Thymidine Metabolism in Cells Treated with DNA‐Suppressing Factor (DSF)

Abstract

Inhibition of thymidine incorporation into DNA in cells treated with DNA-suppressing factor (DSF) has been studied. After 16 hr treatment with DSF, transport of labeled thymidine across the cell membrane was not inhibited, since equilibrium of labeled thymidine with the acid-soluble pool occurred at the same rate and the radioactivity was at the same level as in untreated cells. The values of Vmax and Km in the kinetics of transport of exogenous thymidine were not changed by DSF. Phosphorylation of labeled thymidine to deoxythymidine triphosphate (dTTP) was not inhibited by DSF. After a chase of labeled thymidine, radioactivity of the acid-soluble fraction in DSF-treated cells decreased more rapidly but that of the acid-insoluble fraction remained at a lower level than in untreated cells. It was assumed that DSF might block the entry of dTTP into DNA.As described in our previous paper (6), DNA-suppressing factor (DSF) inhibited cellular DNA synthesis only when added to cells in the early G1-phase, and did not affect the stability of double-stranded DNA or the chain-elongation of singlestranded DNA in HeLa cells. We discussed the mechanisms of the inhibition of DNA synthesis by DSF and suggested the following possibilities: 1) DSF might inhibit transport of exogenous thymidine across the cell membrane, 2) DSF might inhibit phosphorylation of thymidine, or 3) DSF might inhibit the initiation of DNA synthesis. Recently, Hand(3) reported that two mechanisms underlay the inhibition of DNA synthesis in L-929 cells by Newcastle disease virus (NDV) ; NDV inhibited DNA synthesis directly and, in addition, decreased thymidine transport. The present paper describes the metabolism of exogenous thymidine in DSF-treated cells. MATERIALS AND METHODSCell culture. Mouse tumor cells (Meth-A) derived from ascitic tumor induced in a BALB/c mouse by 20-methylcholanthrene was cultivated in suspension in RPMI-1640 medium supplemented with 5% fetal calf serum. As previously described by Yamamoto et al (8), Meth-A cells were more sensitive to DSF than HeLa or NC-37 cells. Therefore, Meth-A cells were used in the present experiments. Meth-A cells were kindly provided by Dr. Kobayashi, Cancer Institute, Hokkaido University.Host DNA-suppressing factor (DSF). The preparation and purification of DSF were described in previous papers (5,8), and a pure preparation (302 unit/mg of 133

The Effect of the DNA‐Suppressing Factor (DSF) on Host DNA Synthesis in Synchronized Cell Cultures

“…Therefore, in the inhibition of host DNA synthesis by DSF, the following steps might be considered: 1) DSF might inhibit the transport of exogeneous thymidine across the cell membrane, 2) DSF might inhibit the phosphorylation of thymidine in the cytoplasmic pool, or 3) DSF might inhibit the initiation of cellular DNA synthesis. These possibilities will be examined in the next paper (5).…”