[CANCER RESE-ARCI! 55. .TWO-.W», September 1. 1995] Isolation of a Gene Encoding a Human Reduced Folate Carrier (RFC1 ) and Analysis of Its Expression in Transport-deficient, Methotrexate-resistant Human Breast Cancer Cells Jeffrey A. Moscow,1 Maokai Gong, Rui He, Magdalene K. Sgagias, Katharine H. Dixon, Sarah L. Anzick, Paul S. Meltzer, and Kenneth H. Cowan Medical Breast Cancer Section. Medicine Branch, National Cancer Institute [J. A. M., M. G., R. H., M. K. S., K. H. D., K. H. C.I and Laboratory of Cancer Genetics. National Center for Human Genome Research. ¡S.L A.. P. S. M.¡.NIH. Bethesda. Maryland 20892 ABSTRACT Our laboratory has previously reported the isolation of a murine cDNA which restores reduced folate carrier (RFC) activity and methotrexate (MTX) sensitivity to a MTX-resistant, transport-déficient human breast cancer cell line (MTXR ZR-75-1) (K. H. Dixon et al., J. Biol. Chem., 269: 17-20, 1994). Using this murine cDNA as a probe, we have isolated two homologous overlapping partial cDNAs from a human testis cDNA li brary. In addition, using human cDNA as a probe, we have isolated a 20-kb human genomic fragment which contains RFC coding regions. Analysis of the nucleotide sequence of these clones revealed that the human RFC gene, RfCI, is approximately 65% homologous to the murine and hamster genes. Using a human genomic PI plasmid clone containing RFC!, we mapped the location of HI (I by fluorescence in situ hybrid ization to the end of the long arm of chromosome 21 (21q22.2-q22.3). Fluorescence in situ hybridization analysis also showed that two copies of RFCI were present in MTXK ZR-75-1 cells, and showed no evidence of rearrangement of this gene. Northern blot analysis of MTXR ZR-75-1 cells demonstrated a marked decrease in the level of the 3-kb RFC I transcript relative to the parental cell line, and Western blot analysis using a polyclonal antibody raised against a peptide generated from the RI'CI sequence showed decreased expression of an approximately M, 56,000 protein in MTXK ZR-75-1 cells. Finally, MTXR ZR-75-1 cells transfected with an RFCI gene showed increased MTX uptake, which was more sensitive to competition by folinic acid than by folie acid. Therefore, decreased RFCI expression appears to be the molecular mechanism of decreased MTX uptake in this MTX-resistant cell line. INTRODUCTION Animal cells, in contrast to bacteria, are unable to synthesize folate compounds and must therefore possess mechanisms for folate uptake. Two classes of folate transporters have been identified. Human cDNAs for the folate receptor, which has a higher affinity for folie acid than for reduced folates, have been isolated from cell lines and tissues and have been extensively characterized (1-4). The other folate transporter is the RFC2, which has been identified in tissue culture cells by its characteristic greater affinity for reduced folates than for folie acid. Both systems are capable of transporting antifo- lates, including MTX, into cells. Decreased RFC activity has been observed in several MTX-resistant cell lines (5-11). Our laboratory described a human breast cancer cell line with acquired MTX resistance (MTXR ZR-75-1 ) which had undetectable levels of both folate receptor and RFC activity (5, 12). We isolated a murine cDNA encoding a RFC by expressing an L1210 cDNA library in MTXR ZR-75-1 cells and selecting the transfected cells with trimetrexate, a Received 2/6/95; accepted 6/3(1/95. The costs of publication of this article were defrayed in pan 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. 1To whom requests for reprints should be addressed, at Building K). Room 12N226, NIH. Bethesda. MD 20H92. : The abbreviations used are: RFC, reduced folate carrier; MTX. methotrexate; RACE, rapid amplification of cDNA ends; RT. reverse transcriptase; FISH, fluorescence in situ hybridization: GTG. Giemsa trypsin G banding. lipophilic antifolate analogue which enters cells by diffusion, in the presence of low concentrations of folinic acid (13). A homologous cDNA for a hamster RFC gene was also recently isolated by screening a cosmid library for complementation of defective MTX uptake in a MTX-resistant Chinese hamster ovary cell line (6), and then screening a cDNA library with the isolated genomic fragment (14). We used the murine RFC cDNA to isolate cDNA and genomic clones encoding a homologous human gene. We have determined the chromosomal location of this gene and have examined its RNA and protein expression in MTXR ZR-75-1 cells. Finally, we cloned the RFCI coding region by RT-PCR and transfected it into MTXR ZR- 75-1 cells to demonstrate its ability to mediate MTX transport. MATERIALS AND METHODS cDNA and Genomic Cloning. Using the murine RFC cDNA as a probe, we isolated two overlapping partial cDNA clones after screening 600,000 plaques from a human testis cDNA library (Clontech) using a protocol sup plied by the manufacturer. Inserts from both clones were ligated into pGEM-3Z (Promega). Nuclcotidc sequence analysis demonstrated that the 5' clone, pRFC-T2 overlapped the 3' cDNA clone, pRFC-Tl 1, by 130 bp (Fig. 1). Several attempts to obtain a full-length cDNA clone containing the 5' end of the gene by screening other libraries were unsuccessful. Therefore, we screened a human placenta genomic AFIXII library (Stratagene) using pRFC-T2 and isolated a single 20-kb genomic fragment by plaque hybridiza tion according to the manufacturer's instructions. The 20-kb insert was cut by Xbul into three fragments of 4, 6, and 10 kb and subcloned into pGEM-3Z. The 6-kb genomic fragment, called pRFC-G5, was determined to contain at least part of the upstream human cDNA pRFC-T2 sequence by Southern blot hybridization with the pRFC-T2 cDNA clone. The 10-kb genomic fragment, called pRFC-G3, was determined to contain the 5' end of the coding region by Southern blot hybridization with the 5' end of the mouse cDNA RFC probe. We used 5' RACE to determine the nucleotide sequence of the 5' region of RFCI as well as to confirm the nucleotide sequence of the RFCI exon present on the genomic fragment pRFC-G5 (Fig. 1). RACE-ready cDNA prepared from human liver mRNA (Clontech) was amplified with nested, AFC/-specific downstream primers according to the manufacturer's directions. The sequence isolated by this technique did not contain the start of translation, but did contain sequences upstream of the 5' intron-exon border on pRFC-G5 which were present on pRFC-G3. Primers corresponding to the RACE sequence permitted nucleic acid sequencing of the 5' exon present on pRFC-G3, which confirmed the RACE cDNA sequence. The nucleotide sequences of the cloned cDNA and genomic fragments were determined by dideoxynucleotide sequencing using Sequenase (United States Biochemical) according the to the manufacturer's instructions. The reactions were size fractionated on a 6% denaturing acrylamide gel (National Diagnos tics). Custom oligonucleotide primers for sequencing were purchased from the Midland Certified Reagent Company (Midland, TX). Subsequently, the 5' end of the RFC coding region was also amplified by RT-PCR with RNA from ZR-75-1 cells using primers derived from the genomic sequence of pRFC-G3. The nucleotide sequence of the RT-PCR fragment was determined using a thermal cycle sequencing kit (Stratagene) according to the manufacturer's instructions with primers end labeled with [-y-"P]ATP and T4 DNA polynucleotide kinase (Promega). The sequencing reactions were performed in a Perkin Elmer 9600 thermal cycler and size 3790 on July 21, 2015. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from