Multidrug-resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotherapeutic agents. It often involves overexpression of P- glycoprotein, a plasma-membrane located protein of around 1280 amino acids, composed of two duplicated halves, each of which contains six predicted transmembrane helices and one nucleotide-binding site. There is firm evidence that P-glycoprotein acts in an ATP-dependent manner to exclude drugs and a wide variety of other hydrophobic compounds from cells. We and others have established that P-glycoprotein displays substantial drug-stimulated ATPase activity, and the most widely- considered current hypothesis is that P-glycoprotein acts as an ATP- driven drug-efflux pump. We have recently generated a Chinese hamster ovary cell line that grows vigorously and constitutively overexpress P-glycoprotein, up to 32% (w/w) in isolated plasma membranes. Using this system as source material, we propose to carry out a thorough biochemical investigation of P-glycoprotein, involving purification to homogeneity, reconstitution in liposomes, characterization of transport properties, detailed study of the nucleotide binding sites and the catalysis of ATP hydrolysis, and characterization of inhibitors of catalysis. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein in cells and overcome multidrug-resistance in patients.
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