Inosine monophosphate dehydrogenase (IMPDH) is the rate limiting enzyme in the synthesis of guanine nucleotides from IMP and has been demonstrated to be a critical determinant of overall guanine nucleotide levels in most cell types. Inhibitors of this enzyme have demonstrated usefulness as immunosuppressive agents and may be of use in the chemotherapy of malignancy. The two isoenzymes of IMDH, I and II, are highly similar in amino acid composition and function, but appear to be regulated differently at the molecular level. The overall objective of this proposal is to elucidate the mechanisms regulating the expression of these two isoenzymes and to develop new approaches to the modulation the expression of the two gene products. In addition, we propose to investigate in greater detail the effects of guanine nucleotide depletion on DNA synthesis and cell cycle progression. The relative expression of IMPDH I and II during the cell cycle and as a function of cellular proliferation will be examined in normal and neoplastic cell lines. Genomic clones containing the regulatory regions for the two genes have been isolated and will be characterized in terms of transcriptional elements important for the constitutive expression of the two enzymes, as well as factors responsible for their up-regulation with pharmacologic stimuli and with cell proliferation. To achieve these goals, the structures of the two genes will be determined, DNase I hypersensitive sites will be mapped to detect potential regulatory regions within the genes, and the promoters will be analyzed for similarities and differences in structure and transcription factor binding sites. We will analyze the two expressed and purified IMPDH proteins for differences in structure that could be exploited in the development of new inhibitors. In addition, antisense oligonucleotides will be used to determine whether the expression of IMPDH I and II genes can be differentially inhibited and whether such inhibition affects T lymphocyte proliferation. Finally, we will attempt to gain new insights into the role of guanine nucleotide depletion in inhibiting cellular proliferation by examining the effects of IMPDH inhibitors on factors influencing the progression of cells from 01 to s phase in the cell cycle, including the expression of p53, the retinoblastoma suppressor protein (pRb), and enzymes directly required for initiating DNA synthesis. We anticipate that the results of these experiments will shed further light on both the mechanisms generating increased levels of guanine nucleotides in proliferating cells and the sequelae of reductions in guanine nucleotides for cell growth. These data should be of use in developing new approaches to immunosuppression and chemotherapy based on more selective manipulation of IMPDH expression and guanine nucleotide synthesis.
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