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The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis. more...

Two proteins with M(r) values of 170 kDa and 200 kDa, respectively, were identified in HOB1 lymphoma cells resistant to 1.0 microM vincristine (designated HOB1/VCR1.0) by Western blot. Using anti-P-glycoprotein monoclonal antibody to immunoprecipitate the protein from the 32P-labeled extract of HOB1/VCR1.0 cells, the major form of the protein was in the area of 200 kDa. When the cells were cultured in 0.5 microM vincristine for 72 h, the major form shifted to the area of 170 kDa. Northern blot analysis of the mdr transcription showed the gene had been overexpressed to a maximum in the cells resistant to 0.5 microM vincristine. [3H]Vincristine uptake study showed the cells with hyperphosphorylated P-glycoprotein accumulated only half the amount of the agent after 60 min of incubation as compared with those with hypophosphorylated protein. The current study suggests hyperphosphorylated P-glycoprotein is a form by which the cells can effectively exploit the protein when it can not be induced any more. more...

Of the compounds that have now been shown to circumvent acquired cellular multidrug resistance, little or no structure-activity relationship has been found, although their proposed mechanism of action is through modulation of function of p-glycoprotein. While it has been suggested that this inhibition is a direct binding to p-glycoprotein, we show here that such a model seriously neglects the effects many of these compounds have on lipid physical properties. We have characterized the interactions between 16 structurally diverse pharmacological agents (nine of which are known to reverse multidrug resistance) and a variety of lipids. Potent modulators inhibit the membrane binding of rhodamine 6G, and we have observed a correlation of the measured Ki values with the effectiveness of the compounds in situ. We have determined the effects of the compounds on detergent micellization, and have shown substantial changes on the critical micelle concentration of detergents in the presence of modulators. Finally, we have examined the changes in model membrane 'viscosity' induced by the compounds. These results indicate that both direct p-glycoprotein and indirect lipid interactions of modulators should be considered in the mechanism by which these compounds reverse multidrug resistance. more...

P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for the multidrug resistant (MDR) phenotype in various cancer cells. It has been shown that P-gp transports various kinds of anti-cancer agents as well as hydrophobic chemicals. Although P-gp is also expressed in normal human tissues, such as liver, kidney, and adrenal gland, its function and transporting substrates in these tissues are still unknown. In previous work, we demonstrated that some compounds in human plasma modulate the transporting activity of P-gp. We also found that P-gp is expressed at a high level in the bovine adrenal gland and that this tissue contains large amount of compounds which inhibit the transporting activity of P-gp. We purified such compounds from the adrenal gland by monitoring the ability to enhance the accumulation of [3H]vincristine in MDR cells. Two major compounds were purified and identified as progesterone and pregnenolone by nuclear magnetic resonance (NMR) analysis. Progesterone was the most potent and abundant compound that inhibited the transporting activity of P-gp among the compounds extracted from bovine adrenal gland with methanol. We also found that six authentic progesterone metabolites in the 5 beta-metabolic pathway but none in the 5 alpha-metabolic pathway were able to enhance the accumulation of [3H]vincristine in MDR cells and to inhibit [3H]azidopine photolabeling of P-gp in the adrenal gland. These results indicate that some progesterone metabolites can interact with P-gp and that stereoisomerism around carbon 5 of the progesterone metabolites is important for them to be recognized by P-gp. more...

The influence of lipid composition, internal pH and internal buffering capacity on the retention properties of vincristine loaded into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been assessed. It is shown that increasing the (saturated) acyl chain length of the phosphatidylcholine molecule, increasing the internal buffering capacity, and decreasing the internal pH all result in increased drug retention. Further, a study of the pH dependence on the rates of accumulation indicate that uptake proceeds via the neutral form of the vincristine molecule. This uptake is associated with an activation energy of 37 kcal/mol for DSPC/Chol LUVs. It is shown that the major improvement in drug retention in vitro is achieved by employing low initial internal pH values, where 90% retention is obtained over 24 h for an initial internal pH of 2. Improved retention in vivo was also observed where a drug-to-lipid ratio approx. 4-fold greater at 24 h was maintained. more...