TY - JOUR
T1 - Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells
AU - Chu, Edward
AU - Drake, James C.
AU - Boarman, Donna
AU - Baram, Jacob
AU - Allegra, Carmen J.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1990/5/25
Y1 - 1990/5/25
N2 - We have studied the roles of 5, 10-methylenetetrahydrofolate (5, 10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2′-deoxyuridine-5′-monophosphate radioenzymatic binding assay, we determined that the 5, 10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 μm MTX for up to 21 h. Similar alterations in the 5, 10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 μm MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 μm MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 μm MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5, 10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.
AB - We have studied the roles of 5, 10-methylenetetrahydrofolate (5, 10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2′-deoxyuridine-5′-monophosphate radioenzymatic binding assay, we determined that the 5, 10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 μm MTX for up to 21 h. Similar alterations in the 5, 10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 μm MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 μm MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 μm MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5, 10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.
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M3 - Article
C2 - 2341391
AN - SCOPUS:0025310519
SN - 0021-9258
VL - 265
SP - 8470
EP - 8478
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 15
ER -