H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

Abstract

Background: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS).

Methods: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response.

Results: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay.

Conclusion: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Figure 1

Dose response of γH2AX focus formation in lymphoblast cell lines (LCLs). Immunocytochemistry (ICC ) was used to capture representative γH2AX focus images from asynchronous log-phase non-radiosensitive (RS) control cells (control-5) before (A) and 1 h after 1 Gy (B), 2 Gy (C) and 4 Gy (D) of ionising radiation (IR). (E) A more detailed view of γH2AX focus distribution, apparently not limited to either euchromatin or heterochromatin in a cell exposed to 2 Gy of IR. Slides were counterstained with DAPI to visualise nuclei (left panels). γH2AX foci were quantified as foci per nucleus for each dose (F). Error bars are the standard error of the mean (s.e.m.) of γH2AX foci number per nucleus from three separate experiments, wherein at least 100 nuclei cells were scored per dose.

Figure 2

Time-course kinetics of DNA repair-deficient LCLs. γH2AX foci per nucleus are shown for controls (n=11; open bars), homozygous ATM mutant (three experimental replicates; grey bars) and ligase IV knock-out (three experimental replicates; black bars) human cell lines. γH2AX foci number was counted in cells before (0 h) and 1 and 8 h after exposure to 2 Gy of IR. Error bars represent s.e.m. of γH2AX foci number per nucleus.

Figure 3

Time-course kinetics of IR-induced γH2AX foci in LCLs derived from clinically RS individuals (n=18) vs control LCLs (n=11). γH2AX focus number was measured in control (open bars) and RS (filled bars) cell lines before (0 h) and at various times after 2 Gy of IR. Error bars represent s.e.m. of foci number per nucleus based on 4–5 fields of approximately 20–25 cells per field. Data have been graphed from 0 to 8 h for all RS and CL cell lines (controls: n=11; RS: n=18), and at 24 h for a random subset of cell lines (controls: n=5 [CL1, CL3, CL4, CL9, CL11] RS: n=13 [A1, A2, A3, A4, A6, A7, L3, L5, L6, L7, L9, L11]) (Table 1).

Figure 4

Time-course kinetics of γH2AX focus induction and repair for each LCL following IR. γH2AX focus numbers per nucleus before (0 h) and at various times after 2 Gy are plotted for each control cell line (open squares), acute (filled triangles) and late (filled circles) RS patients. RS1 is designated as an open triangle.

Figure 5

Dynamics of DNA repair in LCLs from an RS individual (RS1) following IR. (A) γH2AX focus numbers for unirradiated (0 h) cells and irradiated cells at various time intervals after 2 Gy of IR are shown for control cell lines (open bars) and the RS1 cell line from two separate experiments (filled bars). Error bars represent s.e.m. of foci number per nucleus determined from 11 control cell lines and two replicates of RS1. (B) Representative γH2AX ICC focus images are shown for asynchronous log-phase RS1 cells before and at the indicated times (hours) after 2 Gy of IR. (C) Pulsed-field gel electrophoresis (PFGE) was carried out on RS1 (filled triangles), and a ligase IV-deficient cell line (filled squares) after various time points (in hours) along with six controls (open circles; CL1, CL2, CL5, CL9, CL10, CL11) following 40 Gy of radiation. Error bars represent the differences in mean from two separate experiments, each determined from two replicates run on separate gels.