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Role of Substrate Depletion in the Inhibition of Thymidylate Biosynthesis by the Dihydrofolate Reductase Inhibitor Trimetrexate in Cultured Hepatoma Cells¹

Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509 [M. S. R., M. B., G. F. M., F. M., J. G.], and Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29424 [M. B., D. G. P.]

The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH₂H₄PteGlu_n) from 2.0-0.2 µM, respectively. This is accompanied by an increase in H₂PteGlu_n from 1.2 µM in control cultures to 4.7 µM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH₂H₄PteGlu_n in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH₂H₄PteGlu_n in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH₂H₄PteGlu_n) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H₂PteGlu_n for thymidylate synthase (K_i = 5.0 µM) suggest that H₂PteGlu_n accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.

¹ This work was supported by NIH grants CA22754 (D. G. P.), CA25933 (J. G.), CA44355 (F. M.), and CA46126 (J. G.) and National Science Foundation grant DMB 86-16273 (G. F. M.).

² To whom requests for reprints should be addressed, at Wadsworth Center, NY State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509.

Received 11/ 2/89. Revised 2/26/90.

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