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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 5 2051-2055
Copyright © 2001 by The Endocrine Society

Original Studies

Loss of Estrogen Receptor ß Expression in Malignant Human Prostate Cells in Primary Cultures and in Prostate Cancer Tissues1

Daniela Pasquali, Valentina Rossi, Dario Esposito, Ciro Abbondanza, Giovanni A. Puca, Antonio Bellastella and Antonio A. Sinisi

Istituto di Endocrinologia (D.P., V.R., D.E., A.B., A.A.S.) and Istituto di Patologia Generale ed Oncologia (C.A., G.A.P.), Seconda Università di Napoli, 80131 Naples, Italy

Address all correspondence and requests for reprints to: Antonio A. Sinisi, M.D., Istituto di Endocrinologia, Seconda Università di Napoli, Building 16, Via Pansini 5, 80131 Naples, Italy. E-mail: antonio.sinisi{at}unina2.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of this study was to investigate the expression of estrogen receptor (ER) ß and {alpha} genes in normal (N) and malignant (C) primary cultures of human prostate epithelial cells (PEC) and fibroblasts (PFC) and in the prostate tissue donors. Both ERß and ER{alpha} messenger ribonucleic acids were found by RT-PCR analysis in six NPECs and normal prostate tissues and in only one of six CPECs and in the respective cancer tissue donor. The other five CPECs and related cancer tissue donors and all normal and cancer PFCs expressed ER{alpha} messenger ribonucleic acid alone. Immunoblot analysis, using a polyclonal anti-ERß (C-terminal) antibody, demonstrated ERß protein in all NPEC lysates and in one of the six CPECs. ER{alpha} protein was expressed in both NPECs and CPECs when a polyclonal antibody directed against the ER{alpha} N-terminal domain was used. In contrast, ER{alpha} protein was not detected in two of the six CPEC lysates when ER{alpha} C-terminal monoclonal antibodies were used. Using a set of primers designed to amplify the region from exons 6–8, RT-PCR analysis demonstrated the absence of the expected transcript in these cells. The present study shows that the ERß gene is expressed together with ER{alpha} in normal prostates and NPECs, whereas it is barely detectable in prostate cancer and CPECs. Moreover, in some CPECs, the ER{alpha} gene may be transcribed in a changed protein, resulting from the expression of a deletion variant. Together, these data suggest that prostate malignancy is associated with a potential disorder of ER-mediated pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A BODY OF evidence in humans and animal models has suggested a role for estrogens in the regulatory mechanisms of prostate gland growth and in the treatment of prostate cancer (1, 2, 3, 4). The nature of estrogen influence on the human prostate remains unclear and is currently the subject of debate. The findings suggest that estrogens exert either inhibitory or stimulatory effects on the prostate. In fact, estrogens have been implicated not only in the promotion of aberrant prostate growth, such as benign hyperplasia and malignant lesions (5, 6), but also in the control of cell growth and programmed cell death in prostate cancer cells (7, 8). It is still controversial whether estrogens influence prostate cells directly through receptor-mediated effects or act through the hypothalamic-pituitary testicular axis. Until recently the classic estrogen receptor {alpha} (ER{alpha}) was thought to mediate the genomic effects of 17ß-estradiol in mammalian tissues. Several studies have consistently demonstrated that ER{alpha} is present in the stromal compartment and at low levels in basal epithelial cells of normal human prostate (9, 10, 11, 12, 13), suggesting that estrogen-induced epithelial changes could be explained by a paracrine interaction between stromal and epithelial cells. However, the cloning and description of a gene encoding a second type of ER, ERß, in the rat (14), mouse (15), and human (16) suggested a reevaluation of the estrogen-signaling system and function in the prostate. The newly identified ERß is highly expressed in the epithelial components of the rat prostate gland (17), but there is little evidence for its expression in human prostate. Northern blot analysis failed to demonstrate ERß messenger ribonucleic acid (mRNA) (16), and in situ hybridization demonstrated a low level of ERß expression in the epithelial cells (18). Using RT-PCR we (19) and others (20) found the ERß transcript in prostate epithelial cell cultures. The presence of ERß protein in human prostate remains controversial. Bonkhoff et al. (13) failed to demonstrate detectable ERß by immunohistochemistry in normal and malignant tissues, whereas a recent preliminary report immunolocalized the protein in basal epithelial cells in normal tissue samples (21).

Here we evaluated ER{alpha} and ERß expression in prostate tissues and in the stromal and epithelial cells from apparently pure primary cultures prepared from human normal and cancerous prostate specimens. We demonstrated a differential expression pattern of ERß and ER{alpha} mRNAs and proteins between normal and cancerous prostate cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Prostate tissue specimen and primary cultures

Normal human prostatic 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). After prostatectomy, a wedge-shaped specimen of fresh prostate was removed. A sample of the tissue underwent pathological examination to confirm the prostatic origin, the diagnosis, and the absence of other diseases. Only specimens containing 100% normal or cancer prostate cells were used to establish primary cultures according to a previously described method (22). Briefly, prostate epithelial cells (EC) and fibroblasts (FC) were separated by different centrifugations of minced and collagenase (collagenase IV, Life Technologies, Inc.-BRL, Milan, Italy; 10 mg/mL)-digested tissues. The EC were plated on keratinocyte-serum-free medium (Life Technologies, Inc.-BRL, Milan, Italy) supplemented with bovine pituitary extract (10 mg/mL), epidermal growth factor (10 ng/mL), cholera toxin (10 ng/mL), 5% FCS, and antibiotics (fungizon and penicillin-streptomycin). The FC were cultured in MEM (Life Technologies, Inc.-BRL) supplemented with 10% FCS and antibiotics. At confluence, cultures were grown after ethylenediamine tetraacetate-trypsin treatment. Cell cultures were stained with monoclonal antibodies (Roche, Mannheim, Germany) specific for cytokeratin as a marker for EC and for vimentin for FC. At the first passage cultures were considered 100% pure if cytokeratin or vimentin immunostaining was positive in nearly 100% of cells for EC or FC, respectively. Both normal and malignant PECs showed positive immunoreactivity with cytokeratin 8 monoclonal antibody (Mab 35BH11, DAKO Corp., Milan, Italy), indicating their glandular origin (23). Basal cell-specific high molecular weight cytokeratin immunoreactivity (Mab 34BE12, DAKO Corp.) was found in normal cell cultures, demonstrating the presence of cells endowed with basal features (23). High molecular weight cytokeratin immunoreactivity was completely absent from the cells derived from prostate carcinoma. The prostate-specific antigen protein detection from conditioned medium by specific immunoassay and the immunoreactivity of cell monolayers to Mab clone ErPr8 (BioGenex Laboratories, Inc., San Ramon, CA) demonstrated that both normal and malignant short-term PECs retain prostate-specific antigen secretory function. The malignant nature of cells derived from prostate carcinomas was confirmed by a high expression of proliferative antigen Ki67 and, particularly, by a high expression of mutated p53 protein, demonstrated by immunoreactivity with monoclonal antibody clone Ki-67 (DAKO Corp.) and Pab 240 (Serotec, Delta Biological, Milan, Italy), respectively. Six cell strains from normal prostates and six from prostate cancer specimens were used in the experimental protocols that were repeated at least three times. All cultures were performed at 37 C in a humidified 5% CO2 atmosphere in air.

mRNA isolation and RT-PCR

Total RNA was isolated from the cultures at the first passage and from tissues. Total RNA was recovered with the RNAzol B kit (Cinna/Biotecx Laboratories, Houston, TX). Residual DNA was removed by ribonuclease-free deoxyribonuclease I treatment (Promega Corp., Florence, Italy). RT-PCR was carried out as previously described (22). To obtain a negative control for the amplification reactions we carried out an RNA transcription without addition of reverse transcriptase. RNAs were reverse transcribed using 5 µg total RNA in the presence of reverse transcriptase (Superscript -BRL-200 U) at 37 C for 1.5 h. The reaction was stopped by incubation at 95 C for 5 min. Complementary DNA (600 ng) from RT of RNAs was amplified in a total volume of 50 µL of 10 mmol Tris-HCl, 1.5 mmol MgCl2, 50 mmol KCl (pH 8.3), 100 ng of primers, 0.2 mmol deoxynucleotides triphosphate, and 2.5 U Taq DNA polymerase (Roche). A DNA thermal cycler (Perkin-Elmer Corp./Cetus, Milan, Italy) was used for the reaction. The following PCR conditions were used: 35 cycles of amplification, 94 C for 1 min, 58 C for 1 min, and 72 C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (100 ng) were added to each PCR reaction as an internal control, and the 876-bp product of GAPDH was detected in each PCR reaction. The 5'-3' oligonucleotides for ER{alpha}, ERß (24, 25), and GAPDH (22) are shown in Table 1Go. The PCR products were analyzed by electrophoresis on a 1.2% agarose gel and by comparing their sizes with the size expected from the gene sequence. The identity of the products from representative reactions was confirmed by direct sequencing of PCR products. The treatment with deoxyribonuclease and the coamplification of the GAPDH gene containing introns such as ER{alpha} and ERß genes excluded genomic DNA contamination. Granulosa cell mRNA was used for the positive control.


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  		Table 1. Oligonucleotide sequences used for RT-PCR

 
Western blot analysis

ER{alpha} and ERß protein levels were evaluated by Western blot analysis of protein extracts made from six different strains of NPEC and CPEC. Electrophoresis and immunoblotting procedures were performed as previously described (22). For each cell strain 40 µg total protein lysate were loaded. We used the following antibodies for the primary immunoreactions: a polyclonal anti-ERß antibody [human COOH-terminal (PAI-313 ABR, Inalco, Milan, Italy) or 210–180-C050 (Alexis-Italia, Florence, Italy)], a rabbit polyclonal antibody raised against N-terminal part of ER{alpha} (amino acids 154–171, Sigma, Milan, Italy), and a mixture of two monoclonal antibodies (1602 and 1603) raised against a C-terminal complementary DNA recombinant fragment of ER{alpha} (26). Antibody reaction was revealed by a 45-min incubation at room temperature with horseradish peroxide-coupled antigoat or antimouse IgG serum (Amersham Pharmacia Biotech, Milan, Italy), 1:10,000 diluted in Western blot buffer (pH 8.00) containing 1% nonfat dried milk and 0.25% BSA. This was followed by a washing cycle using chemiluminescent substrate (ECL, Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The visualization was obtained by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Using RT-PCR, we found ERß transcript in all six NPECs and in the normal prostate tissue donors (Figs. 1Go and 2Go). We detected the ERß mRNA in only one of six CPECs (see Fig. 1Go, sample CPEC4) and in the corresponding tissue donor, who had a low grade malignancy (Gleasons’ score 4) cancer. Moreover, using a set of primers designed to amplify the ER{alpha} region between exons 4 and 6, we found the ER{alpha} transcript in all epithelial and stromal cell primary cultures and in all benign and malignant tissue samples. The relative levels of ER{alpha}, ERß, and GAPDH gene products in representative samples from normal and cancer prostate tissues and from epithelial and stromal cell cultures are depicted in Fig. 2Go. Both ER subtypes were highly expressed in benign samples, whereas only ER{alpha} was highly present in prostate cancer tissue and epithelial cells. Immunoblot analysis of ERß protein in the EC culture lysates was consistent with the RT-PCR data. In fact, we found ERß protein in all NPECs (Figs. 3Go and 4Go) and in only one of six CPECs, the same sample that had the ERß transcript (see Figs. 1Go and 4Go, sample CPEC4). To screen cell lysates for the ER{alpha} protein we used a polyclonal N-terminal and two monoclonal C-terminal anti-ER{alpha} antibodies. Representative results are shown in Figs. 5Go and 6Go. ER{alpha} protein was found in all normal and malignant epithelial cell primary cultures with the N-terminal antibody (Fig. 5Go). However, ER{alpha} protein was undetectable in two CPECs when we used a mixture of two antibodies directed against the C-terminal portion (Fig. 6Go). In addition, we were unable to detect by RT-PCR the ER{alpha} transcript in these two cell strains, when using a set of primers designed to amplify the region between exons 6 and 8 of the ER{alpha} gene, coding for the distal tract of the ligand-binding domain (Fig. 7Go). We found a difference in the expression of both ERs within normal and cancer epithelial cell cultures coming, respectively, from the normal and the cancer tissue isolated from the same prostate specimen (NPEC2 and CPEC2 in Figs. 1Go, 3Go, and 6BGo).



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  		Figure 1. ERß and GAPDH gene products by RT-PCR from five normal (NPEC2–5) and five cancer (CPEC2–5) prostate epithelial cell cultures. PCR products were separated on a 1.2% agarose gel. M, 100-bp DNA size marker; granulosa cells, positive control cells; -RT, negative control as described in Materials and Methods.

 


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  		Figure 2. Evaluation of ER{alpha} and ERß mRNA levels by RT-PCR. ER{alpha} (483 bp) and ERß (259 bp) and GAPDH gene products in representative samples from normal (NPEC1) and cancer (CPEC1) EC primary cultures, normal (NPFC1) and cancer (CPFC1) stromal cell primary cultures, and donor prostate tissue specimens. PCR products were separated on a 1.2% agarose gel. Granulosa cells, positive control cells; -RT, negative control as described in Materials and Methods.

 


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  		Figure 3. Western blot analysis by a C-terminal polyclonal anti-ERß showing the nearly 55-kDa band corresponding to ERß protein in the cell lysates from five normal epithelial cell primary cultures (NPEC2–5). For each sample, 40 µg protein lysate were loaded. CPEC2 is a representative cancer epithelial cell primary culture that was negative. Note that NPEC2 and CPEC2 are normal and cancer epithelial cell cultures coming, respectively, from normal and cancer tissue isolated from the same prostate specimen.

 


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  		Figure 4. Western blot analysis by a C-terminal polyclonal anti-ERß showing the nearly 55-kDa band corresponding to ERß protein in the cell lysates from a normal epithelial cell primary culture (NPEC1) and a cancer epithelial cell primary culture (CPEC4). CPEC1 is a representative cancer epithelial cell primary culture that was negative.

 


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  		Figure 5. Western blot analysis by N-terminal polyantibody of ER{alpha} (67 kDa) protein expression in representative samples of normal (NPEC1) and cancer (CPEC1) prostate epithelial cell primary cultures. For each sample, 40 µg protein lysate were loaded.

 


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  		Figure 6. Western blot analysis of ER{alpha} protein expression by C-terminal monoclonal antibodies 1602 and 1603. For each sample, 40 µg protein lysate were loaded. The specific 67-kDa protein band is absent in cell lysates from two cancer epithelial cell culture (CPEC1,2). It is evident in representative normal samples (NPEC1,2) and in the MCF7 mammary cell line used as a positive control. A second short band (nearly 54 kDa) is revealed by the antibody mix used and may be due to the presence of a spliced variant reported in several normal and pathological cells. Note that NPEC2 and CPEC2 are normal and cancer epithelial cell cultures coming, respectively, from the normal and the cancer tissue isolated from the same prostate specimen.

 


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  		Figure 7. RT-PCR analysis of ER{alpha} gene using a set of primers designed to amplify exon region 5–8. The transcript is absent in two prostate cancer epithelial cell primary cultures CPEC1 and CPEC2; it is evident in a representative normal sample (NPEC1). PCR products were separated on a 1.2-% agarose gel. M, 100-bp DNA size marker.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides evidence for the coexpression of both ER{alpha} and ERß genes in normal prostate specimens and in normal epithelial cells in primary cultures. The absence of ERß transcripts in the pure culture of isolated fibroblasts is in agreement with the preferential expression of ERß in the epithelial compartment of the human prostate gland. This study also shows that ERß gene expression is frequently lost in malignant prostate epithelial cell primary cultures and in prostate cancer. ERß expression in human prostate has been addressed in very few studies. Using RT-PCR, Lau et al. (20) recently found the transcript of ERß alone in five primary cultures of normal prostate epithelial cells and showed that malignant prostate cell lines (PC-3, DU145, and LNCaP) express ERß associated variably with the ER{alpha} subtype. The discrepancy between these data and ours may be due to the different kinds of samples examined. In fact, we used whole prostate tissue from surgical specimens to establish primary cultures, while Lau used peripheral zone samples obtained by ultrasound-guided biopsy. Moreover, we analyzed malignant epithelial cell primary cultures, whereas they included only prostate cancer cell lines. Although more attention has been focused on the prostate cell lines, they remain a model system and not a perfect in vivo model. We found the same ER expression pattern in the primary cell cultures and in the tissue donors, confirming that primary cultures at the first passages may closely reflect the characteristics of tissues in vivo.

We demonstrated by immunoblotting that ERß protein is expressed preferentially in normal epithelial cell primary cultures. The pattern is consistent with the RT-PCR data. However, the levels of expression of the protein are different among the samples examined. This may be due to either transcriptional or posttranscriptional mechanisms. Current immunohistochemistry studies of ERß in human prostate are scarce and controversial. A preliminary report showed ERß protein immunolocalization on epithelial cells of normal prostate sections (21). Another study failed to demonstrate it in both normal and malignant human prostates (13). It is well known that imperfect antibody specificity, ineffective antigen retrieval and tissue-processing methods, or the presence of unknown isoforms may affect immunohistochemistry performance. Indeed, we found ER{alpha} transcript and protein in all prostate cells analyzed, including malignant ones. Several studies (9, 10, 11, 12, 27, 28, 29, 30) failed to demonstrate ER{alpha} expression in prostate cancer. However, a recent report (13) demonstrated ER{alpha} transcript and protein in premalignant lesions and prostate adenocarcinomas. Our data are in agreement with this finding and suggest that prostate epithelial cancer cells potentially retain estrogen responsiveness via ER{alpha}. Moreover, in two of six malignant epithelial cells in primary culture, we showed an ER{alpha} gene expression variant involving exons 6–8, i.e. the region coding for the distal portion of the ligand-binding domain. The functional significance of ER{alpha} variants that we found in primary cultured cancer cells and that others saw in prostate cancer cell lines (20) remains unknown. It has been suggested that ER variants found in breast cancer might be of clinical significance in the development and progression of the disease (31).

Our observation implies that both ER subtypes may mediate possible direct effects of estrogens on normal epithelial prostate cells. Neoplastic transformation, however, is frequently associated with an impairment of estrogen action due to loss of ERß.

Two recent reports described a hypermethylated status of ER{alpha} gene in prostate cancer cell lines that may be functionally relevant in ER{alpha} gene down-regulation (20, 32). To date, there are no data on ERß gene methylation and its potential role in ERß silencing in prostate cancer cells.

The exact roles of two ERs in human prostate are still unclear, and the significance of the disappearance of ERß expression in human prostate cancer remains to be determined. Distinct effects of the two ERs have been demonstrated at AP-1-containing promoters, suggesting that the balance of ER{alpha} and ERß may be crucial in the physiological response to estrogens in several tissues (33, 34). A possible implication of ERß in neoplastic growth control is suggested by the findings of a selective loss of ERß protein in colon adenocarcinoma and ovarian cancer (35). ERß seems to have a role in the control of proliferation and the prevention of hyperplasia in the rodent prostate, as ERß knockout mice show prostatic hyperplasia on aging (36). The down-regulation of ERß might also be associated with the loss of the antiproliferative effects of estrogens on human prostate. This is probably mediated by the novel ER subtype.

In conclusion, the present study suggests that estrogens may exert their effects on human prostate through ERß. Malignancy seems to be associated with a potential disorder of ERß-mediated pathways. Further investigations on estrogen action and ERß function may help us understand their roles in prostate carcinogenesis.


   Acknowledgments
 
We are grateful to Prof. T. Lotti (Istituto di Urologia, Università Federico II, Naples, Italy) for providing the prostate tissue samples, and to Prof. G. De Rosa and Dr. S. Staibano (Dipartimento di Scienze Biomorfologiche e Funzionali, Università Federico II, Naples, Italy) for cytological characterization and immunostaining of prostate cells.


   Footnotes
 
1 This work was supported by grants from MURST (PRIN 9806102297-003; to A.B.) and from the Italian Association for Cancer (AIRC 1994-1997; to A.A.S.). Back

Received November 14, 2000.

Revised January 9, 2001.

Accepted January 17, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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The relevance of estrogen receptor-{beta} expression to the antiproliferative effects observed with histone deacetylase inhibitors and phytoestrogens in prostate cancer treatment
Mol. Cancer Ther., October 1, 2007; 6(10): 2626 - 2633.
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G. P Risbridger, S. J Ellem, and S. J McPherson
Estrogen action on the prostate gland: a critical mix of endocrine and paracrine signaling
J. Mol. Endocrinol., September 1, 2007; 39(3): 183 - 188.
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D M Peehl
Primary cell cultures as models of prostate cancer development
Endocr. Relat. Cancer, March 1, 2005; 12(1): 19 - 47.
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L.-C. Li, P. R. Carroll, and R. Dahiya
Epigenetic Changes in Prostate Cancer: Implication for Diagnosis and Treatment
J Natl Cancer Inst, January 19, 2005; 97(2): 103 - 115.
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D. M. Selva, O. M. Tirado, N. Toran, C. A. Suarez-Quian, J. Reventos, and F. Munell
Estrogen Receptor {beta} Expression and Apoptosis of Spermatocytes of Mice Overexpressing a Rat Androgen-Binding Protein Transgene
Biol Reprod, November 1, 2004; 71(5): 1461 - 1468.
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A Bardin, N Boulle, G Lazennec, F Vignon, and P Pujol
Loss of ER{beta} expression as a common step in estrogen-dependent tumor progression
Endocr. Relat. Cancer, September 1, 2004; 11(3): 537 - 551.
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O. Imamov, A. Morani, G.-J. Shim, Y. Omoto, C. Thulin-Andersson, M. Warner, and J.-A. Gustafsson
Estrogen receptor {beta} regulates epithelial cellular differentiation in the mouse ventral prostate
PNAS, June 22, 2004; 101(25): 9375 - 9380.
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O. M. Tirado, D. M. Selva, N. Toran, C. A. Suarez-Quian, M. Jansen, D. P. McDonnell, J. Reventos, and F. Munell
Increased Expression of Estrogen Receptor {beta} in Pachytene Spermatocytes After Short-Term Methoxyacetic Acid Administration
J Androl, January 1, 2004; 25(1): 84 - 94.
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 Clin. Cancer Res.Home page
V. Pais, I. Leav, K.-M. Lau, Z. Jiang, and S.-M. Ho
Estrogen Receptor-{beta} Expression in Human Testicular Germ Cell Tumors
Clin. Cancer Res., October 1, 2003; 9(12): 4475 - 4482.
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M.-M. Liu, C. Albanese, C. M. Anderson, K. Hilty, P. Webb, R. M. Uht, R. H. Price Jr., R. G. Pestell, and P. J. Kushner
Opposing Action of Estrogen Receptors alpha and beta on Cyclin D1 Gene Expression
J. Biol. Chem., June 28, 2002; 277(27): 24353 - 24360.
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E. K. Lobenhofer, L. Bennett, P. L. Cable, L. Li, P. R. Bushel, and C. A. Afshari
Regulation of DNA Replication Fork Genes by 17{beta}-Estradiol
Mol. Endocrinol., June 1, 2002; 16(6): 1215 - 1229.
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N. Kuwajerwala, E. Cifuentes, S. Gautam, M. Menon, E. R. Barrack, and G. P. V. Reddy
Resveratrol Induces Prostate Cancer Cell Entry into S Phase and Inhibits DNA Synthesis
Cancer Res., May 1, 2002; 62(9): 2488 - 2492.
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