Methotrexate-resistant human promyelocytic leukemia (HL-60) cells express a dihydrofolate reductase with altered properties associated with increased enzyme activity.
Dihydrofolate reductase from a MTX-resistant human promyelocytic leukemia cell line (HL-60 R4-29) had previously been found to have a higher specific activity than the DHFR from the parent MTX-sensitive cell line, in the absence of enzyme overproduction (Dedhar et al, Biochem. J. 225, 609-617, 1985). The enzymes from these two cell lines have been purified to apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The dihydrofolate reductase from the sensitive cells has an apparent molecular weight of 42,000 daltons, whereas that from the resistant cells has an apparent molecular weight of 21,000 daltons. The dihydrofolate reductase activity from the resistant cells is characterized by marked heat instability and substantially higher Vmax and Km values for the dihydrofolic acid/NADPH combination. The enzyme from the resistant cells can be protected against heat inactivation by either dihydrofolic acid or NADPH, or both. A 50 fold higher concentration of methotrexate was required to totally inhibit the R4-29 dihydrofolate reductase activity as compared to the (S) activity when the enzymes were assayed using equivalent amounts of enzyme protein. These data show that the increased dihydrofolate reductase specific activity present in the (R4-29) cells is associated with an alteration in the structure of this enzyme.
D002460:Cell Line; D004351:Drug Resistance; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology*; D008970:Molecular Weight; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate