Correlation between DNA damage responses of skin to a test dose of radiation and late adverse effects of earlier breast radiotherapy.

AIM: To correlate residual double strand breaks (DSB) 24h after 4Gy test doses to skin in vivo and to lymphocytes in vitro with adverse effects of earlier breast radiotherapy (RT). PATIENTS AND METHODS: Patients given whole breast RT ⩾5years earlier were identified on the basis of moderate/marked or minimal/no adverse effects despite the absence ('RT-Sensitive', RT-S) or presence ('RT-Resistant', RT-R) of variables predisposing to late adverse effects. Residual DSB were quantified in skin 24h after a 4Gy test dose in 20 RT-S and 15 RT-R patients. Residual DSB were quantified in lymphocytes irradiated with 4Gy in vitro in 30/35 patients. RESULTS: Mean foci per dermal fibroblast were 3.29 (RT-S) vs 2.80 (RT-R) (p=0.137); 3.28 (RT-S) vs 2.60 (RT-R) in endothelium (p=0.158); 2.50 (RT-S) vs 2.41 (RT-R) in suprabasal keratinocytes (p=0.633); 2.70 (RT-S) vs 2.35 (RT-R) in basal epidermis (p=0.419); 12.1 (RT-S) vs 10.3 (RT-R) in lymphocytes (p=0.0052). CONCLUSIONS: Residual DSB in skin following a 4Gy dose were not significantly associated with risk of late adverse effects of breast radiotherapy, although exploratory analyses suggested an association in severely affected individuals. By contrast, a significant association was detected based on the in vitro response of lymphocytes.

Inter-individual and inter-cell type variation in residual DNA damage after in vivo irradiation of human skin.

PURPOSE: The aim of this study was to compare inter-individual and inter-cell type variation in DNA double-strand break (DSB) repair following in vivo irradiation of human skin. MATERIALS AND METHODS: Duplicate 4mm core biopsies of irradiated and unirradiated skin were collected from 35 patients 24h after 4Gy exposure using 6MeV electrons. Residual DSB were quantified by scoring 53BP1 foci in dermal fibroblasts, endothelial cells, superficial keratinocytes and basal epidermal cells. RESULTS: Coefficients of inter-individual variation for levels of residual foci 24h after in vivo irradiation of skin were 39.9% in dermal fibroblasts, 44.3% in endothelial cells, 32.9% in superficial keratinocytes and 46.4% in basal epidermal cells (p<0.001, ANOVA). In contrast, the coefficient of inter-cell type variation for residual foci levels was only 11.3% in human skin between the different epidermal and dermal cells (p=0.034, ANOVA). Foci levels between the different skin cell types were correlated (Pearson's R=0.855-0.955, p<0.001). CONCLUSIONS: Patient-specific factors appear to be more important than cell type-specific factors in determining residual foci levels following in vivo irradiation of human skin.

Rad51 inhibition is an effective means of targeting DNA repair in glioma models and CD133+ tumor-derived cells.

High grade gliomas (HGGs) are characterized by resistance to radiotherapy and chemotherapy. Targeting Rad51-dependent homologous recombination repair may be an effective target for chemo- and radiosensitization. In this study we assessed the role of Rad51-dependent repair on sensitivity to radiation and temozolomide (TMZ) as single agents or in combination. Repair protein levels in established glioma cell lines, early passage glioblastoma multiforme (GBM) cell lines, and normal human astrocytes (NHAs) were measured using western blot. Viability and clonogenic survival assays were used to measure the effects of Rad51 knockdown with radiation (XR) and TMZ. Immunocytochemistry was used to evaluate kinetics of Rad51 and γ-H2AX repair foci. Immunohistochemistry was used to assess Rad51 protein levels in glioma specimens. Repair proteins including Rad51 are upregulated in HGG cells compared with NHA. Established glioma cell lines show a dose-dependent increase in Rad51 foci formation after XR and TMZ. Rad51 levels are inversely correlated with radiosensitivity, and downregulation markedly increases the cytotoxicity of TMZ. Rad51 knockdown also promotes more residual γ-H2AX foci 24 h after combined treatment. Newly established GBM cell lines also have high Rad51 levels and are extremely sensitive to Rad51 knockdown. Clinical samples from recently resected gliomas of varying grades demonstrate that Rad51 levels do not correlate with tumor grade. Rad51-dependent repair makes a significant contribution to DNA repair in glioma cells and contributes to resistance to both XR and TMZ. Agents targeting Rad51-dependent repair would be effective adjuvants in standard combination regimens.

DNA repair after irradiation in glioma cells and normal human astrocytes.

We examined DNA damage responses and repair in four human glioma cell lines (A7, U87, T98G, and U373) and normal human astrocytes (NHAs) after clinically relevant radiation doses to establish whether we could identify differences among them that might suggest new approaches to selective radiosensitization. We used phosphorylation of histone H2AX visualized by immunocytochemistry to assess DNA double-strand break (DSB) formation and resolution. Fluorescence immunocytochemistry was used to visualize and quantify repair foci. Western blotting was used to quantify repair protein levels in the different cell lines before and after irradiation and during different cell cycle phases. Mitotic labeling was used to measure cell cycle parameters after irradiation. We found that the glioma cell lines repaired DSBs more slowly and less effectively than did NHAs in the clinically relevant dose range, as assessed by induction and resolution of H2AX phosphorylation, and this was most marked in the three TP53-mutated cell lines (T98G, A7, and U373). The glioma cells also expressed relatively high repair-protein levels compared with NHAs that were not altered by irradiation. High levels of the repair protein Rad51 in these cells persisted throughout the cell cycle, and a marked increase in Rad51 foci formation, which was not restricted to cells in G2/S phase, occurred at early time points after irradiation. TP53-mutated glioma cell lines demonstrated a very prominent dose-responsive G2 checkpoint and were sensitized to radiation by caffeine, which inhibits G2/S phase checkpoint activation. In conclusion, DNA repair events differed in these four glioma cell lines compared with NHAs. In particular, the three TP53-mutated glioma cell lines exhibited markedly increased Rad51 protein levels and marked, dose-dependent Rad51 foci formation after low radiation doses. This suggests that agents that disrupt Rad51-dependent repair or prevent G2 checkpoint activation may selectively sensitize these cells.