Radiation Sensitivity, H2AX Phosphorylation, and Kinetics of Repair of DNA Strand Breaks in Irradiated Cervical Cancer Cell Lines

Abstract

Six human cervical cancer cell lines [five human papillomavirus (HPV) positive, one HPV negative] for induction and rejoining of DNA strand breaks and for kinetics of formation and loss of serine 139 phosphorylated histone H2AX (␥H2AX). X-rays induced the same level of DNA breakage for all cell lines. By 8 hours after 20 Gy, <2% of the initial single-strand breaks remained and no double-strand breaks could be detected. In contrast, 24 hours after irradiation, ␥H2AX representing up to 30% of the initial signal still present. SW756 cells showed almost four times higher background levels of ␥H2AX and no residual ␥H2AX compared with the most radiosensitive HPV-negative C33A cells that showed the lowest background and retained 30% of the maximum level of ␥H2AX. Radiation sensitivity, measured as clonogenic-surviving fraction after 2 Gy, was correlated with the fraction of ␥H2AX remaining 24 hours after irradiation. A substantial correlation with ␥H2AX loss half-time measured over the first 4 hours was seen only when cervical cell lines were included in a larger series of p53-deficient cell lines. Interestingly, p53 wild-type cell lines consistently showed faster ␥H2AX loss half-times than p53-deficient cell lines. We conclude that cell line-dependent differences in loss of ␥H2AX after irradiation are related in part to intrinsic radiosensitivity. The possibility that the presence of ␥H2AX foci may not always signify the presence of a physical break, notably in some tumor cell lines, is also supported by these results.

Cyclin G1 overcomes radiation-induced G2 arrest and increases cell death through transcriptional activation of cyclin B1

“…At 48 h after 5 Gy radiation, cell death shown by PI staining was significantly increased (Figure 1d). Since the presence and the rate of loss of histone H2AX foci are correlated with cellular radiosensitivity, 19 phosphorylated H2AX on serine 139 was examined by Western blotting analysis: Because phosphorylated H2AX can be visualized as foci by immunofluorescence using phosphospecific antibodies, foci formation was also examined. Phosphorylated H2AX was increased at 24 and 48 h after radiation with further potentiation by cyclin G1.…”

Targeting of Rad51-dependent homologous recombination: implications for the radiation sensitivity of human lung cancer cell lines

“…One of the earliest steps in the cellular response to DSBs is the phosphorylation of serine 139 of g-H2AX. A quantitative similarity between the induction and repair of DSBs and the formation and disappearance of g-H2AX foci has been found (Banath et al, 2004). Thus, we used g-H2AX foci formation and a gel-electrophoresis assay to determine whether persisting Rad51 foci were indicative of nonrepaired Dsb.…”

“…In order to explore the possibility that residual Rad51 foci reflect nonrepaired DNA double-strand breaks, phosphorylation of histone g-H2AX in response to DNA double-strand breaks produced by IR has been measured, which is proposed to concentrate repair factors at sites of DNA damage (Celeste et al, 2003). Although differences in the loss of g-H2AX foci have been found to be related in part to the intrinsic radiosensitivity of cervical cancer cell lines (Banath et al, 2004), our data did not support these observations. A co-localisation frequency of more than 80% for Rad51 and g-H2AX foci elucidate that persisting Rad51 foci mostly contains DNA double-strand breaks.…”

Overt Increase of Oxidative Stress and DNA Damage in Murine and Human Colitis and Colitis-Associated Neoplasia

“…Evaluating H2AX activation was already tested in various malignancies such as gastric, lung-and cervical cancer and it is discussed as a potential marker for cancer progression as well as for radiation-and chemotherapy response. (45,(56)(57)(58) In IBD, the risk of developing CAC is heterogeneous and relates to duration, extent and severity of disease. γH2AX-staining of biopsies from surveillance endoscopy may help separating those individuals at risk from those without.…”