UBC Theses and Dissertations
An investigation of the G2 checkpoint Curman, Darko
Current approaches to treating cancers often involve the use of drugs that are toxic to both cancer cells and normal cells. Over the past decade, advances have been made in understanding the genetic changes underlying the transformation of normal cells into cancerous cells that has led to treatment models that would selectively target the cancer cells. It has been found that cancer cells with mutations in p53 that are exposed to DNA damage are hypersensitive to agents that inhibit the G2 checkpoint. To find novel G2 checkpoint inhibitors, mitotic ELISA assays were used to screen 1500 marine bacterial and sponge extracts. Four G2 checkpoint inhibitors were isolated and identified: staurosporine and its oxazolidone derivative, debromohymenialdisine and isogranulatimide. It was found that debromohymenialdisine and isogranulatimide induce entry into mitosis and this is dependent on the activation of cdc25 phosphatase and cdc2 kinase. To gain a better understanding of the G2 checkpoint pathway, attempts were made to identify the in vivo targets of debromohymenialdisine and isogranulatimide by affinity chromatography, overlay assays and immunocytochemistry, without success. However, in vitro kinase assays have shown that debromohymenialdisine and isogranulatimide potently inhibit the kinase GSK-3β. Further studies involving β-catenin, a key downstream component of GSK-3β, provided evidence that the compounds inhibit GSK-3β in vivo as well. The results suggest that GSK-3β and β-catenin may play a yet unidentified role in the G2 checkpoint pathway.