Tiazofurin decreases Ras-GTP complex in K562 cells.
The ras oncogene product (p21ras, Ras) is a GTP-binding protein and is thought to transduce signals regulating cellular proliferation and differentiation. The active form Ras-GTP is inactivated by hydrolyzing bound GTP to GDP. Tiazofurin, a specific inhibitor of IMP dehydrogenase, decreased cellular GTP pools and downregulated c-ras gene expression, leading to differentiation (Olah, E. et al., Proc. Natl. Acad. Sci. USA 85: 6533-6537, 1988; Weber et al., Cancer Commun. 3:61-66, 1991). To clarify the link between the action of tiazofurin on metabolic alterations and the induction of differentiation, we examined the effect of tiazofurin on the ratio of active Ras-GTP to total Ras in K562 cells in culture. Cells were labeled for 6 h with [32P]Pi in phosphate-free RPMI 1640. Tiazofurin (100 or 200 microM) was added to cells, and samples were taken at 0, 2, 4, 6 and 12 h of incubation. Cell lysates were immunoprecipitated with monoclonal anti-p21 antibody (Y13-259), then developed on thin layer chromatography. GTP and GDP bound to Ras were visualized by autoradiography. Tiazofurin treatment decreased Ras-GTP concentration in a time- and dose-dependent fashion. In the untreated K562 cells the Ras-GTP concentration was 26.3 +/- 1.4, and tiazofurin (200 microM) decreased it at 6 h to 16.6 +/- 2.9 and at 12 h to 10.6 +/- 2.1%. Inhibition of the GTP salvage pathway with hypoxanthine (100 or 200 microM) enhanced the tiazofurin-induced decrease of Ras-GTP, whereas addition of guanosine (100 microM) prevented the Ras-GTP decrease.(ABSTRACT TRUNCATED AT 250 WORDS)
Hata
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Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis 46202-5200.
Natsumeda
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Y
Weber
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G
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D006151:Guanosine / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism* / Q000473:pathology; D015689:Oncogene Protein p21(ras) / Q000187:drug effects* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012254:Ribavirin / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D007042:Hypoxanthines; D006151:Guanosine; D006153:Guanosine Diphosphate; D019271:Hypoxanthine; D012254:Ribavirin; D006160:Guanosine Triphosphate; D015689:Oncogene Protein p21(ras); C033706:tiazofurin