Disposition of Endogenous Homocysteine by Mouse Fibroblast C3H/1 OT1 /2 CI 8 and the Chemically Transformed C3H/1 OT1 /2 MCA CI16 Cells Following Methotrexate Exposure 1,2 Per Magne Ueland, 3,4 Helga Refsum, 3 Rune Male, 5 and Johan R. Lillehaug 5,6 ABSTRACT-The tumorigenic cell line termed "MCA CI 16" was derived from C3H/10T1/2 clone (CI) 8 cells by chemical trans- formation in the presence of 3-methylcholanthrene [(MCA) CAS: 56-49-5]. Transformed (CI16) cells were more sensitive toward the cytotoxic effect of methotrexate (MTX) than their normal counter- part CI 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 in- creased after MTX exposure (0.5-10 JlM), 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 (>10 JlM) 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.-JNCI 1986; 77:283-289. The antifolate drug MTX has been found useful in the treatment of acute lymphoblastic leukemia and sev- eral solid tumors (1). The ubiquitous enzyme DHFR seems to be the primary molecular target of MTX. This enzyme is responsible for the regeneration of THF from DHF. Inhibition of DHFR leads to cellular depletion of THF and related reduced folates, including 5-methyl- THF, and thereby blocks the various THF coenzymes- dependent synthesis of proteins, DNA, and RNA (2,3). However, several questions on the action mode of MTX, as for example, the biochemical basis of several side effects of MTX (4) and the selective kill of sensitive tumor cells (5), remain to be answered. This has stimu- lated research on the effects of MTX on numerous bio- logical processes, including transport of glucose (6, 7), folates (8, 9), and amino acids (10), and the interaction of the drug with enzymes other than DHFR (2, 11-13). There has been a growing interest in the amino acid Hcy because altered cellular disposition of this com- pound has been suggested to play an important role in the pathogenesis of various processes, including homo- cystinuria (14), atherosclerosis (15), methionine-depen- dent cancer (16), and the hepatic injury induced by MTX(3). Hcy is not supplied by food but is formed from the endogenous transmethylase inhibitor AdoHcy through the action of the enzyme AdoHcy hydrolase (17). Once formed, Hcy is either metabolized to cystathionine via the so-called transsulfuration pathway, or it is salvaged to methionine. The conversion to methionine is in most tissues catalyzed by an enzyme that requires 5-methyl- THF as methyl donor (14). This reaction links Hcy metabolism to the biological effect of the antifolate drug MTX, which might be expected to block methionine synthesis from Hcy. These metabolic relations are de- picted in text-figure 1. In the present paper we investigate the disposition of endogenous Hcy in a nonmalignant (C3H/IOTl/2 Cl 8) and malignant (C3H/lOTl/2 MCA Cl 16) cell line dur- ing MTX exposure. The malignant cells (MCA Cl 16) are derived from the nonmalignant cells (Cl 8) by chemi- cal transformation in the presence of MCA and are tumorigenic upon implantation in immunosuppressed syngeneic mice (18). MATERIALS AND METHODS Chemicals and drugs.-DL-Homocysteine, Hcy, L- methionine, adenosine, and AdoHcy were obtained from Sigma Chemical Co., St. Louis, MO, and AdoMet was obtained from Koch-Light Laboratories, Colnbrook, England. MTX and leucovorin (5-formyl-THF) were purchased from Nyegaard & Co. (Nyco), Oslo, Norway. [14C]Adenosine (0.59 Ci/mmol) and [ 35 S]methionine (1,070 Ci/mmol) were obtained from the Radiochemical ABBREVIATIONS USED: AdoHcy=S-adenosyl-L-homocysteine; AdoMet= S-adenosylmethionine; CI=clone; DHF=dihydrofolate; DHFR=di- hydrofolate reductase; Hcy = L-homocysteine; HPLC == high-perfor- mance liquid chromatography; LD==lethal dose; MCA=3-methyl- cholanthrene; MTX==methotrexate; ODS==octadecylsilyl; THF==tetra- hydrofolate. I Received September 27, 1985; accepted March 5, 1986. 2 Supported by grants from the Norwegian Society for Fighting Cancer and by the Norwegian Cancer Society. 3Clinical Pharmacology Unit, Department of Pharmacology, Uni- versity of Bergen, 5000 Bergen, Norway. 4 Address reprint requests to Dr. Ueland, Clinical Pharmacology Unit, Central Laboratory, University of Bergen, N-5016 Haukeland Hospital, Norway. 5 Department of Biochemistry, University of Bergen. 6 The technical assistance of H. Bergesen and H. Kanestr</>m IS highly appreciated. 283 JNCI. VOL. 77. 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