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Multiple forms of L1210 dihydrofolate reductase differing in affinity for methotrexate
Biochemical and Biophysical Research Communications
|February 29, 1984
Summary
Two forms of dihydrofolate reductase were identified in Methotrexate-resistant L1210 cells. These forms exhibit distinct drug affinities and biochemical properties, impacting Methotrexate efficacy in leukemia.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism.
- Methotrexate is a key chemotherapeutic agent targeting DHFR.
- Drug resistance in cancer often involves alterations in target enzymes.
Purpose of the Study:
- To characterize the forms of dihydrofolate reductase in Methotrexate-resistant L1210 mouse leukemia cells.
- To investigate the biochemical differences between these DHFR forms.
- To understand the implications for Methotrexate binding and efficacy.
Main Methods:
- Purification of dihydrofolate reductase from L1210 cell subline R6.
- Isoelectric focusing for separation and characterization of enzyme forms.
- Enzyme activity assays and drug binding studies using [3H]Methotrexate.
Main Results:
- Two distinct forms of DHFR (Form 1 and Form 2) were purified.
- Form 1 showed higher sensitivity to Methotrexate inhibition and higher specific activity.
- Form 2 was more heat-labile but could be stabilized; it exhibited lower drug affinity.
- Isoelectric points (pI) were determined as 7.4 for Form 1 and 8.2 for Form 2.
- [3H]Methotrexate binding revealed KD values of approximately 1 nM for Form 1 and 27 nM for Form 2.
Conclusions:
- Methotrexate-resistant L1210 cells possess heterogeneous dihydrofolate reductase.
- The two DHFR forms differ significantly in their biochemical properties and drug binding affinities.
- These variations likely contribute to the observed Methotrexate resistance in this leukemia model.