Simultaneous expression of two P-glycoprotein genes in drug-sensitive Chinese hamster ovary cells.

Abstract

Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells. We analyzed four Pglycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line. We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites. A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species. These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.The presence of multidrug-resistant cells in human tumors and their selection and proliferation during chemotherapy may be major obstacles to cancer treatment (9). Mammalian cell lines selected for resistance to a single cytotoxic agent often develop pleiotropic resistance to unrelated drugs (9). Since many of the drugs involved are commonly used in chemotherapy, such cell lines have proven useful as in vitro models for the study of multidrug resistance in human tumors.A consistent alteration in multidrug-resistant cells is the overexpression of P-glycoprotein, an integral membrane protein of 170 kilodaltons (9). A P-glycoprotein cDNA clone, pCHP1 (19), has been isolated by using a P-glycoproteinspecific monoclonal antibody (14). This clone was used to isolate the cDNA clones discussed in this paper. Independently, an in-gel renaturation technique (20) has also been used to isolate amplified sequences from a drug-resistant hamster cell line (21). These sequences were used to isolate homologous human (2) and mouse (10) cDNA clones. The human cDNA sequence (mdrl) was reconstructed from several overlapping clones isolated from a multidrug-resistant cell line cDNA library. The mouse sequence was determined from a single full-length cDNA clone isolated from a drug-sensitive cell line. The human cDNA sequence (mdrl) encodes a protein detected by P-glycoprotein-specific monoclonal antibodies (26). We show that the human and mouse genes are homologs of different hamster P-glycoprotein (pgp) genes. We refer to the human and mouse mdr sequences as pgp genes in this communication to facilitate comparison of the P-glycoprotein genes across species.P-glycoprotein contains a tandem duplication in structure, with each half consisting of six transmembrane domains and a cytoplasmic domain containing the consensus sequences for an ATP-binding site (Fig.

The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues.

“…A central issue in understanding the normal functions of the P-glycoprotein has been the development of data indicating that the gene encoding the P-glycoprotein is a member of a multigene family (11,15,18,30). Probes from the amplified DNA domain in multidrug-resistant cell lines cross-hybridized to multiple genomic sequences (16,25) and identified two homologous but distinct mouse cDNAs (17).…”

“…Drug-resistant cell lines selected by stepwise selection using a single cytotoxic compound are often crossresistant to a broad spectrum of structurally and functionally unrelated chemotherapeutic agents (5,6,9,21,23). Multidrug-resistant cultured cell lines express increased levels of the P-glycoprotein (22), a 170-kilodalton membrane glycoprotein encoded by a gene termed mdr (16,24,25) or pgp (11). P-glycoprotein appears to cause multidrug resistance via an energy-dependent drug efflux mechanism (10,26).…”

Molecular analysis of the multidrug transporter, P-glycoprotein

“…Hence, only class 1 includes the drug-transporting P-glycoproteins or multidrug transporters which are the focus of this review. The human MDR1 (Chen et al, 1986), the mouse mdr3 (or mdr1a) (Devault and Gros, 1990; and mdr1 (or mdr1b) (Gros et al, 1986b), the hamster pgp1 and pgp2 (Endicott et al, 1987;Gros et al, 1986c), and the rat pgp1 and pgp2 (or mdr1b) gene products (Deuchars et al, 1992;Silverman et al, 1991) all belong to this class, while non drug-transporting P-glycoproteins, such as the human MDR2, or an alternative splice form thereof referred to as MDR3 (Schinkel et al, 1991;Van der Bliek et al, 1987), the mouse mdr2 (Buschman et al, 1992;Gros et al, 1988), the hamster pgp3 (Endicott et al, 1991), and the rat mdr2/mdr3 (Brown et al, 1993;Furuya et al, 1994) gene products belong to class 2 (which sometimes is also described as class 3). Class 2 mdr gene products are primarily expressed in the liver bile canaliculi (Buschman et al, 1992;CordonCardo et al, 1990;Smit et al, 1994;van der Valk et al, 1990) and analyses of mdr2 knock-out mice have indicated that class 2 P-glycoproteins represent phosphatidylcholine transporters (Smit et al, 1993).…”

Identification of members of the P-glycoprotein multigene family.

“…Transfection experiments (8,16,36) have demonstrated that overproduction of P-glycoprotein is sufficient to cause MDR. Analyses of partial and full-length cDNAs encoding P-glycoprotein from hamster (11,12), mouse (15,17), and human (3, 37; A. M. van der Bliek, P. M. Kooiman, C. Schneider, and P. Borst, Gene, in press) cells have shown that P-glycoprotein is a highly conserved protein which appears to be tandemly duplicated. Each duplicated half consists of six potential membrane-spanning regions and a putative nucleotidebinding site, features compatible with the proposed role of P-glycoprotein as an energy-dependent pump (3,12,15).…”

“…A family of closely related genes, referred to as pgp genes in hamsters (11) and as mdr genes in mice and humans (3,15,17,37), encodes P-glycoprotein (29). These genes are frequently differentially amplified in cell lines selected for resistance to different drugs (7,29,32).…”