The proposed studies will seek to determine whether antitumor drugs exert direct actions on the phospholipase C second messenger system. Stimulation of phospholipase C leads to elevation of Ca++ and activation of protein kinase C, effects that are intimately involved with the regulation of proliferation, differentiation and immune responses. Signal transduction from agonist-stimulated receptors to phospholipase C is mediated by GTP-binding and hydrolyzing protein(s). Antimetabolites that deplete cellular GTP, i.e., selenazofurin and mycophenolic acid, or that form GTP analogs, i.e., 6-thioguanine, 3-deazaguanine and 8-azaguanine, may interfere with the activity of this class of stimulatory G protein(s). Agents that deplete CTP, such as pyrazofurin and 3-deazauridine, and 5-azacytidine, which forms 5- azaCTP, may interfere with synthesis of the CDP-diacylglycerol precursor of phosphatidylinositol. Our preliminary results show that selenazofurin reduces the agonist-stimulated inositol phosphate levels of proliferating and monocyte-induced HL-60 cells. The actions of these antimetabolites on the agonist- stimulated release of radiolabeled inositol phosphates, diacylglycerol and glucosamine will be determined by Dowex column chromatography, HPLC and TLC methods. Transport and metabolic uptake of (3H)inositol will be measured in proliferating and differentiated cells in the presence of CTP modulators. The response of the phospholipase C system to various putative growth factors, hormones and lymphokines will be characterized initally in the HL-60 cell line, using proliferating cells as well as cells that have been induced to mature toward graulocytic or moncytic phenotypes with dibutyryl cAMP, phorbol ester and 1 alpha, 25=dihydroxyvitamin D3. Later studies will compare the HOL-60 cell response with those of other types of inducible human leukemia cells. Drugs that alter basal or agonist-stimulated second messenger levels will be tested for effects on primary cellular responses such as ornithine decarboxylase activation in proliferating cells and superoxide anion production in differentiated cells. These studies may provide insights into the cytotoxic, differentiation-inducing and immunosuppressive effects of these antimetabolites and suggest ways to overcome host toxicities or enhance therapeutic efficacy.
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