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ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells
M Horio1, M M Gottesman, I Pastan
1Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
Abstract:
Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the "MDR1" gene, whose product is the plasma membrane P-glycoprotein. Plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulate [3H]vinblastine in an ATP-dependent manner. This transport is osmotically sensitive, with an apparent Km of 38 microM for ATP and of approximately equal to 2 microM for vinblastine. The nonhydrolyzable analog adenosine 5'-[beta, gamma-imido]triphosphate does not substitute for ATP but is a competitive inhibitor of ATP for the transport process. Vanadate, an ATPase inhibitor, is a potent noncompetitive inhibitor of transport. These results indicate that hydrolysis of ATP is probably required for active transport of vinblastine. Several other drugs to which multidrug-resistant cell lines are resistant inhibit transport, with relative potencies as follows: vincristine greater than actinomycin D greater than daunomycin greater than colchicine = puromycin. Verapamil and quinidine, which reverse the multidrug-resistance phenotype, are good inhibitors of the transport process. These results confirm that multidrug-resistant cells express an energy-dependent plasma membrane transporter for hydrophobic drugs, and establish a system for the detailed biochemical analysis of this transport process.
Insights
Multidrug resistance in cancer cells involves P-glycoprotein, which actively transports hydrophobic drugs like vinblastine using ATP. This energy-dependent transporter can be inhibited by other drugs and reversing agents.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Multidrug resistance (MDR) in human cancer cells is a significant clinical challenge.
- Overexpression of the MDR1 gene leads to P-glycoprotein, a plasma membrane efflux pump.
- P-glycoprotein confers resistance to various cytotoxic hydrophobic agents.
Purpose of the Study:
- To investigate the biochemical properties of the P-glycoprotein transporter.
- To confirm the energy dependence and substrate specificity of vinblastine transport.
- To establish a system for analyzing the multidrug resistance transporter.
Main Methods:
- Isolation of plasma membrane vesicles from multidrug-resistant (MDR) and drug-sensitive human KB carcinoma cells.
- Measurement of [3H]vinblastine accumulation in vesicles in an ATP-dependent manner.
- Assays using ATP analogs, ATPase inhibitors (vanadate), and various drugs/reversing agents to characterize transport.
Main Results:
- MDR cells, but not sensitive cells, showed ATP-dependent accumulation of [3H]vinblastine.
- Transport required ATP hydrolysis, was osmotically sensitive, and exhibited specific kinetics for ATP and vinblastine.
- Several MDR drugs (vincristine, actinomycin D, daunomycin) and MDR-reversing agents (verapamil, quinidine) inhibited vinblastine transport.
Conclusions:
- Multidrug-resistant cells possess an energy-dependent plasma membrane transporter for hydrophobic drugs.
- ATP hydrolysis is essential for the active transport function of P-glycoprotein.
- This study provides a system for detailed biochemical analysis of the MDR transporter.