Abstract
N1-S1/FdUrd Novikoff hepatoma cells, which lack thymidine kinase activity, are resistant to 5-fluorouracil (FUra) as well as 5-fluorodeoxyuridine (FdUrd), suggesting that the pathway, FUra leads to FdUrd leads to FdUMP, is utilized for the conversion of FUra to FdUMP. However, the inhibition of thymidylate synthetase activity, the presumed target of FdUMP, by 1 X 10(-4) M FUra in intact N1-S1 Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 X 10(-4) M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase. These results imply that the formation of ribonucleotides and does not involve thymidine kinase. This apparent dichotomy can be explained by the fact that, in addition to the well known lack of thymidine kinase activity, [14C]FUra conversion to ribonucleotides is greatly depressed in the N1-S1/FdUrd cells. Hence, the formation of FdUMP from FUra in Novikoff hepatoma cells apparently proceeds primarily via the intermediate formation of ribonucleotides. The decreased conversion of FUra to ribonucleotides in N1-S1/FdUrd cells decreases not only the ability of the analog to inhibit DNA synthesis, but also its effect on RNA metabolism. FUra, at a concentration of 1 X 10(-5) M, inhibits rRNA maturation in N1-S1 cells, but not in N1-S1/FdUrd cells. Since N1-S1/FdUrd cells are completely resistant to 1 X 10(-5) M FUra, whereas N1-S1 cells are completely inhibited by 1 X 10(-5) M FUra, even in the presence of 1 X 10(-4) M thymidine, the effects of FUra on RNA metabolism appear to contribute significantly to the cytotoxicity of the analog at higher drug concentrations.
Highlights
PROCEDURESNovikoff hepatoma was originally induced as a solid tumor in rats fed 4-dimethylaminoazobenr.ene [13]
FdUMP, by 1 x 10m4M FUra in intact Nl-Sl Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 x 10m4M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase
Novikoff hepatoma cells were incubated for 1 h with [6
Summary
Novikoff hepatoma was originally induced as a solid tumor in rats fed 4-dimethylaminoazobenr.ene [13]. Suspension cultures of the Novikoff cells were grown at 37” in Medium S-69 as described previously [15]. Incorporation of Radioactive Nucleosides- Cells were grown to mid- or late log phase density (0.4 to 1.5 x lo cells/ml), harvested at room temperature by centrifugation (500 x g, 20 min), and resuspended in one-fifth the original volume of fresh Medium S-69. Upon completion of the experimental incubations, which were performed at 37”, the cells were immediately chilled in an ice bath and harvested by centrifugation (500 x g, 5 min) at 4”. Incorporated into Acid-soluble, RNA, and DNA Fructions- Cell pellets, containing approximately. After 10 min, the precipitate was removed by centrifugation and washed twice with 1 ml of 0.3 N perchloric acid. Mmol) was supplied by Moravek Biochemicals (City of Industry, Cal.)
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