MOLECULAR EXPRESSION OF PSMA mRNA AND PROTEIN IN PRIMARY RENAL TUMORS Fre ´de ´ric DUMAS 1 , Jean Luc GALA 2 , Pierre BERTEAU 1 , Francis BRASSEUR 3 , Pascal ESCHWE ` GE 4 , Vale `rie PARADIS 5 , Bernard LACOUR 1 , Marianne PHILIPPE 2 and Sylvain LORIC 1,4,6 * 1 Clinical Chemistry Laboratory, Necker University Hospital, Paris, France 2 Molecular Biology Laboratory, Saint-Luc Clinical University, and Queen Astrid Military Hospital, Brussels, Belgium 3 Ludwig Institute for Cancer Research, Brussels, Belgium 4 Urology Unit and Experimental Surgery Laboratory, Bice ˆtre University Hospital, Le Kremlin-Bice ˆtre, France 5 Pathology Unit, Bice ˆtre University Hospital, Le Kremlin-Bice ˆtre, France 6 Differentiation Laboratory, CNRS URA 1960, Pasteur Institute, Paris, France Human prostate-specific membrane antigen (PSMA), a 100-kDa integral transmembrane glycoprotein, is considered to be a highly specific marker of the prostate gland, and has successfully been used as a marker of circulating prostatic epithelial cells. Extended PSMA homology has been demon- strated with a cDN A found in rat cerebral and renal tissues. In this study, we aimed to evaluate the expression of PSMA mRN A in a variety of human renal cancer tissues(n  20) and cell lines (n  12). Using reverse transcriptase-polymerase chain reaction, DN A sequencing, blottings, and specific anti- PSMA labelling with CYT 351 antibody, we identified PSMA mRNA and protein in normal and in neoplastic renal tissue. The sequence of the polymerase-chain-reaction products is identical to that of PSMA cDN A derived from prostate tissue. Immunological staining with the CYT 351 reveals that PSMA is expressed mainly in tubular cells. Since PSMA does not appear to be restricted to prostatic tissue, this novel biomar- ker may prove useful in the staging of renal cancer and in the search for the hematogenous spread of renal cells. Int. J. Cancer 80:799–803, 1999.  1999 Wiley-Liss, Inc. An estimated 31,000 new cases of renal cancer (RCC) will be diagnosed this year in the United States, leading to the death of 12,000 patients as a consequence of tumor progression (Parker et al., 1996). Radical surgery of organ-confined tumors gives pro- longed long-term survival. Unfortunately, RCC is associated with a reduction of life expectancy to less than 10% at 5 years (Maldazys and de Kernion, 1986). Unlike prostatic carcinoma (CaP), no epithelial or serum markers are so far available to detect or monitor RCC. Today, early diagnosis of organ-confined disease relies mainly on the incidental discovery of clinically silent tumors by abdominal computerized tomography or ultrasound, since RCC is rarely symptomatic before loco-regional or metastatic invasion. Since radical surgery is curative for organ-confined disease only, it is of major interest to evaluate markers for diagnostic and prognostic purposes. PSMA is a 100-kDa prostate transmembrane glycoprotein, initially described by Horoszewicz et al. (1987) from crude membrane extracts of a LNCaP prostate-hormone-responsive can- cer cell line. This protein has been identified by immunodetection with the murine monoclonal antibody (MAb) 7E11C5.3, produced by mouse immunization with LNCaP-cell extracts (Horoszewicz et al., 1983). Using anti-PSMA antibodies (7E11-C.3 and site-specific CYT357), Lopes et al. (1990) and Babain et al. (1994) revealed faint expression in renal tubular cells. The 2700-bp PSMA cDNA is highly divergent from other known genes, except for 54% homol- ogy with the transferrin receptor within 450 bp in the coding region (Israeli et al., 1993). PSMA has been shown to share 86% sequence homology with the 3' end of a rat gene coding for an abundant brain enzyme (Carter et al., 1996) called NAADLAse, acting as a folate-hydrolase-neurocarboxypeptidase with terminal  and -glu- tamate as substrates. Interestingly, this NAADLAse activity has also been identified in rat kidney- and prostate-cancer cells, which tends to indicate that this enzyme and PSMA could be identical proteins (Carter et al., 1996). In this study, we further investigated and characterized the expression of PSMA mRNA in normal kidney, in renal carcinomas and in several in vitro-established cancer cell lines. MATERIAL AND METHODS Human kidney samples Renal tumor samples were collected at the time of radical nephrectomy. In total, 20 specimens containing renal cancer were available (Table I), all with matched tissues from the normal area of the resected kidney. Tissues were snap-frozen in liquid nitrogen, then stored at -80°C. Frozen sections of each specimen were cut and analyzed by 2 pathologists who determined the presence or the absence of renal cancer in the samples. Cells and reagents The following renal-carcinoma cell lines (Table II) were estab- lished at the Ludwig Institute for Cancer Research (Brussels, Belgium): LB996-RCC, LB1047-RCC, LB1358-RCC, BB64-RCC and BB65-RCC. The others were provided as follows: Dr. P. Schrier (Academic Hospital, Leiden, the Netherlands), LE8915- RCC, LE9004-RCC, LE9211-RCC; Dr. A. Knuth (Akademisches Lehrkrankenhaus der Johann-Wolfgang-Goethe Universita ¨t, Frank- furt on Main, Germany), MZ1851-RCC, MZ1257-RCC, MZ1973- RCC; and Dr. D. Rohde (Aachen, Germany), ACHN1. Cells were obtained from primary tumors in all cases. Histologically, they were 3 clear-cell carcinomas, 1 granular-cell carcinoma, all other cells lines being renal-carcinoma. Some of these lines have been described (Brandle et al., 1996; Brouwenstijn et al., 1996). Cells were grown in Iscove’s modified Dulbecco’s medium containing 10% FCS, L-arginine (116 μg/ml), L-asparagine (36 μg/ml), L-glutamine (219 μg/ml) and antibiotics (200 U/ml penicillin and 100 μg/ml streptomycin) in a CO 2 incubator at 37°C. The LNCaP cell line used as an internal control was obtained from the ATCC (Rockville, MD); these cells were grown in RPMI 1640 supple- mented with L-glutamine, non-essential amino acids and 8% FCS (Life Technologies, Eragny, France) in a CO 2 incubator at 37°C. All other chemical reagents were of the highest grade possible, and were obtained from Sigma (Saint-Quentin Fallavier, France). Northern blotting Nylon membranes with UV-fixed poly (A + ) RNA (2 μg per lane) extracted from normal adult tissues (Clontech, Palo Alto, CA) were Grant sponsor: Association pour la Recherche contre le Cancer; Grant number: ARC 1367; Grant sponsor: Belgian Fonds national de la recherche scientifique; Grant number: 3.4574.98. *Correspondence to: Laboratoire de Biochimie A, Ho ˆpital Necker, 149, rue de Se `vres, 75015 Paris, France. Fax: (33) 1-4449-5120. E-mail: sgloric@pasteur.fr Received 12 March 1998; Revised 1 October 1998 Int. J. Cancer: 80, 799–803 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer