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Construction of a model cell line for the assay of MDR1 (multi drug resistance gene-1) substrates/inhibitors using HeLa cells.

ABSTRACT: Cancer cells often become resistant to chemotherapy, and induction of the ABC transporter Multi-drug Resistance gene-1 (MDR1) is a major cause. We established a tool for high-throughput screening of substrates and inhibitors of MDR1, using transformed HeLa cells that over-express MDR1. The cDNA for human MDR1 was subcloned into the eukaryotic expression vector pBK-CMV to produce an MDR1 expression vector, pBK-CMV/MDR1. HeLa cells were transfected with pBK-CMV/MDR1 or the empty vector pBK-CMV. Transfection of the vector sequence for MDR1 and its expression were evaluated by genomic PCR and western blotting, respectively. The efficiency of the MDR1 transporter for pumping a substrate out of the transformed cells was evaluated using rhodamine123 (R-123), a mitochondrial dye that is also an MDR1 substrate. After treatment of the MDR1-expressing HeLa cells with MDR1 substrate vinblastin or inhibitors cyclosporin A and verapamil, the amount of R-123 retained in the cells was increased to 2 to 2.3 times the level in untreated MDR1-expressing HeLa cells. The transfection of empty pBK-CMV had no effect on the R-123 retention in HeLa cells, regardless of drug treatment. In conclusion, we have established a model human carcinoma cell line that expresses functional MDR1 and can be used to screen for substrates and inhibitors of MDR1.

SUBMITTER: Kugawa F

PROVIDER: S-EPMC4370427 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Construction of a model cell line for the assay of MDR1 (multi drug resistance gene-1) substrates/inhibitors using HeLa cells.

Kugawa F F Suzuki T T Miyata M M Tomono K K Tamanoi F F

Die Pharmazie 20090501 5

Cancer cells often become resistant to chemotherapy, and induction of the ABC transporter Multi-drug Resistance gene-1 (MDR1) is a major cause. We established a tool for high-throughput screening of substrates and inhibitors of MDR1, using transformed HeLa cells that over-express MDR1. The cDNA for human MDR1 was subcloned into the eukaryotic expression vector pBK-CMV to produce an MDR1 expression vector, pBK-CMV/MDR1. HeLa cells were transfected with pBK-CMV/MDR1 or the empty vector pBK-CMV. Tr ...[more]

PMID: 19530439

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Dataset Information

Construction of a model cell line for the assay of MDR1 (multi drug resistance gene-1) substrates/inhibitors using HeLa cells.

Publications

Construction of a model cell line for the assay of MDR1 (multi drug resistance gene-1) substrates/inhibitors using HeLa cells.

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