Cells from a wide variety of human tumors lack the ability to undergo apoptosis in response to radiation or drug-induced DNA damage, thus representing a major obstacle to further improvements in cure rates using conventional therapies. Given the substantial evidence linking the aberrant expression of the BCL-2 protein and its pro-survival family members to treatment resistance, efforts to identify cellular targets that could be exploited to disable these central mediators of apoptosis are clearly warranted. The zebrafish, with its close synteny to the human genome and its conserved molecular pathways regulating the development of tissues and organs, offers a powerful tool with which to conduct such research. Thus, after cloning and characterizing the zebrafish bcl-2 gene, we have developed a transgenic zebrafish line in which the rag2 promoter directs the expression of EGFP-bcl-2 in T- and B-lymphoid cells. The superficially localized GFP-positive T cells in this line are easily observed by fluorescence microscopy, and thymocytes in the living rag2-EGFP-bcl-2transgenic fish are resistant to radiation- and dexamethasone-induced apoptosis. This transgenic line will be used in systematic genome-wide screening to identify genes required for bcl-2 and its pro-survival family members to exert their anti-apoptotic effects. The underlying hypothesis is that knowledge of the zebrafish genes able to suppress bcl-2-mediated apoptosis in thymocytes with damaged DNA will implicate novel pathways through which human BCL-2 exerts its anti-apoptotic activity in cancer cells.
In Aim 1, suppressors of bcl-2- mediated resistance to radiation-induced apoptosis in T cells will be identified by screening both (a) a panel of zebrafish lines harboring retroviral insertional mutations in known genes and (b) F1 fish harboring ENU-induced point mutations. The feasibility of this approach has been demonstrated by the discovery of three candidate bcl-2 suppressor mutants in a pilot END mutagenesis screen. Mutations of interest will be analyzed using a rag2-EGFP-mMyc zebrafish model of T- ALL to determine their ability to resensitize bcl-2-expressing leukemic T cells to radiation-induced cell death.
In Aim 2, functional and molecular analyses will be used to investigate the mechanisms by which each mutant gene (selected by rigorous criteria) suppresses the function of bcl-2 and/or one or more of its pro-survival family members. Importantly, these studies will complement ongoing research to understand Bcl-2 function in mice and other mammalian systems. Our long-term goal is to restore normal cell death pathways in cancer cells by targeting pivotal molecules with small molecule inhibitors or antibodies, thus enhancing the effects of therapy with radiation and genotoxic drugs.
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