Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A
Agnieszka Krawczenko1, Aleksandra Bielawska-Pohl1, Karolina Wojtowicz2
1Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
Abstract:
Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.
Insights
Multidrug resistance (MDR) proteins are crucial in endothelial cells, but assessing their expression is challenging. Functional tests proved most conclusive for evaluating MDR protein activity in endothelial cells, despite limited specificity.
Area of Science:
- Cell Biology
- Pharmacology
- Biochemistry
Background:
- Multidrug resistance (MDR) proteins are active transporters found in various cell types, including tumor, stem, and endothelial cells.
- While extensively studied in cancer, the role of MDR proteins in non-tumor cells and their functions beyond xenobiotic removal is less understood.
- Accurate assessment of MDR protein expression is complicated by discrepancies between mRNA and protein levels, and challenges in detecting low physiological expression.
Purpose of the Study:
- To identify the optimal methods for evaluating the expression level and functional activity of five key MDR proteins (MDR1, BCRP, MRP1, MRP4, MRP5).
- To investigate MDR protein expression and function in human endothelial cells (ECs) and their progenitors using various in vitro assays.
Main Methods:
- Quantitative analysis of MDR mRNA expression.
- Detection of MDR protein levels using immunological assays.
- Functional assays to assess the drug-transporting activity of MDR proteins.
- Comparative analysis of different methods for evaluating MDR expression and activity in endothelial cells.
Main Results:
- Endothelial cells exhibited MDR presence at the mRNA level, but this was not consistently confirmed at the protein level or via functional tests.
- Multiple assays were required to comprehensively evaluate MDR protein expression and function in endothelial cells.
- Functional tests were found to be the most conclusive for assessing MDR protein activity, although they lacked high specificity.
Conclusions:
- Evaluating MDR protein expression and function in endothelial cells necessitates a multi-assay approach.
- Functional assays are valuable for determining the biological activity of MDR proteins in endothelial cells.
- Further research is needed to refine methods for specific and accurate assessment of MDR proteins in physiological contexts.
