guanosine-triphosphate and 3-deazaguanine

3-Deazaguanosine containing a 14C label in the ribose moiety was prepared using [U-14C]inosine as the [14C] ribose donor and commercial purine-nucleoside phosphorylase (EC 2.4.2.1) both to degrade the inosine, in the presence of phosphate, and to synthesize [14C-ribosyl]3-deazaguanosine in reduced phosphate and an excess of 3-deazaguanine. Purification was by high-pressure liquid chromatography (HPLC). [14C-ribosyl]3-Deazaguanosine was metabolized by Chinese hamster ovary cells to two metabolites, one major and one minor, eluting in the triphosphate region after HPLC analysis, and appeared to be incorporated into perchloric acid-insoluble material. Cell line TGR-3, deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and resistant to 3-deazaguanine, also formed both metabolites. Line TGR-1/DGRR-9, deficient in hypoxanthine-guanine phosphoribosyltransferase and resistant to both 3-deazaguanine and 3-deazaguanosine, formed greatly reduced levels of the major metabolite. 3-Deazaguanosine 5'-triphosphate, prepared enzymically from authentic 3-deazaguanosine 5'-monophosphate, co-eluted with the major metabolite peak during HPLC analysis. Treatment of a metabolite-containing extract with bacterial alkaline phosphatase (EC 3.1.3.1) resulted in the formation of 3-deazaguanosine. These observations indicate that 3-deazaguanosine can be metabolized, in Chinese hamster ovary cells, to the triphosphate derivative in lieu of the action of hypoxanthine-guanine phosphoribosyltransferase.

Topics: Alkaline Phosphatase; Animals; Cell Line; Chromatography, High Pressure Liquid; Cricetinae; Cricetulus; Female; Formates; Guanine; Guanosine; Guanosine Triphosphate; Hypoxanthine Phosphoribosyltransferase; Inosine; Ovary; Thymidine

3-Deazaguanine, a tumor-inhibitory purine antimetabolite, is cytotoxic to L1210 leukemic cells in culture. The log percentage of viability correlated strongly (r2 = 0.986) with the product of the concentration of 3-deazaguanine, or [3-deazaguanine], and period of exposure (t) when [3-deazaguanine] was between 3 and 50 microM, and t was 12 or 24 h. We wished to relate this cytotoxicity to biochemical effects mediated by 3-deazaguanine. 3-Deazaguanine inhibited both DNA and protein synthesis, and both log DNA synthesis and log protein synthesis correlated inversely with [3-deazaguanine] X t and directly with cell viability (P less than 0.001). L1210 cells accumulated 3-deazaguanine 5'-triphosphate to a level of 1.5 nmol/10(6) cells. 3-Deazaguanine treatment had no effect on intracellular cytidine 5'-triphosphate levels, but reduced adenosine 5'-triphosphate and uridine 5'-triphosphate levels by 40% relative to control and guanosine 5'-triphosphate levels by 85% relative to control at a [3-deazaguanine] X t value at which 3-deazaguanine 5'-triphosphate accumulation was near maximal. Incorporation of 2-14C-labeled 3-deazaguanine into DNA and RNA, separated by Cs2SO4 density gradient centrifugation, was demonstrated. Incorporation into DNA was linear versus [3-deazaguanine] X t and correlated inversely with cell viability (P less than 0.001). These data suggest that 3-deazaguanine is anabolized and incorporated into DNA, and that this incorporation is related to decreased DNA synthesis and cell death. The decrease in protein synthesis and diminution of guanosine 5'-triphosphate levels following drug treatment may also contribute to the growth-inhibitory actions of 3-deazaguanine.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Line; Cell Survival; DNA, Neoplasm; Guanine; Guanosine Triphosphate; Leukemia L1210; Neoplasm Proteins; RNA, Neoplasm