[CANCER RESEARCH 53,227-230, January 15, 1993] Advances in Brief Molecular Cloning of a Complementary DNA Encoding a Prostate-specific Membrane Antigen I Ron S. Israeli, C. Thomas Powell, William R. Fair, and Warren D. W. Heston z Urologic Oncology Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Abstract Recently, a novel Mr 100,000 prostate-specific membrane glycoprotein (PSM) has been detected by the prostate-specific monoclonal antibody 7Ell-C5, raised against the human prostatic carcinoma cell line LNCaP. The PSM antigen is expressed exclusively by normal and neoplastic pros- tate cells and metastases. We now report the molecular cloning of a full-length 2.65-kilobase complementary DNA encoding the PSM antigen from a human LNCaP complementary DNA library by polymerase chain reaction using degenerate oligonucleotide primers. Analysis of the com- plementary DNA sequence has revealed that a portion of the coding region, from nucleotide 1250 to 1700, has 54% homology to the human transferrin receptor mRNA. The deduced polypeptide has a putative transmembrane domain enabling the delineation of intra- and extracellu- lar portions of this antigen. In contrast to prostate-specific antigen and prostatic acid phosphatase which are secreted proteins, PSM as an integral membrane protein may prove to be effective as a target for imaging and cytotoxic targeting modalities. Introduction Prostate cancer represents the most common malignancy in Amer- ican males and is the second leading cause of cancer-related death in the male population (1). The disease has diverse manifestations, from slow growing, indolent primary lesions to aggressive, refractory met- astatic disease, with a predilection toward bone metastases. PAP 3 was one of the earliest serum markers for detecting metastatic spread of prostate cancer (1); this marker has been augmented in recent years by PSA (1). PSA has been shown to correlate with tumor burden, serve as an indicator of metastatic involvement, and provide an excellent parameter for following the response to surgery, irradiation, and an- drogen ablation therapy in patients with prostate cancer. Both of these proteins are secreted and are readily measured in the serum, as well as in prostatic secretions. The LNCaP human prostate cancer cell line was established from a metastatic lymph node from a heavily pre- treated patient with hormone-refractory prostate carcinoma (2). This cell line serves as the best in vitro model for human prostatic carci- noma in that it possesses an aneuploid male karyotype, maintains prostatic differentiation functionality in that it produces PAP and PSA, and expresses a high affinity androgen receptor. Cell membranes were isolated from these cells and mice were immunized with them to form hybridomas. A prostate-specific monoclonal antibody was generated using spleen cells of mice immunized with LNCaP cell membranes and designated 7El 1-C5 (3). The antibody staining exhibited a mem- Received 10/15/92; accepted 11/25/92. 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. ~The MSKCC Microchemistry Core Facility is supported in part by NIH Grant P30-CA-08748. R. S. I. receives partial support from NIH Training Grant CA-09501-07. Genback Accession Number M99487. PSM, Homo sapiens, 2653 base pairs. 2 To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 334, New York, NY 10021. 3 The abbreviations used are: PAP, prostatic acid phosphatase; PSA, prostate-specific antigen; PSM, prostate-specificmembrane glycoprotein; SDS, sodium dodecyl sulfate; cDNA, complementary DNA; MSKCC, MemorialSloan-Kettering Cancer Center; PAGE, polyacrylamidegel electrophoresis;PCR, polymerasechain reaction. brane location with LNCaP cells reacting strongly. Both benign and neoplastic prostate cells stained positively, with more intense staining seen with malignant cells. Lymph node and bone metastases also stain positively with the antibody, with the highest expression seen in hormone-refractory lesions (4). The epitope of the antibody has been shown to include a carbohydrate portion of the PSM antigen and the antigen has an apparent molecular weight of approximately 100,000 on SDS-polyacrylamide gel electrophoresis (5). In this paper, we report the molecular cloning of a full-length cDNA encoding the Mr 100,000 prostate-specific membrane antigen. Materials and Methods Cells and Reagents. The LNCaP, DU-145, and PC-3 cell lines used were obtained from the American Type Culture Collection. Details regarding the development of these cell lines and their characteristics have been published previously (2, 6, 7). Unless specified otherwise, LNCaP cells were grown in RPMI 1640 supplemented with L-glutamine, nonessential amino acids, and 5% fetal calf serum (Gibco-BRL) in a CO2 incubator at 37~ DU-145 and PC-3 cells were grown in minimal essential medium supplemented with 10% fetal calf serum. All media was obtained from the MSKCC Media Preparation Facility. Routine chemical reagents were obtained from Sigma Chemical Com- pany, St. Louis, MO. The modified 7El 1-C5 monoclonal antibody to the PSM antigen (CYT-356) was obtained from Cytogen Corporation, Princeton, NJ. lmmunopreeipitation of the PSM Antigen. LNCaP cells were starved in methionine-depleted RPMI for 2 h, after which [35S]methionine was added at 100 jaCi/ml and the cells were grown for another 16-18 h. Cells were then washed and lysed by addition of 1 ml of lysis buffer [1% Triton X-100, 50 mM Hepes (pH 7.5), 10% glycerol, 15 mM MgC12, 1 rnM phenylmethylsulfonyl fluoride, and 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid] and incu- bated for 20 min at 4~ Lysates were precleared by mixing with Pansorbin cells (Calbiochem) for 90 min at 4~ Cell lysates were then mixed with protein A-Sepharose CL-4B beads (Pharmacia) previously bound with CYT- 356 monoclonal antibody and rabbit anti-mouse IgG (Accurate Scientific) for 4 h at 4~ Beads were then washed with 20 rr~ 4-(2-hydroxyethyl)-1-piper- azineethanesulfonic acid (pH 7.5), 150 mM NaCI, 0.1% Triton X-100, 10% glycerol, and 2 mM sodium o-vanadate buffer, resuspended in Laemmli sample loading buffer, and denatured prior to electrophoresing on a 10% SDS-PAGE gel at 10 mA overnight. Gels were dried down at 60~ in a vacuum dryer and autoradiographed for 1 6-24 h at -70~ For the large scale purification of 5-10 jag of PSM antigen, the above procedure was repeated using approximately 6 x 107 LNCaP cells. The immunoprecipitation product was pooled and loaded into two lanes of a 10% SDS-PAGE gel and electrophoresed for 16 h at 10 mA. Proteins were electroblotted onto nitrocellulose membranes and stained with Ponceau red to visualize the proteins. Peptide Microsequencing. This work was performed with the assistance of the Sloan-Kettering Institute Microchemistry Core Facility: Briefly, the Mr 100,000 PSM antigen band was excised from the membrane, solubilized, and digested proteolytically with trypsin. High performance liquid chromatography was performed on the digested sample using a HPLC Applied Biosystems Model 171C, and clear dominant peptide peaks were selected and sequenced on a modified post-liquid Applied Biosystems Model 477A Protein/Peptide Microsequencer (8). Nine peptides were sequenced ranging in size from 7 to 22 amino acids and all were screened for homology with the Genbank database and found to be unique. A similar technique was used to sequence the amino terminus of the PSM antigen and it was determined that it was in fact blocked, and no protein sequence was obtained. 227 Research. on October 14, 2021. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from