Deoxythymidine catabolism in cultured mammalian cells
Thymidine catabolism has been extensively studied in rat liver. Thymidine is degraded to β-aminoisobutyric acid and CO2 via thymine, dihydrothymine and β-ureidoisobutyric acid in for example liver, kidney, human leukocytes and mouse spleen lymphocytes. The main aim of this project was to investigate thymidine catabolic activity in tissue culture cells, which were hitherto all thought to have only negligible catabolic activity. Lysates prepared from various cell types were assayed for thymidine phosphorylase activity, the enzyme which catalyses the first step in the catabolic pathway; the conversion of thymidine to thymine. Mouse LM cells and Friend cells showed thymidine phosphorylase activity whereas F2K, BHK, PyY, Rat 1 and Vero cells did not appear to have any activity. LM cells were studied further to see if there was any variation in catabolic activity during the cell cycle as had been suggested previously in other systems. The cells were synchronized using the serum step-up method and incubated with [3H] thymidine. At various times after the serum addition, PCA soluble material was extracted from the cells and analysed by high pressure liquid chromatography. The 3H label could be traced to dTTP, dTDP and dTMP at all times after serum step-up and to thymine at 8, 12 and 24h after step-up. DNA synthesis commenced in these cells at 8h after step-up arid therefore catabolism was occurring concomitantly with DNA synthesis. This was contrary to the results of Usher and Reiter (1977) who observed thymidine degradation at stages of the cell cycle outwith DNA synthesis in concanavalin A stimulated mouse spleen lymphocytes. Studies were also carried out on HSV-1 infected BHK cells concerning thymidine metabolism in the presence and absence of viral DNA synthesis. The drug phosphonoecetate, while inhibiting viral DNA synthesis, had no effect on the uptake and phosphorylation of [3H] thymidine. About 20% of the intracellular phosphorylated derivatives of [3H] thymidine were excreted from the cells in the presence of phosphonoacetate while no such excretion occurred in the drugs absence. The fate of radiolabelled host cell DNA was also studied. In both the presence and absence of viral DNA synthesis about 50% of the host cell DNA was rendered acid-soluble and most of this was excreted from the cell. This would suggest that the products of host cell DNA degradation are not available for viral DNA synthesis, and was supported by the absence of radiolabel in viral DNA. These results may provide support for the existence of multiple nucleotide pools, where one pool supplies precursors for DNA replication while other pool(s) are not directly available for DNA synthesis.