Investigation of DNA Damage and Genomic Organization in the Cellular Response to Platinum Chemotherapy

Abstract

Ovarian cancer (OC) is the primary reason for gynecological cancer-related deaths among women. This cancer rarely has specific symptoms making early detection challenging. Platinum compounds have been used as the first-line treatment for OC. However, OC patients often develop chemoresistance to current platinum/taxane chemotherapy regimens upon recurrence, leaving patients with no alternative treatment options. The purpose of this thesis was to investigate the molecular basis of chemoresistance in OC by analyzing the damage of DNA response and gene positioning after treatment exposure. A modified comet assay was used to measure interstrand cross-link (ICL) formation and repair in chemoresistant (CR) (SKOV-3) and chemosensitive (CS) (A2780/OCIP5X) ovarian cancer cell lines. Although the peak of ICL formation for the three cell lines was at 12 h, a significant attenuation of ICL formation in SKOV-3 was observed, compared to the chemosensitive lines. Furthermore, ICL levels in SKOV-3 did not return baseline, in contrast to the chemosensitive cell lines. We further investigated the basis of this differential DNA damage response by examining the potential role of nuclear organization,via the comparison of damage induction in nucleoid bodies versus intact cells. The results demonstrated the same rank order of dose-response in nucleoid bodies as observed in intact cells, with chemoresistant cells being considerably more resistant to damage formation, at all doses of cisplatin. When studying gene positioning, we hypothesized that there is a difference in the organization of chromatin in CS and CR ovarian cancer cell lines. The organization of seventeen genes was tested in CR and CS cells. Non-random organization was observed for the majority of genes (47.06%). BRAF and AKT3 were randomly organized in CS and non-randomly organized in CR. A significantly increased copy number was observed in the CR cell line compared to the CS in the majority of the studied genes. The overall data provide the basis for further investigation to improve our understanding of the differential DNA damage response and gene positioning associated with chemosensitive and chemoresistant ovarian cells. Elucidating the mechanisms of chemoresistance may ultimately lead to novel therapeutic interventions that may resensitize or prevent the development of chemoresistance.