Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines.

3'-azido-3'-deoxythymidine (AZT) has been shown to be a potent inhibitor of thymidine kinase 2 in work from this laboratory. Inhibition results in decreased salvage of thymidine to TTP, which may lead to depletion of the TTP pool and result in the mitochondrial dysfunction and mt-DNA depletion observed with AZT toxicity. The effect of AZT on thymidine phosphorylation in growing cells expressing thymidine kinase 1 has not been shown. Three cell lines were used in these experiments: H9c2, derived from rat cardiomyoblasts; U-937, derived from human monocytes; and Raji, derived from human lymphoblasts. AZT inhibited growth in a concentration-dependent manner in U-937 cells, but not the other cell lines. The phosphorylation of [3H]-thymidine or [3H]-AZT was determined during log growth. All cell lines salvaged and phosphorylated thymidine to TTP, with TTP the major product. The U-937 cells had a much more active salvage pathway than the other cells. All cell lines phosphorylated AZT to the triphosphate, but the major product was AZTMP. The AZT inhibition of growth of the U-937 cells did not correlate with levels of the phosphorylated AZT. In contrast, pro-drug AZT was shown to inhibit thymidine phosphorylation in all lines with 50% inhibition concentrations (IC50) ranging from 4.4 to 21.9muM. Since the U-937 cells expressed higher activity of the salvage pathway than the other cell lines, the U-937 cells may rely more heavily on the salvage pathway for TTP synthesis, accounting for AZT inhibition of growth.

The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance.

Homologues of the bacterial ArsA ATPase are found in nearly every organism. While the enzyme is involved in arsenic detoxification in bacteria, the roles of eukaryotic homologues have not been identified. This article reports the function of the Saccharomyces cerevisiae homologue encoded by ARR4 gene (YDL100c ORF). Disruption of ARR4 was not lethal, but the disrupted strain displayed increased sensitivity to As3+, As5+, Co2+, Cr3+, Cu2+ or VO4(3-) salts and temperature. A plasmid-encoded copy of a wild-type ARR4 gene could complement the heat- or metal-related stress responses. Mutation of a codon within the consensus sequence for the nucleotide-binding site resulted in loss of complementation of the disrupted strain and produced a dominant negative phenotype in a wild type strain. Wild type and mutant Arr4p were purified from Escherichia coli. The wild type protein exhibited a low level of ATPase activity, and the mutant was inactive. The purified ATPase eluted as a dimer of 80-kDa species. A fusion of ARR4 and the GFP (green fluorescent protein) gene was constructed. The gene fusion was able to complement stress-related phenotype of the ARR4 disruption. Under non-stress conditions, GFP fluorescence was found diffusely in the cytosol. Under stress conditions GFP was localized in a few punctate bodies resembling late endosomes. It is proposed that under heat or metal stress, the soluble ATPase becomes membrane-associated, perhaps through interaction with a partner protein, and that this complex is involved in stress tolerance.