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dc.contributor.advisorCarty, Michael
dc.contributor.authorVillalan, Sangamitra
dc.description.abstractPlatinum-based chemotherapeutic drugs such as cisplatin and carboplatin induce intra- and interstrand crosslinks, resulting in replication arrest, and cell death. Translesion synthesis by DNA polymerase eta, is one way in which human cells can tolerate platinum-induced adducts. Because these adduct block replication, the cell cycle stage at the time of drug exposure, and the DNA polymerases expressed could affect the outcome of exposure. The aim of this research was therefore to investigate the effects of cell cycle phase at the time of drug exposure on cell viability, cell cycle progression and activation of DNA damage responses in pol eta-deficient (XP30R0) and pol eta-expressing (TR30-2) human fibroblast cell lines. Using nocodazole arrest and release to generate populations of cells enriched in G1, S or M phases, it was found that XP30R0 cells in S-phase were more sensitive to drug treatment compared to cells that were in G1- and M-phase at the time of treatment. When G1 cells were treated with drug, cell arrest in S- and G2/M-phases was detected by flow cytometry. When S-phase XP30R0 cells were treated with cisplatin, a strong S-phase arrest was detected, however, when treated with carboplatin, arrest in S phase was less pronounced, and there was evidence for S-phase arrest in a second cell cycle. In TR30-2 cells, the extent of S-phase arrest was less than in XP30R0 cells, consistent with a role for pol eta in replication of damaged DNA. In M-phase XP30R0 cells following treatment, cells were delayed entering S and by 24 hours post-treatment, strong S-phase arrest occurred. Using western blotting and immunofluorescence, it was found that DNA damage-induced phosphorylation of key PIK kinase substrates including H2AX, Chk1 and RPA2, was induced following drug treatment of XP30RO cells in all cell cycle phases. Comparison of the timing of Chk1 ser317 and RPA2 ser4/ser8 phosphorylation demonstrated that activation of the Chk1 phosphorylation was an earlier event than RPA2 phosphorylation, independent of the cell cycle phase at the time of exposure. RPA2 phosphorylation was consistent with prolonged replication arrest in S-phase cells. Using small molecule inhibitors, roles for DNA-PK and CDK1/2 in damage-induced RPA2 phosphorylation were identified. The data provides an insight into the molecular events that determine the outcome of drug exposure in cells treated at different phases of the cell cycle, and may be relevant to identification of pathways that can be targeted to improve the efficacy of these drugs in cancer therapy.en_US
dc.subjectDNA polymerase Etaen_US
dc.subjectDNA Damage Responseen_US
dc.subjectCell Cycle Progressionen_US
dc.titleCell Cycle-dependence of cisplatin-and carboplatin-induced dna damageen_US

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