Abstract
Incubation of cultured human fibroblasts in 1 µM methotrexate results in a progressive accumulation of poly-γ-glutamyl derivatives. This accumulation is not dependent on new protein synthesis. No polyglutamate derivatives are detectable until saturation by nonmetabolized methotrexate of a nonexchangeable intracellular fraction occurs. After saturation occurs both methotrexate and methotrexate polyglutamates are found in a freely-exchangeable and a non-exchangeable fraction. After saturation occurs all of the increase in the size of the nonexchangeable fraction is due to the accumulation of polyglutamates. Cells which have been incubated for up to 2 hr in 1 µM methotrexate rapidly regain the ability to incorporate deoxyuridine into DNA when transferred into methotrexate-free medium. With longer times of incubation resulting in the formation of longer and greater concentrations of polyglutamates, deoxyuridine incorporation is inhibited at least 24 h following transfer into methotrexate-free medium. In contrast, thymidine incorporation into DNA is increased over that seen in cells not incubated in methotrexate. We suggest that the metabolism of methotrexate in cultured human fibroblasts occurs according to the following steps: (i) saturation by nonmetabolized methotrexate of a nonexchangeable pool, (ii) addition of γ-glutamyl residues to methotrexate in a freely-exchangeable pool, (iii) appearance of methotrexate polyglutamates in a nonexchangeable pool. The inhibition of deoxyuridine incorporation in the absence of continued methotrexate may be due to the effects of previous sustained exposure to methotrexate, to the total quantity of methotrexate derivatives, or to some undefined differential effect of methotrexate polyglutamates.
ACKNOWLEDGMENTS We thank C. M. Baugh and M. G. Nair for the kind provision of methotrexate polyglutamates. We are also grateful to Huguette Ishmael and Lynne Prevost for assistance in the preparation of the manuscript.
- Copyright © 1978 by Academic Press, Inc.
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