The multidrug-resistance associated protein MRP is a 180-to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-manisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, (20) and pRc/RSV (Invitrogen). All cDNA fragments used for the assembly of the MRP cDNA were sequenced and the integrity of the MRP cDNA fragment in the resulting expression vectors, pJ3fl-MRP and pRc/RSV-MRP (Fig. 1) Abbreviations: MDR, multidrug resistance (resistant); Pgp, P-glycoprotein; SCLC, small-cell lung cancer; pH;, intracellular pH; m-AMSA, 4'-(9-acridinylamino)methanesulfon-m-anisidide. 8822The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Multidrug-resistant cancer cells frequently overexpress the 110-kD LRP protein (originally named Lung Resistance-related Protein). LRP overexpression has been found to predict a poor response to chemotherapy in acute myeloid leukaemia and ovarian carcinoma. We describe the cloning and chromosome localization of the gene coding for this novel protein. The deduced LRP amino acid sequence shows 87.7% identity with the 104-kD rat major vault protein. Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport. The LRP gene is located on chromosome 16, close to the genes coding for multidrug resistance-associated protein and protein kinase C-beta, and may mediate drug resistance, perhaps via a transport process.

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein.Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membranebound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRPlike ATP-binding cassette transporters.Overexpression of the multidrug transporter proteins, Pglycoprotein (MDR1) 1 or the multidrug resistance-associated protein (MRP) provides the molecular basis of the multidrug resistance phenotype in tumor cells. The possible clinical importance fuels an intensive research activity toward a better understanding of the molecular structure and mechanism of action of these membrane transporters (1-3).Both P-glycoprotein and MRP, together with several other bacterial and eukaryotic transporters, are members of the ABC-transporter (ATP-binding cassette) protein family. These proteins share a common molecular architecture, i.e. they contain two large transmembrane domains and two cytoplasmic ATP utilization (ABC) units (4). Due to the difficulty of crystallizing large membrane proteins, no detailed three-dimensional structure of any members of these transporters is currently available, and empirical prediction methods are used to obtain molecular models of their structure, especially to predict the locations and numbers of the membrane-spanning helices. In most cases, these methods identify six short transmembrane segments in each of the two transmembrane domains (1-4). The relevance of the prediction for the membrane topology of CFTR has been confirmed experimentally by insertional mutagenesis (5), thus proving the 2 ϫ 6 transmembrane helix model. The same arrangement of transmembrane helices has been suggested in the case of P-glycoprotein (6, 7), and a large body of experimental data strongly favors this model (8, 9). On the other hand, Ling and co-workers (10, 11), by suggesting an alternative 6-and 4-helix conformation, raised the possibility that P-glycoprotein may exist in two different topological forms in the cell membrane.When the m...

Hyperexpression of MRP is frequently observed in primary NSCLC, especially in the well differentiated squamous cell carcinomas. Further studies are needed to assess the role of MRP in the mechanism of clinical drug resistance in NSCLC.