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Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse mdr1
E Schurr1, M Raymond, J C Bell
1Department of Biochemistry, Faculty of Medicine, McGill University, Montreal, Canada.
Abstract:
Structural features of the multidrug resistance protein encoded by the mouse mdr1 gene were studied in multidrug-resistant cell clones stably transfected with a biologically active cDNA clone. Independently derived transfectant cell clones, initially selected in Adriamycin, were shown to be cross-resistant to several drugs, including actinomycin D, amsacrine, mitoxantrone, VP-16, and vinblastine but remained sensitive to cis-platinum, 5-fluorouracil, arabinocytosine, and bleomycin. In drug-resistant transfectants the mdr1 gene product was greatly overexpressed as a polypeptide of apparent molecular weight 160,000-170,000. This protein was present in membrane enriched fractions and could be metabolically labeled with [3H )glucosamine, confirming that the transfected mdr1 gene encodes a membrane glycoprotein. The protein was found phosphorylated on serine residues and was shown to be photolabeled by both the calcium antagonist azidopine and the ATP analogue 8-azido ATP. Tryptic mapping of the ATP-photoaffinity labeled protein indicated that ATP crosslinking was site-specific and limited to two discrete peptide fragments of the protein, suggesting that the overexpressed mdr protein is capable of direct and specific ATP binding.
Insights
The mouse mdr1 gene product, a membrane glycoprotein, is overexpressed in multidrug-resistant cells. This protein directly binds ATP, contributing to cellular resistance against various chemotherapy drugs.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Multidrug resistance (MDR) is a major challenge in cancer chemotherapy.
- The mouse mdr1 gene encodes a protein implicated in MDR.
- Understanding the structural and functional properties of this protein is crucial.
Purpose of the Study:
- To characterize the structural features of the mouse mdr1 gene product.
- To investigate the protein's role in multidrug resistance.
- To determine the drug-binding and ATP-binding capabilities of the mdr1 protein.
Main Methods:
- Stable transfection of mouse cell lines with mdr1 cDNA.
- Drug resistance assays (Adriamycin, actinomycin D, etc.).
- Protein expression analysis (Western blotting, metabolic labeling).
- Photolabeling with azidopine and 8-azido ATP.
- Tryptic mapping of photolabeled protein.
Main Results:
- Transfected cells exhibited cross-resistance to multiple drugs.
- Overexpression of a 160,000-170,000 MW membrane glycoprotein encoded by mdr1.
- The protein is a glycoprotein, phosphorylated on serine residues.
- Photolabeling confirmed binding of calcium antagonist and ATP analogue.
- ATP crosslinking was site-specific, indicating direct ATP binding.
Conclusions:
- The mdr1 gene product is a membrane glycoprotein overexpressed in multidrug-resistant cells.
- This protein directly binds ATP, suggesting a role in drug efflux mechanisms.
- The findings provide insights into the molecular basis of multidrug resistance.