Effects of overexpression of gamma-glutamyl hydrolase on methotrexate metabolism and resistance

Intracellular metabolism of methotrexate (MTX) to MTX-polyglutamates (MTXPG ) is one determinant of cytotoxicity, Steady-state accumulation of MTXPG se ems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma -glutamyl hydrolase (GGH) , which removes them. Overexpression of GGH would be expected to decrease i ntracellular MTXPG, thereby increasing efflux of MTX and decreasing cytotox icity, Increased expression of GGH has been shown to be associated with res istance to MTX in human sarcoma cell lines and a rat hepatoma cell line. To clarify the specific role of GGH in determining MTX sensitivity, we invest igated the phenotype produced by forced GGH overexpression in two cell type s. Furthermore, because MTX and folic acid share metabolic pathways, we mea sured the effects of GGH overexpression on folic acid metabolism. The full- length cDNA for GGH, subcloned into a constitutive expression vector, was t ransfected into a human fibrosarcoma (HT-1080) and a human breast carcinoma (MCF-7) cell line. Compared with the clones containing an empty vector, th e GGH-overexpressing cells express 15- to 30-fold more GGH mRNA, more GGH p rotein, and 15- to 90-fold more GGH enzyme activity. GGH overexpression alt ered MTX accumulation and metabolism to long-chain polyglutamates. In contr ast to expectations, however, GGH overexpression did not confer resistance to short MTX exposures in either cell line. Changes in MTX metabolism were found to be balanced by alterations in accummulation and metabolism of foli c acid. The ratio of MTX:folate accumulation may be a better predictor of M TX cytotoxicity than the accumulation of either alone. We conclude that, at least for these two cell lines, GGH overexpression alone is insufficient t o produce clinical resistance to MTX.