The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

Increased expression of P-glycoprotein, a plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (170K), occurs in a wide variety of cell lines that exhibit pleiotropic resistance to unrelated drugs. The presence of P-glycoprotein in human cancers refractory to chemotherapy suggests that tumour cells with multidrug resistance can arise during malignant progression. We have discovered striking homology between P-glycoprotein and the HlyB protein, a 66K Escherichia coli membrane protein required for the export of haemolysin (protein of Mr 107K). P-glycoprotein can be viewed as a tandem duplication of the HlyB protein. The hydropathy profiles of the two proteins are similar and reveal an extensive transmembrane region resembling those found in pore-forming plasma membrane proteins. The C-terminal region of P-glycoprotein and the HlyB protein contain sequences homologous to the nucleotide-binding domains of a group of closely related bacterial ATP-binding proteins. We propose a model for multidrug resistance in which P-glycoprotein functions as an energy-dependent export pump to reduce intracellular levels of anticancer drugs.

Multidrug resistance is frequently characterized by an ATPdependent reduction in cellular drug accumulation. This phenotype can occur in mammalian cells by overexpression of either the multidrug resistance protein (MRP) 1 or P-glycoprotein (MDR1) (1-5). MRP and P-glycoprotein belong to the ATPbinding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identity (1). Nevertheless, both proteins confer resistance to a broad range of cytotoxic xenobiotics including doxorubicin, vincristine, and VP-16 (etoposide), drugs that are widely used in the treatment of many human cancers. However, there is growing evidence that the mechanisms by which MRP and P-glycoprotein reduce cellular drug accumulation are not the same, suggesting that there are major differences in the drug-protein interactions of these two molecules (6 -8).Like most eukaryotic ABC proteins, MRP and P-glycoprotein contain hydrophobic membrane spanning domains (MSDs) and cytoplasmic nucleotide binding domains (NBDs) (9). To understand how drugs interact with P-glycoprotein, there has been considerable interest in determining the precise topology of this integral membrane protein. Investigations in most experimental systems support a model in which P-glycoprotein is organized as a symmetrically arranged, tandemly duplicated molecule with each half consisting of six transmembrane segments followed by a NBD (10), but alternate models have also been proposed (11,12).At present, little is known about the membrane topology of MRP. The topological model we proposed when MRP was cloned in 1992 was based on computer-assisted hydropathy analyses of its deduced amino acid sequence and alignment with the predicted structure of ltpgpA (1). LtpgpA is an ABC protein cloned from Leishmania tarentolae which was the most closely related protein to MRP known at that time (13). In the original model, we suggested that MRP consisted of eight transmembrane segments and an NBD in its NH 2 -proximal half and only four transmembrane segments and an NBD in its COOH-proximal half (1). More recently, alignment of the hydropathy profiles of human MRP with those of its murine ortholog (14) and several members of the ABC superfamily (including the related sulfonylurea receptor, SUR (15), and the yeast cadmium resistance factor, YCF1 (16), as well as Pglycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)) suggested to us a different topology for MRP. In this later model, we predicted that MRP contains two MSDs of six transmembrane helices in a "6 ϩ 6" configuration typical of several eukaryotic ABC transporters (9), plus an extremely hydrophobic NH 2 -terminal MSD of approximately 220 amino acids (4,14,17,18). This additional hydrophobic domain is predicted to contain four to six transmembrane segments and is not present in ABC proteins such as P-glycoprotein and CFTR. Thus it is a characteristic feature of members of the MRP branch of the ABC transporter superfamily (4,14).