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Flow cytometric double labeling technique for screening of multidrug resistance
E E Gheuens1, D R van Bockstaele, M van der Keur
1Lab of Cancer Research and Clinical Oncology, Antwerp University, Wilrijk, Belgium.
Abstract:
We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR-content were not related to differences in DNA-content. In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.
Insights
Flow cytometry effectively analyzes multidrug resistance (MDR) in ovarian cancer cells. Combining daunorubicin uptake and P-glycoprotein expression reveals aberrant cell subpopulations missed by single-assay methods.
Area of Science:
- Oncology
- Cell Biology
- Biochemistry
Background:
- Multidrug resistance (MDR) is a significant challenge in ovarian cancer chemotherapy.
- P-glycoprotein (P-gp) is a key efflux pump contributing to MDR.
- Accurate assessment of MDR mechanisms is crucial for effective treatment strategies.
Purpose of the Study:
- To evaluate the utility of flow cytometry for analyzing MDR in ovarian cancer.
- To combine functional daunorubicin (DNR) uptake assays with P-gp expression analysis.
- To assess the ability of this multiparametric approach to detect aberrant cell subpopulations.
Main Methods:
- Utilized flow cytometry to analyze a multidrug resistant (MDR) ovarian cancer cell line (2780AD) and its drug-sensitive parental line (A2780).
- Employed a functional assay with daunorubicin (DNR) as a fluorescent probe to measure drug accumulation.
- Combined DNR assay with immunofluorescence detection of P-glycoprotein (P-gp) using the MRK-16 antibody.
Main Results:
- MDR cells exhibited significantly lower intracellular DNR content compared to sensitive cells.
- Verapamil treatment increased DNR content in MDR cells, but did not reach levels seen in sensitive cells.
- P-gp expression levels were inversely correlated with DNR content; MDR cells showed high P-gp and low DNR, while sensitive cells showed the opposite.
- Multiparametric analysis revealed aberrant subpopulations in clinical samples undetectable by single-parameter assays.
Conclusions:
- Flow cytometry, combining DNR uptake and P-gp expression, is a powerful tool for characterizing MDR in ovarian cancer.
- This multiparametric approach enhances the detection of heterogeneous cell populations within clinical samples.
- The findings support the use of combined flow cytometric assays for a more comprehensive understanding of MDR mechanisms and patient stratification.