DHFR MEDIATED ANTIFOLATE RESISTANCE

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The wide use of the antifolate drug methotrexate (NM) as a chemotherapeutic agent requires that knowledge of its target enzyme, dihydrofolate reductase (DHFR), be extensive and that the enzymes' biochemical and genetic characteristics be understood. Moreover, since the effectiveness of MTX is often comprimised by intrinsic or acquired resistance, a better understanding of the mechanism(s) of such resistance might provide insights into more effective treatment protocols. It has been found that the Chinese hamster lung cell line DC-3F is heterozygous at the DHFR locus and contains 2 polymorphic DHFR alleles. Among 58 independently derived, gene amplification mediated DHFR overproducing MTX-resistant sublines, 44 amplify and overexpress one of the 2 allelic forms of DHFR and 14 the other. Since gene amplification is thought to be a random phenomenon, one of the goals of this proposal is to determine, by use of pulse field gel electrophoresis, Southern blotting and cDNA transfection analysis, the genetic basis for the observed differential allelic amplification. In addition, recent determination that commonly observed minor isoelectric forms of DHFR (which can account for 10-15% of the cellular enzyme) are derived by N-terminal acetylation of the major DHFR proteins, suggests that these forms may serve a specific biological function. With a combination of NM-affinity column and isoelectrofocusing purification techniques, enzyme kinetic analysis and monoclonal antibody preparation for immunocytochemical detection we propose to clarify the biological significance of those long recognized but poorly understood DHFRS.