Disposition of Endogenous Homocysteine by Mouse Fibroblast C3H/10T1/2 CI 8 and the Chemically Transformed C3H/10T1/2 MCA CI 16 Cells Following Methotrexate Exposure<xref ref-type="fn" rid="FN1">2</xref>

Abstract

The tumorigenic cell line termed "MCA Cl 16" was derived from C3H/10T1/2 clone (Cl) 8 cells by chemical transformation in the presence of 3-methylcholanthrene [(MCA) CAS: 56-49-5]. Transformed (Cl 16) cells were more sensitive toward the cytotoxic effect of methotrexate (MTX) than their normal counterpart Cl 8 cells. The disposition of endogenous L-homocysteine (Hcy) was investigated in these two cell lines after MTX exposure. Both nonmalignant and transformed cells exported Hcy into the extracellular medium, and only small amounts were retained within the cells. The Hcy efflux from the malignant cells was markedly increased after MTX exposure (0.5-10 microM), and this effect was almost completely prevented by 5-formyl-tetrahydrofolate (THF), whereas treatment with thymidine plus hypoxanthine did not inhibit the MTX-dependent Hcy efflux. Cytotoxic concentration of MCA reduced rather than increased the Hcy efflux from these cells. High concentrations of MTX (greater than 10 microM) were required to increase the release of Hcy from nonmalignant cells. The enhancement of Hcy export from the malignant cells in the presence of MTX was not associated with cellular build-up of S-adenosyl-L-homocysteine (AdoHcy), indicating that the amount of intracellular Hcy was kept below the level required for inhibition or reversion of the AdoHcy hydrolase reaction. MTX-dependent Hcy efflux probably reflects cellular deficiency of 5-methyl-THF required for the salvage of Hcy to methionine and may therefore be a measure of lack of this reduced folate relative to the metabolic demand.