TY - JOUR
T1 - Further studies on the pharmacologic effects of the 7-hydroxy catabolite of methotrexate in the L1210 murine leukemia cell
AU - Seither, Richard L.
AU - Rape, Tracy J.
AU - Goldman, I. David
N1 - Funding Information:
* This investigation was supported by USPHS Grant CA-39807 from the National Cancer Institute. National Institutes of Health. t Supported by USPHS Training Grant CA-09340 from the National Cancer Institute, National Institutes of Health. $ Correspondence: Dr. I. David Goldman, Division of Hematology/Oncology, Medical College of Virginia, P.O. Box 230, Richmond, VA 23298. 8 Abbreviations: MTX, methotrexate; 7-OH-MTX, 7-hydroxymethotrexate; DHFR, dihydrofolate reductase; FdUrd, 5-fluorodeoxyuridine; AICAR, aminoimidazolecarboxamide ribonucleotide; and TCA, trichloroacetic acid.
PY - 1989/3/1
Y1 - 1989/3/1
N2 - This paper describes studies that further explore the pharmacologic activity of the 7-hydroxy catabolite of methotrexate (7-OH-MTX). A 3-hr exposure of L1210 leukemia cells to 100 μM 7-OH-MTX produced negligible suppression of cell growth despite the build-up of intracellular polyglutamyl congeners to levels 2.7 times greater than the dihydrofolate reductase (DHFR) binding capacity. There was no evidence for direct inhibition of DHFR under these conditions based upon measurements of cellular tetrahydrofolate cofactor and dihydrofolate levels, nor was there suppression of [3H]deoxyuridine incorporation into DNA or [14C]formate incorporation into purines. When the interval of exposure to 100 μM 7-OH-MTX was increased to 6 hr, cell growth was inhibited by 60% and there was mild (~50%) inhibition of purine and thymidylate biosynthesis associated with a small increase in cellular dihydrofolate and a small decline in cellular tetrahydrofolates. Consistent with weak inhibition of DHFR was the absence of significant binding of 7-OH-MTX polyglutamates to DHFR as assessed by gel filtration of cell extracts. Mild direct inhibition of purine biosynthetics by 7-OH-MTX- or MTX-polyglutamyl congeners was demonstrated based upon inhibition of [14C]formate incorporation into purines in cells pretreated with fluorodeoxyuridine so as to prevent tetrahydrofolate cofactor depletion or dihydrofolate polyglutamate build-up. Effects of a 6-hr exposure of cells to 100 μM 7-OH MTX on cell growth were reversed completely by 10 μM leucovorin; effects on cells containing comparable levels of MTX polyglutamyl congeners were unaffected by leucovorin. These studies demonstrate very weak inhibition of L1210 leukemia cell growth and purine, pyrimidine and tetrahydrofolate synthesis by the polyglutamyl congeners of 7-OH-MTX. The data suggest that effects of 7-OH-MTX polyglutamates on folate-requiring enzymes are not likely to play an important role in moderate-dose MTX regimens. However, pharmacologic activity may be expressed in high-dose MTX protocols when high blood levels of 7-OH-MTX are sustained over long intervals to the extent to which polyglutamate congeners accumulate in tumor cells and add to the much more potent inhibitory effects of MTX polyglutamates already present. Pharmacologic activity, however, would be diminished, if not completely reversed, by the concurrent administration of leucovorin.
AB - This paper describes studies that further explore the pharmacologic activity of the 7-hydroxy catabolite of methotrexate (7-OH-MTX). A 3-hr exposure of L1210 leukemia cells to 100 μM 7-OH-MTX produced negligible suppression of cell growth despite the build-up of intracellular polyglutamyl congeners to levels 2.7 times greater than the dihydrofolate reductase (DHFR) binding capacity. There was no evidence for direct inhibition of DHFR under these conditions based upon measurements of cellular tetrahydrofolate cofactor and dihydrofolate levels, nor was there suppression of [3H]deoxyuridine incorporation into DNA or [14C]formate incorporation into purines. When the interval of exposure to 100 μM 7-OH-MTX was increased to 6 hr, cell growth was inhibited by 60% and there was mild (~50%) inhibition of purine and thymidylate biosynthesis associated with a small increase in cellular dihydrofolate and a small decline in cellular tetrahydrofolates. Consistent with weak inhibition of DHFR was the absence of significant binding of 7-OH-MTX polyglutamates to DHFR as assessed by gel filtration of cell extracts. Mild direct inhibition of purine biosynthetics by 7-OH-MTX- or MTX-polyglutamyl congeners was demonstrated based upon inhibition of [14C]formate incorporation into purines in cells pretreated with fluorodeoxyuridine so as to prevent tetrahydrofolate cofactor depletion or dihydrofolate polyglutamate build-up. Effects of a 6-hr exposure of cells to 100 μM 7-OH MTX on cell growth were reversed completely by 10 μM leucovorin; effects on cells containing comparable levels of MTX polyglutamyl congeners were unaffected by leucovorin. These studies demonstrate very weak inhibition of L1210 leukemia cell growth and purine, pyrimidine and tetrahydrofolate synthesis by the polyglutamyl congeners of 7-OH-MTX. The data suggest that effects of 7-OH-MTX polyglutamates on folate-requiring enzymes are not likely to play an important role in moderate-dose MTX regimens. However, pharmacologic activity may be expressed in high-dose MTX protocols when high blood levels of 7-OH-MTX are sustained over long intervals to the extent to which polyglutamate congeners accumulate in tumor cells and add to the much more potent inhibitory effects of MTX polyglutamates already present. Pharmacologic activity, however, would be diminished, if not completely reversed, by the concurrent administration of leucovorin.
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U2 - 10.1016/0006-2952(89)90236-0
DO - 10.1016/0006-2952(89)90236-0
M3 - Article
C2 - 2467676
AN - SCOPUS:0024523045
SN - 0006-2952
VL - 38
SP - 815
EP - 822
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 5
ER -