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 gH2AX) 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 gH2AX 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 gH2AX 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: gH2AX 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.

Non-homologous end-joining protein expression screen from radiosensitive cancer patients yields a novel DNA double strand break repair phenotype

“…DNA DSBs are key radiation-induced cellular lesions (10,(16)(17)(18). The NHEJ pathway of DNA DSB repair is the fastest and most prominent DNA DSB repair mechanism in mammalian cells [(19) and reviewed in (20)], operative in all cell cycle phases (18).…”

“…Control and RS patient characteristics have been previously described (10,13). In summary, clinically RS patients had Radiation Therapy Oncology Group (RTOG) (2) Grade 3 or 4 toxicities, whereas those radiotherapy patients without significant toxicity (RTOG ≤1) were controls.…”

“…In summary, clinically RS patients had Radiation Therapy Oncology Group (RTOG) (2) Grade 3 or 4 toxicities, whereas those radiotherapy patients without significant toxicity (RTOG ≤1) were controls. We accessioned pathoclinical details and biological samples from cancer patients who had developed clinical hypersensitivity reactions to radiotherapy (RS cases) (10,13). RS was in the clinical form of either severe acute or severe late reactions, or so-called consequential late effects, which result from severe and persisting acute effects (14).…”

“…We and others have taken different approaches in an attempt to discern which genes may be responsible. One is a candidate molecule approach, where dysfunction of genes and their products or regulators linked to radiosensitivity in other contexts/organisms, are tested for dysfunction in RS cancer patients (6)(7)(8)(9)(10). Another are genome-wide association studies (11), which to date have yielded some radiosensitivity susceptibility loci/genes, e.g., TANC1 (12).…”

In vitro prediction of breast cancer therapy toxicity

“…These include using a candidate gene/protein approach and studying molecules known or suspected to influence radiosensitivity in other species, but using IR-sensitive patient cells/tissues. There are a number of examples of such studies (17)(18)(19)(20)(21). Increasingly, gene expression profiling is giving insights into human disease and such studies are emerging in the field of radiosensitivity (22)(23)(24).…”