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Flow cytometric characterization of antifolate resistance in cultured mammalian cells using fluoresceinated
S W Sherwood1, Y G Assaraf, A Molina
1Department of Biological Sciences, Stanford University, California 94305.
Abstract:
We have studied antifolate-resistant rodent cell lines with respect to dihydrofolate reductase gene expression and expression of the "classic" multidrug resistance phenotype by flow cytometry. Using a series of antifolate-resistant and colchicine-resistant Chinese hamster ovary cell lines obtained by single-step and stepwise selection protocols, we show that viable cell staining with fluoresceinated methotrexate and daunorubicin correlates well with drug resistance and expression of dihydrofolate reductase protein and the "classic" MDR phenotype in these cell lines. We show that flow cytometric analysis makes it possible to rapidly assess the potentially complex drug resistance phenotype(s) of cells selected with hydrophilic and lipophilic antifolates.
Insights
Flow cytometry rapidly assesses drug resistance in rodent cell lines. This method correlates cell staining with antifolate resistance and multidrug resistance (MDR) phenotypes, aiding in understanding complex cellular responses.
Area of Science:
- Cell Biology
- Pharmacology
- Biochemistry
Background:
- Antifolate resistance is a significant challenge in chemotherapy.
- Multidrug resistance (MDR) complicates treatment efficacy.
- Understanding resistance mechanisms requires robust analytical methods.
Purpose of the Study:
- To evaluate flow cytometry for assessing antifolate resistance.
- To correlate drug resistance with dihydrofolate reductase (DHFR) expression.
- To analyze the multidrug resistance (MDR) phenotype in resistant cell lines.
Main Methods:
- Utilized antifolate-resistant and colchicine-resistant Chinese hamster ovary (CHO) cell lines.
- Employed single-step and stepwise selection protocols.
- Applied flow cytometry with fluoresceinated methotrexate and daunorubicin for cell staining.
Main Results:
- Viable cell staining correlated well with drug resistance levels.
- Fluorescent staining accurately reflected DHFR protein expression.
- Flow cytometry effectively identified the classic MDR phenotype.
Conclusions:
- Flow cytometry provides a rapid and reliable method for assessing complex drug resistance phenotypes.
- This technique is valuable for studying cells selected with various antifolates.
- The study demonstrates the utility of flow cytometry in characterizing cellular drug resistance.