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Small hydrophobic peptides were studied as possible substrates of the multidrug resistance protein (MRP)-1 (ABCC1) transmembrane transporter molecule. As observed earlier for P-glycoprotein- (Pgp; ABCB1) overexpressing cells, MRP1-overexpressing cells, including cells stably transfected with the MRP1 cDNA, showed distinct resistance to the cytotoxic peptide N-acetyl-Leu-Leu-norleucinal (ALLN). Resistance to this peptide and another toxic peptide derivative, which is based on a Thr-His-Thr-Nle-Glu-Gly backbone conjugated to butyl and benzyl groups (4A6), could be reversed by MRP1 inhibitors. The reduced toxicity of 4A6 in MRP1-overexpressing cells was found to be associated with lower accumulation of a fluorescein-labeled derivative of this peptide. Glutathione (GSH) depletion had a clear effect on resistance to ALLN but hardly affected 4A6 resistance. In a limited structure-activity study using peptides that are analogous to 4A6, MRP1-overexpressing cells were found to be resistant to these peptides as well. Remarkably, when selecting A2780 ovarian cancer cells for resistance to ALLN, even in the absence of Pgp blockers, resulting cell lines had up-regulated MRP1, rather than any of the other currently known multidrug resistance transporter molecules including Pgp, MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCCS), and the breast cancer resistance protein ABCG2. ALLN-resistant, MRP1-overexpressing cells were found to be cross-resistant to 4A6 and the classical multidrug resistance drugs doxorubicin, vincristine, and etoposide. This establishes MRP1 as a transporter for small hydrophobic peptides. More extensive structure-activity relationship studies should allow the identification of clinically useful peptide antagonists of MRP1.

Vaults are 13 megadalton ribonucleoprotein particles composed largely of the major vault protein (MVP) and two high molecular weight proteins, p240 and p193, and a small vault RNA (vRNA). Increased levels of MVP expression, vault-associated vRNA, and vaults have been linked directly to multidrug resistance (MDR). To further define the putative role of vaults in MDR, we produced monoclonal antibodies against the Mr 193,000 vault protein and studied its expression levels in various multidrug-resistant cell lines. We find that, like MVP, p193 mRNA and protein levels are increased in various multidrug-resistant cell lines. Subcellular fractionation of vault particles revealed that vault-associated p193 levels are increased in multidrug-resistant cells as compared with the parental, drug-sensitive cells. Furthermore, protein analysis of postnuclear supernatants and co-immunoprecipitation studies show that drug-sensitive MVP-transfected tumor cells lack this up-regulation in vault-associated p193. Our observations indicate that vault formation is limited not only by the expression of the MVP but also by the expression or assembly of at least one of the other vault proteins.