DHFR MEDIATED ANTIFOLATE RESISTANCE IN MAMMALIAN CELLS

Project Details

Description

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.
StatusFinished
Effective start/end date5/1/9012/31/03

Funding

  • National Institutes of Health: $238,981.00
  • National Institutes of Health: $218,116.00
  • National Institutes of Health
  • National Institutes of Health: $199,782.00
  • National Institutes of Health: $232,024.00
  • National Institutes of Health: $225,263.00
  • National Institutes of Health: $235,915.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $192,590.00
  • National Institutes of Health
  • National Institutes of Health: $171,891.00

ASJC

  • Medicine(all)

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