hyperplasia and normal prostate; and (2) P-cadherin expression in benign prostatic ..... In BPH (Figure 16), and prostatic carcinoma with microglandular pattern ...
The Histochemical Journal 32: 659–667, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
E-, N- and P-cadherin, and α-, β- and γ-catenin protein expression in normal, hyperplastic and carcinomatous human prostate Mar´ıa I. Arenas, Eva Romo, Mar Royuela, Benito Fraile & Ricardo Paniagua∗ Department of Cell Biology and Genetics, University of Alcal´a, E-28871 Alcal´a de Henares, Madrid, Spain ∗
Author for correspondence
Received 23 May 2000 and in revised form 22 September 2000
Summary The expression of E-, N- and P-cadherin, α-, β- and γ -catenin, and actin was studied by immunohistochemistry, ELISA, and Western blot analysis in normal prostates, and in the prostates of men with benign prostatic hyperplasia and men with prostatic carcinoma, in order to evaluate their possible role in the pathogenesis of these diseases. Present results reveal that the immunophenotype of hyperplastic prostates differs from those of both normal and carcinomatous prostates in the intracellular distribution (observed by immunohistochemistry) and the intensity (measured by ELISA) of immunoreactions to cadherins, catenins, and actin. Hyperplastic prostates differ form normal prostates in the weaker immunoreaction to the three cadherin types, the two catenins, and actin, as well as in the intracellular distribution of P-cadherin, β- and γ -catenin, and actin. Differences between benign prostatic hyperplasia and prostatic carcinoma are less marked because hyperplastic prostates differ from carcinomatous prostates only in the weaker immunoreactions to P-cadherin, and α-catenin. The most remarkable findings in this study were: (1) α-catenin production was elevated in prostatic carcinoma in comparison with benign prostatic hyperplasia and normal prostate; and (2) P-cadherin expression in benign prostatic hyperplasia is reduced with regard to those of normal and carcinomatous prostates. It may be concluded that a decreased immunoreaction to cadherins, catenins, and actin, as well as changes in the intracellular distribution of actin in prostatic cells are not necessarily suggestive of malignancy, because these alterations are also present in BPH, and thus, the loss of cadherin–catenin-mediated adhesion alone is not sufficient to establish an invasive phenotype.
Introduction Cell–cell junctions are specialised macromolecular structures that are essential for both intercellular adhesion and communication (Woods & Bryant 1993). Adherens junctions and desmosomes play an adhesive as well as an architectural role in the epithelium by providing a link between cellsurface adhesion molecules and the cytoskeleton (Kirkpatrick & Pfeifer 1995). One of the most important and ubiquitous types of adhesive interactions required for the maintenance of solid tissues is that mediated by the classic cadherin adhesion molecules. Cadherins are transmembrane Ca2+ -dependent homophylic adhesion receptors which play important roles in cell recognition and cell sorting during development (Takeichi 1991). Cadherins genes are considered tumour suppressor genes (Hedrick et al. 1993) and defects in their expression or function have been associated with tumour progression (Behrens et al. 1989). The expression of cadherins in tumour cells can serve to trace the histogenetic origin of tumours and can be used as differential diagnostic markers between tumours of similar phenotype but different histogenesis (Peralta Soler et al. 1995, 1997). Cadherins are localised in specialised cell-to-cell adhesion sites that are termed
adherence junctions; at these sites cadherins can establish linkages with the actin-containing cytoskeleton. The classical cadherins include E-, N- and P-cadherin. E-cadherin is expressed in most epithelial tissues at points of cell– cell contact (Boller et al. 1985), and acts as an important suppressor of epithelial tumour cell invasiveness and metastasis (Birchmeier & Behrens 1994). N-cadherin is expressed in neuroectodermal and mesodermal-derived tissues (Hatta et al. 1987). P-cadherin is found in mouse placenta (Nose & Takeichi 1986), lung epithelia (Hirai et al. 1989a,b), basal cells of the skin (Shimoyama et al. 1989) and myoepithelial cells of the mammary gland (Daniel et al. 1995). The expression of P-cadherin in epithelial tissues is characteristic of cell populations with proliferative potential, and its expression decreases as cells differentiate (Shimoyama et al. 1989). Cadherins associate with a group of intracellular proteins termed catenins, which link the cadherin molecules to the actin microfilaments, and mediate signal transduction mechanisms regulating cell growth and differentiation (Ozawa et al. 1989). Three catenins have been identified: α-, β- and γ -catenin (also termed plakoglobin). β- and γ -catenin form mutually exclusive complexes with α-catenin and bind to the carboxy-terminal cytoplasmic domain of the cadherin molecules (Jou et al. 1995). The association of
660 catenins to cadherins is a key step in the function of intact adhesion complexes and alterations in catenin molecules can lead to disruption of the cell–cell adhesion, resulting in tumour aggressiveness and invasiveness in neoplastic diseases (Behrens et al. 1989). The prostate gland is composed of tubulo-alveoli which are lined by a biestratified epithelium and embedded within a fibromuscular stroma. The epithelium consists of three major cell types: secretory cells, basal cells, and neuroendocrine cells. The secretory epithelial cells are tall columnar cells which express E-cadherin mainly in the areas of cell–cell contact (Umbas et al. 1992). Basal cells are small, round cells which rest on the basal membrane of the tubulo-alveoli, and express E- and P-cadherins as well as α- and β-catenin (Paul et al. 1997). Numerous studies about the expression of cadherins and catenins in prostatic carcinoma (PC) have been reported. In vitro and in vivo studies have shown a E-cadherin dysfunction associated with an invasive phenotype (Otto et al. 1993, Richmond et al. 1997). A correlation between reduction of E-cadherin levels and aberrant α-catenin expression has been reported (Morton et al. 1993, Richmond et al. 1997). Low expression of catenins has been associated with increased tumour dedifferentiation, invasion, and metastasis (Shiozaki et al. 1996). Particularly, the down-regulation of β-catenin seems to be associated with malignant transformation (Morin 1999). However, quantitative studies have not been reported. In addition, the expression of cadherins and catenins has hardly been studied in benign prostatic hyperplasia (BPH) because it has been assumed that it is as in normal prostate (Murant et al. 1997). The aim of the present work was to study by immunohistochemical and semiquantitative methods the expression of E-, N- and P-cadherin, and α-, βand γ -catenin, in human BPH, and to compare these results with those of normal and carcinomatous prostates, in order to obtain a more comprehensive understanding of the potential significance of prostatic hyperplasia and to evaluate the possible role of these adhesion molecules in the pathogenesis of this disease. The results reveal that immunoreactions to cadherins and catenins in BPH differ from those of normal prostate and PC.
Materials and methods The prostates from 30 men (between 55 and 85 years of age) were obtained from prostatectomies and transurethral resections. Fifteen of these men were clinically and histopathologically diagnosed as BPH, and the other 15 men presented prostatic adenocarcinoma, dominant Gleason grade 3, Gleason score 5–7. The patients neither received presurgical treatments such as androgen-deprivation therapy, chemotherapy or radiation therapy nor were diagnosed for metastatic cancer. In addition, 15 prostates from 20- to 50-year-old men without reproductive, endocrine and related diseases were obtained from autopsies between 8 and 10 h after death. Seven of these men showed BPH and the other eight men presented
M.I. Arenas et al. histologically normal prostates. Each sample was longitudinally sectioned in three equal portions; to ensure that the tissue of concern was present, one frozen cryostat section of each portion was stained with haematoxylin and eosin and diagnosed by a pathologist. One portion was immediately processed for immunohistochemistry, and another two portions were frozen in liquid nitrogen and maintained at −80 ◦ C for Western immunoblot analysis and ELISA. Goat polyclonal antibodies anti-E-, N- and P-cadherin, and α-, β- and γ -catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, Ca, USA). A mouse monoclonal anti-actin antibody, which recognises actin microfilaments in skeletal muscle cells, myoblasts, fibroblasts and HeLa cells was purchased from Amersham Iberica (Madrid, Spain). The specificity of primary antibodies was tested by Western blotting analysis, as described by Towbin et al. (1979). The prostates were homogenised in 0.5 M Tris–HCl buffer (pH 7.4) containing 1 mM EDTA, 12 mM 2-mercaptoethanol, 1 mM benzamidine, 0.5% NP-40, and 1 mM phenylmethylsulphonyl fluoride (PMSF). Homogenates were centrifuged at 10,000 g for 30 min. After boiling for 2 min at 98 ◦ C, aliquots of 200 µg protein were separated in SDS-polyacrylamide (9%, w/v) slab minigels, according to the procedure of Laemmli (1970). Separated proteins were transferred for 4 h at 0.25 Å to nitrocellulose paper and, thereafter, the nitrocellulose sheets were soaked in blocking solution (1 mM glucose, 1% bovine serum albumin [BSA], 0.5% Tween-20, 10% glycerol in phosphate-buffered saline [PBS], pH 7.3) overnight at 37 ◦ C, and then incubated with the primary antibodies, all at 1 : 500 dilution in blocking solution for 4 h. After extensive washing with PBS–Tween-20 the sheets were incubated with a peroxidase-labelled secondary antibody (rabbit anti-goat or rabbit anti-mouse biotinylated immunoglobulin, Dako, Barcelona, Spain) at 1 : 3000 dilution in blocking solution. The filters were developed with a chemiluminescence ECL Western blotting detection reagent, following the procedure described by the manufacturer (Amersham Iberica). The staining intensity (optical density) of each band was measured with an automatic image analyser (MIP4 version 4.4, Consulting Image Digital, Barcelona, Spain) in order to compare the expression of each protein between the different groups of prostates. For enzyme-linked immunoassay (ELISA) the protein concentration of each prostate was calculated by the Bradford method (Bradford 1976), and were diluted to 18.2 µg/µl. Serial dilutions of proteins from each prostate were made and incubated on 96 well multiplates overnight at 37 ◦ C. The plates were washed with TBS containing 0.05% Tween 20, blocked with 1% BSA in TBS for 1 h at room temperature, and incubated with the first antibody at 1 : 2000 dilution for 3 h also at room temperature. After a new wash, the peroxidase-conjugated anti-goat or antimouse immunoglobulins (Dako) at 1 : 300 dilution were added to each well. The interactions were visualised with 0.05% 2,20 -azino-bis-3-ethylbenzthiazoline sulphonic acid (ABTS) (Sigma, Barcelona, Spain) in 100 mM citrate buffer, pH 5. Optical density values at 405 nm were obtained in
Cadherins and catenins in human prostate a spectrophotometer (Multiskan Bichromatic, Labsystems, Finland). The means ±SD from each dilution were calculated and represented. The optical density values from 18.2 µg/µl of protein from each group (normal, BPH, and PC) were compared by ANOVA and the significance of differences between groups were evaluated by the Fisher and Behrens test. The intra-assay and inter-assay variation coefficients obtained for each antibody were: E-cadherin: 5.54% and 9.19%; Ncadherin: 4.09% and 8.41%; P-cadherin: 4.25% and 6.09%; α-catenin: 7.01% and 8.59%; β-catenin: 6.65% and 8.77%; γ -catenin: 5.46% and 8.14%; and actin: 5.59% and 7.91%, respectively. For light microscopy immunohistochemistry, the prostates were fixed for 24 h at room temperature in 0.1 M phosphatebuffered 10% formaldehyde, dehydrated and embedded in paraffin wax. Sections, 5-µm-thick, were processed following the avidin–biotin-peroxidase complex (ABC) method (Hsu et al. 1981). Following deparaffinisation, sections were hydrated, incubated for 30 min in 0.3% H2 O2 diluted in methanol to reduce endogenous peroxidase activity and to retrieve the antigen, the sections were incubated with 0.1 M citrate buffer (pH 6) for 5 min in a conventional pressure cooker (Norton et al. 1994). After rinsing in TBS buffer, the slides were incubated with normal donkey serum at 1 : 5 diluted in TBS containing 5% BSA for 30 min to prevent non-specific binding of the first antibody. Thereafter, the primary antibodies were applied at a dilution of 1 : 20 in TBS–BSA at 37 ◦ C overnight. Afterwards, the sections were washed twice in TBS and then incubated with rabbit antigoat biotinylated immunoglobulin (Dako) at 1 : 500 dilution. After 1 h of incubation with the secondary antibody, the sections were incubated with a standard streptavidin–biotin complex (Dako) and developed with 3,30 -diaminobenzidine (DAB), using the glucose oxidase–DAB–nickel intensification method (Hsu & Soban 1982). In some sections, a double immunostaining method to detect a cadherin and actin or a catenin and actin in the same section was used: one antigen was detected with the glucose oxidase–DAB– nickel method while the other antigen was detected either with the (DAB–H2 O2 ) method, without intensification (Hsu et al. 1981, Kren´acs et al. 1990) or with nitroblue tetrazolium (NBT). To prevent the binding of the secondary antibody from the second labelling step with free binding sites of primary antibody from the first immunostaining, the binding sites of unreacted immunoglobulins were denatured using hot paraformaldehyde vapour (Wang and Larson 1985). To assess the immunostaining specificity in both light and electron microscope methods, negative controls either omitting primary antibody or using this antibody preabsorbed with an excess of purified antigens were also analysed. For double immunohistochemistry, control sections omitting the primary antibody in either the first or the second step were used. A histological semiquantitative comparison of immunolabelling density in normal, hyperplastic and neoplastic prostates was performed for each of the seven antibodies. Of each prostate, six histologic sections of each region (central, transitional and peripheral) were selected at random, and
661 the staining intensity (optical density) of the epithelium was measured with an automatic image analyser in five microscopic fields using the ×40 objective. In the study, the number of sections and microscopic fields in each section necessary for calculation were determined by successive approaches to obtain the minimum number required to reach the lowest SD. The statistical significance between means was assessed by the Fisher and Behrens test.
Results Comparison between post-mortem specimens showing prostatic hyperplasia and surgical specimens from men with prostatic hyperplasia revealed neither histological nor quantitative histochemical changes caused by autolysis. Western blot The results of Western blot analysis are shown in Table 1 and Figure 1. The antibodies used showed a single band, at approximately 120, 138, 118, 102, 92, 82, and 45 kDa for E-, N- and P-cadherin, α-, β- and γ -catenin, and actin, respectively, in the three groups of men studied. These results agree with those obtained by ELISA and immunohistochemistry; the reactivities to antibodies showed differences of intensity between the homogenates from normal prostate, BPH and carcinomatous prostate. E- and N-cadherin antibodies showed an intense reaction in normal prostates compared with homogenates from BPH and carcinoma. P-cadherin reaction was lower in BPH than normal prostates and carcinoma. The expression of α-catenin showed a higher intensity in homogenates from carcinoma than in normal and hyperplastic prostates. The expression of β- and γ -catenin was more intense in normal prostates than in hyperplasia and PC. Reaction to actin was similar in BPH and PC and higher in normal prostates. ELISA The results of the ELISA study showed a linear correlation between the increasing concentrations of the homogenised Table 1. Immunostaining intensities of the Western blot analysis (measured as optical density) in normal prostate, benign prostatic hyperplasia, and prostatic carcinoma.
E-cadherin N-cadherin P-cadherin α-catenin β-catenin γ -catenin Actin
Normal prostate
Benign prostatic hyperplasia
Prostatic carcinoma
4.045 ± 0.3a 2.757 ± 0.19a 1.882 ± 0.07a 1.669 ± 0.06a 3.046 ± 0.14a 1.519 ± 0.12a 2.259 ± 0.2a
1.832 ± 0.07b 1.674 ± 0.13b 0.645 ± 0.05b 2.151 ± 0.18b 1.297 ± 0.11b 0.828 ± 0.06b 1.590 ± 0.14b
1.651 ± 0.09b 1.460 ± 0.03b 1.854 ± 0.06a 2.421 ± 0.19b 1.376 ± 0.12b 1.115 ± 0.10c 1.587 ± 0.13b
For each antibody, values with different superscript letters differ significantly between them (p ≤ 0.05).
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E2CAD
N2CAD
P2CAD
250 160 105 75 50 35 30 N
H
C
α2CAT
N
H
C
β2CAT
N
H
C
γ2CAT
N
H
C
ACTIN
250 160 105 75 50 35 30 N
H
C
N
H
C
N
H
C
N
H
C
Figure 1. Western blot analysis of E-cadherin (E-cad), N-cadherin (Ncad), P-cadherin (P-cad), α-catenin (α-cat), β-catenin (β-cat), γ -catenin (γ -cat), and actin, in normal (N), hyperplastic (H), and carcinomatous (C) human prostates, after polyacrylamide gel electrophoresis (9% w/v). TP: total proteins. Each blot is representative of its respective group.
tissues and their respective optic densities. The optical density of the E- and N-cadherin from 18.2 µg/µl of protein was significantly decreased (p ≤ 0.05) in both PC and BPH with respect to normal prostates. Comparison between both groups of patients revealed that optical density of P-cadherin was significantly lower (p ≤ 0.05) in BPH, whereas the optical density of E- and N-cadherin was similar in both patient groups (Figure 2). The reactivity to α-catenin at saturation was significantly higher (p ≤ 0.05) in homogenates from PC than in hyperplasia and normal prostates. The optical density of β-catenin was significantly higher in normal prostates than in the other two groups which showed similar optical densities. The optical density of γ -catenin was also significantly higher in normal prostates than in the other two groups. Comparison between the two groups of patients revealed a lower optical density in BPH (Figure 3). The results for optical density of actin were similar to those obtained for E-cadherin (Figure 3). Immunohistochemistry The results of light and electron microscopy immunohistochemistry are shown in Table 2 and Figures 4–19. No
Figure 2. ELISA study of E-, N- and P-cadherin in normal prostates (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PC). Antibody binding was followed at 405 nm, using peroxidase-conjugated anti-goat IgG antibody. Figures show the average values from 18.2 µg/µl of protein for each group of prostates.
immunostaining appeared in negative controls in which the primary antibody was omitted (Figure 4). Expression of E-cadherin was observed in the borders of the secretory cells, mainly in the apical zone; this expression was more intense in normal prostates (Figure 5) than in hyperplastic (Figure 6) and carcinomatous prostates (Figure 7). Immunostaining to N-cadherin was observed in the lateral borders of epithelial cells, and was more intense in normal prostates (Figure 8) than in BPH (Figure 9) and PC (Figure 10). Immunostaining to P-cadherin was intense along the lateral borders of epithelial cells in normal prostates (Figure 11). In the two groups of patients, immunostaining to
Cadherins and catenins in human prostate
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Figure 3. ELISA study of α-, β-, γ -catenin, and actin, in normal prostates (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PC). Antibody binding was followed at 405 nm, using peroxidase-conjugated anti-goat or anti-mouse IgG antibody. Figures show the average values from 18.2 µg/µl of protein for each group of prostates. Table 2. Comparison of immunostaining intensities of the prostatic epithelium (measured as optical density) in normal prostate, benign prostatic hyperplasia, and prostatic carcinoma.
E-cadherin N-cadherin P-cadherin α-catenin β-catenin γ -catenin Actin Negative control
Normal prostate
Benign prostatic hyperplasia
Prostatic carcinoma
3.461 ± 0.14a 2.863 ± 0.18a 3.090 ± 0.18a 1.369 ± 0.01a 2.734 ± 0.05a 2.543 ± 0.06a 2.944 ± 0.18a 0.893 ± 0.01a
1.274 ± 0.12b 1.431 ± 0.13b 1.881 ± 0.02b 1.742 ± 0.07b 2.232 ± 0.09b 1.932 ± 0.04b 1.774 ± 0.13b 0.739 ± 0.02a
1.352 ± 0.11b 1.260 ± 0.11b 2.960 ± 0.03a 1.830 ± 0.16b 2.067 ± 0.02b 2.014 ± 0.07b 1.745 ± 0.07b 0.801 ± 0.01a
For each antibody, values with different superscript letters differ significantly between them (p ≤ 0.05). Given values were obtained after subtraction of negative control values.
this cadherin appeared in discrete areas of contact between cells and diffusely in the cytoplasm, although staining was weaker in BPH (Figure 12) than in PC (Figure 13). Antibody for α-catenin immunostained the basolateral borders of epithelial cells in the three groups of specimens; the reaction to this antibody was higher in PC than in hyperplastic and normal prostates (Figures 14–17). Immunostaining to β-catenin was observed in the lateral borders of epithelial cells in normal prostates (Figure 18) and PC. In BPH, the immunostaining to this catenin was observed in discrete areas of intercellular junctions as well as in the apical cytoplasm (Figure 19). Reaction to γ -catenin showed a similar distribution and staining intensity to that to β-catenin.
Changes in the distribution of actin-labelling have been observed using a double immunostaining to α-catenin (black) and actin (blue). In normal prostates, actin immunostaining was mainly found in the apical cytoplasm (Figure 15), whereas in BPH and prostatic carcinoma, immunostaining was weaker and uniformly distributed throughout the cytoplasm (Figures 16 and 17). The diminution of actin labelling in carcinoma was correlated with the increase of α-catenin reactivity. Discussion Our results reveal that the immunophenotype of hyperplastic prostates differs from those of both normal and carcinomatous prostates in the intracellular distribution (observed by immunohistochemistry) and the intensity (measured by ELISA) of immunoreactions to cadherins, catenins, and actin. Hyperplastic prostates differ form normal prostates in the weaker immunoreaction to the three cadherin types, β- and γ -catenin, and actin, as well as in the intracellular distribution of P-cadherin, β- and γ -catenin, and actin. Differences between BPH and PC are less marked because hyperplastic prostates differ from carcinomatous prostates only in the weaker immunoreactions to P-cadherin, and α-catenin. E-Cadherin deficiency in PC has been reported previously (Isaacs et al. 1995, Richmond et al. 1997). Dysfunctions of E-cadherin distribution in several neoplasias have been explained by a failure in either the translocation or the anchorage of this protein to the cell membrane (Vessey et al. 1995).
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Figures 4–19. 4. Control section of normal prostate after omitting the primary antibody. Bar: 25 µm. 5–7. Immunostaining to E-cadherin. In normal prostate (Figure 5) labelling is visualised in the borders of secretory cells. Hyperplastic glands show a lower reactivity than normal prostate localised in the cellular borders (Figure 6). In well-differentiated prostatic carcinoma reaction is limited to discrete areas of contact between adjacent cells (Figure 7). Bar: 25 µm. 8–10. Immunostaining to N-cadherin. The reaction is intense in the lateral borders of epithelial cells of normal glands (Figure 8). In benign hyperplasia (Figure 9) and carcinoma (Figure 10) this protein shows a lower reaction. Bar: 25 µm.
Cadherins and catenins in human prostate The anchorage failure has been attributed to a functional defect of the E-cadherin cytoplasmic tail, which cannot bind the intracellular catenins (Bringuier et al. 1993). In PC and also in BPH, the cytoplasmic distribution of E-cadherin does not change, although there is a pronounced loss of immunoexpression. This loss has led to the suggestion that the E-cadherin pathway is frequently disrupted at different levels (Richmond et al. 1997). Recent allelic mapping experiments in prostatic cancer have implicated abnormalities in the long arm of chromosome 16, in the region of the E-cadherin gene (Isaacs et al. 1995). In highly invasive breast tumours, N-cadherin was shown to replace E-cadherin at cell–cell contacts and it has been proposed that N-cadherin mediates carcinoma cell interaction with mammary stromal cells (Hazan et al. 1997). It has also been suggested that this cadherin would be involved in the promotion of breast cancer metastasis by facilitating carcinoma cell migration through the mammary stroma as well as in reestablishment of homophyllic cell–cell adhesion in metastasis (Hagios et al. 1998). This assertion may not be generalised for most tumours. In ovarian cancer a decrease in N-cadherin is observed during the transition from borderline to overt malignant tumour (Dara¨ı et al. 1997). No previous references to the N-cadherin of prostatic tissue have been reported and, in the present study, we have observed a decreased expression of N-cadherin in both PC and BPH. Paul et al. (1997) and Peralta Soler et al. (1997) have found P-cadherin only in basal cells of normal prostates, and absence of this cadherin in PC, which is devoid of true basal cells. There were no references to BPH in these reports. In the present immunohistochemical study we have observed that P-cadherin is present in both basal and columnar cells in normal prostates, and that the immunoreaction to this protein (evaluated by ELISA and Western blotting) decreased without disappearing in prostatic cancer and BPH. Moreover, in both patient groups a weak immunostaining appeared innerly located in the cytoplasm, and this suggests a role of P-cadherin in cellular functions other than cell–cell adhesion. Murant et al. (1997) reported that in prostatic cancer epithelium and tissues showing lost or reduced E-cadherin expression, there was also a loss of α-catenin expression. However, in our study, we have encountered that the decrease in E-cadherin expression was not associated with the loss of α-catenin; conversely, α-catenin expression was higher in prostatic cancer than in normal prostate and BPH. This increase in α-catenin expression does not mean an increase in the number of adherens junctions because the expression of β- and γ -catenin is diminished in BPH and PC, and both
665 catenins form the bridge that joins α-catenin to E-cadherin. Our results are more similar to those of Mialhe et al. (1997) in human bladder carcinomas. These authors observed a decreased expression of E-cadherin associated to a decreased expression of β- and γ -catenin but not of α-catenin. In the present study, immunostainings to β- and γ -catenin were localised associated to the cell membrane but, in BPH, staining also appeared innerly located in the cytoplasm. βCatenin is involved in cell adhesion but also in intracellular signalling (Ilyas et al. 1997). This catenin forms complexes with the transcription factors Tcf-4 (to inhibit apoptosis and to stimulate cell proliferation), or LEF-1 (to regulate E-cadherin gene expression via binding to the promoter region of the Ecadherin gene) (Peifer 1997). β-Catenin is also able to bind to the gene product of a tumour suppressor gene called adenomatous polyposis coli (APC), and an increase in the free form of β-catenin has been demonstrated in several cancer cell types in which the APC gene was mutated (Ilyas et al. 1997). In pervanadate-treated leukaemia cell (K562) culture, Ozawa and Kemler (1998) have observed that, β- and γ -catenins exhibit increased tyrosine phosphorylation and conformational changes, and that these changes are correlated with reduced association of α-catenin to E-cadherin. It has also been reported that epidermal growth factor (EGF) induces the tyrosine phosphorylation of β-catenin (Hoschuetzky et al. 1994, Aberle et al. 1996). Present observation of a reduced expression of β-catenin in BPH agrees with the results of De Miguel et al. (1999) who observed that the expression of EGF was increased in epithelial cells of BPH. These results suggest that there is a correlation between the increase of EGF and the phosphorylation of β-catenin and the decrease of its expression. As tyrosine phosphorylation of β-catenin works as a signal transduction pathway, β-catenin might have the effect of down-regulating for E-cadherin function. The presence of apical actin in normal glands could be related to the secretion process (throughout prostasomes) and the occurrence of adherens junctions. It has been reported that, in PC and BPH, actin immunostaining changes its distribution and appears more widespread in the cytoplasm (Brancolini et al. 1997). These authors have interpreted this change in relation to the apoptosis process that occurs in PC. In the present study we have observed this change of actin distribution not only in PC but also in BPH, and thus, actin changes might be related to aspects other than apoptosis, such as changes in the adhesion, shape, and proliferative activity of prostatic cells. The actin deficiency might be a
11–13. Immunostaining to P-cadherin. In normal prostate (Figure 11) immunoreaction is intense and appeared in the lateral borders of epithelial cells. In BPH the reaction is weaker (Figure 12) and in well-differentiated prostatic carcinoma (Figure 13) labelling also appeared in the apical cytoplasm. Bar: 25 µm. 14. Immunostaining to α-catenin in BPH. Staining is observed in the basolateral borders of epithelial cells. Bar: 25 µm. 15–17. Double immunostaining to actin (blue) and α-catenin (brown). In normal prostate (Figure 15) immunoreaction to actin is intense and appears in the apical cytoplasm. In BPH (Figure 16), and prostatic carcinoma with microglandular pattern (Figure 17) actin immunostaining is weaker and more diffusely distributed. Immunostaining to α-catenin is more intense in prostatic carcinoma than in normal prostate. Bar: 25 µm. 18–19. Immunoreaction to β-catenin. In normal prostate (Figure 18), staining is observed in focal contacts between adjacent cells. In BPH (Figure 19), staining is also observed in the apical cytoplasm Bar: 25 µm.
666 consequence of the deficiencies in cadherins and/or catenins. Cortical actin microfilaments associate with cadherin complexes at the plasma membrane to form the adherens junction (Pavalko & Otey 1994), and this association is mediated by catenins (Cowin & Burke 1996). Since cadherins, and β- and γ -catenin are decreased in BPH and prostatic cancer, microfilaments lose their attachment to the adherens junctions and become widespread in the cytoplasm. Other factors such as the increase of EGF and its receptors reported in BPH (De Miguel et al. 1999) could also be involved in this process, since it has been shown that activation of EGF receptors induces microfilament disassembly followed by reassembly (Chang et al. 1995). Sommers (1996) observed, in breast carcinoma, that Ecadherin and catenin deficiencies are non-invasive, indicating that loss of E-cadherin-mediated adhesion alone is not sufficient to establish an invasive phenotype. In our study, it may be concluded that a decreased immunoreaction to cadherins, catenins, and actin, as well as changes in the intracellular distribution of P-cadherin, β- and γ -catenin, and actin in prostatic cells are not necessarily suggestive of malignancy because these alterations are also present in BPH.
Acknowledgements This work was supported by grants from the Fondo de Investigaciones Sanitarias (98/0820), Sociedad Urol´ogica Madrile˜na, and University of Alcal´a.
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