Investigation of impact of non-homologous end joining (NHEJ) DNA repair pathway in the development of doxorubicin resistance in MCF7 cell lines

Non-homologous end joining DNA repair mechanism is assoicated with many of cell precesses. Multidrug resistance and cell devision involves diverse cellular pathways. Aim of this study was to investigate involvement of NHEJ repair mechanism in the development of an anthracycline type doxorubicin resistance in MCF7 cell lines. Doxorubicin (4µM) were applied to sensitive and resistant sublines, and all parameters were tested after 1, 6, 12, and 24h of applications. MTT was used to evaluate time-dependent cytotoxicity of doxorubicin. Doxorubicin induced genotoxicity was tested by single cell alkaline gel electrophoresis assay. Immunoflorescent labeling of 53BP1 protein performed for the analysis of NHEJ. Expression of the genes coding the essential components of the NHEJ pathway, XRCC4, XRCC5, XRCC6, XRCC7, LIG4 and XLF, and two control miRNAs of the pathway, miR-101 and miR502, were analyzed by real-time PCR analysis. Results demonstrated that cell proliferation decreased in MCF7/S, MCF7/400Dox, and MCF7/1000Dox cells, due to 24h doxorubicin application. Doxorubicin induced genotoxic damage after 6h incubation in all cells. After 24h of incubation, doxorubicin caused higher genotoxic damage in sensitive cells in comparison to resistant cells. NHEJ pathway was activated after 1h of doxorubicin application in sensitive cells, whereas activity was observed after 12h in resistant cells. Though, expression of NHEJ components was higher in control group sensitive cells in comparison to respective resistant cells, expression of these genes were significantly upregulated in MCF7/1000Dox cells in response to doxorubicin application. miR-101 and miR502 alterations were correlated to XRCC7, and XRCC5, LIG4 and XLF gene expressions, respectively in sensitive cells. Conclusively, NHEJ pathway components were upregulated in 24h of doxorubicin application. Doxorubicin induced NHEJ response were more rapid and high in MCF7/1000Dox cells, although pathway did not seem to be under the control of proposed miRNAs. Investigation of timing and level of NHEJ DNA damage response in cells having varying resistance indices and different resistance mechanisms, provided a better understanding of the repair of doxorubicin induced genotoxic damage and development of drug resistance in these cells. The results of the thesis will provide basic knowledge for further studies.