Date of Award
Open Access Dissertation
Pharmacology, Physiology and Neuroscience
Michael D. Wyatt
When a mammalian cell suffers DNA damage, DNA damage signaling responses and repair pathways are invoked. The phosphorylation of histone variant H2AX (γH2AX) and of replication protein A (pRPA) are two well-documented damage signals, marking double strand breaks and stalled replication forks, respectively. Inhibitors of thymidylate synthase (TS) and ribonucleotide reductase (RNR) are chemotherapeutics that act by depriving cells of the deoxynucleotides necessary for DNA synthesis, which causes damage. The response and repair pathways activated by these chemotherapeutics have been studied for a number of years but there remain unanswered questions as to how cancer cells perceive this damage, the kinases active in the response, and the promotion of damage repair. To investigate the damage response and the necessity of H2AX phosphorylation during TS inhibition, we utilized cell models in which H2AX has been knocked out genetically as well as a shRNA H2AX knockdown cell line. Cell survival assays and immunofluorescence for the homologous recombination (HR) protein RAD51 showed that H2AX mutation or deficiency do not affect cell sensitivity or HR damage response. However, significant differences in the kinetics of pRPA were noted: pRPA was seen as early as 4 h post TS inhibition in H2AX deficient cells compared to up to 24 h in H2AX proficient cells. The data suggests that H2AX signaling is not involved in the response to TS inhibition but may affect the repair pathway selection. A kinase known to be over-expressed in cancer and act in mitosis, polo-like kinase 1 (PLK1), has recently gained attention for its reported activity in S-phase stress. Therefore, we examined how PLK1 inhibition during deoxynucleotide deprivation affected response and repair of DNA damage. Our research suggests that inhibition of PLK1 decreases pRPA during replication stress as well as affects the formation of RAD51 foci in response to damage. Overall, the data presented here re-enforce the notion that though replication stress inducers have been researched and used in chemotherapy for decades, aspects of the damage response are still unknown. As well, our data highlights the potential for new chemotherapeutic combinations of replication stress inducers with drugs that inhibit pRPA or PLK1 inhibitors.
Brady, K.(2014). INVESTIGATION OF THE RESPONSE AND REPAIR OF REPLICATION. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/2776