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High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants
1Institute of Applied Microbiology, University of Tokyo, Japan.
Abstract:
Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidicolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase alpha from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparent Ki value for aphidicolin of AC3 polymerase alpha was three to four times that of the enzyme from the parent cells, but the apparent Km values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase alpha that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.
Insights
Researchers identified two mechanisms for aphidicolin resistance in mouse cells. These involve altered DNA polymerase alpha and expanded DNA precursor pools, impacting DNA synthesis and drug resistance.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Aphidicolin is a potent inhibitor of DNA synthesis.
- Understanding drug resistance mechanisms is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the mechanisms of aphidicolin resistance in mouse FM3A cells.
- To characterize the biochemical and molecular alterations in resistant cell lines.
Main Methods:
- Multistep selection of spontaneous aphidicolin-resistant mutants (AC1, AC2, AC3).
- Assays for DNA synthesizing activity in permeabilized cells and nuclear extracts.
- Partial purification and characterization of DNA polymerase alpha.
- Analysis of deoxyribonucleoside triphosphate pools and protein expression via gel electrophoresis.
Main Results:
- Mutants exhibited varying degrees of aphidicolin resistance.
- DNA polymerase alpha from AC1 and AC3 showed resistance, while AC2 did not.
- AC3 DNA polymerase alpha had altered kinetic parameters for aphidicolin.
- All mutants displayed cross-resistance to other nucleoside analogs.
- AC3 showed expanded deoxyribonucleoside triphosphate pools.
- A novel 40 kDa protein was observed in AC1.
Conclusions:
- Mammalian cells possess at least two distinct mechanisms for aphidicolin resistance.
- One mechanism involves aphidicolin-resistant DNA polymerase alpha.
- Another mechanism involves the expansion of cellular pools of DNA precursors.