Rational design of cancer treatment protocols using chemotherapeutic agents has been hampered by the fact that this group of toxic drugs has a wide range of effects on both normal and transformed cells. Although the specific biochemical mechanisms of action of many of these drugs are understood, their broader effects on cellular processes remain unclear. The action of antimetabolites, which inhibit specific enzymes required for DNA or RNA synthesis, can be abrogated by increased production of the target enzymes. We have used the dihydrofolate reductase (DHFR) gene as a model system to demonstrate that treatment of cells with methotrexate (MTX) and with other antimetabolites stimulates DHFR transcription in a transient transfection assay (Eastman, et al., Proc. Natl. Acad. Sci., USA, In Press). We plan to extend these studies by examining rates of transcription of endogenous genes in response to antimetabolites. To further explore the response, we will determine whether the effect is unique to the DFW promoter, we will study the effects of other drugs (e.g. fluorodeoxyuridine, hydroxyurea, and 2'deoxycoformycin) on both DHFR and their respective target genes (thymidylate synthetase, ribonucleotide reductase, and adenosine deaminase) by nuclear run-on assays and by promoter driven expression in transient transfection assays. The relationship between stimulated transcription and a well-known cause of drug resistance, gene amplification, will also be examined. We propose to determine the mechanism whereby MTX stimulates DHFR transcription by studying relevant transcription factors and their interactions with the DHFR promoter and with the cellular transcription machinery. We would then extend our findings to other antimetabolites and their target genes. Our preliminary work shows that pre-treatment of cells with cisplatin prevents the stimulatory effect of MTX on the DHFR promoter. On the basis of these preliminary data, we propose to test several hypotheses concerning the mechanism of cisplatin action that may abrogate the increased transcription caused by MTX. The proposed research plan will enhance our understanding of drug resistance and cross-resistance, and will thereby lead to the development of more effective protocols for the treatment of cancers with these agents.
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