Paul L. Chello

Growth inhibitory, transport and biochemical properties of the γ-glutamyl and γ-aspartyl peptides of methotrexate inL1210 leukemia cells in vitro

Abstract Both methotrexate-γ-glutamate and methotrexate-γ-aspartate are equivalent to metho-trexate as inhibitors of L1210 cell dihydrofolate reductase. However, the initial influx of both peptides into L1210 cells during transport studies is substantially lower than that of methotrexate. The apparent K m tor influx of methotrexate-γ-glutamate and methotrexate-γ-aspartate is 15-fold and 100-fold greater than methotrexate respectively. Efflux measurements, which were possible only for methotrexate-γ-glutamate, showed a similar rate for this peptide and methotrexate. The intracelluiar accumulation and subsequent metabolism to methotrexate of methotrexate-γ-glutamate, but not of methotrexate-γ-aspartate, were confirmed by bioautographic analysis of cell extracts. After correction for extracellular cleavage of both peptides mediated by enzymes in calf serum supplementing the culture medium, the relative growth (L1210 cell)-inhibitory potency for the three agents was 1:18:210 for methotrexate, methotrexate-γ-glutamate and methotrexate-γ-aspartate respectively. Both the relative inhibitory potency and the difference in absolute inhibitory concentration among the three agents were predictable solely from the data on the influx of each measured during transport studies. Methotrexate-γ-aspartate is apparently more resistant to enzymic cleavage than is methotrexate-γ-glutamate.

Effect of methionine deprivation on L5178Y murine leukemia cells in culture interference with the antineoplastic effect of methotrexate.

Abstract L5178Y cells in culture have a requirement for l -methionine which cannot be satisfied by supplying the components necessary for de novo biosynthesis [1]. Methionine deprivation produced a rapid and progressive loss of cell viability (30°o by 6 hr: 90°o by 24 hr). Cells which remained viable after being deprived of methionine could be rescued by l -methionine supplementation. L5178Y cells in culture were also highly sensitive to the folate antagonist, methotrexate. Exposure to a concentration of 10−6 M for 6 hr resulted in a 95–97°o loss of viability. However, if cells were deprived of methionine for 6 hr before exposure to methotrexate, the methotrexate effect was reduced. The cell-killing effect of melhotrexate was blocked by longer intervals of methionine deprivation. If the deprived cells were resupplied with the amino acid, the effect of methotrexate was still reduced for at least 12 hr following the methionine resupplementation.

Improved purification of tetrahydrofolate dehydrogenase from L1210 leukemia by affinity chromatography

Abstract Tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate:NADP + oxidoreductase, EC 1.5.1.3; formerly known as dihydrofolate reductase) from a high enzyme mutant of L1210 mouse leukemia was purified to homogeneity by a simple two step procedure involving pH 5.1 precipitation of inert protein, and affinity chromatography employing a methotrexate-agarose column. By raising the pH and ionic strength of the eluting buffer from 0.05 M citrate (pH 6.0) to 0.05 M Tris-HCl (pH 8.5) containing 0.1 M KCl, a peak was eluted containing pure enzyme (spec. act. 2800 μmoles/h per mg). Purity was confirmed by methotrexate titration and polyacrylamide disc electrophoresis. A second small peak containing tetrahydrofolate dehydrogenase activity was eluted by further increasing the pH of the 0.05 M Tris-HCl buffer to 9.0, and increasing the KCl concentration to 0.4 M. The material in this peak showed an absorbance at 258 nm, suggesting that a nucleotide was bound to the enzyme. The identify of this material has yet not been established.

Sequential combination of methotrexate and vindesine in previously treated children with acute leukemia. A phase I-II study.

A therapeutic synergistic effect is seen in animal models when vinca alkaloids are administered after methotrexate. To examine further this interaction in clinical studies, a phase I-II trial was conducted in children with hematologic malignancies in the Department of Pediatrics at Memorial Sloan-Kettering Cancer Center. A schedule of sequential of methotrexate and vindesine was developed which showed effect in acute lymphoblastic leukemia in children in relapse and which was relatively nontoxic. The regimen has also been useful for reinduction for patients who are candidates for bone marrow transplant.