Glutamylation of methotrexate in hepatoma cells in vitro: regulation and the development of specific inhibitors.
Methotrexate is glutamylated in cultured hepatoma cells to derivatives that contain a total of 2 to 5 gamma-glutamyl residues. The rate of polyglutamate formation and extent of accumulation are saturable with respect to both medium concentration of methotrexate and time. Maximal rates of glutamylation and accumulation of methotrexate polyglutamates at steady state occur at approximately 10 microM extracellular methotrexate. Inclusion of physiologic concentrations of insulin or removal of folate from the medium each cause a doubling of the rate of glutamylation, and these effects are additive. Insulin and folate restriction also enhance the accumulation of methotrexate polyglutamates. In combination they result in a doubling in the intracellular methotrexate polyglutamate pool at steady state and a shift in the polyglutamate distribution to longer-chain-length species. The importance of the longer-chain-length polyglutamates is apparent from the 6-hr retention of the polyglutamate species: Glu2, 15%; Glu3, 21%; Glu4, 50%; and Glu5, 83%. In probing the glutamylation reaction, a new series of inhibitors have been initiated. These are based upon replacing the incoming glutamate with 4-fluoroglutamate or synthesizing methotrexate with the glutamate replaced by 4-fluoroglutamate. The 4-fluoroglutamyl analogs of methotrexate are effective inhibitors of dihydrofolate reductase but cannot be glutamylated. They will be utilized to probe the role of glutamylation in antifolate activity and folate metabolism.
Galivan
J
J
Nimec
Z
Z
Coward
J K
JK
McGuire
J J
JJ
D000818:Animals; D002478:Cells, Cultured; D005492:Folic Acid / Q000494:pharmacology; D005493:Folic Acid Antagonists; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D008114:Liver Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D005493:Folic Acid Antagonists; D005971:Glutamates; D007328:Insulin; D011099:Polyglutamic Acid; C038473:4-fluoroglutamic acid; C014085:methotrexate polyglutamate; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate