Role of DNA repair, ROS and apoptosis in daunorubicin chemotherapy of leukaemia

Submission note: A thesis submitted in total fulfilment of the requirement for the degree of Doctor of Philosophy to the Department of Pharmacy and Applied Science, School of Molecular Sciences, College of Sciences, Health and Engineering, La Trobe University, Victoria.<br><br>Daunorubicin (DNR) has been used for decades to treat acute leukaemias. Over this time there has been extensive research into its mechanism of action and associated toxicities. Recent discoveries regarding the interaction between reactive oxygen species (ROS) and ataxia‐telangiectasia mutated (ATM), an initiator of DNA repair and an activator of both p53 and CHK2 with implications for cell cycle arrest and apoptosis, offer intriguing new insights into these mechanisms. In this study, the DNA damaging chemotherapeutic drug daunorubicin was evaluated. The primary aim was to elucidate the fundamental mechanism of cell kill by daunorubicin in acute leukaemia models. Results revealed no mutations in the ATM coding region of MOLT-4 and CCFR-CEM (both Acute T lymphoblastic leukaemia) and SUP-B15 (Acute B lymphoblastic leukaemia) cell lines. ATM and DNA-dependent Protein Kinase (DNA-PK) were activated in all cell lines. ATM‘s downstream targets p53, BRCA1 and MnSOD were activated in MOLT-4 and CCRF-CEM cell lines, whereas p53 and BRCA1 were not activated in SUP-B15 cells. Daunorubicin induced apoptosis in all cell lines but there were variations in ROS levels in response to daunorubicin. MOLT-4 and CCRF-CEM cells, responded by initiating both the intrinsic and extrinsic pathways. In contrast, SUP-B15, which lacks p53 activity, apoptosis stimulation was isolated to the extrinsic pathway. Both MOLT-4 and CCFRCEM cells utilised non-homologous end joining (NHEJ) and homologous recombination (HR) as double stranded break repair pathways. Whereas, SUP-B15 cells utilised only NHEJ pathway, this can be attributed to BRCA1 deficiency which can impair HR pathway. Overall, higher sensitivity to daunorubicin was seen in MOLT-4 and CCFR-CEM cells (Acute T lymphoblastic leukaemia). Whereas, SUPB15 (Acute B lymphoblastic leukaemia) cell lines showed low sensitivity to. daunorubicin treatment and this could be due to variations in DNA repair pathways, Bcl-2 overexpression or anti-oxidant pathways occurring independently of ATM.