In vitro formation of polyglutamyl derivatives of methotrexate and 7-hydroxymethotrexate in human lymphoblastic leukemia cells.

Abstract

The intracellular synthesis of polyglutamyl derivatives of both methotrexate (4-amino-N-10-methylpteroylglutamic acid) and 7-hydroxymethotrexate, the primary plasma metabolite of methotrexate in humans, was evaluated in a methotrexate-sensitive, acute lymphoblastic leukemia cell line, MOLT 4. These studies were performed using a highly specific ion-pairing high-pressure liquid chromatography method which permits the simultaneous determination of methotrexate, 7-hydroxymethotrexate, and their corresponding polyglutamyl derivatives. When MOLT 4 cells were exposed to 1 microM methotrexate, the monoglutamate attained a steady state after 30 min, and polyglutamyl derivatives having from one to 4 additional glutamyl residues were observed over 4 hr. Four additional metabolites were also detected upon incubation with 1 microM 7-hydroxymethotrexate. On the basis of the retention times for these compounds relative to methotrexate polyglutamyl standards and since these metabolites reverted to 7-hydroxymethotrexate upon treatment with a preparation of hog kidney conjugase, they were identified as polyglutamyl derivatives of 7-hydroxymethotrexate. The identification of 7-hydroxymethotrexate polyglutamyl derivatives in vitro raises the possibility of an important new dimension in the pharmacological action of methotrexate. We investigated the effect of extracellular 7-hydroxymethotrexate on net methotrexate uptake and metabolism when cells were exposed simultaneously to 1 microM [3H]-methotrexate and unlabeled 7-hydroxymethotrexate. A decrease in the levels of both intracellular methotrexate and the corresponding polyglutamyl derivatives was noted for cells treated with 1 or 10 microM 7-hydroxymethotrexate. However, no appreciable effect of 7-hydroxymethotrexate on the amount of polyglutamyl derivatives formed relative to the total intracellular antifolate was noted. These studies show that in MOLT 4 cells (a) both methotrexate and 7-hydroxymethotrexate are rapidly converted to polyglutamyl derivatives, and (b) 7-hydroxymethotrexate interferes with net methotrexate accumulation and metabolism when present simultaneously in the extracellular medium. These results, moreover, suggest a potential role for 7-hydroxymethotrexate in modulating the biochemical effects of methotrexate in vivo.

• Authors: G Fabre, L H Matherly, R Favre, J Catalin, J P Cano
• Journal: Cancer research (Cancer Res) Vol. 43 Issue 10 Pg. 4648-52 (Oct 1983) ISSN: 0008-5472 [Print] United States
• PMID: 6192907 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Peptides
• Polyglutamic Acid
• methotrexate polyglutamate
• 7-hydroxymethotrexate
• Methotrexate

Topics

• Cell Line
• Chromatography, High Pressure Liquid
• Humans
• Leukemia, Lymphoid (metabolism)
• Methotrexate (analogs & derivatives, metabolism)
• Peptides (metabolism)
• Polyglutamic Acid (analogs & derivatives, metabolism)