tiazofurin and Melanoma

A human melanoma cell line (MM96L) had a spontaneous mutation rate at the HGPRT locus of approx. 7 times normal. The cells had elevated dATP and dGTP pools, lacked purine nucleoside phosphorylase (PNP) and were sensitive to killing by deoxyadenosine, deoxyinosine and related purines but not to inosine or hypoxanthine. Four other melanoma cell lines exhibited a range of nucleoside sensitivities and dNTP pool sizes. Failure of intact MM96L cells to degrade exogenous deoxyadenosine and deoxyinosine to hypoxanthine was confirmed by NMR of culture medium. Normal melanocytes were PNP+ and were insensitive to deoxyinosine. Comparison of the metabolites of [14C]deoxyinosine from MM96L and a PNP+ cell line of similar doubling time (HeLa) showed that both cell types produced 14C-labelled guanine and adenine nucleotides, with [14C]dATP and [14C]dADP being found in MM96L. This indicates that human sAMP synthetase or a similar enzyme catalyses the conversion of dIMP to dAMP, the resultant elevation of dATP causing base misincorporation and a mutator phenotype.

Topics: Alanine; Antibiotics, Antineoplastic; Antineoplastic Agents; Cell Survival; Chromatography, High Pressure Liquid; Deoxyadenosines; Deoxyribonucleotides; HeLa Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Inosine; Magnetic Resonance Spectroscopy; Melanoma; Mutation; Ribavirin; Thioguanine; Tumor Cells, Cultured

Anti-tumor effects of agents known to intervene with signal transduction pathways (ras and protein kinase c cascades) were examined in the B16 melanoma cell model. The compounds examined included: lovastatin, an inhibitor of HMG-CoA reductase, which interferes with membrane localization of p21 ras protein; H-7, a classic inhibitor of protein kinase C; and tiazofurin, a GTP depleting agent, that might affect the GTP/GDP ratio on p21ras. The three agents were found to inhibit the proliferation of B16 melanoma cells. Only tiazofurin, as expected, induced a significant decrease in GTP levels. Lovastatin and H-7 altered p21 subcellular localization. They reduced membrane expression of p21 ras, while increasing its expression in the cytosol. Following tiazofurin treatment a trend towards increased membranal p21 was observed. These results suggest that p21 is a target for the action of signal transduction inhibitors. However, the relationship between growth inhibition and altered p21 expression is not yet clear.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Antineoplastic Agents; Cell Division; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Lovastatin; Melanoma; Proto-Oncogene Proteins p21(ras); Ribavirin; Signal Transduction; Tumor Cells, Cultured

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.

Topics: Biomarkers; Gene Expression Regulation, Neoplastic; Guanosine; Humans; IMP Dehydrogenase; Melanins; Melanoma; Monophenol Monooxygenase; Mycophenolic Acid; Neoplasm Proteins; Ribavirin; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured