DNA damaging anticancer agents induce arrest at various checkpoints throughout the cell cycle. This protective mechanism allows cells time to repair damage before progressing. UCN-01 (7-hydroxy-staurosporine) was identified as a potent inhibitor of the DNA damage-induced S and G2 arrest, thereby causing a marked enhancement in cell killing. UCN-01-mediated abrogation of normal cells is prevented by the p53 tumor suppressor protein. Thus, UCN-01 may selectively enhance chemotherapy in the tumor while sparing normal tissue. However, some p53-defective tumors are also resistant to UCN-01, while some p53-wildtype tumors are sensitive. This leads to the major question in this proposal: what are the determinants of response to checkpoint inhibitors? Aim 1 will focus on p53-defective tumor cell lines and investigate the role of Chk1 (inhibited by UCN-01) and other checkpoint kinases in arresting cell cycle progression. The response of various cell lines to different checkpoint inhibitors will be assessed. Three resistant tumor cell models will be analyzed for alternate kinases that explain their resistance to Chk1 and Chk2 inhibitors;candidate kinases include hSAD1, PLK3, MAPKAPK2 and JNK. To confirm the role of each kinase in checkpoint regulation, cell lines will be generated in which the kinase expression is prevented by siRNA.
Aim 2 will address the question as to why some p53-wildtype tumors retain sensitivity to Chk1 inhibitors despite the fact that non-tumorigenic lines are resistant. Recent results demonstrate that p53 regulates the checkpoint through both gene activation (p21waf1) and repression (cyclin B) and that regulation of both of these proteins is defective in UCN-01-sensitive p53 wildtype tumors;p21 fails to be induced during S phase arrest, while cyclin B fails to be repressed during G2 arrest. The transcriptional and post-translational regulation of these two proteins will be studied and contributors to their differential regulation assessed. Experimental approaches will include dissection of the pathways through promoter analysis and chromatin immunoprecipitation assays. As novel checkpoint inhibitors enter clinical trial, the results of these studies will provide a basis upon which to stratify patients and thereby enhance the probability of developing a successful therapeutic regimen. For those tumors in which a response is not indicated, these experiments will likely identify alternate targets for drug discovery.
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