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Pyrimidine Metabolism in Tissue Culture Cells Derived from Rat Hepatomas

II. Thymidine Uptake in Suspension Cultures Derived from the Novikoff Hepatoma¹

( McArdle Memorial Laboratory Medical Center, University of Wisconsin, Madison, Wisconsin)

Novikoff tissue culture cells were made totally dependent on thymidine supplied via the medium, by blocking with amethopterin. Radioactive thymidine then was supplied in various molarities, and after incubation periods of varying length the cultures were fractionated into medium, intracellular acid-soluble compounds, and deoxyribonucleic acid (DNA), and the fractions assayed for radioactivity. Several observations were made.

a) The cells rapidly concentrated thymidine from the medium into the acid-soluble fraction, reaching equilibrium in less than 10 min.

b) Radiochromatographic studies showed that the intracellular thymidine was largely (80–90%) in the form of thymidine triphosphate, with some (10–20%) thymidine diphosphate and little, if any, thymidine monophosphate or thymidine.

c) Increasing the concentration of thymidine in the medium caused an increase in the intracellular concentration of thymidine compounds.

d) For cell multiplication, the optimal intracellular concentration of thymidine triphosphate was roughly 5–10 x 10^-5 M. This was inferred from growth rates in various external concentrations of thymidine, and the relationship of extracellular to intracellular concentrations of thymidine and thymidine compounds.

e) While the over-all rate (but not onset) of DNA synthesis of Novikoff tissue culture cells can be modified by limiting the intracellular thymidine triphosphate concentration, the importance of this mechanism in vivo remains to be demonstrated.

¹ This work was supported in part by grant CA-00646 from the National Cancer Institute, NIH, USPHS.

² Present address: Department of Microbiology, The University of Mississippi Medical Center, Jackson, Miss.

³ Present address: Department of Biochemistry, The Ohio State University, Columbus, Ohio.

Received 6/29/64. Revised 12/11/64.