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Elimination of permeability mutants from selections for drug resistance in mammalian cells
M J Schibler1, S B Barlow, F Cabral
1Department of Pharmacology, University of Texas Medical School, Houston 77225.
Abstract:
Chinese hamster ovary (CHO) cells exhibit increased sensitivity to a wide variety of microtubule inhibitory drugs when verapamil is present in the growth medium. The extent of this increased sensitivity is drug specific: some drugs such as taxol and vinblastine respond greatly to the presence of verapamil, whereas other drugs such as griseofulvin respond very poorly. For the majority of drugs examined, however, a 2- to 10-fold increase in drug sensitivity is observed in the presence of verapamil at 5 micrograms/ml. The effects of verapamil are even more dramatic when drug-resistant mutant cells with a presumed alteration in membrane permeability are examined. In the presence of appropriate levels of verapamil, these mutants demonstrate a level of drug sensitivity comparable to that of the wild-type parental cells. Drug-resistant cells from similar selections but with well-defined alterations in alpha- or beta-tubulin and no evidence of alterations in membrane permeability, however, continue to exhibit increased resistance to the selecting drug even in the presence of verapamil. These studies support the conclusion that verapamil affects the membrane permeability to or transport of a wide variety of hydrophobic drugs. In addition, we have used this information to devise selections that virtually eliminate the isolation of drug-resistant permeability mutants. This methodology should be generally applicable to genetic studies of drug action that are complicated by the isolation of large numbers of mutants with permeability alterations.
Insights
Verapamil enhances the sensitivity of Chinese hamster ovary (CHO) cells to microtubule inhibitory drugs by increasing membrane permeability. This finding aids in developing new methods for genetic studies of drug action.
Area of Science:
- Cell Biology
- Pharmacology
- Genetics
Background:
- Chinese hamster ovary (CHO) cells are a common model for studying drug effects.
- Drug resistance in cell lines can arise from various mechanisms, including altered membrane permeability.
- Verapamil is a known calcium channel blocker with potential effects on cellular transport.
Purpose of the Study:
- To investigate the effect of verapamil on the sensitivity of CHO cells to microtubule inhibitory drugs.
- To determine if verapamil influences drug resistance mechanisms related to membrane permeability.
- To develop improved selection strategies for genetic studies of drug action.
Main Methods:
- Treatment of CHO cells with various microtubule inhibitory drugs in the presence and absence of verapamil.
- Examination of drug sensitivity in wild-type and drug-resistant mutant CHO cell lines.
- Analysis of drug resistance mechanisms, distinguishing between permeability alterations and target protein mutations.
Main Results:
- Verapamil significantly increased sensitivity to many hydrophobic drugs in CHO cells (2- to 10-fold).
- Verapamil restored drug sensitivity in mutants with presumed membrane permeability defects to wild-type levels.
- Mutants with altered tubulin but intact permeability remained resistant to verapamil's effects.
Conclusions:
- Verapamil enhances the uptake or transport of various hydrophobic drugs across the cell membrane in CHO cells.
- This mechanism is distinct from direct effects on drug targets like tubulin.
- The findings enable the development of selection methods to minimize the isolation of drug-resistant permeability mutants in genetic studies.