Relationship between phosphorylated histone H2AX formation and cell survival in human microvascular endothelial cells (HMEC) as a function of ionizing radiation exposure in the presence or absence of thiol-containing drugs

Abstract

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (gamma-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. gamma-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated gamma-H2AX antibody and nuclear DAPI staining. gamma-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of gamma-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for gamma-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on gamma-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against gamma-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on gamma-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against gamma-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of gamma-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of gamma-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit gamma-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced gamma-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of gamma-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.

FIG. 1

Radiation dose response of γ-H2AX-positive cells using bivariant flow cytometry analysis. Confluent HMEC were irradiated with 0–16 Gy X rays and the percentages of γ-H2AX-positive cells were assessed 1, 4 and 24 h after irradiation. The percentages of γ-H2AX-positive HMEC ± 1 SEM are plotted as a function of dose and time after irradiation.

FIG. 2

Radiation dose response of γ-H2AX-positive cells 1 h after treatment as a function of radiation dose and the presence or absence of 4 mM WR1065 (WR) during irradiation. All cells were exposed to FITC-γ-H2AX antibody and stained with DAPI. Panel A: Two-dimensional flow cytometry histograms showing the percentages of γ-H2AX-positive cells after bivariant analysis. Panel B: Relative changes in the percentages of γ-H2AX-positive cells ±SEM as a function of radiation dose (IR) and WR1065 treatment. Panel C: Percentage of γ-H2AX-positive HMEC as a function of radiation dose in the absence of WR1065. Panel D: Percentage of γ-H2AX-positive HMEC as a function of radiation dose in the presence of 4 mM WR1065.

FIG. 3

Relative effectiveness of amifostine’s prodrug (WR2721), free thiol (WR1065), and disulfide (WR33278) forms, each administered 30 min prior to irradiation at a concentration of 4 mM, on the induction of γ-H2AX-positive cells by 8 Gy. Error bars represent ±SEM. Statistical analysis of γ-H2AX-positive in the presence or absence of each of the drug forms tested was performed using ANOVA with Tukey-Kramer multiple comparisons (not significant = ns; ** = <0.01; *** = <0.001).

FIG. 4

Effect of WR1065 on the induction of γ-H2AX-positive cells by 8 Gy as a function of the timing of administration before (B) or various times starting immediately (I) after (A) irradiation. Cells were exposed to 4 mM WR1065 for 30 min in all cases. Error bars represent ±SEM. Significance was determined after pairwise comparisons of the percentage of γ-H2AX-positive cells at 8 Gy with each of the WR1065 treatments (not significant = ns; *** = <0.001).

FIG. 5

Effect of the free thiol forms of amifostine (WR1065) and phosphonol (WR255591) and the drugs N-acetyl-l-cysteine (NAC), captopril and mesna on the induction of γ-H2AX-positive cells (panel A) and as a function of their relative position within the cell cycle (panel B). All thiols were administered 30 min prior to radiation exposure at a dose of 4 mM. Error bars represent ±SEM. Significance was determined after pairwise comparisons of the percentage of γ-H2AX-positive cells at 8 Gy with each of the thiol treatment conditions (* = <0.05; ** = <0.01; *** = <0.001).

FIG. 6

Bar graphs on a semilogarithmic plot depicting the surviving fraction of HMEC exposed to (panel A) 4 Gy and (panel B) 8 Gy of 250 kVp X rays after a 30-min treatment with 4 mM of WR1065, WR255591, NAC, captopril and mesna. Each bar represents the mean ±SEM of three separate experiments. Significance was determined after pairwise comparisons of surviving fractions at 4 or 8 Gy, respectively, with each of their corresponding thiol treatment conditions (not significant = ns; *** = <0.001).