Abstract
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.
Highlights
MTX treatment, suggesting that the mitochondrialfol- folylpoly-y-glutamate synthetase (FPGS) activity, but, in other cases, FPGS activity appeared ate transport system is specific for reduced folates
Theeffect of FPGS activity on the metabis a potent inhibitorof dihydrofolate reductase and inhibition olism and cytotoxicity of MTX was investigated using model of this enzyme prevents the reduction of dihydrofolate Chinese hamster ovary (CHO)cells expressing different levels (H2PteGlu)that accumulates in cells actively synthesizing of human FPGS activity
The model cells used in this study allowed an assessment of the role of FPGS level in MTXmetabolism and cytotoxicity
Summary
After 72 h, the medium was removed, the cell layer was washed twice with PBS (2 ml), and the cells were detached with 0.1% trypsin-EDTA (0.1 ml) and counted using a Folate-depleted cells (1X IO6)were cultured for 24 h in DMEM/ FBS+GHT medium containing 5 pM [3H]MTX and thenincubated for a further 18 h in identical medium lacking MTX. The column was eluted by a buffer gradient from 15% B to 25% B over 50 min at tabolites by hamster cells expressing human FPGSwas influenced by the level of FPGS activity (Table I). Subcellular Distribution of Folate and MTX Metabolites-CHO cells were cultured as described previously in 150-cm2flasks [28]. Postnuclear supernatants were fractionated on a mM chloroquine or 0.4 mM MMGA, and MTX derivatives in self-forming continuous Percoll density gradient as described previ- cell extracts were separated according to glutamate chain ously [28]
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