Inhibitors of purine nucleoside phosphorylase (PNP) have been sought as specific lympholytic agents because of the association of T cell deficiency states with inherited deficiency of PNP enzyme acitivity. The depletion of T cells in this disorder has been attributed to the selective metabolism of the PNP substrate, 2'-deoxyguanosine, to dGTP. Thus, analogs of 2'-deoxyguanosine which are not substrates for PNP are also of potential utility as immunosuppressive agents and in the chemotherapy of T cell lymphoproliferative disorders. We propose to utilize 9-Beta-D-arabinosylguanine (ara-G), an analog of 2'-deoxyguanosine which is not readily degraded by PNP activity, to sort out the metabolism and selective toxicity of 2'-deoxyguanosine for T cells. We will a) examine the sensitivity of fresh and cultured leukemic cells to ara-G; b) determine the effects of this drug on mature proliferating T4 and T8 cell populations, as well as on mixed lymphocyte populations; c) determine the relative roles of two 2'-deoxyguanosine phosphorylating enzymes in ara-G phosphorylation; d) isolate T lymphoblast cDNA clones corresponding to these phosphorylating enzymes; and e) further examine the tissue specificity of kinase expression at the mRNA level. We will also examine the metabolic sequelae of partial inhibition of PNP activity induced by weak inhibitors of the enzyme. Recent studies have documented that partial inhibition of PNP ativity in cultured lymphoid cells can result in increases in guanine ribo-, rather than deoxyribonucleotide pools. Guanine ribonucleotide accumulation inhibits cell growth and may have major relevance for the clinical use of these inhibitors. We therefore intend to study the effects of elevations and depletions in guanine ribonucleotide pools on lymphoid and leukemic cell viability. We will extend these studies to further investigation of the effects of guanine ribonucleotide depletion and cell differentiation. These studies should elucidate some of the biochemical effects of perturbations in guanine ribo- and deoxyribonucleotide pools on immature and mature lymphoid cells and should assist in the development of clinically useful lympholytic agents.
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