The primary goal of this proposal is to define (at least in part) the mechanism(s) of tumor progression. To achieve this, we plan to further analyse the role of DNA hypomethylation in the progression of tumors from the benign to the malignant state. Because it is possible that hypomethylation in itself may be insufficient to achieve this transition (i.e., secondary agents likely play a role), the central focus of this proposal is to study the role of genomic destabilization in tumor progression. We believe that agents such as 5-aza-d-Cyd and DFMO, which respectively cause DNA hypomethylation and polyamine depletion, can broadly destabilize DNA and result in augmented genomic injury induced by secondary agents. The agents which we have chosen to provide 'secondary' injury are MNNG (a monofunctional alkylating agent) and m-AMSA, an intercalating agent. The key factor in these studies is that the effects of each of these agents, 5-aza-d-Cyd, DFMO, MNNG and m-AMSA, can be quantitated biochemically. In addition, we have chosen to study tumor cell lines with known characteristics so that specific phenotypic changes induced by each agent alone or in defined combinations can be readily assessed. Four phenotypic characteristics will be analyzed including the generation of drug resistant mutants, metastatic variants, immunogenic variants, and changes in isozymes. Chromosomal rearrangements will also be assessed. These protocols will therefore allow for a systematic analysis (using the parameters outlined) of the role of genomic instability in the generation of phenotypic diversity. A secondary goal of these studies is to address the role of DNA hypomethylation in carcinogenesis. Both an in vivo and in vitro model for potential murine lymphoma/leukemia generation are outlined.
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