Two C-terminal BRCT motifs and XRCC4-binding sites (grey) are required for interaction with XRCC4. Localization of truncation mutations found in the LIG4 is labeled in the panel.
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Two mutations in the LIG4 gene were identified in both genomic DNA and cDNA sequencing (Fig. 8 and Fig. One mutation is a single base deletion at coding nucleotide 613 (613DeltaT), which results in a reading frame shift at amino acid L205 (L205FS) and generates a new stop codon at amino acid 232 (Fig. 8b). The other is a single base mutation (2440C>T) that generates a stop codon (R814X) (Fig. 8a). As shown in Figure 8c, it contains a DNA binding domain (amino acids 1-241), and two BRCT motifs (amino acids 658-743 and 801-911). Two nonsense mutations (R580X and R814X) and a frame-shift (K424FS) that cause protein truncations have been reported (Fig. 8c), and all other mutations cause amino acid substitutions and deletion. CoDa cells is relieved to the same level as normal fibroblasts at 8 hours after irradiation (Fig. 10). Collectively, these data (Figs. 8, 9, 10) suggest that the CoDa cells' radiation sensitivity and defective DSB repair are solely caused by the LIG4 mutations.
Expression of wild type LIG4 complements the cellular sensitivity of CoDa cells to DNA damage agents and rescues DSB repair defect.. CoDa and FBCL cells were infected with retrovirus that express Myc-LIG4 or Myc (negative control) and selected with puromycin for 1 week. The cells were used for cellular sensitivity analysis or fluorescent immunostaining. Panel (a) shows the survival curve with irradiation treatment measured with colony formation assay. Panel (b) shows the cellular sensitivity to doxorubicin treatment assessed by growth inhibition assay (see Materilas and Method for details). For immunostaining, the cells grown on coverglass were fixed at various time points after 2 Gy IR and co-stained with anti-gammaH2AX (Red) and anti-53BP1 (Green) antibodies. DAPI staining (Blue) shows the location of nuclear. The numbers of gammaH2AX and 53BP1 foci were counted in at least 200 cells from each slide and the experiments were repeated twice. Shown are the averages nuclear focus numbers of gammaH2AX (Panel c) and 53BP1 (Panel d). Error bars were the standard deviations. Panel e is representative images taken at 30 min, 8 hour and 24 hours after 2 Gy irradiation.
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The cellular sensitivity to doxorubicin was also rescued by wild-type LIG4 as determined with growth inhibition assay (Fig. 9b). This is accompanied by reduced residual gammaH2AX and 53BP1 nuclear foci in the CoDa cells at later time point (8 and 24 hours) after irradiation, to a similar level as the normal fibroblasts (Fig. 9c, d, e). CoDa cells is relieved to the same level as normal fibroblasts at 8 hours after irradiation (Fig. 10). Collectively, these data (Figs. 8, 9, 10) suggest that the CoDa cells' radiation sensitivity and defective DSB repair are solely caused by the LIG4 mutations.
expression of LIG4 in CoDa cells does not affect the initial activation of ATM, p53, and H2AX, but eliminates the excessive residual level of phosphorylated ATM, p53, and H2AX after irradiation.. After irradiation with 2 Gy of gamma-rays, whole cell extracts were collected at various time points and subjected to western-blot analyses to measure the phosphorylations of ATM (Ser1981), p53 (Ser15), and H2AX (Ser139), and beta-actin was used as loading control. Myc or Myc-LIG4 represents the cells were infected with retrovirus expressing Myc or My-LIG4, respectively. Protein samples from FBCL cells are labeled as "N", from CoDa cells are labeled as "P".
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CoDa cells is relieved to the same level as normal fibroblasts at 8 hours after irradiation (Fig. 10). Collectively, these data (Figs. 8, 9, 10) suggest that the CoDa cells' radiation sensitivity and defective DSB repair are solely caused by the LIG4 mutations.