levoleucovorin and Carcinoma--Ehrlich-Tumor

5-Formyltetrahydrofolate promotes the net dissociation of methotrexate bound to dihydrofolate reductase in the Ehrlich ascites tumor (L. H. Matherly et al., Cancer Res., 43: 2694-2699, 1983). Treatment of Ehrlich tumor cells with glucose or inhibitors of electron transfer stabilized the association of the antifolate with dihydrofolate reductase as reflected by a 2-fold increased fraction of dihydrofolate reductase-bound methotrexate and an abolition of the 5-formyltetrahydrofolate-induced dissociation of the inhibitor-enzyme complex. Glucose and azide were also found to increase the intracellular ratio of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP+) in the tumor approximately 8- and 11-fold, respectively. However, other agents which enhanced the association between methotrexate and its target enzyme were less effective in increasing the intracellular level of NADPH relative to NADP+. Micromolar concentrations of NADPH promoted methotrexate binding to the purified Ehrlich tumor dihydrofolate reductase. Bound methotrexate could be dissociated from the purified enzyme by 5-methyltetrahydrofolate but less readily by 5-formyltetrahydrofolate and only in the presence of reduced levels of NADPH relative to NADP+. The tetraglutamate derivative of 5-methyltetrahydrofolate was even more effective than the underivatized compound in dissociating methotrexate from dihydrofolate reductase. These findings suggest a critical role for the cellular oxidation-reduction state in determining the affinity of dihydrofolate reductase for methotrexate and thus the cellular sensitivity to the antifolate. In addition, the data are consistent with the possibility that dihydrofolate reductase is a key locus for intracellular competitive interactions between reduced folates and methotrexate during leucovorin rescue from the pharmacological effects of the antifolate.

Topics: Animals; Azides; Carcinoma, Ehrlich Tumor; Folic Acid Antagonists; Glucose; Indicators and Reagents; Kinetics; Leucovorin; Methotrexate; Mice; NAD; NADP; Oxidation-Reduction; Potassium Cyanide; Protein Binding; Rotenone; Sodium Azide; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase

5-Formyltetrahydrofolate was found to reverse the binding of methotrexate to dihydrofolate reductase in the Ehrlich ascites tumor in vitro. When cells pretreated with methotrexate were resuspended in methotrexate-free buffer containing 5-formyltetrahydrofolate (or 5-methyltetrahydrofolate), net dissociation of the antifolate from the enzyme was observed. Methotrexate associated with the enzyme under these conditions was below the enzyme binding capacity. However, glucose or azide increased the fraction of dihydrofolate reductase associated with methotrexate and abolished the effect of tetrahydrofolates on this intracellular component. Addition of 5-fluoro-2'-deoxyuridine had no effect on this response to the reduced folate, thereby precluding a direct role for the thymidylate synthase-dependent generation of dihydrofolate in this dissociation of methotrexate from dihydrofolate reductase. Enzyme-bound methotrexate could also be reduced by exposure to 5-formyltetrahydrofolate prior to uptake and efflux of free methotrexate. When cells were incubated under conditions which favored formation of methotrexate polyglutamate derivatives, subsequent treatment with 5-formyltetrahydrofolate had no effect on the binding of the conjugated antifolate to dihydrofolate reductase. These findings support a role for dihydrofolate reductase as a locus for competitive binding interactions between reduced folates and methotrexate that may be a basis for the ability of 5-formyltetrahydrofolate to prevent the biochemical effects of this antifolate. These data suggest that the presence of methotrexate polyglutamate derivatives and cellular energy metabolism may be critical determinants of the responsiveness of methotrexate-treated cells to reduced folates and may play important roles in the selectivity of 5-formyltetrahydrofolate rescue.

Topics: Animals; Binding, Competitive; Carcinoma, Ehrlich Tumor; Energy Metabolism; Leucovorin; Male; Methotrexate; Mice; Peptides; Polyglutamic Acid; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase

The selective toxicity of antifolates for a variety of cancers can be improved, as illustrated by the combined administration of N5-methyltetrahydrofolate and methotrexate in tissue culture. When a variety of neoplastic cell types characterized by a deficiency of vitamin B12-dependent N5-methyltetrahydrofolate methyltransferase (5-methyltetrahydropteroyl-L-glutamate:L-homocysteine S-methyltransferase, EC 2.1.1.13) and normal adult cells are grown in media containing methotrexate and either N5-methyltetrahydrofolate or N5-formyltetrahydrofolate, not only is the selective toxicity of methotrexate demonstrated, but the advantage of using N5-methyltetrahydrofolate in place of N5-formyltetrahydrofolate is also revealed. The implications and applications of this particular combination in the treatment of human cancer are discussed.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Division; Cells, Cultured; Female; Fibroblasts; Humans; Leucovorin; Leukemia L1210; Leukemia, Myeloid; Liver; Methotrexate; Mice; Neoplasms, Experimental; Rats; Tetrahydrofolates

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell-Free System; Chromatography, Ion Exchange; Cricetinae; Culture Techniques; Deoxyuridine; DNA; Embryo, Mammalian; Escherichia coli; Folic Acid; Histocytochemistry; Kinetics; L Cells; Leucovorin; Methotrexate; Mice; Nucleotides; RNA; Thymidine; Transferases; Tritium

Topics: Animals; Antimalarials; Antineoplastic Agents; Biguanides; Boron Compounds; Carcinoma, Ehrlich Tumor; Dogs; Folic Acid Antagonists; Hematopoiesis; Leucovorin; Leukopenia; Mice; Rabbits; Rats; Sarcoma 180; Species Specificity