| The biological functions of the tumor suppressor gene, ING1, have been studied extensively in the last few years since it was cloned. Four alternatively spliced forms of ING1, named p47 ING1, p33ING1, p27 ING1, and p24ING1, have been identified and found to share many biological functions with those of p53. Some of these isoforms have previously been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, and chemosensitivity. Functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. In this thesis, we sought to characterize further a number of important biological functions of the p33ING1 isoform. We first investigated how the expression of ING1 is regulated in normal and stress conditions. Using a p53-knockout mouse model and various cell lines differing in p53 status and cell type, we found that the expression of p33ING1 is independent of p53 status and induced by ultraviolet (UV) irradiation in a dose-time-dependent and tissue-specific manner. These findings subsequently prompted us to investigate if p33ING1 plays a role in UV-stress response, such as repair of UV-damaged DNA. Using both in vitro (host-cell-reactivation assay) and in vivo (radioimmunoassay) methods, we found that p33ING1 enhances the repair of UV-damaged DNA in collaboration with p53 in melanoma cells and that GADD45 may participate in the process. Next we investigated the molecular pathways of p33ING1 enhancement in UV-induced apoptosis in melanoma cells using various survival and apoptotic assays. We demonstrated that overexpression of p33ING1 increases the apoptotic rate in melanoma cells after UV irradiation and that p53 has a synergistic effect on this process. Moreover, we found that p33 ING1 enhances the expression of endogenous Bax and alters mitochondrial membrane potential, suggesting that p33ING1 cooperates with p53 in UV-induced apoptosis via the mitochondrial cell death pathway in melanoma cells. Lastly, we examined the role of p33 ING1 isoform in melanoma chemosensitivity because previous findings indicate that the isoform p24ING1 is capable of enhancing chemosensitivity in human fibroblasts. Using a number of survival and apoptotic assays to quantitate cell death in melanoma cells, we showed that neither overexpressing p33ING1 alone nor coexpression of p33ING1 and p53 had an effect on the frequency of cell death induced by the chemodrug, camptothecin. We therefore demonstrated that p33ING1 does not enhance camptothecin-induced cell death in melanoma cells. In conclusion, we have elucidated in this thesis some of the novel functions of p33 ING1 and the importance of this gene in the context of tumor suppression. |
