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Cholesterol increases in a nonlinear way with the level of chemoresistance of the human CEM acute lymphoblastic leukemia and the membrane cholesterol can modulate the P-glycoprotein ATPase activity. The analysis highlights the complex effect of cholesterol on the multidrug resistance (MDR) phenotype and also the possibility of chemosensitizing resistant cells by the modulation of their membrane cholesterol content.

A study was conducted to analyze the overexpression and purification of the C-terminal nucleotide-binding domain of a parasite Pgp-like multidrug transporter that is encoded by the ltrmdr I of Leishmania tropica. Direction interaction with various classes of flavonoids and reversal of the multidrug resistance was also examined. Results indicated that flavonoids that bind with high affinity to the cytosolic domain of the transporter increases daunomycin accumulation in a L. tropica line overexpressing the transporter.

A hexahistidine-tagged C-terminal nucleotide-binding domain ([H.sub.6]-NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring of high-affinity binding of 2[prime](3[prime])-N-methylanthraniloyl-ATP (MANT-ATP), a fluorescent ATP derivative that induces a marked quenching correlated to fluorescence resonance-energy transfer. [H.sub.6]-NBD2 also bound all flavonoids known to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells, with similar affinities and relative efficiencies. Flavones (like quercetin or apigenin) bound more strongly than flavanones (naringenin), isoflavones (genistein), or glycosylated derivatives (rutin). Kaempferide, a 4[prime]-methoxy 3,5,7-trihydroxy flavone, was even more reactive and induced a complete quenching of [H.sub.6]-NBD2 intrinsic fluorescence. Kaempferide binding was partly prevented by preincubation with ATP, or partly displaced upon ATP addition. Interestingly, kaempferide was also able to partly prevent the binding of the antiprogestin RU 486 to a hydrophobic region similar to that recently found, close to the ATP site, in the N-terminal cytosolic domain. Conversely, RU 486 partly prevented kaempferide binding, the effect being additive to the partial prevention by ATP. Furthermore, MANT-ATP binding, which occurred at the ATP site and extended to the vicinal steroid-interacting hydrophobic region, was completely prevented or displaced by kaempferide. All results indicate that flavonoids constitute a new class of modulators with bifunctional interactions at vicinal ATP-binding site and steroid-interacting region within a cytosolic domain of P-glycoprotein.

Experiment was conducted to prove the hypothesis that a longer 371-705 domain, including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacts with steroids that have been proven to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Results prove that both ATP and steroid-binding regions are present on the cytosolic extended-NBD1 domain of P-glycoprotein which constitutes a promising tool for the identification of amino acids involved in both types of binding.