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Immunohistochemical Detection of the Retinoid X Receptors , ß, and in Human Prostate

MARIA V. T. LOBO

From the Department of ^* Cell Biology and Genetics, University of Alcalá, 28871, Alcalá de Henares, Spain; and Molecular Oncology Laboratory, Hospital Ramón y Cajal, Madrid, Spain.

Correspondence to: Dr María I. Arenas, Department of Cell Biology and Genetics, University of Alcalá, 28871 Alcalá de Henares, Madrid, Spain (e-mail: misabel.arenas{at}uah.es).

Received for publication April 16, 2002; accepted for publication July 29, 2002.

	Abstract

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The aim of this study was to evaluate the presence and distribution of retinoid X receptors (RXRs) , ß, and in normal, hyperplastic (nodular, basal cell, and atrophic hyperplasia), and carcinomatous human prostates in order to elucidate the relationship among these receptors and the onset and development of prostatic adenocarcinoma. RXR and RXR were immunodetected in all samples of normal, nodular, and basal cell hyperplasia, as well as carcinomatous prostates. In atrophic glands, the expression of both receptors was found in 22.5% of samples. Positive immunostaining for RXRß was observed in 53.3% of normal prostates, 100% of samples showed basal cell hyperplasia, and were negative in nodular and atrophic hyperplasia. In prostatic adenocarcinoma, only 3 of 25 samples (the 3 diagnosed as well-differentiated) were positive for RXRß. Results suggest that diminished RXRß expression might be related to prostate cancer progression and because the responsiveness to retinoic acid treatments depends on the expression of different receptors, it is important to study their expression before therapy.

     Key words: Immunohistochemistry, RxRs, prostate cancer

Studies on the molecular mechanisms of retinoid action have revealed that retinoic acid binds to two receptor types: retinoic acid receptors (RARs) and retinoid X receptors (RXRs; Petrovick et al, 1987; Mangelsdorf et al, 1992). Both receptor types belong to the steroid/thyroid hormone nuclear receptor superfamily, and are characterized by their ligand- and DNA-binding abilities, and also by their possible dimerization partners (Kastner et al, 1994; Mangelsdorf and Evans, 1995). Each class of receptor is composed of three gene products (RAR , ß, and ; and RXR , ß, and ), the transcription of which results in several isoforms as a result of the action of different promoters and messenger RNA (mRNA) splicing (Kastner et al, 1994; Brocard et al, 1996). Two forms of retinoic acid, named all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA), can bind to RARs, but only 9-cis-RA is able to bind to RXRs (Mangelsdorf et al, 1992).

In addition to the occurrence of different ligands and receptors, the complexity of retinoid signaling is increased by the possible formation of different homodimer and heterodimer receptors. These dimers can bind to different hormone response elements (HREs) in the promoters of certain genes, and act as transcription factors (Kastner et al, 1994; Mangelsdorf and Evans, 1995). In general, when an HRE is bound to a nuclear hormone receptor, this may either activate or repress the transcription, depending on the presence of ligand, cell type, promoter, response element, or other signals (Vos et al, 1997).

Few studies have examined the expression of RXRs in the human prostate. Using immunohistochemistry, Kikugawa et al (2000) detected much more expression of RXR and RXR than of RXRß in human prostatic adenocarcinoma cells. In addition, Lotan et al (2000) found RXR and RXR mRNA expression in normal and carcinomatous human prostates. However, these authors observed that the intensity of the in situ hybridization signal was much weaker for RXRß than for the other receptor probes.

Further studies are needed to investigate the possible role of these receptors in the physiological behavior of human prostatic cells. Thus, the aim of this study was to evaluate the presence and distribution of RXR , ß, and in normal, hyperplastic and carcinomatous human prostates, using immunohistochemistry and Western blot analysis, in order to elucidate the relationship among these receptors and the onset and development of prostatic adenocarcinoma.

	Materials and Methods

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Materials

We used 1) histologically normal prostates obtained at autopsy (8-10 hours after death); 2) tissues obtained via prostatectomy and transurethral resections from 25 men (aged 53 to 88 years) clinically and histopathologically diagnosed with benign prostatic hyperplasia (BPH), including 8 nodular hyperplasias, 8 basal cell hyperplasias, and 9 atrophy samples; and 3) tissues obtained via prostatectomy and transurethral resections from 25 men (aged from 54 to 69 years) diagnosed with prostatic cancer (PC), including 9 well-differentiated (Gleason score 2-4), 8 moderately differentiated (5-7), and 8 poorly differentiated (8-9) samples. The patients received neither hormonal therapy before surgery nor were they diagnosed with metastatic cancer. Each prostatic sample was divided into two approximately equal portions; one was immediately processed for immunohistochemistry, and another was frozen in liquid nitrogen and maintained at -80°C for Western blot analysis. Removal of tissues and autopsy samples for study were taken with the consent of the patients' relatives and permission of the ethics committee of the hospital.

Immunohistochemistry

For immunohistochemistry, tissues were fixed in 10% (v/v) formaldehyde in phosphate-buffered saline (pH 7.4) for 24 h, dehydrated, and embedded in paraffin. Sections (5 µm) were processed following the avidin-biotin-peroxidase complex method (Hsu et al, 1981). Following deparaffination, sections were hydrated and incubated for 30 minutes in 0.3% H₂O₂, diluted in methanol to inhibit endogenous peroxidase activity. To retrieve the antigen the sections were incubated with 0.1 M citrate buffer (pH 6) for 5 minutes in a conventional pressure cooker (Norton et al, 1994). After rinsing in Tris-buffered saline (TBS), the slides were incubated with normal donkey serum (NDS) at 1:10 diluted in TBS containing 5% bovine serum albumin for 30 minutes to prevent nonspecific binding of the first antibody. Afterward, the sections were incubated overnight at 37°C with the following primary antibodies (all from Santa Cruz Biotechnologies, Santa Cruz, Calif) diluted in TBS containing 10% NDS: rabbit polyclonal antibody against RXR (1:20); mouse monoclonal immunoglobulin G₁ antibody against RXRß (1:20); and rabbit polyclonal antibody against RXR (1:20). The sections were then washed in TBS and incubated with either swine anti-rabbit (for RXR and RXR), or rabbit anti-mouse (for RXRß) biotinylated immunoglobulins (DAKO, Barcelona, Spain), all at 1:500 dilution. After 1 hour of incubation with the second antibody, the sections were incubated with avidin-biotin-peroxidase complex (DAKO) and developed with 3,3'-diaminobenzidine using the glucose oxidase-DAB-nickel intensification method (Hsu and Soban, 1982). After this, sections were dehydrated and mounted in DePex (Probus, Badalona, Spain). Care was always taken to develop the pathological and nonpathological sections in precisely the same way each time for each immunohistochemical reaction.

The specificity of immunohistochemical procedures was checked by using negative and positive control sections. For negative control of immunoreactions, adjacent sections of each type (normal, BPH, and PC) were incubated with either preimmune rabbit or mouse serum according to the first antibody, or by using the antibody preabsorbed with an excess of purified antigens. As positive controls, sections of human skin and liver (for all receptors) were incubated with the same antibody.

In order to determine whether the source of material (surgery or autopsy) could be responsible for changes in the immunohistochemical pattern, five prostatic biopsies (taken because of suspected prostatic disease, but their histological study revealed a normal histological pattern) were processed for immunohistochemistry. The results of the quantitative immunohistochemical study in these biopsies were compared with those performed in autopsy prostates.

Western Blotting

For Western blot analysis, tissues (including skin, liver, and prostate) were homogenized in the extraction buffer (0.05 M Tris-HCl pH 8) with the addition of a cocktail of protease inhibitors (10 mM iodoacetamide, 100 mM phenylmethyl sulfonic fluoride, 0.01 mg/mL of soybean trypsin inhibitor, and 1 µL/mL of leupeptin) in the presence of 0.5 % Triton X-100. Homogenates were centrifuged for 10 minutes at 6000 x g. Supernatants were mixed with an equivalent volume of sodium dodecyl sulfate (SDS) buffer (10% SDS in Tris/HCl pH 8 containing 50% glycerol, 0.1 mM 2-beta-mercaptoetanol, and 0.1% bromphenol blue). Then the mixture was denatured for 5 minutes at 100°C, and aliquots of 80 µg of protein were separated in SDS-polyacrylamide slab minigels (9% w/v). Separated proteins were transferred to nitrocellulose membranes (0.2 µm) in the transfer buffer (25 mM Tris-HCl, 192 mM glycine, 0.1% SDS, and 20% methanol). Membranes were blocked with 5% Blotto (Santa Cruz) dissolved in 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 pH 8 for 1 h, and then incubated with the primary antibodies at 1:100 (RXRß) and 1:200 (RXR and RXR) dilution in blocking solution (TBS, 1% BSA, and 10% NDS) overnight at 37°C. After extensive washing with TBS/Tween-20, the membranes were incubated with the following peroxidase-conjugated secondary antibodies: goat anti-rabbit or goat anti-mouse (Chemicon, Temecula, Calif) at 1:4000 dilution in blocking solution. The membranes were developed with an enhanced chemiluminescence kit, following the procedure described by the manufacturer (Amersham, Buckinghamshire, United Kingdom).

	Results

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Western Blot Analysis

The results of Western blot analysis are shown in Figure 1. Only a single band was found for each antibody at the corresponding molecular weight: 60 kd for RXR, ß, and receptors. For RXR and RXR, the observed expression was similar in the three samples types, however, for RXRß, the expression was weaker in prostate tissues exhibited cancer than in normal and hyperplastic tissues.

View larger version (9K): [in this window] [in a new window]   Figure 1. Western blot analysis of RXR, RXRß, and RXR. L, liver (positive control); NP, normal prostate; BPH, benign prostatic hyperplasia; PC, prostate cancer. Each blot is representative of its respective group. Molecular weights are represented on the left.

Immunohistochemical Study of Control Sections

The immunohistochemical study showed no reaction in the negative controls incubated with the preimmune serum or using the antibody preabsorbed with an excess purified antigens (Figure 2a). Immunostaining of human skin sections were always positive (Figure 2b).

View larger version (138K): [in this window] [in a new window]   Figure 2. (a) Section from prostatic nodular hyperplasia showing no immunoreaction when it was incubated with preimmune serum. Bar, 25 µm. (b) Control section of human skin. The nuclei of keratinocytes (arrow) were intensely labeled for RXR. Bar, 25 µm. (c) Positive immunoreaction to RXR in the nucleus of both basal cells (arrowhead) and secretory epithelial cells (arrow) from a normal prostate. Immunoreaction was more intense in basal cells. Bar, 25 µm. (d) In well-differentiated carcinoma, RXR was observed in the cytoplasm. Bar, 25 µm. (e) Poorly differentiated adenocarcinoma showing nuclear label (arrow) to RXR. Bar, 25 µm. (f) Basal cell hyperplasia presenting strong reaction to RXR in the nucleus and cytoplasm of basal cells. Bar, 25 µm. (g) The nuclei (arrow) of cells that aligned the atrophic prostatic glands were intensely labeled to RXR. Bar, 25 µm. (h) Expression of RXRß in normal prostate. Immunoreaction was located in both the cytoplasm and the nucleus of basal cells (arrowhead) and in a lesser degree, in principal cells. Bar, 50 µm. The inset shows a higher magnification of the epithelium. Arrowhead, basal cell. Bar, 10 µm. (i) In patients with basal cell hyperplasia, the RXRß label was more intense in the cytoplasm than in the nucleus (arrow). Bar, 25 µm. (j) Cytoplasmic location of RXRß in well-differentiated adenocarcinoma. Bar, 25 µm. (k) Well-differentiated adenocarcinoma showing cytoplasmic immunoreaction to RXR. Bar, 25 µm. (l) Reaction to RXR in periurethral glands was observed in the cytoplasm. Bar, 25 µm.

No histological or quantitative immunohistochemical differences between the two subgroups of normal prostates (biopsies and autopsies) were observed.

RXR Immunohistochemistry

The table lists the number of prostates that were positively immunostained for the three types of the RXRs in normal, hyperplastic, and carcinomatous prostates.

RXR was detected in all samples from normal and carcinomatous prostates. The cellular distribution of this receptor in normal prostates was observed in both basal cells and secretory epithelial cell nuclei, the former showed a more intense reaction (Figure 2c). In well-differentiated adenocarcinomas, RXR was detected in the cytoplasm (Figure 2d), but in moderate and poorly differentiated carcinoma, the expression was similar to that of normal prostate (Figure 2e).

In nodular and basal cell hyperplasia immunoreaction to RXR appeared almost exclusively in basal cells in all samples, with a nuclear and cytoplasmic location (Figure 2f). However, in atrophic glands, immunoreaction was found in only 22.2% of samples, and showed a nuclear location (Figure 2g).

RXRß immunoexpression was detected in 53.3% of normal prostates, appearing almost exclusively in basal cells and with a weaker expression than the other retinoid receptors. The intracellular distribution of this receptor was nuclear and cytoplasmic (Figure 2h). In prostates from patients diagnosed with BPH, only those presenting basal cell hyperplasia showed immunoreaction to this receptor in all samples studied. Immunostaining appeared in both the nucleus and the cytoplasm of basal cells, and reactivity more intense in the cytoplasm (Figure 2i). In prostatic adenocarcinoma, RXRß was detected in only 3 of 25 samples (12%), and showed a cytoplasmic location. Positive samples for RXRß belong to well-differentiated carcinomas (Figure 2j).

RXR label was similar to that found for RXR (Figure 2k), however, in nodular hyperplasia, the expression of RXR was reduced to 87.5% of cases.

It was interesting that immunoreaction to all antibodies was also found in the cytoplasm of periurethral gland cells (Figure 2l).

	Discussion

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Retinoic acid effects are mediated by specific RAR and RXR subtypes and modulated by binding proteins at the tissue level (Gudas et al, 1994; Guiguere, 1994; Hong and Itri, 1994). These receptors may play a role in controlling cell proliferation and apoptosis, and the aberrant expression of one or more RARs and RXRs might result in abrogated retinoid signaling and greater cell transformation in several cancer types, including prostate, breast, and lung (Liu et al, 1996; Xu et al, 1997; Campbell et al, 1998). The identification of a molecular alteration in the RAR gene in human acute promyelocytic leukemia and the report of a lower expression of RARß in lung cancer (de The et al, 1990; Gebert et al, 1991) agree with this hypothesis.

Gyftopoulos et al (2000) studied the distribution of RAR in neoplastic and nonneoplastic human prostate. They observed a low expression of this receptor in hyperplastic prostate tissue, in which 3 of 24 cases were completely negative. In contrast, RAR-positive cells were present in all cases of prostatic carcinoma.

The expression of retinoid receptors may depend on the level of retinoids in prostate tissue. Pasquali et al (1996) reported that prostate cancer tissues have five to eight times less retinoic acid than normal prostate or those exhibiting BPH. In primary cultures of prostate cells, ATRA is implicated in the control of growth and induction of apoptosis, and these effects are mediated by specific RAR subtypes, RAR and RARß. In these cells, the expression of messenger RARß was increased, whereas bcl-2 protein levels were decreased (Pasquali et al, 1999). However, in a clinical trial, Trump et al (1997) concluded that ATRA was not active in patients with hormone refractory prostate cancer. These authors proposed that the failure of this agent in hormone refractory prostate cancer might be related to a failure of drug delivery and associated with enhanced mechanisms of ATRA clearance, which occur within a few days of beginning ATRA treatment.

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been shown to induce apoptosis in various malignant cells including human prostate carcinoma cell lines; this induction is mediated by nuclear RARs; by increasing the reactive oxygen species activity; expression of p53, p21, and c-jun genes; and decreasing the expression of the c-myc gene (Sun et al, 1999). However, in patients treated with 4-HPR for 28 days before radical prostatectomy, this synthetic retinoid was ineffective because retinoic acid concentrations in serum and in prostates were not significantly altered (Thaller et al, 2000).

To bind DNA, RARs require heterodimerization with RXRs; the latter can act as homodimer or heterodimer partners of a number of nuclear receptors (Mangelsdorf and Evans, 1995). Because of the role of RXRs as pivotal mediators in several signaling pathways, the aim of this work was to study by immunohistochemistry the presence and distribution of RXRs in normal, hyperplastic, and carcinomatous prostates.

In this study we found both nuclear and cytoplasmic locations for the three types of receptors in some cases. It is known that retinoid receptors belong to the class of receptors (ie, thyroid hormone receptors, retinoid receptors, PPAR, etc) that are constitutively found in the nucleus, regardless of whether the ligand is bound or not bound to the receptor (Reichrath et al, 1997). However, some studies suggest that the intracellular location of retinoic acid nuclear receptors may be regulated by retinoic acid and protein kinase C (Tahayato et al, 1993; Weis et al, 1994; Akmal et al, 1998). In this sense, Akmal et al (1998) reported that depletion of vitamin A leads to a change in the location of RAR from the nucleus to the cytoplasm in rat germ cells. Moreover, down-regulation of protein kinase C, a molecule that is not a ligand for these receptors, is able to increase the cytoplasmic location of RAR in COS-7 cells (Tahayato et al, 1993). Also, Liu et al (2000) encountered RXR in the cytoplasm and nucleus of LAPC-4 cells and PC3 cells and, after the cells were treated with the RXR ligand LG1069, cytoplasmic RXR translocated to the nucleus. Moreover, it has been shown that some nuclear receptors (steroids receptors) are found as an inactive cytoplasmic form in a complex with heat shock proteins (Pratt and Toft, 1997). Thus, it is possible that either inactive retinoic acid nuclear receptors were forming a similar complex together with heat shock proteins or they are located in the cytoplasm in ligand absence.

We observed that expression of RXR and RXR decreased in atrophic hyperplasia in comparison to normal prostatic tissue. However, in nodular and basal cell hyperplasia, the expression was maintained. In prostatic adenocarcinoma, RXR and RXR immunoexpression did not suffer variation compared with normal tissue. The absence of changes in the expression of these receptors could suggest that they do not play a significant role in prostate carcinogenesis. This hypothesis is in agreement with the weak inhibition of prostate cancer cell proliferation induced by RXR selective synthetic ligands (Vos et al, 1997).

In situ hybridization studies by Lotan et al (2000) reported a selective and significant reduction of RXRß mRNA in prostate cancer and in normal prostate tissue adjacent to carcinoma. Also, Kikuwaga et al (2000) detected a lower expression of RXRß protein in prostatic cancer tissue. We have also observed a reduction of RXRß (it was expressed only in three cases classified as well-differentiated carcinomas of 25 prostatic carcinoma samples); however, Kikuwaga et al (2000) observed RXRß expression in moderate and poorly differentiated carcinomas.

This reduced expression of RXRß could be involved in the onset of prostate carcinogenesis and has also been related (in advanced disease) to the ineffectiveness of retinoic acid treatment in some patients with androgen-in-dependent or -dependent prostate cancer (Trump et al, 1997; Kelly et al, 2000), but this hypothesis remains to be investigated.

We have also observed that RXRß was expressed in 8 of 15 (53.3%) normal prostates and in 32% of the hyperplastic prostates studied, all of them diagnosed as basal cell hyperplasia. These data, together with those observed for the other RXRs, lead us to suggest that patients with basal cell hyperplasia are potential targets for receiving treatment with retinoic acid due to the presence of the three types of receptors. However, in those patients who suffer atrophic and nodular hyperplasia, it is probable that this treatment will be unsuccessful because of the low amount of the three types of RXRs (and a complete absence of RXRß).

At present, many of the markers in use are not useful for a precise and early diagnosis of prostatic adenocarcinoma. Qiu et al (1999) have proposed that the loss of RARß expression is an early event associated with esophageal carcinogenesis and the status of squamous differentiation. They have also suggested that loss of this receptor is a common event across cancers of different sites and etiologies.

We propose that the study of RXRß expression, together with RARs in prostatic tissue, could be considered in the near future as key factors in the responsiveness to retinoic acid-based therapies. However, further studies are needed to elucidate the mechanisms of these receptors in order to improve their usefulness in prostate cancer.

	Footnotes

 
Supported by grants from University of Alcalá.

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