Increased γ-H2AX and Rad51 DNA Repair Biomarker Expression in Human Cell Lines Resistant to the Chemotherapeutic Agents Nitrogen Mustard and Cisplatin

Abstract

Chemotherapeutic anticancer drugs mediate cytotoxicity by a number of mechanisms. However, alkylating agents which induce DNA interstrand crosslinks (ICL) are amongst the most effective anticancer agents and often form the mainstay of many anticancer therapies. The effectiveness of these drugs can be limited by the development of drug resistance in cancer cells and many studies have demonstrated that alterations in DNA repair kinetics are responsible for drug resistance. In this study we developed two cell lines resistant to the alkylating agents nitrogen mustard (HN2) and cisplatin (Pt). To determine if drug resistance was associated with enhanced ICL DNA repair we used immunocytochemistry and imaging flow cytometry to quantitate the number of γ-H2AX and Rad51 foci in the nuclei of cells after drug exposure. γ-H2AX was used to evaluate DNA strand breaks caused by repair incision nucleases and Rad51 was used to measure the activity of homologous recombination in the repair of ICL. In the drug-resistant derivative cell lines there was overall a significant increase in the number and persistence of both γ-H2AX and Rad51 foci in the nuclei of cells over a 72-hour period, when compared to the non-resistant parental cell lines (ANOVA p < 0.0001). In a Pt-resistant ovarian cancer cell line (A2780cis^R) a similar enhancement of DNA repair was observed when compared to the non-drug-resistant wild-type ovarian cancer cells (A2780) following exposure to HN2. Our data suggest that using DNA repair biomarkers to evaluate mechanisms of resistance in cancer cell lines and human tumours may be of experimental and clinical benefit. We concede, however, that examination of a larger population of cell lines and tumours is required to fully evaluate the validity of this approach.

Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages

“…As γ-H2AX is a well-established DSB marker, such results implicated that there may be a higher basal level of DNA damage in MCF-7 TR5 cells. Similar results were also observed in human ovarian cancer cell lines resistant to nitrogen mustard and cisplatin [40]. In general, the DSB repair mechanisms mainly include the HR repair, an error free DNA repair process, and the NHEJ repair, an error prone DNA repair process [31].…”

Identification of a pyridine derivative inducing senescence in ovarian cancer cell lines via P21 activation

“…SN‐38, a topoisomerase I and II inhibitor, was reported to induce cellular senescence in colon, breast and ovarian cancer cells by activating p53‐p21 pathway through producing excessive DNA‐damage in cells . In our experiment, FPTHQ also caused DNA‐damage accumulation in A2780 cells, which are sensitive to different culture stress . From our data, we speculate that at normal culture conditions, A2780 cells are capable to fix these DNA damages by themselves, so the cells are vital and proliferating.…”

USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

“…Histone H2AX phosphorylation on γH2AX is a sensitive marker for DNA DSBs (Rogakou et al, 1999). It has been reported that γH2AX is associated with resistance to radiation and multiple chemotherapy drugs including cisplatin (Adam-Zahir et al, 2014; van Oorschot et al, 2016). Our study suggested that, the level of γH2AX was upregulated in cisplatin-resistant A549/CDDP cells ( Figure 2B ) and increased significantly after cisplatin treatment ( Figure 2C ).…”