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Analysis of random recombination between human MDR1 and mouse mdr1a cDNA in a pHaMDR-dihydrofolate reductase
1Laboratory of Cell Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
Human P-glycoprotein (Pgp) confers multidrug resistance (MDR) to otherwise sensitive cells. The homologous mouse Pgps, which are encoded by mouse mdr1a (also known as mdr3) and mdr1b (also known as mdr1), confer different degrees of resistance to the same MDR drugs and inhibitors. To create recombinants for the study of sequences responsible for these differences in drug-resistance, chimeric cDNA libraries can be constructed by homologous recombination of pools of related sequences. This mutagenesis approach is called DNA shuffling. To select for chimeric Pgp with an altered resistance profile, DNA shuffling between the homologous but not identical drug interacting transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a was used. The chimeric proteins were expressed in human KB-3-1 cells. One recombinant Pgp (clone 3-4) with a novel phenotype was analyzed in detail. Inhibitors of Pgp, including verapamil and cyclosporin A, were less effective in reversing resistance of the chimeric Pgp compared with wild-type Pgp, for certain drugs. However, [125I]iodoarylazidoprazosin photoaffinity labeling of the chimeric Pgp and its binding competition with cyclosporin A, showed that cyclosporin A competed for the photoaffinity labeling. The chimeric Pgp cells stained less well with human-specific anti-Pgp mAb MRK16 than wild-type Pgp, despite having the described epitopes for MRK16. Staining with human-specific mAb UIC2 was increased when the chimeric protein was compared with wild-type Pgp. These results suggest an alteration in exposure of human Pgp specific epitopes in this chimeric Pgp, as well as a change in the interaction of reversing agents with the chimeric protein.
Insights
DNA shuffling created a chimeric P-glycoprotein (Pgp) with altered drug resistance. This novel Pgp showed reduced sensitivity to inhibitors like verapamil and cyclosporin A, indicating changes in drug interaction and epitope exposure.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Human P-glycoprotein (Pgp) is a key mediator of multidrug resistance (MDR) in cancer cells.
- Mouse Pgps (mdr1a and mdr1b) exhibit different resistance profiles compared to human Pgp.
- Understanding sequence variations is crucial for overcoming drug resistance.
Purpose of the Study:
- To generate and characterize chimeric Pgp variants with altered drug resistance profiles.
- To investigate the role of specific Pgp domains in drug interaction and inhibitor sensitivity.
- To explore changes in Pgp epitope exposure and drug binding interactions.
Main Methods:
- DNA shuffling was employed to create homologous recombination between human MDR1 and mouse mdr1a Pgp domains.
- Chimeric Pgp proteins were expressed in human KB-3-1 cells for functional analysis.
- Drug resistance assays, photoaffinity labeling with [125I]iodoarylazidoprazosin, and antibody staining (mAbs MRK16 and UIC2) were utilized.
Main Results:
- A novel chimeric Pgp (clone 3-4) exhibited altered drug resistance.
- Inhibitors like verapamil and cyclosporin A were less effective against the chimeric Pgp for certain drugs.
- Cyclosporin A competed for photoaffinity labeling, suggesting altered binding interactions.
- Chimeric Pgp showed reduced staining with mAb MRK16 but increased staining with mAb UIC2, indicating altered epitope exposure.
Conclusions:
- DNA shuffling successfully generated a chimeric Pgp with a modified resistance phenotype.
- The study highlights alterations in drug-reversing agent interactions and Pgp epitope presentation.
- These findings provide insights into Pgp structure-function relationships relevant to MDR.