Synthesis and properties of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells in vitro

G. Fabre, I. Fabre, L. H. Matherly, J. P. Cano, I. David Goldman

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Abstract

The synthesis of poly-γ-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-4-NH2-10-CH3-pteroyl-glutamic acid (PteGlu1)) was evaluated by direct hydroxylation of the tetraglutamyl derivative of methotrexate (4-NH2-10-CH3-PteGlu4) by a cell-free preparation of rabbit liver aldehyde oxidase and by polyglutamylation of 7-OH-methotrexate in Ehrlich ascites tumor cells in vitro. The polyglutamyl derivatives of 7-OH-methotrexate rapidly accumulate in cells to the 7-OH-4-NH2-10-CH3-PteGlu4. While 7-OH-methotrexate monoglutamate does not bind dihydrofolate reductase, 7-OH-4-NH2-10-CH3-PteGlu4 does bind to the enzyme as established by gel filtration analysis of cell extracts and by use of purified dihydrofolate reductase from Ehrlich cells. Within cells, the rate of formation of 7-OH-methotrexate polyglutamyl derivatives exceeds that for methotrexate by a factor of 2.7 at comparable free monoglutamyl substrate levels, suggesting that 7-OH-methotrexate may be a better substrate than methotrexate for the folylpolyglutamate synthetase. 7-OH-methotrexate slows the rate of methotrexate polyglutamylation in cells, a consequence of the inhibition of methotrexate transport with reduced methotrexate substrate available for polyglutamylation. When 7-OH-methotrexate polyglutamyl derivatives were accumulated inside the cells following which extracellular 7-OH-methotrexate was removed, the monoglutamate, and to a lesser extent the diglutamate, exited the cells whereas the majority of the longer polyglutamyl derivatives were retained and continued to be metabolized to higher forms. These studies suggest that 7-OH-methotrexate and its polyglutamyl derivatives may play a role in modulating methotrexate action, either by their own inhibitory effects on folate-dependent enzymes or by their effects on methotrexate transport and metabolism within cells.

Original languageEnglish (US)
Pages (from-to)5066-5072
Number of pages7
JournalJournal of Biological Chemistry
Volume259
Issue number8
StatePublished - 1984
Externally publishedYes

Fingerprint

7-hydroxymethotrexate
Ehrlich Tumor Carcinoma
Tumors
Cells
Derivatives
Methotrexate
Tetrahydrofolate Dehydrogenase
hydroxide ion
In Vitro Techniques
Substrates
Aldehyde Oxidase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Synthesis and properties of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells in vitro. / Fabre, G.; Fabre, I.; Matherly, L. H.; Cano, J. P.; Goldman, I. David.

In: Journal of Biological Chemistry, Vol. 259, No. 8, 1984, p. 5066-5072.

Research output: Contribution to journalArticle

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AU - Goldman, I. David

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