Persistence Of gamma-H2AX Foci Research Articles

Role of Inhibition of DNA Double-Strand Break Repair and Autophagy in Pancreatic Cancer Cytotoxicity and Radiation-Induced Cell Killing

Low survival rates of pancreatic ductal adenocarcinoma (PDAC) are attributed to its detection at advanced stage and high chemotherapy and radiation resistance. Previous work in the lab has shown that resistance to radiation therapy can be decreased through simultaneous inhibition of the anti-apoptotic NF-kB cell survival signaling pathway by dimethyl-amino-parthenolide (DMAPT) and inhibition of Warburg cancer metabolism by dichloroacetate (DCA). We sought to determine the mechanism by which dual treatment with DMAPT and DCA increases cytotoxicity and radiation-induced cell killing in pancreatic cancer. Established pancreatic cancer cell lines, MIA-PaCa-2, Panc-1, and AsPc-1 cells were plated in T-25 flasks. For single fraction experiments, cells were treated with no drugs or both DMAPT and DCA at 24 and 48 hours after plating and irradiated with 0-6Gy with 160 kVp X-rays. To assess split dose repair, experimental groups were 5Gy, 2 x 2.5Gy, 5Gy with DMAPT and DCA, and 2x2.5Gy with DMAPT and DCA. Colonies were counted after 10 days of growth, and plating efficiency and survival fractions were calculated. Western blots of autophagy specific ATGs, LC-3a/b, and P62- SQSTM1 proteins were also determined. To assess radiation DNA damage response (DDR), cells were grown on chambered slides, treated with no drugs or dual DMAPT and DCA, irradiated at 2Gy, and fixed at 0hr, 1hr, and 24hrs after irradiation. After labeling with Gamma-H2Ax antibody, 53BP1 antibody, and DAPI, cells were imaged on a Leica Sp8 confocal microscope. Foci quantification was performed using ImageJ software. Forty-eight hours of DMAPT and DCA dual treatment significantly reduced plating efficiency of MIA-PaCa-2 cells from 0.406±0.092 (40.6%) to 0.253±.0.10 (25.3%), of Panc-1 cells from 0.528±0.11 (52.8%) to 0.0755±0.044 (7.55%), and of AsPc-1 cells from 0.294±0.16 (29.4%) to 0.0300±0.031 (3.0%). Cell death involves alterations of autophagy regulatory proteins. Treatment of cells with two 2.5Gy fractions given four hours apart with DMAPT and DCA dual treatment significantly reduced cell survival fraction compared to split-dose fractionated cells without drug treatment in MIA-PaCA-2 (0.224±0.098 to 0.0716±0.064), Panc-1(0.260±0.040 to 0.0130±0.012), and AsPc-1 cells (0.121±0.011 to 0.0370±0.026). Furthermore, for all three cell lines, persistence of gamma-H2Ax foci was seen at 24 hours post 2Gy irradiation in DMAPT and DCA dual treated cells compared to 2 Gy irradiated only cells, where foci levels returned to baseline levels. Dual DMAPT and DCA treatment of pancreatic cancer decreases plating efficiency, inhibits autophagy, inhibits split dose repair, and results in persistence of gamma-H2Ax foci at 24 hours post irradiation. We conclude that dual DMAPT and DCA treatment of pancreatic cancer increases radiation-induced cell killing through inhibition of DNA double-strand break repair.

Persistence of Gamma-H2AX Foci in Bronchial Cells Correlates with Susceptibility to Radiation Associated Lung Cancer in Mice.

The risk of developing radiation-induced lung cancer differs between different strains of mice, but the underlying cause of the strain differences is unknown. Strains of mice also differ in how quickly they repair radiation-induced DNA double-strand breaks (DSBs). We assayed mouse strains from the CcS/Dem recombinant congenic strain set for their efficacy in repairing DNA DSBs during protracted irradiation. We measured unrepaired γ-H2AX radiation-induced foci (RIF), which persisted after chronic 24-h gamma irradiation, as a surrogate marker for repair efficiency in bronchial epithelial cells for 17 of the CcS/Dem strains and the BALB/c founder strain. We observed a very strong correlation (R2 = 79.18%, P < 0.001) between the level of unrepaired RIF and radiogenic lung cancer incidence measured in the same strains. Interestingly, spontaneous levels of foci in nonirradiated mice also showed good correlation with lung cancer incidence when incidence data from male and female mice were combined. These results suggest that genetic differences in DNA repair capacity largely account for differing susceptibilities to radiation-induced lung cancer among CcS/Dem mouse strains, and that high levels of spontaneous DNA damage are also a relatively good marker of cancer predisposition. In a smaller pilot study, we found that the repair capacity measured in peripheral blood leucocytes also correlated well with radiogenic lung cancer susceptibility, raising the possibility that the assay could be used to detect radiogenic lung cancer susceptibility in humans.

Induction of gamma-H2AX Foci in Lymphocytes, Fibroblasts and Tumor Cells by Single and Pulsed High Dose Rate X-rays

Purpose and background: Variation in radiosensitivity of normal tissues is related to genetic susceptibility (Svensson et al. 2006). Persistence of gamma-H2AX foci after ionizing radiation is higher in DNA-DSB repair deficient cells than in normal cells. We investigated whether differences in number of ?-H2AX foci may be useful as a predictor of radiosensitivity. Materials and methods: Induction of gamma-H2AX foci was measured in various cell types: freshly isolated lymphocytes, fibroblast cells (AT+/+, AT+/-, AT-/-), DNA-DSB repair deficient and normal hamster cells and human tumour cells (lung carcinoma, SW-1573 and prostate cancer cell lines with different TP53 status: LNCaP: TP53 wt, PC3: TP53 null and DU145: TP53 mutant). A radiation dose of 2.4 Gy was given as a single dose or as pulsed doses of 100 mGy in 24 h (PDR: 240 pulses of 100mGy, 1 pulse every 6 min). The number of gamma-H2AX foci was determined 30 min and 24 h after single dose and directly after PDR. Results: Maximal number of γ-H2AX foci occurred after single dose at 30 min after irradiation without significant differences in numbers of foci between different cell lines. The number of gamma-H2AX foci was radiation dose dependent and linearly correlated with clonogenic survival. Both 24 h after a single dose and after PDR more remaining foci are observed in repair deficient cells than in normal cells. In addition the TP53 null cells have more remaining foci than the mutant and wt TP53 cells. Conclusions: The detection gamma-H2AX after in vitro irradiation of lymphocytes and cell lines is simple, reproducible and sensitive. The number of residual gamma-H2AX foci after single dose as well as after PDR enables us to distinguish cells with differences in DNA-DSB repair capacities. However, it remains to be established whether gamma-H2AX assay applied in lymphocytes can be used to predict for late normal tissue toxicity after radiotherapy. Sponsored by the Dutch Cancer Foundation UVA2008-4019

Low-Dose Hyper-radiosensitivity is not Caused by a Failure to Recognize DNA Double-Strand Breaks

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.