Abstract
Methotrexate-resistant (MTXR) human breast cancer cells have been obtained which are 1000-fold less sensitive to this drug than the wild type MCF-7 cells from which they were derived. The resistant cells contain approximately a 25-fold increase in the activity of the target enzyme dihydrofolate (DHF) reductase. Enzyme inhibition studies and methotrexate affinity studies fail to reveal any difference in the DHF reductase present in the MTXR cells compared to wild type MCF-7 cells. Cytogenetic analysis demonstrates the presence of elongated marker chromosomes in the resistant cells which are not found in the parental cell line. Analysis of the DNA from MTXR and wild type MCF-7 cells using Southern blot hybridization indicates that the MTXR MCF-7 cells contain more copies of DHF reductase gene sequences than do wild type MCF-7 cells. These experiments also suggest that the amplified DHF reductase gene sequences in MTXR cells may have undergone a uniform structural rearrangement involving the 5' flanking sequences during the process of amplification. MTXR MCF-7 cells respond to estradiol by increasing cell growth, and the level of DHF reductase in the MTXR cells is further induced following administration of estradiol. Radiolabeling studies demonstrate that estrogen stimulates the actual synthesis of DHF reductase in human breast cancer cells.
Highlights
Methotrexate, a potentinhibitor of dihydrofolate reductase (EC 1.5.1.3),is an effective antineoplastic agent with activity in a wide variety of human malignancies
One study of patients with acute myelocytic leukemia suggested that the natural resistance of these patients to therapy with methotrexate is associated with the inherent ability of acute myelocytic leukemia cells to rapidly synthesize DHF reductase and accumulate intracellular methotrexate [26]
In order to facilitate the study of the mechanisms of resistance to methotrexate in human cells, we developed methotrexateresistant human breast cancer cellsby serial passage of a human breast cancer cell line (MCF-7)in stepwise increasing concentrations of methotrexate
Summary
Methotrexate, a potentinhibitor of dihydrofolate reductase (EC 1.5.1.3),is an effective antineoplastic agent with activity in a wide variety of human malignancies. Resistance to metho- demonstrates the presence of only one protein band which is trexate in these human breast cancer cells is associated pri- in marked higher concentrations in the MTXRcells compared marily with the presence of increased concentrations of DHF to the wild type MCF-7 cells and this band co-migrates with reductase.
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