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P-glycoprotein (Pgp) and vaults are associated with multidrug resistance in tumor cells, but their physiological functions are not yet clear. Pgp, the prototypical transmembrane transporter molecule, may also facilitate the migration of skin dendritic cells (DC). Vaults--ribonucleoprotein cell organelles, frequently overexpressed in Pgp-negative drug-resistant tumor cells--have also been associated with intracellular transport processes. Given the pivotal role of DC in dealing with exposure to potentially harmful substances, the present study was set out to examine the expression of Pgp and vaults during differentiation and maturation of DC. DC were obtained from different sources, including blood-derived monocytes, CD34(+) mononuclear cells, and chronic myeloid leukemia cells. Whereas flow cytometric and immunocytochemical analyses showed slightly augmented levels of Pgp, up-regulation of vault expression during DC culturing was strong, readily confirmed by Western blotting, and independent of the source of DC. In further exploring the functional significance of vault expression, it was found that supplementing DC cultures with polyclonal or mAbs against the major vault protein led to lower viabilities of LPS- or TNF-alpha-matured monocytes-DC. Moreover, expression of critical differentiation, maturation, and costimulatory molecules, including CD1a and CD83, was reduced and their capacity to induce Ag-specific T cell proliferative and IFN-gamma release responses was impaired. These data point to a role for vaults in both DC survival and functioning as APC.

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.

Tumor cells that are insensitive to anticancer drugs frequently have a multidrug-resistant (MDR) phenotype. Proteins that can be involved in this phenomenon are transport-associated proteins such as P-glycoprotein, multidrug-resistance protein 1, breast cancer resistance protein, and lung resistance-related protein (LRP). LRP was identified as the major vault protein (MVP), the main component of multimeric vault particles. With the recent identification of the two minor vault proteins as telomerase-associated protein (TEP1) and vault-poly (ADP-ribose) polymerase (VPARP), and with high-resolution three-dimensional imaging, the composition of vaults is almost unraveled. Although the first direct evidence for a causal relationship between LRP/MVP expression and drug resistance has been obtained, many functional aspects of vaults in normal physiology and in MDR still need to be clarified. The current clinical data on LRP/MVP detection indicate that LRP/MVP expression can be of high clinical value to predict the response to chemotherapy of several tumor types.

Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (MDRI) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported solely using mRNA data. In this study, we describe a BCRP-specific monoclonal antibody, BXP-34, obtained from mice, immunized with mitoxantrone-resistant, BCRP mRNA-positive MCF-7 MR human breast cancer cells. BCRP was detected in BCRP-transfected cells and in several mitoxantrone- and topotecan-selected tumor cell sublines. Pronounced staining of the cell membranes showed that the transporter is mainly present at the plasma membrane. In a panel of human tumors, including primary tumors as well as drug-treated breast cancer and acute myeloid leukemia samples, BCRP was low or undetectable. Extended studies will be required to analyze the possible contribution of BCRP to clinical multidrug resistance.

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.

Aim: To analyse the expression of multidrug resistance (MDR) related proteins at different steps in colorectal carcinogenesis., Methods: The presence of three MDR related proteins (Pgp, MRP1, and LRP/MVP) was studied by means of immunohistochemistry in normal, adenomatous, and malignant colorectal epithelium. Formaldehyde fixed, paraffin embedded tissue sections of 17 samples of colorectal tissue were used (normal mucosa, n = 4; adjacent mucosa, n = 5; adenoma, n = 5; carcinoma, n = 3)., Results: For all three proteins, expression was found in the surface epithelium and the upper parts of the crypts in normal colon. In the adenomas, staining was seen along the complete length of the crypts. In the carcinomas analysed, all epithelium showed positive staining. Mucosa adjacent to either carcinoma or adenoma showed staining patterns mostly resembling those of normal mucosa, but sometimes some extension of staining was seen along the crypt., Conclusions: These proteins already show increased expression in the adenoma stage. In the absence of adequate mucin production in adenomas, MDR related proteins could be an important factor in protecting the epithelium against further environmentally induced genetic damage. This could be one of the reasons why only about 5% of colorectal adenomas will actually progress to carcinomas.

In this review we analyze the data supporting the notion that vault-related MDR, as reflected by LRP/MVP overexpression, represents a marker of drug resistance in vitro and in the clinic. Vaults, besides playing a fundamental biological role, may be involved in a novel mechanism of MDR.

Multidrug resistant cancer cells frequently overexpress the 110-kd lung resistance-related protein (LRP) as detected with the monoclonal antibody (MAb) LRP-56. Recently, we identified LRP as the major vault protein (MVP), which is the major constituent of vaults, multisubunit cellular organelles. Clinically, LRP/MVP expression in cancer at time of diagnosis provided a strong and independent prognostic factor for response to chemotherapy and outcome in different tumor types, notably acute myeloid leukemia and ovarian cancer. To facilitate additional immunohistopathological studies, we have optimized LRP/MVP detection in paraffin-embedded tissues using two monoclonal antibodies, LRP-56 and LMR-5. Blocking experiments showed that LRP-56 and LMR-5 MAbs detect different epitopes of LRP/MVP. Immunohistochemical studies with both MAbs in a panel of human multidrug resistant tumor cell lines, normal tissues, and colorectal tumors showed that LRP/MVP expression can be reliably detected after formalin-fixation and paraffm-embedding using overnight incubation at 4 degrees C with the primary MAbs at 5- to 10-fold higher concentrations (ie, 1 to 10 microg/ml) as currently used with frozen sections. Both streptavidin-biotin complex and alkaline phosphatase-anti-alkaline phosphatase techniques could be successfully used for signal-amplification. Staining quality did not benefit from antigen-retrieval pretreatments. The optimized staining methodology facilitates studies in archival material on the putative role of LRP/MVP in clinical drug resistance.

The human multidrug resistance protein (MRP) is a 190 kd membrane glycoprotein that can cause resistance of human tumor cells to various anticancer drugs, by extruding these drugs out of the cell. Three different monoclonal antibodies, directed against different domains of MRP, allowed us to determine the localization of MRP in a panel of normal human tissues and malignant tumors. Whereas in malignant tumors strong plasma membrane MRP staining was frequently observed, in normal human tissues MRP staining was predominantly cytoplasmatic. Here, MRP was detected in several types of epithelia, muscle cells, and macrophages. From the presence of MRP in many epithelia we infer that MRP, like MDR1 P-glycoprotein, may have an excretory function in protecting the organism against xenobiotics. Recent studies indicate a role for MRP as a carrier for transport of glutathione-conjugated endo- and xenobiotics. The presence of MRP in bronchiolar epithelium, heart muscle, and macrophages would agree with the glutathione S-conjugate carrier activity previously detected in these cells. Furthermore, in 46 of 119 untreated tumors from various histogenetic origins MRP staining was seen. In these tumors MRP may contribute to the intrinsic resistance against treatment with chemotherapeutic drugs.