[CANCER RESEARCH 58, 4160-4167. September 15. 1998] Taxol Resistance Mediated by Transfection of the Liver-specific Sister Gene of P-Glycoprotein1 Sarah Childs, Richard Lin Yeh, David Hui, and Victor Ling2 BC Cancer Research Center. BC Cancer Agencv, University of British Columbia. Vancouver, British Columbia, V5Z IL3 Canada ABSTRACT The sister gene of P-glycoprotein (Spgp) is a liver-specific ATP-binding cassette protein highly related to the P-glycoprotein (Pgp) family (S. Childs et al, Cancer Res., 55: 2029-2034, 1995). Spgp appears to be related to the Pgp family by an ancient duplication occurring before the division of fish and mammals. P-Glycoproteins have diverse functions including broad specificity multidrug resistance in cell lines and tumors, detoxification of tissues such as the intestine and blood-brain barrier, and phosphatidylcholine transport in liver. Spgp is a ,17, —170,000 glycosy- lated plasma membrane protein localized to the canalicular surface of hepatocytes in the rat liver. The full-length cDNA of Spgp was isolated from rat, and its expression was characterized in situ and in transfected cells. The expression of Spgp correlates with the differentiation of hepa tocytes and is seen only in late liver development. It is not observed in hepatoma cell lines. The physiological function of Spgp in liver is un known, but it maps to 2q31 in humans, in the vicinity of liver transport disorders for bile acids and cholesterol. Spgp may therefore be involved in some aspect of bile acid or cholesterol metabolism. Spgp transfectants have a low level resistance to Taxol but not to other drugs that form part of the multidrug resistance phenotype. This resistance is reversible by the Pgp-reversing agents cyclosporin A, PSC833, and verapamil, suggesting a conservation in some functions of Pgps across large evolutionary distance. INTRODUCTION A new member of the P-glycoprotein family of genes called Spgp3 was first identified in pig liver from a partial cDNA sequence (1). It appears to be a highly liver-specific protein. Its relationship to the Pgp family is close enough that some nucleic acid probes and antibodies cross-react with Spgp. However, Spgp is distinct from the Pgp mul- tigene family and exists as an independent gene in species as distantly related to mammals as the fish, winter flounder, and minnow (2).4 Thus, the divergence of Spgp and the Pgps probably preceeded the emergence of the Pgp multigene family (class I, II, and III genes) in mammalian species. The function of Spgp is unknown, but its evo lutionary relationship to the P-glycoproteins suggests that it could be capable of transporting drugs similar to the class I and class II isoforms, or it could have a basic role in liver physiology similar to the class III isoform. Although in sequence Spgp does not resemble any Pgp family member more than any other, its unique liver-specific expression pattern most closely resembles that of mdr2 encoding the class III isoform (l). In liver, the class III isoform of Pgp is a bile canalicular membrane protein (3). Its physiological function was discovered by analysis of Received 3/10/98: accepted 7/20/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the National Cancer Institute of Canada (to V. L.). a National Cancer Institute of Canada Sieve Fonyo Fellowship (to S. C.), and a Canadian Breast Cancer Foundation Studentship (to D. H.). 2 To whom requests for reprints should be addressed, at BC Cancer Research Centre, 601 West 10th Avenue. Vancouver, British Columbia, V5Z 1L3 Canada. Phone: (604) 877-6151: Fax: (604) 877-6150; E-mail: vling@bccancer.bc.ca. 3 The abbreviations used are: Spgp. sister gene of P-glycoprotein; Pgp. P-glycoprotein; PC, phosphatidylcholine; CsA. cyclosporin A: MDR. multidrug resistance: FISH, fluo rescence in situ hybridization: PAC. PI-derived artificial chromosome; DAPI, 4',6- diamidino-2-phenylindole; UTR. untranslated region: PFIC2. progressive familial intra- hepatic cholestasis type 2; cMOAT, canalicular multiple organic anión. 4 P. S. Cooper, personal communication. mice in which the gene had been "knocked out." These mice exhibited abnormal liver pathology including proliferation of bile ducts and a complete absence of PC in bile (4). Subsequent in vitro experiments have shown that mdr2 encodes a PC translocase, transferring PC from the inner leaflet of the membrane to the outer leaflet (5). PC, but not other phospholipids, is an essential component of bile, and by forming mixed micelles with bile acids, it is thought to prevent the detergent action of bile from damaging the cells lining the bile ducts. In contrast, the other members of the Pgp family have been found to confer broad specificity MDR, transporting a myriad of seemingly unrelated drugs out of cells using the energy supplied by the hydrol ysis of ATP (reviewed in Ref. 6). In humans, the overexpression of MDR 1 has been shown to correlate with poor response to chemother apy, while coadministering drugs that inhibit Pgp during chemother apy can improve survival (7, 8). Studies with mice lacking one or both Pgp genes associated with MDR have shown that these molecules contribute significantly in normal physiology to the prevention of drugs crossing the blood-brain barrier and to the clearance of drugs through the intestine and liver (9, 10). One surprising result from these studies is that Taxol (paclitaxel) elimination from the liver is normal in the knockout mice, whereas Taxol elimination from the gut is impaired. This suggested that the liver has an additional non-Pgp mechanism for transporting Taxol into bile (10-12). Here we describe the cloning and characterization of Spgp from rat to gain further insight into its transport specificity and function to see if it might play a pharmacological role in the elimi nation of drugs from the liver. At the same time, the specificity of expression of Spgp in the liver across the entire range of vertebrate phylogeny suggests that it has a conserved ancient function in the liver. We have used FISH mapping as a first step to identify putative physiological functions and substrates. MATERIALS AND METHODS Probe Design and cDNA Isolation. The 109-bp GAPIS probe was gen erated by PCR amplification of the pig spgp sequence5 using the primers 5'-TGCCGTCATGTCACAGGG-3' and 5'-TCAACTGATGGGGGCTCC-3'. Total RNA was isolated from an adult male Sprague Dawley rat liver by the cesium chloride density gradient method (13). mRNA was then purified on an oligo-dT cellulose column (Cedarlane Labs), and the cDNA was synthesized (Superscript II; Life Technologies, Inc.), ligated into ÀZAP phage (Strat- agene), and screened with GAPIS on Hybond-N membrane (Amersham), according to the manufacturer. 5' rapid amplification of cDNA ends was used to obtain the 5' end of spgp using the Matchmaker protocol (Clontech). Spgp-specific primers 5'- GAAAAAGTCCCAAGGAGCTGGCTG-3' and 5'-ACCCACACATAGC- CGTCG-3' were used in the first and second amplifications, respectively. Independent PCRs resulted in a fragment of 1 kb in length, which was subcloned as a 900-bp Noti-Sali fragment. Two independent PCR fragments were completely sequenced, and a third and fourth clone were also sequenced at one position of difference to determine the correct sequence. The full-length cDNA was cloned by ligating this fragment with a 4.5-kb, Sall-EcoRl-cut cDNA into the Noti and EcoRl sites of pBluescript (Stratagene). Oligonucleotide primers were designed at ~300-bp intervals to sequence full-length spgp on both strands from a minimum of two clones by the dideoxy 5 The sequence has been deposited with accession number AFOI0597 in GenBank. 4160 Research. on November 21, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from