Regulation of folate and one-carbon metabolism in mammalian cells. IV. Role of folylpoly-gamma-glutamate synthetase in methotrexate metabolism and cytotoxicity.

Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of methotrexate (MTX). CHO cells expressing human FPGS metabolized MTX to polyglutamates characteristic of human cells. Cellular MTX accumulation and metabolism to polyglutamates were dependent on the level of FPGS activity and were unaffected by putative gamma-glutamyl hydrolase inhibitors. The sensitivity of cells continuously exposed to MTX was not influenced by FPGS activity. After short term exposure to MTX, cells expressing higher levels of FPGS were more sensitive to the drug. MTX was not transported into the mitochondria and MTX treatment had no effect on preexisting mitochondrial folates while cytosolic folates were converted to oxidized forms. Mitochondrial folate accumulation was significantly impaired by MTX treatment, suggesting that the mitochondrial folate transport system is specific for reduced folates.