Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta

Abstract

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control.

Fig. 1.

Pol η-deficiency is associated with decreased sensitivity to ionizing radiation. (A) Clonogenic survival assay showing increased survival of XP30RO human fibroblasts compared to XP30RO/pol η cells after treatment with IR. Each data point represents three independent experiments. Error bars show standard error of the mean. Statistical analysis performed by paired Student’s t-test; **P < 0.01. (B) Western blot showing levels of pol η protein in XP30RO cell lines. (C) Viability assay in pol η-proficient BL-2 and pol η-knockout (cl-82 and cl-123) cell lines after IR. Each data point represents five independent experiments. Error bars show standard error of the mean. Statistical analysis performed by paired Student’s t-test; **P < 0.01. (D) Western blot showing pol η protein levels in POLH wild-type and two POLH knockout BL-2 cell clones.

Fig. 2.

Pol η knockdown increases resistance of cancer cells to IR. Clonogenic survival in (A) HCT116 and (C) SQ20B cells after pol η knockdown. Cells were either transfected with non-targeting siRNA or siRNA against pol η for 48h before irradiation. Each data point represents three independent experiments. Error bars show standard error of the mean. Statistical analysis performed by Student’s t-test; *P < 0.05, **P <0.01. B, D. Western blot analyses showing efficiency of pol η protein knockdown after 48 and 72h. Knockdown efficiency was above 75% for both HCT116 and SQ20B cells (bar diagrams).

Fig. 3.

Pol η deficiency is associated with accumulation of cells in S-phase. (A) Cell cycle profiles of un-irradiated POLH wild-type and POLH^−/− BL-2 (cl-82) cells. (B) POLH wild-type and POLH^−/− BL-2 (cl-82) were treated with 10 μM BrdU 5.5h after IR (10 Gy) and cells were collected 30min later. As compared with the profile in mock-irradiated cells (upper panel), PolH^−/− BL-2 cells had a higher proportion of cells in mid-S-phase cells post-IR than the POLH wild-type cells (lower panel). (C) FACS analysis showing S-phase population in untreated XP30RO and XP30RO/pol η fibroblast cells. (D) Cell cycle profile of HCT116 either transfected with 10nM non-targeting siRNA or POLH siRNA. Knockdown efficiency was confirmed by western blot analysis. Data from the original cell cycle histogram data were pooled and depicted as bar diagrams. Error bars show the standard of the mean of at least five independent experiments. Statistical analysis was performed by Student’s t-test; *P < 0.05.

Fig. 4.

DNA repair protein foci formation in XP30R0 cells after irradiation. (A, B) Pol η-deficient XP30R0 cells and the same cells transfected with pol η vector were irradiated with 5 Gy and fixed at the time points shown. γH2AX foci were visualized by immunostaining; analysis was performed using the automated IN Cell Analyzer system. Cells with more than 9 γH2AX foci were counted as positive, and the percentage of positive cells is plotted against time after IR. Data points represent three independent experiments. (C, D) For RAD51 foci analysis, cells were treated with 5 Gy irradiation dose and fixed and stained at the time points shown. Cells with more than 5 RAD51 foci were counted as positive. Bars represent three independent experiments. Statistical analysis was performed by two-sided Student’s t-test; *P < 0.05, ***P < 0.001. Sample pictures were prepared using ImageJ software.

Fig. 5.

Abrogation of radioresistance of pol η-deficiency by knockdown of the HR protein, XRCC3. (A) Clonogenic survival assays were performed in XP30RO and XP30RO/pol η cells after XRCC3 knockdown for 48h and then treatment with IR. Controls used mock transfection and non-targeting siRNA. Each data point represents three independent experiments; error bars show the standard error of the mean. Statistical analysis performed using Student’s t-test; **P < 0.01. (B) Western blot analysis showing knockdown efficiency of XRCC3 siRNA after 48 and 72h. Quantification shows more than 70% knockdown efficiency for both cell lines.

Fig. 6.

Hypothetical model for the increase in radioresistance mediated by pol η deficiency. Cells proficient in pol η-mediated translesion synthesis progress through S-phase and have a higher proportion of cells in radiosensitive phases of the cell cycle. In contrast, cells lacking pol η are delayed in S-phase, presumably since they need to employ other S-phase-specific DNA repair mechanisms to deal with damage to DNA by endogenous and exogenous factors. Redistribution of pol η-deficient cells towards the relatively radioresistant stages of S-phase decreases overall radiosensitivity, probably mediated via a dependence on homologous recombination repair.