A control for the day-to-day normalization of the flow cytometry γ-H2AX assay for clinical routine.

BACKGROUND: The phosphorylation of histone H2AX (γ-H2AX) at the DNA double-strand break (DSB) site is frequently used for quantifying DSBs and may be useful as a biomarker for clinical applications. We have previously reported a flow cytometry-based quantification of γ-H2AX for clinical routine. One major challenge, however, is the lack of a control sample for normalization of the day-to-day variation of the flow cytometry γ-H2AX assay. METHODS: Here, we report development of a mix-control sample containing peripheral blood mononuclear cells (PBMC) from 10 control individuals, for normalization of day-to-day variation of the flow cytometry-γ-H2AX assay. RESULTS: We showed that control individuals sampled on different days show an average day-to-day variation (CV) of 34%, which was reduced to 12% after normalization to the control sample. The normalization allowed detection of radiosensitivity of lymphoblastoid cell lines from ataxia telangiectasia patients, sampled over three days. CONCLUSION: The mix-control sample, consisting of 10 control individuals' PBMC, can be used as a control sample to normalize for day-to-day variation of the γ-H2AX assay. The use of this sample will facilitate integration of the γ-H2AX assay into clinical routine. © 2018 International Clinical Cytometry Society.

A flow cytometry assay that measures cellular sensitivity to DNA-damaging agents, customized for clinical routine laboratories.

OBJECTIVES: The clonogenic assay examines cell sensitivity to toxic agents and has been shown to correlate with normal tissue sensitivity to radiotherapy in cancer patients. The clonogenic assay is not clinically applicable due to its intra-individual variability and the time frame of the protocol. We aimed to develop a clinically applicable assay that correlated with the clonogenic assay. DESIGN AND METHODS: We have developed a faster and less labor-intensive cell division assay (CD assay) using flow cytometry and incorporation of a fluorescent thymidine analogue. The CD assay was calibrated to the clonogenic assay and optimized for peripheral blood lymphocytes. RESULTS: Following ionizing radiation of primary human skin fibroblasts, the four-day CD assay gave similar results as the 14-day clonogenic survival assay. In lymphocytes isolated from patient blood samples, the CD assay was able to detect increased radiosensitivity in ataxia telangiectasia patients and increased radiosensitivity after in vitro treatment with DNA-PK and ATM inhibitors. The CD assay found a variation in the intrinsic radiosensitivity of lymphocytes isolated from healthy control samples. The CD assay was able to measure the anti-proliferation effect of different chemotherapeutic drugs in lymphocytes. CONCLUSIONS: Our results indicate that the CD assay is a fast and reliable method to measure the anti-proliferation effect of DNA-damaging agents with a potential to find the most sensitive patients in the work-up before cancer treatment.

Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation.

Most of the cytotoxicity induced by ionizing radiation is mediated by radical-induced DNA double-strand breaks. Cellular protection from free radicals can be stimulated several fold by sulforaphane-mediated activation of the transcription factor Nrf2 that regulates more than 50 genes involved in the detoxification of reactive substances and radicals. Here, we report that repeated sulforaphane treatment increases radioresistance in primary human skin fibroblasts. Cells were either treated with sulforaphane for four hours once or with four-hour treatments repeatedly for three consecutive days prior to radiation exposure. Fibroblasts exposed to repeated-sulforaphane treatment showed a more pronounced dose-dependent induction of Nrf2-regulated mRNA and reduced amount of radiation-induced free radicals compared with cells treated once with sulforaphane. In addition, radiation- induced DNA double-strand breaks measured by gamma-H2AX foci were attenuated following repeated sulforaphane treatment. As a result, cellular protection from ionizing radiation measured by the 5-ethynyl-2'-deoxyuridine (EdU) assay was increased, specifically in cells exposed to repeated sulforaphane treatment. Sulforaphane treatment was unable to protect Nrf2 knockout mouse embryonic fibroblasts, indicating that the sulforaphane-induced radioprotection was Nrf2-dependent. Moreover, radioprotection by repeated sulforaphane treatment was dose-dependent with an optimal effect at 10 uM, whereas both lower and higher concentrations resulted in lower levels of radioprotection. Our data indicate that the Nrf2 system can be trained to provide further protection from radical damage.