Inhibition of methionine uptake by methotrexate in mouse leukemia L1210 cells

Section: And Discussionsupporting

confidence: 70%

Section: And Discussionmentioning

confidence: 99%

Section: And Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Elevated expression of thymidylate synthase cycle genes in cisplatin-resistant human ovarian carcinoma A2780 cells.

Scanlon

Kashani‐Sabet

1988

“…dTMP Synthase Assay. dTMP synthase activity was measured in extracts of A2780 cells by the amount of tritium released to H2O from [5-3H]dUMP as described (13), except that 1% Triton X-100 was included in the buffer to extract the cells.…”

mentioning

confidence: 99%

Biochemical basis for cisplatin and 5-fluorouracil synergism in human ovarian carcinoma cells.

Scanlon¹,

Newman²,

Lu³

et al. 1986

338

166

The human ovarian cell line A2780 was exposed to either cisplatin (10 ,uM) or 5-fluorouracil (5FUra) (5 ,uM) for 1 hr. Cytotoxicity was less than 14% with either agent alone. Cisplatin (10 j#M) and 5FUra (5 jzM) in combination for 1 hr caused a 76% reduction in cell growth. Thymidine (dThd, 10 jM), if given concomitantly with the combination of cisplatin and 5FUra. completely protected the tumor cells. A 30-min exposure to cisplatin increased the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate 2.5-fold.The capacity of intact cells to form 5-fluorodeoxyuridylate (FdUMP)-thymidylate (dTMP) synthase complex when incubated with fluorodeoxyuridine (FdUrd) was enhanced 2.5-fold when the cells were pretreated with cisplatin. These experiments demonstrate that cisplatin can increase the availability of the reduced folate necessary for tight binding of FdUMP to dTMP synthase, thus enhancing the cytotoxicity of the cisplatin and 5FUra combination.5-Fluorouracil (5FUra) and cis-diamminedichloroplatinum-(II) (cisplatin) in combination have been shown to have synergistic cytotoxicity against both murine and human neoplasms (1, 2). The mechanism ofthis synergy has not been elucidated. SFUra is metabolized within cells to 5-fluorodeoxyuridine monophosphate (FdUMP). FdUMP can covalently bind to dTMP synthase in the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate) and inhibit DNA synthesis by depleting the cells of dTMP. 5FUra is also metabolized to 5-fluorouridine triphosphate (FUTP), which is incorporated into RNA. One or both of these 5FUra metabolites account for the antineoplastic activity of SFUra in experimental models (3). The antitumor activity of the second agent, cisplatin, may be explained in part by DNA crosslinking (4, 5), interactions with the cell-surface nucleic acids (6) or with the plasma membrane (7-9). Also, cisplatin can inhibit methionine uptake into tumor cells and cause perturbation of the methionine pools (7-9). Cells may respond by increasing methionine biosynthesis and increasing the pools of folate cofactors (10). The interaction of these two chemotherapeutic drugs was studied in patients with human ovarian cancer (11). Data presented in this report suggest that cisplatin treatment does increase the CH2-H4folate pool and the binding of FdUMP to dTMP synthase. The resultant inhibition of dTMP synthesis appears to be responsible for the cytotoxicity of the SFUra and cisplatin combination. MATERIALS AND METHODSThe following chemicals were obtained from these companies: thymidine, Sigma; SFUra, Hoffmann-La Roche; deoxyuridine, P-L Biochemicals; cisplatin and 5-fluorodeoxy- Cell Culture. The human ovarian cancer cell line A2780 was obtained from R. Ozols, National Cancer Institute (12) and maintained in RPMI 1640 with 10% (vol/vol) fetal calf serum, penicillin (62.9 mg/liter) and streptomycin (100 mg/ liter) (GIBCO). Cells were exposed to chemotherapeutic agents for only 60 min in RPMI 1640 medium and 10% (vol/vol) fetal calf serum and then washed three times wi...

“…If the activity of this enzyme, as measured in vitro, reflects the activity in vivo, then an increased flux of one-carbon units through this step into the folate pathway in monkeys may lead to an accumulation of hepatic HCOH4folate at the expense of H4folate. The results of studies of cultured mammalian cells suggest that the activity of serine hydroxyrmethyltransferase is directly related to the incorporation of the serine carbon-3 into DNA via folate-dependent reactions (36,37).…”

Section: Discussionmentioning

confidence: 99%

Role of hepatic tetrahydrofolate in the species difference in methanol toxicity.

Black¹,

Eells²,

Noker³

et al. 1985

The susceptibility of various species to methanol toxicity is inversely related to the rate of tetrahydrofolate (H4olate)-dependent formate oxidation to carbon dioxide.Thus, the levels of various folate derivatives and folatedependent enzyme activities present in the livers of monkeys, which are sensitive to methanol, and rats, which are not, were compared in order to investigate the biochemical basis of this species difference. Hepatic H4folate levels in monkeys were 60% of those in rats, and formylated-H4folate derivatives were 2-fold higher in monkeys than in rats. No significant difference between monkeys and rats in the levels of total hepatic folate or 5-methyl-H4folate was observed. The activities of formylH4folate synthetase (EC 6.3.4.3) and formyl-H4folate dehydrogenase (EC 1.5.1.6) were 4-and 2-fold higher, respectively, in monkeys than in rats. There was no significant difference between monkeys and rats in methionine synthetase activity (EC 2.1.1.13). Dihydrofolate reductase activity (EC 1.5.1.3) in monkeys was 20% of that in rats. 5,10-Methylene-H4folate reductase (NADPH) activity (EC 1.1.1.171) in monkeys was 40% and 25% of that in rats when the rates of the forward and reverse reactions, respectively, were compared. Serine hydroxymethyltransferase activity (EC 2.1.2.1) was 2-fold higher in monkeys than in rats. The differences in the activities of methylene-H4folate reductase and serine hydroxymethyltransferase between monkeys and rats may have contributed to the difference in hepatic Il4folate levels. The 40% lower level of hepatic H4folate in monkeys, as compared to rats, relates well to the 50% lower maximal rate of formate oxidation in monkeys. Thus, the species difference in susceptibility to methanol may be explained by the difference in the level of hepatic H4folate.A species difference exists in the susceptibility to methanol toxicity (1-3), which appears to be related to the regulation of folate-dependent one-carbon metabolism (4-7). The syndrome of methanol poisoning in humans and monkeys is characterized by a mild central nervous system depression which is followed by a latent period, metabolic acidosis, ocular toxicity, and death (1-3). In contrast, rats and other rodents display only a mild central nervous system depression after methanol administration (1-3). This difference in response to methanol is related to elevated levels of formic acid in species susceptible to methanol poisoning (8-13). Thus, marked elevations of formate in body fluids and tissues occur in humans and monkeys receiving toxic doses of methanol (8, 9, 11-13); but in rats, no significant accumulation of formate is observed (8, 9). The species variation in formate accumulation is explained by the observation that, over a range of formate dose levels, the rate of formate oxidation to carbon dioxide in vivo in rats is at least twice that observed in monkeys (4). In both rats and monkeys, the major route of formate oxidation in vivo is through a folatedependent pathway (4, 5) (Fig. 1) (Fig. 1, reaction 1) (1...