Molecular and Cellular Endocrinology 178 (2001) 47 – 50 www.elsevier.com/locate/mce
Estrogen receptor b expression in human prostate tissue Daniela Pasquali a, Stefania Staibano b, Domenico Prezioso c, Renato Franco b, Dario Esposito a, Antonio Notaro a, Gaetano De Rosa b, Antonio Bellastella a, Antonio A. Sinisi a,* a
b
Istituto di Endocrinologia, Seconda Uni6ersita` di Napoli, Via Pansini 5, 80131 Naples, Italy Department of Biomorphological Sciences, Section of Pathology, Uni6ersita` Federico II, Naples, Italy c Department of Urology, Uni6ersita` Federico II, Naples, Italy
Abstract Estrogen receptor subtype b (ERb) is highly expressed in rat prostate epithelium, but its presence in human prostate needs to be confirmed. Here we investigated the expression of ERb in five benign (normal and/or hyperplastic) and 10 malignant (Gleasons’ score 2–7) prostate tissue specimens using immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections, using a commercially available ERb polyclonal antibody developed against the C-terminal amino acid residue. Nuclear ERb expression was found in the nuclei of glandular epithelium of benign prostate tissue specimens; faint nuclear ERb positivity was also present in a few stromal cells around normal epithelium. Nuclear ERb specific immunostaining was undetectable in all prostate cancer sections. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Estrogen receptors; Estrogen receptor b; Prostate; Prostate cancer
1. Introduction It is well established that androgens and other hormones interact with nuclear receptors to regulate growth, structure and function of the prostate gland. Among these hormones, estrogens seem to be involved in normal and abnormal prostate growth, although their exact role is still controversial. Estrogens exert both inhibitory and/or stimulatory effects on the prostate gland (Levine et al., 1991; Santti et al., 1994; Jin et al., 1996; Chang and Prins, 1999). They may act by inhibiting the hypothalamic – pituitary testicular axis or directly at the prostate through receptor-mediated effects. The presence of estrogen receptors (ERs) in the human prostate gland is still a matter of debate. A low level of classic ERa has been demonstrated in the stromal compartment and in basal
* Corresponding author. Tel.: + 39-081-5666627; fax: +39-0815666628. E-mail address:
[email protected] (A.A. Sinisi).
epithelial cells of normal human prostate (Shulze and Claus, 1990; Brolin et al., 1992; Ehara et al., 1995; Hiramatsu et al., 1996; Bonkhoff et al., 1999). In prostate cancer, ERa expression was absent in the studies reported so far (Shulze and Claus, 1990; Brolin et al., 1992; Ehara et al., 1995; Hiramatsu et al., 1996; Kruithof-Dekker et al., 1996; Hobisch et al., 1997), except for a recent report, demonstrating ERa positivity in premalignant lesions and prostate adenocarcinomas. This was more evident in high grade and metastatic tumours (Bonkhoff et al., 1999). The novel ERb has been cloned in the rat prostate, where it is highly expressed in the epithelial components of the gland (Kuiper et al., 1996, 1997). The ERb transcript is expressed to a small degree (Enmark et al., 1997) or absent in the human prostate (Mosselman et al., 1996). ERb protein expression in human prostate has not been confirmed by immunohistochemistry (Bonkhoff et al., 1999). In this study we investigated ERb expression in human prostate sections from normal and cancer specimens, using a polyclonal antibody directed against the C-terminal domain of the protein.
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D. Pasquali et al. / Molecular and Cellular Endocrinology 178 (2001) 47–50
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of Fast Red. For ABC, a conventional avidin–biotin procedure was performed (Hsu et al., 1981). Negative controls were made using normal mouse serum instead of the primary antibody. Positive controls for protein were carried out on sections of granulosa cell tumor (Enmark et al., 1997). The slides were then counterstained with Mayers haematoxylin and mounted in an aqueous medium for APAAP and in a synthetic medium for ABC. Immunostaining was evaluated independently by two observers. Only cells with a purplishred precipitate in the nucleus for APAAP and/or brown nuclear precipitate for ABC were considered positive for anti-ERb. The proportion of positive cells was ranked on a semiquantitative scale from 1 to +3; I, negative (0B 5% stained cells); II, (\ 10–25% stained cells); III, (\ 25% stained cells).
2. Methods
2.1. Prostate tissue specimen We selected 15 prostate specimens (five normal and/ or hyperplastic, 10 prostate cancer tissues), for which clinical data and a follow-up of at least a year were available, from the archives of the Department of Biomorphological Sciences, Section of Pathology at Universita` Federico II of Naples. Benign prostate tissues were collected from patients who had undergone radical cystectomy for bladder cancer. Prostate cancer tissues were obtained from patients who had undergone radical prostatectomy (Gleasons’ score 2– 7). None received hormone treatment before surgery. A paraffin block holding a representative area of the prostate gland or tumour was cut in serial sections of 4 mm in thickness. A colored section with hematoxylin– eosin was examined using a Leitz Laborlux K microscope (Leica Imaging System Inc., Cambridge, UK), to confirm the original diagnosis.
3. Results We found nuclear expression of ERb protein in normal and benign hyperplastic prostate tissues but not in malignant specimens (Table 1). ERb expression was found in the nuclei of glandular epithelium of normal prostate tissue specimens; moreover ERb nuclear positivity was found in a few stromal cells around normal epithelium (Fig. 1). In all prostate cancer sections, ERb specific nuclear immunostaining was undetectable (Figs. 2 and 3). A faint cytoplasmatic positivity was detected in normal, hyperplastic and in well-differentiated prostate cancers (Figs. 1 and 2).
2.2. Immunohistochemistry For each paraffin embedded sample, 4-mm serial sections mounted on slides pretreated for immunohistochemistry were dewaxed in xylene and brought through ethanol to deionized distilled water. Before performing the immunohistochemical staining, sections were incubated in a 750 W microwave oven for 15 min in 10 mM, pH 6.0 buffered citrate, to allow antigen unmasking (Shi et al., 1991). Non specific binding was blocked by incubating with non-immune serum (1% Tris– bovine albumin for 15 min at room temperature). Incubation with the primary antibody, a polyclonal antibody, PA1-313 (Affinity Bioreagents, Golden, CO), raised against synthetic peptide corresponding to the C-terminal part of ERb, was carried out overnight, at 1:50 dilution. We used the alkaline phosphatase (APAAP) Envision system and/or conventional avidin– biotin complex (ABC) staining as revelation systems. For APAAP, slides were gently washed in Tris buffer, then incubated with Alkaline Phosphatase Labeled Polymer for 30 min at room temperature and the reaction was then developed with a chromogen solution
4. Discussion The present data demonstrate that ERb is expressed in the nuclei of the epithelial cells as well as in some cells from the stromal compartment of normal or hyperplastic human prostates. Moreover, we found no nuclear ERb localization in sections from prostate cancers, irrespectively from tumour differentiation. Whether the difference between benign and malignant prostate samples occurs as a consequence of transcriptional or post-transcriptional mechanisms remains to be established. In a preliminary study on primary epithe-
Table 1
Gleasons’ score ERb positivity Score
Cases with normal/hyperplastic prostate tissue
Cases with prostate cancer tissue
1
2
3
4
5
1
2
3
4
5
6
7
8
9
10
+ II
+ III
+ II
+ III
+ III
3 – 0
2 – 0
5 – 0
4 – 0
5 – 0
7 – 0
7 – 0
3 – 0
6 – 0
7 – 0
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Fig. 1. Expression of ERb in normal and hyperplastic prostate epithelia. Paraffin-embedded sections of human normal and hyperplastic prostate were stained with a polyclonal antibody, PA1-313. Specific nuclear ERb expression is evident in the periglandular epithelium ( ×400).
lial cell cultures from normal and cancer prostates, we detected ERb mRNA only in normal cell cultures. This suggests that ERb expression is regulated at the transcriptional level in human prostate (Sinisi AA, pers. commun., Endo ‘99, June 12– 15, 1999, San Diego, CA). The ERb expression in human prostate has been addressed in limited studies. ERb gene transcript has not been found using Northern blot analysis (Mosselman et al., 1996), whereas a low mRNA level has been demonstrated using in situ hybridization (Enmark et al., 1997). Recently, an ERb isoform, ERbcx, was shown in human prostate tissues to overlap with the ERa transcript (Ogawa et al., 1998). Bonkhoff et al. (1999) failed to demonstrate the ERb protein using immunohistochemistry in normal and malignant human prostates. These conflicting findings may be due to the low sensitivity of the techniques used, limited antibody specificity, ineffective antigen retrieval methods and tissue processing, or presence of unknown isoforms. Knowledge of ERb distribution in human tissues is still limited and its exact role remains unclear. Distinct effects of the two ERs have been demonstrated at AP-1 containing promoters, suggesting that the ERa and ERb balance may be crucial in the physiological response to estrogens (Paech et al., 1997). An estrogendominant environment after middle age is associated with prostate disease development (Santti et al., 1994). Estrogen treatment in mature men induces complex and contrasting effects on the prostate gland, including atrophy, basal cell hyperplasia and squamous cell metaplasia, or direct stimulatory effects (Levine et al., 1991; Jin et al., 1996). Moreover, experimental data from animal models seem to support a potential implication of estrogens in prostate carcinogenesis (Santti et al., 1994). Our study suggests that estrogens may even exert their effects on human prostate through the novel ERb. Malignancy seems to be associated with the disappear-
Fig. 2. Expression of ERb in prostate cancer tissues. Paraffin-embedded sections of prostate cancer were stained with a polyclonal antibody, PA1-313. ERb was not expressed in prostate cancer ( × 250).
Fig. 3. Expression of ERb in prostate cancer tissues. Paraffin-embedded sections of prostate cancer were stained with PA1-313 polyclonal antibody. ERb was not expressed in prostate cancer ( × 400).
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ance of ERb expression in prostate tissues. The significance of this observation remains to be determined. In other tumors, such as colon adenocarcinoma, a selective loss of ERb protein has been reported, suggesting that neoplastic growth may be associated with ER-mediated pathway changes, at least in some tissues (Foley et al., 2000).
Acknowledgements This work was supported by a grant from MURST, Italy, 1998–1999 to A. Bellastella.
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