[CANCER RESEARCH 54, 6344-6347, December 15, 1994] Advances in Brief Molecular Characterization of Prostate-specific Antigen Messenger RNA Expressed in Breast Tumors1 Maria Monne, Carlo M. Croce,2 He Yu, and Eleftherios P. Diamandis Department of Microbiology-Immunology and Jefferson Cancer Institute, Jefferson Medical College, Thomas Jefferson University. Philadelphia. Pennsylvania 19107 (M. M., C. M. C.], and Department of Clinical Biochemistry. University of Toronto, Toronto, Ontario M5G 11.5. Canada [H. Y., E. P. D.J Abstract Prostate-specific antigen (PSA) Is considered a highly specific blochem Ical marker of the prostate gland and is currently used for prostate cancer diagnosis and monitoring of patients with prostate adenocarcinoma. We recently demonstrated, however, that about 30% of female breast tumors produce a Mr 33,000 protein that has striking similarities to seminal PSA. In this study we characterized the presence ofPSA in 6 breast tumors and in the testosterone-stimulated T47D breast cancer cell line at the mRNA level. Using reverse transcriptase-polymerase chain reaction and DNA sequencing techniques we identified PSA mRNA In Immunoreactive PSA positive breast tumors but not in immunoreactive PSA-negative breast tumors. The sequence of the generated polymerase chain reaction prod ucts was identical to the sequence of the PSA complementary DNA derived from prostate tissue. The data presented here support the notion that breast tumors produce a Mr 33,000 protein which is identical to PSA produced by the prostate gland. Our study suggests that the presence of PSA in breast tumors may be used as a new additional biochemical marker for breast cancer prognosis, for the spreading of hematogenous micrometastases, and/or for response to adjuvant treatment. Introduction PSA3 is a Mr 33,000 glycoprotein produced almost exclusively by the epithelial cells of the prostate and is currently used as a marker for diagnosis and monitoring of prostate cancer (1—3).PSA detection in tumors of nonprostatic origin has been reported as a rare event, in a few instances, using immunohistochemical techniques (4). We have recently reported that PSA immunoreactivity can be detected in about 30% of breast tumors and that breast cancer cells in culture can produce IRPSA after stimulation by steroid hormones (5—7). This immunoreactive PSA was shown to be associated with the presence of steroid hormone receptors, early disease stage, and younger patient age (5). Although the results from previous studies suggested that IRPSA in breast cancer cells seems identical to the seminal PSA, a final proof has not yet been provided. In this study we report the molecular characterization of the PSA detected in breast tumors and in the testosterone-stimulated T47D breast cancer cell line. Six breast tumors were tested for PSA immunoreactivity and then analyzed for PSA gene expression by reverse transcriptase-PCR and DNA sequence was determined on the PCR fragments. DNA se quence analysis demonstrated that PSA mRNA derived from breast tumors is identical to the PSA from prostate tissue. The PSA production by 30% of breast tumors underscores the Received 9/6/94; accepted 11/2/94. 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. I This work was supported by a NIH Outstanding Investigator Grant CA39860 to C. M. C. 2 To whom requests for reprints should be addressed, at Department of Microbiology and Immunology, Jefferson Cancer Institute, Jefferson Medical College, Thomas Jefferson University, 1ith and Walnut Streets, Philadelphia, PA 19107. 3 The abbreviations used are: PSA, prostate-specific antigen; IGFBP, insulin growth factor-binding protein; IRPSA, immunoreactive PSA; eDNA, complementary DNA PCR, polymerase chain reaction. similarities between breast and prostate tissues and holds promise of being used in breast cancer for prognosis, in selection of therapy, or for devising new therapeutic interventions. Materials and Methods Tissue Specimens. Six breast cancer specimens were obtained from women undergoing surgery for primary breast cancer. The breast tumor tissue was stored in liquid nitrogen immediately after surgical resection, transported to the laboratory, and subsequently stored at —70°C until protein and R.NA extraction was performed. Frozen surgical specimens from various other tumors were obtained from the Toronto Hospital, Toronto, Ontario, Canada (3 ovarian and 2 lung carcinomas), from the Hospital of Padova, Padua, Italy (1 gastric and 1 colon carcinomas), and from the Jefferson Hospital, Philadel phia, PA (1 melanoma). All tumors were primary lesions except for one ovarian carcinoma, which was metastatic from breast cancer, and one lung carcinoma, which was metastatic from prostate cancer. One normal breast tissue specimen was obtained from a woman undergoing breast cosmetic surgery, and peripheral blood leukocytes obtained from a healthy volunteer were prepared from 10 ml venous blood. Tissue Culture and Stimulation with Steroids. The steroid hormone receptor-positive breast carcinoma cell line T47D was obtained from the American Type Culture Collection, Rockville, MD. The cell line was grown in flasks at 37°C and 5% CO2 in RPMI 1640 supplemented with 0.2 Hi of bovine insulin/ml, 29 g/liter glutamine, and 10% FCS. When the cells were grown to 90% confluencythey were washedwith isotonic saline and maintainedin the medium as above but without the presence of FCS. Stimulation experiments with steroids were performed by adding in the culture medium various steroids in alcoholatfinalconcentrationsof iO@ M.Thefinalconcentrationof alcohol was always <0.1% of the total volume. Control experiments with alcohol alone were performed in parallel. In this work we used T47D cells stimulated with either solvent or testosterone. T47D cells stimulated with testosterone produce PSA which is secreted in the tissue culture supernatant and which is measurable by the immunofluoro metric procedure. After 4 days of stimulation the cells were detached by trypsin-EDTA treatment and were kept frozen at —70°C until RNA extraction was performed. Preparation ofTumor CytosolicExtracts. Approximately0.5goftumor tissue was weighed out, fragmented with a hammer if necessary, and pulver ized in a Thermovac tissue pulverizer at liquid nitrogen temperature. The resulting powder was transferred into 50-mi plastic tubes along with 10 ml of extraction buffer [10 mmol/liter Tris-HC1 (pH 7.40), 1.5 mmol/liter EDTA, 5 mmol/liter sodium molybdate]. The suspendedtissue powder was solubilized on ice with a single 5-s burst of a Polytron homogenizer. The particulate material was pelleted by centrifugation at 105,000 X g for 1 h. The interme diate layer (cytosol extract) was collected without disturbing the lipid or particulate layers. The cytosols were then assayed for PSA with the immun ofluorometric procedure. PSA Assay in Tumor Cytosols and Tissue Culture Supernatants. PSA in the cytosolic breast extracts and tissue culture supernatants was measured with a highly sensitive and specific immunofluorometric technique described in detail elsewhere (7). Briefly, the PSA assay uses a mouse monoclonal anti-PSA capture antibody coated to polystyrene microtiter wells, a biotiny lated polyclonal rabbit detection antibody, and alkaline phosphatase-labeled streptavidin. In the assay 50 ml of sample are first incubated with the coating antibody in the presence of 50 ml of assay buffer. After 3 h incubation and 6 6344 on May 15, 2021. © 1994 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from