Androgens are classically acting in androgen-receptor positive tissues, like the human prostate, as selective transcription factors, after binding to specific intracellular receptors, translocation to the nucleus and transcription of androgen related genes, as the prostate specific antigen (PSA). In recent years, however, evidences have been accumulated that steroid, including androgen, action can also be mediated by non-classical, membrane receptor sites. These latter are biochemically distinguishable from intracellular androgen receptors, and their activation results in rapid non-genomic effects 12345, elicited minutes after binding. Classical and non-classical binding sites can coexist in the same cells 12.

Recently we have reported the identification of a functional membrane testosterone binding site on LNCaP prostate cancer cell line 6. Its activation results in the rapid (within minutes) secretion of stored PSA, through a specific signaling cascade (Papakonstanti et al, submitted) leading to actin cytoskeleton rearrangements. The identification of this membrane testosterone binding site is a constant characteristic of androgen receptor positive and negative cell lines, independently of the expression of intracellular receptors (Kogia et al in preparation). In the present work, we investigated whether membrane testosterone sites are expressed in human prostate carcinomas. In addition, we have investigated whether these sites are differentially expressed in malignant and non-malignant (benigh prostate hyperplasia and non-malignant prostate cells) prostate epithelial cells, using a number of methods. We show that membrane testosterone binding sites are preferentially expressed in carcinomas, and therefore they could provide additional evidences for the diagnostic discrimination of prostate cancer.

Mechanical dissociation of cells from selected regions of prostate specimens yielded mainly epithelial cells, as revealed by cytokeratin and PSA staining. We prefered this mild dissociation method, as enzymatic treatment of tissue specimens could modify membrane structures, leading to a loss of membrane testosterone binding. By touching preparations we ensured about the quality (not necrotic tissue, representative sample) of the tissue specimens obtained, while by immunostaining we ensured that the dissociation of tissues yielded epithelial cells, and the suspensions contained only malignant or non-malignant cells, as per case. Epithelial cells accounted for more than 85% of total cells, in all studied specimens. On confocal microscopy and flow cytometry, only carcinoma cells exhibited detectable reactivity with testosterone-BSA-FITC. A typical result is presented in Figure 1. As shown, a 85% maximal specific staining with testosterone-BSA-FITC was found, compatible with the percentage of epithelial cells in our preparations.

In Figure 2, typical cases of testosterone-BSA-FITC binding on cells prepared from prostate carcinoma (left panel) and benign prostate hyperplasia (right panel) are presented. As shown, Testosterone-BSA-FITC binds only to malignant cells, showing a peripheral annular staining. Although some unavoidable, slight, background reaction is evident with different intensity in both panels, no cell staining can be seen on the right one (BPH case). In addition, non-malignant cells, isolated from prostatectomy specimens, do not show any membrane testosterone binding. As in the case of LNCaP cells, the identification of membrane testosterone receptors can be obtained in short incubation times. It is evident after one minute incubation, and maximum binding is observed after 15 min (Fig. 1). Thereafter, fluorescence intensity remains constant for times up to one hour. As reported before for LNCaP cells 6, no internalization of the tracer was observed in any case of malignant or non-malignant cells, confirming that testosterone-BSA analog binds exclusively to membrane testosterone sites. Although the selectivity of the binding was not assayed in the present study, our previous results 6 indicate that this site is selective for androgens.

Figure 3 depicts the flow cytometry analysis of membrane testosterone binding in cases of malignant and non-malignant cells isolated from surgical specimens of the prostate. Due probably to the higher discrimination potency of the method, a specific testosterone-BSA binding was revealed in all cells. The intensity of testosterone-BSA-FITC staining was different depending on the nature of cells examined: for normal cells and benign prostate hyperplasia the staining was very low, while cells from prostate cancer were intensively stained. Quantification of flow cytometry results showed that testosterone-BSA-FITC binding was significantly higher in prostate carcinomas as compared to BPH (t = 7.65, p < 0.001) and normal tissue (t = 7.65, p < 0.001). These two latter groups showed similar results (Table 1).

In order to be useful in everyday practice, the detection of membrane testosterone binding sites should be performed on routine histology slides. In this aim, we have developed a method for the identification of the testosterone membrane binding sites on formalin-fixed, paraffin-embedded routine prostate tissue sections, as described in Material and Methods. Applying this protocol, membrane testosterone binding sites could be identified in tissue sections obtained from the same cases studied by flow cytometry. As shown in Figure 4, carcinoma cells and cells of PIN fixed the testosterone tracer intensely and consistently, and the pattern of reaction was peripheral and annular. In addition, due to the very fine thickness of slices (2 μm), a nuclear staining is also obtained, making the method also useful for the detection of classical androgen receptors, in addition of membrane receptors. A slight membrane staining was also observed in some cases of BPH and in some normal epithelia of the gland (not shown). In some cases, in spite of a preincubation with BSA, stromal structures could fix low amounts of tracer, which in no way interfere with the epithelial structure staining estimation, and do not restrict the efficiency of the method.

Prostate carcinoma is, at least initially, a hormone-regulated neoplasm, but under therapy eventually escapes and relapses 211121314151617. Androgen effects are classically exerted through binding of the hormone to intracellular specific receptors 1819, which consequently dimerize, translocate to the nucleus and bind to specific elements of DNA, affecting thus the transcription of androgen-responsive genes 14. In recent years, evidence has been accumulated, indicating that androgen can alternatively exert on respective target cells short-time non-genomic effects mediated through membrane binding sites and subsequent changing in Ca²⁺ concentration 452021, in cell types presenting 12 or lacking intracellular androgen receptors 345. Quite recently we have described a membrane testosterone binding site in the hormone positive human prostate cancer cell line LNCaP 6. We have shown that activation of this site initiates within minutes the rearrangement of actin cytoskeleton through a specific signaling cascade (Papakonstanti et al, submitted) and increases the secretion of PSA. This site was selective for androgens, and immunologically different from the intracellular classical testosterone receptor. In addition, no internalization of membrane testosterone sites was obvious after incubation times less than one hour.

The results of the present study indicate that membrane testosterone binding sites can also be identified in freshly prepared human prostate cells. This binding was restrained to epithelial cells and is completed within minutes (Figure 1). Neoplastic cells, morphologically normal prostate epithelium cells and BPH epithelia bind testosterone-BSA analog (Figure 3, Table 1) but with a different intensity. In this respect, as presented in Figure 3, a clear discrimination of malignant and non-malignant cells could be obtained by membrane testosterone binding. This is also evident in touching preparations (Figure 2) and histological preparations of prostate cancer (Figure 4). It is of interest to note that cells underwent prostate intraepithelial neoplasia (PIN) are also intensely stained with testosterone-BSA, indicating that the expression of these sites might correlate with a neoplastic and premalignant (PIN) phenotype. With more sensitive methods however (flow cytometry), some slight staining-reaction of normal and BPH epithelia was also evidenced. In this respect, the difference between neoplastic and non-neoplastic cells is merely a quantitative differential expression. Indeed, two cases of transurethral resection (Table 1) in which high testosterone-BSA-FITC staining was obtained, were revealed to present foci of PIN after a thorough histological examination.

Traditionally the differential diagnosis, grading and staging of prostate carcinoma has been based predominantly on morphological features 92223. A number of immunological markers have been proposed in prostate carcinoma or premalignant conditions (such as PIN), but they lack selectivity and specificity 92223242526272829. Indeed, a good biomarker can be defined as one that adds independent information to standard prognostic indicators, and which can be reliably reproduced in the laboratory, aiding in diagnostic and/or therapeutic decisions. Our results indicate that membrane testosterone binding sites could fulfill this definition, as they can aid in discrimination between benign and malignant/premalignant prostate conditions. As shown in Figure 3, in only one case of morphologically normal epithelial cells the values overlapped with those obtained in the carcinoma group. It is possible, however, that a number of tumor cells could interfere with the measurement. In addition, two samples, initially considered as BPH, but finally diagnosed as harboring PIN lesions were positively stained for membrane testosterone binding sites, indicating the potential diagnostic usefulness of membrane testosterone binding sites identification and/or measurement. Although further investigations in large series are needed, our results indicate that membrane testosterone binding sites could be a valuable addition in prostate cancer differential diagnosis, and a possible target for novel therapies.