[CANCER RESEARCH 53. 3221-3225. July 15. 1993] Advances in Brief Localization of a Novel Multidrug Resistance-associated Gene in the HT1080/DR4 and H69AR Human 1\imor Cell Lines1 Marilyn L. Slovak,2 Jennifer Pelkey Ho, Gabu Bhardwaj, Ebba U. Kurz, Roger G. Deeley, and Susan P.C. Cole Department of Cytogenetics, City of Hope National Medical Center, Duane, California 91010 ¡M.L. S., J. P. H.¡,and Cancer Research Laboratories, Queen's University, Kingston, Ontario. Canada K7L 3N6 ¡G.B., E. U. K., R. G. D., S. P. C C.¡ Abstract Two doxorubicin-selected human tumor cell lines, H69AR and HT1080/ DR4, display a multidrug resistance phenotype but do not overexpress P-glycoprotein. Recently, a 6.5-kilobase mRNA encoding a novel member of the ATP-binding cassette superfamily of transport proteins, designated multidrug resistance-associated protein (MRP), has been identified in the H69AR cell line. In the present study, the levels of MRP mRNA were found to be 14-fold higher in HT1080/DR4 cells relative to sensitive HT1080 cells. Southern blotting indicates that gene amplification contributes to the overexpression of MRP in HT1080/DR4 cells. Using a 4-kilobase MRP complementary DNA probe, MRP genes were localized to 2-5 chromo somes bearing homogeneously staining regions and to multiple double minute chromosomes in H69AR cells. Resistant H69AR cells also con tained a new der(16) with a structural aberration affecting 16pl3.1, the normal cellular locus of the MRP gene. The MRP probe hybridized to two small homogeneously staining regions (hsr) in HT1080/DR4 cells including hsr(7)(pl2pl5). MRP localization was restricted to the normal cellular locus, 16pl3.1, in the parental H69 and HT1080 cells and the drug- sensitive H69PR revertan! cells. Our data provide combined evidence that amplification of the MRP gene is associated with the expression of drug resistance in selected solid tumor cell lines. Introduction Multidrug resistance is a complex and multifactorial phenomenon that limits the efficacy of cancer chemotherapy. Overexpression of a Mr 170,000 plasma membrane protein, P-gp,3 is found frequently in many drug-resistant cell lines (1). However, it is apparent that reduced drug accumulation mediated by P-gp constitutes only one of several biological mechanisms involved in the development of a multidrug- resistant phenotype. HT1080/DR4 is a DOX-selected multidrug-resis- tant human fibrosarcoma cell line that does not overexpress P-gp (2, 3) and the mechanisms responsible for resistance in this cell line remain unclear (4). The multidrug-resistant small cell lung carcinoma cell line, H69AR, also does not overexpress P-gp (5, 6) but recently, a cDNA encoding a novel MRP has been cloned from this cell line (7). MRP belongs to the superfamily of ATP-binding cassette transport systems. Other members of this superfamily include the cystic fibrosis transmembrane conductance regulator, P-glycoprotein, and the MHC class H-linked peptide transporters (7, 8). The 6.5-kilobase MRP mRNA is increased approximately 100-fold in H69AR cells relative to Received 4/9/93; accepted 6/14/93. 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 in part by Grants CA-33572 (M. L. S.). and the Medical Research Council (MRC) of Canada (R. G. D., S. P. C. C.). E. U. K. is the recipient of an MRC studentship. 2 To whom requests for reprints should be addressed, at City of Hope National Medical Center. Department of Cytogenetics. Northwest Bldg.. Room 2255. 1500 E. Duarte Road. Duarte, CA 91010. 3 The abbreviations used are: P-gp, P-glycoprotein; hsr, homogeneously staining re gion; dmins, double minute chromosomes; MRP, multidrug resistance-associated protein; DOX. doxorubicin; SDS, sodium dodecyl sulfate; FISH, fluorescent in situ hybridization; GTG, Giemsa-trypsin; der, derivative chromosome; cDNA, complementary DNA; SSC, standard saline-citrate (IX SSC = 150 rriM NaCl-15 HIMsodium citrate, pH 7.0); add, additional material of unknown origin. drug-sensitive parental H69 and revenant H69PR cells. This increase is associated with a 40-50-fold amplification of the MRP gene which maps in normal cells to chromosome band, 16pl3.1 (7). In the present study, we have found that the MRP gene is also amplified in resistant HT1080/DR4 cells relative to parental HT 1080 cells. To identify the cytogenetic alterations associated with amplification of the MRP gene, we have carried out cytogenetic and FISH analyses on the sensitive and resistant HT1080 cell lines and the sensitive, resistant, and rever- tant H69 cell lines. Materials and Methods Cell Lines. The two DOX-selected multidrug-resistant cell lines used in this study have been described previously (2, 5, 9). The 200-fold DOX- resistant HT1080/DR4 fibrosarcoma cell line was derived from parental HT1080 cells and exhibits a multidrug-resistant phenotype (2. 9). H69AR is a 50-fold DOX-resistant small cell lung carcinoma cell line that also displays a typical multidrug-resistant phenotype (5, 10). H69PR, a drug-sensitive rever- tant cell line, was derived from H69AR by maintaining H69AR cells in DOX-free medium for 36 months (11). The resistant HT1080/DR4 and H69AR cell lines do not overexpress P-gp (2-6). RNA and DNA Analyses of HT1080 Fibrosarcoma Cell Lines. Polyade- nylated RNA was obtained with a Micro-FastTrack mRNA isolation kit (In- vitrogen, San Diego, CA) and 1 (j.g from each cell line was separated by electrophoresis on a formaldehyde-agarose denaturing gel. The RNA was transferred to a nylon membrane (Zetaprobe; Bio-Rad, Mississauga, Ontario, Canada), prehybridized in 50% formamide, 5x SSC-5X Denhardt's (50x = 1% bovine serum albumin, 1% polyvinylpyrrolidone, and 1% Ficoll), 1% SDS, and sheared and denatured herring testes DNA (100 u,g/ml) for 4-6 h at 42°C. The blot was hybridized with a 1.8-kilobase cDNA fragment of MRP labeled to a specific activity of >5 X IO8 cpm/u,g DNA with [a-12P]dCTP (3000 Ci/mmol; NEN-Dupont, Mississauga, Ontario, Canada) by the random priming method (12). Hybridization was carried out for 16-20 h at 42°C.Blots were washed once in 2X SSC for 10 min at room temperature and then three times in 0.1% SDS and 0.1X SSC for 30 min each at 52°Cfollowed by autorad- iography. To estimate variation in RNA loading of the gel, the blots were stripped and reprobed with a 32P-labeled cDNA for /3-actin (201pBv2.2) (13). Relative levels of MRP and ß-actinmRNAs were determined by densitometry (7). For Southern analyses, genomic DNA ( 10 fig) from HT1080 and HT1080/ DR4 cells was digested with EcoRl and BamHl and electrophoresed through a 0.8% agarose gel. After blotting onto a nylon membrane (Zetaprobe), prehy- bridization was carried out for 4 h at 42°Cin 50% formamide, 5X standard saline-phosphate-EDTA (IX = 3 M NaCl-0.2 M NaH2PO4-0.02 M EDTA, pH 7.4), 0.5% SDS, 4x Denhardt's, and herring testes DNA (100 u.g/ml). The blot was then hybridized for 20 h at 42°Cwith a 4-kilobase MRP cDNA fragment labeled by random priming with [a-3;2P]dCTPas described above. The blot was washed once with 1x SSC for 20 min at room temperature, and twice with O.lx SSC and 0.1% SDS for 20 min at 42°Cand then exposed to film. Densitometry was carried out as before (7). Classical Cytogenetics. Cytogenetic analyses were performed according to the standard method using 50 /ig/ml Colcemid for 45 min before cell harvest ing. GTG-banding was used to identify the individual chromosomes and the chromosomes were classified according to the Guidelines for Cancer Cytoge netics, Supplement to An International System for Human Cytogenetic No menclature (14). 3221 Research. on December 7, 2021. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from