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Centre for Urological Research (S.J.E., J.F.S., J.S.P., G.P.R.), Monash Institute of Reproduction and Development, Clayton, Victoria 3168, Australia; and Urology Department (M.F.), Monash Medical Centre, Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Professor Gail P. Risbridger, 27–31 Wright Street, Clayton, Victoria 3168, Australia. E-mail: Gail.Risbridger{at}med.monash.edu.au.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogens are recognized as carcinogens by the International Agency for Research on Cancer, and induce tumors in various organs of a number of different species (Ref. 4 and references cited therein). If androgens are locally converted to estrogens, then the synthesis and regulation of estrogen production in the prostate and the subsequent effects on the prostate gland require evaluation.
Human aromatase is encoded by a single copy of the cyp19 gene, localized to chromosome 15q21.2 (5). The complex expression and regulation of aromatase is achieved through the use of multiple exon I that encodes the 5' untranslated region. Each exon I is used in a tissue-specific fashion by alternative splicing and is flanked by its own unique promoter region. Exon I is spliced into exon II at the same splice junction immediately upstream of the start of translation. Therefore, the open reading frame of each transcript, and thus the aromatase protein, is the same irrespective of the site of expression and promoter used. A schematic of the gene is presented in Fig. 1
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The heterogeneous nature of the prostate is a confounding aspect of the previous studies. Multiple pathologies are commonly present in biopsies from men with benign or malignant disease, such that benign tissue may be observed adjacent to premalignant and/or low- or high-grade tumor. Any analysis of the biopsies or extracts thereof will represent multiple cell types and pathologies. Laser capture microdissection (LCM) directly visualizes tissue sections and permits the rapid isolation of specific cell types (16). Analysis of homogeneous samples procured using this technique provides better comparison between specific cell populations.
To examine aromatase gene expression and cellular localization in human prostate tissues and cell lines, we have used RT-PCR and immunohistochemistry. Enzyme activity was examined by biochemical assay in prostate cell lines. Using LCM, expression of aromatase in pure populations of stroma and epithelium from nonmalignant and malignant tissues was examined. Alternative exon I and promoter use was examined by relative real-time RT-PCR in microdissected human tissues and prostate cell lines.
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Materials and Methods |
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For immunohistochemistry, formalin-fixed, paraffin-embedded human prostate, placenta, and breast tissues were obtained from the Melbourne Pathology Archives (Collingwood, Australia). Samples from two placenta, 10 breast cancer, 20 BPH, and 20 PCa patients were collected.
Human prostate samples for RT-PCR, LCM, and Western blot analysis were obtained by needle biopsy from men diagnosed with BPH (n = 12) or PCa (n = 6). Biopsies were collected under sterile conditions during transurethral resection (BPH) or radical prostatectomy (PCa). Biopsies (a minimum of two) were collected from each patient. Samples for LCM were immediately frozen in optimal cutting temperature media; all other samples were snap-frozen in liquid nitrogen. Term placental tissue was kindly provided by the Prince Henry’s Breast Cancer Research Group (Monash Medical Center, Victoria, Australia). All tissues were stored at –70 C until required.
All tissues were obtained in accordance with the requirements and approval of the Committee for Human Ethics and Experimentation at Monash Medical Centre and Monash University.
Culture of human cell lines
Human PCa cell lines DU145, PC3, and LNCaP were obtained from American Type Culture Collection (Rockville, MD). Cells were cultured in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (CSL Ltd., Parkville, Victoria, Australia), 100 IU/ml penicillin, and 10 µg/ml streptomycin (CSL Ltd.).
A benign prostate epithelial cell (PrEC) line was obtained from Clonetics (Clonetics Limited, BioWhittaker, Walkersville, MD). Cells were cultured per the manufacturer’s instructions and were not used beyond passage 10.
Human placental choriocarcinoma JEG-3 cells were kindly provided by the Department of Obstetrics and Gynaecology (Monash Medical Centre, Clayton, Australia). Cells were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 10 µg/ml streptomycin.
All cell cultures were grown in 75-cm2 culture flasks (Falcon; Becton Dickinson and Co., Franklin Lakes, NJ) at 37 C in a humidified incubator with 5% CO2.
LCM of prostate epithelia or stroma
Six-micrometer frozen prostate sections were cut from frozen optimal cutting temperature media blocks at –20 C onto uncoated glass slides cleaned with 100% ethanol. Sections were fixed in 70% ethanol for 3 min, stained with hematoxylin and eosin as previously described (17), dehydrated in graded ethanol washes, and washed in xylene. Sections were air-dried (2–3 min), and microdissection was performed immediately to ensure minimal RNA degradation. Cells were isolated from the slides using the PixCell II LCM System (Arcturus Engineering, Mountain View, CA).
Specific cell types microdissected include stroma (from both BPH and PCa; not adjacent to or near epithelial or tumor cells), benign epithelia, and high-grade tumor cells (Gleason grade 4+; identified morphologically and later confirmed by a pathologist). Cells collected for each sample type varied from approximately 2,500 (tumor cells) to approximately 10,000 (stroma). Specific cell types were microdissected using a 7.5- to 30-µm laser aperture with power between 25 and 35 mW and a pulse duration of 5 msec.
Sections used for LCM sample preparation were used once only. A minimum of three samples was prepared for each cell type from each patient examined.
RNA purification and RT-PCR
Total RNA was extracted from prostate and placental tissues, prostate cell lines, and LCM cells using the TRIzol reagent according to the manufacturer’s protocol (Life Technologies, Rockville, MD). Whole tissues were homogenized in TRIzol, whereas cell lines and microdissected cells were lysed directly in TRIzol reagent. The protocol for microdissected cells was modified after phase separation by pooling the supernatant of five samples and by adding 5 µg of linear acrylamide (Ambion, Austin, TX).
Potential genomic DNA contamination was removed by digestion in a 20-µl solution containing 2 U deoxyribonuclease (DNase) I (Ambion), 1x DNase I reaction buffer (Ambion), and 10 µl of extracted RNA (to a maximum of 2 µg). DNase was subsequently removed by phenol:chloroform purification, and RNA was stored at –70 C.
Total RNA from prostate cell lines, whole placenta, whole prostate (3 µg), or from microdissected cells (100 ng) was reverse-transcribed using Superscript II reverse transcriptase (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol.
PCR primers for 18S were 18SF (5'-TCA AGA ACG AAA GTC GGA GGT TCG-3') and 18SR (5'-GTC TGT GAT GCC CTT AGA TGT CC-3'). Aromatase coding sequence primers were CDSF (5'-CAC AAT CAT TAC AGC TCT CGA TTC G-3'), CDSR (5'-CGT CGT GTC ATG CTG GAC AC-3'), and CDSD (5'-CGG GCT ATG TGG ACG TGT TG-3'). Primers specific for aromatase exon I were modified from those used by Agarwal et al. (18) and were I.1 (5'-GCT GAA CAC GTG GAG GCA AAC-3'), I.2 (5'-CCT CTG AGG TCA AGG AAC AC-3'), I.3 (5'-CAA GAT GAT AAG GTT CTA TCA GAC C-3'), I.4 (5'-AAC GTG ACC AAC TGG AGC CTG-3'), PII (5'-CTC TGA AGC AAC AGG AGC TAT AGA T-3'), and EX2R (5'-CAT CAC CAG CAT CGT GCC TG-3'). Specific exon I was detected using exon I-specific sense primers and the EX2R primer. The position and orientation of the aromatase primers are depicted in Fig. 1
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PCRs were prepared on ice for each sample as follows: 2 mM MgCl2 (PerkinElmer, Boston, MA) 1x PCR buffer (PerkinElmer), 0.25 mM each deoxy (d)ATP, dCTP, dGTP, deoxythymidine triphosphate (PerkinElmer), 0.5 pmol each of forward and reverse primers, 0.02 U/µl Taq DNA polymerase, and 1 µl of cDNA (5% of reverse transcriptase reaction). Reaction volume was made up to 25 µl with nuclease-free H2O. PCRs consisted of 95 C for 5 min followed by 30 (18S) or 40 (Aromatase CDS and exon I) cycles of 95 C for 30 sec, 60 C (18S), 56 C (Aromatase CDS) or 54 C (Aromatase exon I) for 30 sec, and 72 C for 30 sec with a final incubation of 72 C for 10 min.
Real-time PCR was carried out using the Roche LightCycler and SYBR Green protocol (Roche Diagnostics). Individual reactions were prepared in capillary cuvettes (Roche Diagnostics) according to the manufacturer’s instructions. cDNA was diluted a minimum of 20-fold before use. All LightCycler PCRs were run for 45 cycles with the aforementioned annealing temperatures. The aromatase coding sequence was used to generate a standard curve to enable the relative quantitation of each promoter. Promoter levels were expressed as a percentage of total aromatase expression.
All real-time experiments were performed a minimum of three times, with each sample run in triplicate (n = 9).
Southern transfer and autoradiography
PCR products were separated by agarose gel electrophoresis and were then alkaline-capillary-blotted onto Hybond N+ membrane (Amersham Life Science, Little Chalfont, Buckinghamshire, UK) as previously described (19).
The CDSD oligonucleotide was radioactively 5' end-labeled using [
-32P]ATP and T4 polynucleotide kinase (Roche Molecular Biochemicals, Castle Hill, New South Wales, Australia). After successive washes in 0.2x saline sodium citrate/0.1% sodium dodecyl sulfate (SDS) and 0.1x saline sodium citrate/0.1% SDS, the membrane was exposed to XOMAT AR film (Eastman Kodak, Rochester, NY) for up to 48 h at room temperature and developed.
Immunohistochemical staining for aromatase protein
Two antiaromatase antibodies were examined; a monoclonal antibody kindly provided by Dr. Nigel P. Groome (20) and a polyclonal antibody purchased from Dr. Y. Osawa and colleagues (21).
Sections were dewaxed and rehydrated in graded ethanol washes. For antigen retrieval, sections were placed in either a 5% citrate solution (Groome) or Target Retrieval Solution (Osawa; Dako, Carpinteria, CA) then heated to 90 C in a 1350-W microwave, maintained for 5 min, and cooled for an additional 20 min. Sections were treated with 6% H2O2 for 30 min to block endogenous peroxidase activity and incubated in CAS blocking solution (Zymed Laboratories, San Francisco, CA) for 1 h at room temperature.
The sections were incubated overnight at 4 C in either a 1:10 (Groome) or 1:200 (Osawa) dilution of primary antibody. Control sections were incubated in a matched dilution of mouse IgG (Groome), primary antibody preincubated with immunizing peptide (10x excess concentration) (Groome) or normal rabbit serum (Osawa). After washes in 1x PBS, sections were incubated for 1 h in a 1:250 dilution of either biotinylated horse antimouse IgG (Groome; Vector Laboratories, Burlingame, CA) or biotinylated goat antirabbit IgG (Osawa; Vector Laboratories). All sections were then washed in PBS, incubated for 1 h with the Vectastain Elite ABC kit (Vector Laboratories), and color-reacted with 3,3'-diaminobenzidine tetrahydrochloride (DAB Liquid Substrate Kit, Zymed Laboratories). The reactions were stopped in water and sections were counterstained with Mayer’s hematoxylin (Sigma Diagnostics, St. Louis, MO), dehydrated, cleared, and mounted.
Tritiated water release assay
Aromatase activity was measured by the rate of formation of 3HOH from [1]3H-androstenedione (25.9 Ci/µl) using a modified version of the procedure originally described by Ackerman et al. (22). Cell lines were cultured in 12-well plates until approximately 60% confluent. After being starved in serum-free media for 24 h, cultures were incubated for 6 h in serum-free media (1 ml per well) containing 60 pmol of [1]3H-androstenedione. Media were subsequently collected and, after the addition of trichloroacetic acid, steroids were extracted with chloroform and adsorbed with dextran-coated charcoal. The specific activity of aromatase was expressed as the rate of formation of 3HOH per mg protein per 6 h. Protein was assayed using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA).
Samples were run in triplicate, with experiments repeated a minimum of three times (n = 9).
SDS-PAGE and Western immunoblotting
Total cellular protein was extracted from whole tissue samples (human placenta and BPH) and prostate cell lines by homogenization in the presence of 0.15 M NaCl, 1% Nonidet P-40, 0.25 mM Na-deoxycholate, 1% SDS, 0.05 M Tris, and Complete Cocktail Inhibitor (Roche Molecular Biochemicals). Cellular debris was removed by centrifugation. Protein extracts (5 µg placenta, 60 µg all other samples) were separated on denaturing 12.5% SDS-PAGE gels in a Mini-Proten 3 Gel Tank (Bio-Rad) at 120 V for 2 h in the presence of 25 mM Tris, 250 mM glycine (pH 8.3), and 0.1% SDS and were blotted onto Immobilon P transfer membranes (Millipore, Bedford, MA) as previously described (23). Membranes were blocked for 4 h at room temperature using 5% nonfat milk powder dissolved in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (TBST) and incubated overnight at 4 C in aromatase antiserum (diluted 1:4000 or 1:400 in TBST containing 1% nonfat milk powder and 5% normal goat serum for Groome and Osawa antibodies, respectively), TBST with 1% nonfat milk powder, and 5% normal goat serum (antibody control) or antibody preincubated with immunizing peptide (10x excess concentration; Groome only).
Membranes were then incubated for 1 h at room temperature in horseradish peroxidase-conjugated goat antimouse (diluted 1:15,000 in TBST with 1% nonfat milk powder; Groome; Dako) or horseradish peroxidase-conjugated goat antirabbit (diluted 1:10,000 in TBST with 1% nonfat milk powder; Osawa; Dako) antisera. Bound antibody was visualized on XOMAT AR film (Eastman Kodak) using the ECL Plus Western blotting detection system (Amersham Life Science).
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Results |
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) and biopsy specimens from men diagnosed with BPH and PCa (Fig. 2B) consistently demonstrated the presence of the aromatase coding sequence. mRNA for the aromatase gene was detected in the placental control tissue but was not present in a primary PrEC line. In contrast to nonmalignant epithelial cells, aromatase mRNA was expressed in all of the human prostate tumor cell lines, i.e. LNCaP, DU145, and PC3 (Fig. 2A). Aromatase mRNA was reliably and consistently detected in all human prostate biopsy specimens (18 of 18, both BPH and PCa; nine BPH shown), and confirmed by Southern hybridization, autoradiography (Fig. 2B), and sequencing (data not shown). Although aromatase mRNA was readily detectable in the tumor cell lines and patient biopsies, low expression levels were noted, particularly in patient tissues.
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shows the immunolocalization of aromatase protein in placenta (Fig. 3, A, D, and E), but not in breast (Fig. 3B) or prostate (Fig. 3C) specimens. The localization of immunoreactivity for aromatase to the syncytiotrophoblasts within the placenta using the Groome monoclonal antibody was identical with results obtained using the polyclonal antibody from Osawa and colleagues (data not shown). Both antibodies failed to detect aromatase protein in either breast or prostate tissues (Fig. 3, B and C). In contrast, Western blot analysis detected protein in placenta, whole prostate, and all prostate tumor cell lines but not PrECs (Fig. 3F). Controls run without primary antibody (not shown) or with excess antigenic peptide (Fig. 3G; placenta only) were clear.
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). The control JEG-3 cells demonstrated a high activity of 41.79 ± 1.25 pmol/mg protein·6 h that is comparable to levels previously reported (24, 25, 26). In the prostate lines, activity was significant (P < 0.001) and highest in DU145 and PC3 cells, being 734.9 ± 77.7 and 318.3 ± 59.8 fmol/mg protein·6 h, respectively. Comparatively, the activity measured in LNCaP cells was low, 26.7 ± 6.0 fmol/mg protein·6 h, but was still significantly above background (P < 0.01). PrECs consistently demonstrated no measurable activity.
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). In contrast, the androgen-responsive LNCaP prostate tumor cells used PII as well as I.4 and, to a lesser extent, I.3 (Fig. 4). Promoter use in the androgen-independent PC3 and DU145 differed to that of LNCaP cells with predominant use of promoter I.4 with I.3 also being detectable. PII was undetectable in these cells.
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). In contrast to the observations in the tumor cell lines, promoter PII was exclusively used in the microdissected stromal tissue of all five patients studied (Fig. 5B), with identical expression in samples isolated from either BPH or PCa sections. After the onset of aromatase expression in the malignant epithelia, a different promoter use to stroma was observed, and it now included promoters I.4 and I.3 in addition to PII (Fig. 5C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Most significantly, the levels we have observed are within the range that has been reported for the breast. These data provide additional evidence for a direct intraprostatic effect of estrogen on the prostate gland. In breast cancer, aromatase and its aberrant expression are significant (28, 29, 30). After the induction of malignancy in this system, there is a shift in promoter usage from promoter I.4 to PII or I.3. Subsequently, aromatase expression increases at the tumor site, and the resultant elevated local estrogen levels produce a positive feedback loop that drives tumor cell proliferation (31). Aromatase inhibitors have successfully been used to treat breast cancer, and it has been postulated that they may replace tamoxifen as the first therapeutic option (32, 33, 34). We have demonstrated that analogous changes in promoter usage occur in prostate malignancy, potentially associated with the progression to androgen independence. As shown in the cell lines, this change from PII to promoter I.4 would result in altered regulation and is consistent with the role cytokines are believed to play once prostate tumor cells reach an androgen-independent state. PII is responsive to cAMP and factors such as prostaglandin E2 (35), dexamethasone, and phorbol esters (36). Alternately, promoter I.4 is induced by TGFß (37), IL-6, IL-11, oncostatin M, and leukemia inhibitory factor (38). These factors are recognized to be involved in carcinogenesis in many tissue types, including PCa. Therefore, one of the mechanisms through which factors such as TGFß, the ILs, and leukemia inhibitory factor could mediate tumorigenic actions in PCa may be through an induction of estrogen production in epithelia. Although the alternate promoter use seen in the androgen-independent cell lines would imply that androgens are directly or indirectly involved in the regulation of these promoters, subsequent experiments on cells cultured with and without testosterone failed to show any significant change (data not shown).
Additional parallels between the prostate and breast were noted after the examination of aromatase activity in malignant tissues. Both the presence of the aromatase enzyme and its activity was detected in prostate epithelial tumor cell lines but not in a benign epithelial cell line. This confirms that local conversion of testosterone to estrogen occurs in prostate tumor cells, and, given the aberrant promoter regulation observed in these tissues, this would potentially result in elevated local estrogen levels at the tumor site. Although the activity measured in the prostate tumor cell lines was low overall, all levels detected were within the activity range that has been reported in breast tumors [(2.5 fmol/mg protein·h up to 2.6 pmol/mg protein·h; (39, 40, 41)].
Although estrogens can regulate androgen levels indirectly via the hypothalamic-pituitary-gonadal axis (27), mounting evidence indicates that they can also act directly on the prostate gland. Two estrogen receptor (ER) subtypes have been localized to the prostate; ER
predominantly in stroma and also in epithelia (42, 43, 44) and ERß solely in epithelia (45). At maturity, estrogens administered in combination with a constant level of androgens cause metaplastic growth of the prostate, whereas estrogens alone are directly growth inhibititory in vitro (46, 47, 48). Additionally, exposure to high levels of estrogens during the neonatal period results in permanent alterations in prostate growth as well as predisposing the gland to hyperplasia and severe dysplasia similar to the human pathology, prostatic intraepithelial neoplasia (43, 49). Understanding the role of estrogen in the prostate is further complicated by data demonstrating that low levels of estrogen can induce proliferation, both during development and at maturity (50, 51).
Collectively, these reports emphasize the importance of estrogens in the prostate, and we have conclusively demonstrated that the gland also has the capacity to locally synthesize estrogens, something that has been a matter of contention to date. A major confounding aspect of previous studies may be the heterogeneity of the gland itself because needle biopsies from tissues from men with benign disease will contain tumor cells, and vice versa. Ergo, any conclusion drawn from the examination of such samples will not be definitive. The heterogeneity of the gland, in addition to low levels of aromatase expression, are the likely reasons for the discrepancies in the literature and the difficulty in generating reliable and reproducible results. We have overcome these inherent problems by using LCM to analyze patient samples in conjunction with a number of different human prostate cell lines, and this study is the first to examine aromatase expression in homogeneous prostate samples prepared directly from patient tissues.
The action of estrogen in prostatic stroma and epithelia is principally mediated through ER, other than ERß, which, to date, has no apparent role (52). Not only have we detected aromatase expression in 100% (18 of 18; BPH and PCa) of patient biopsies examined, but in samples prepared by LCM we have found that aromatase expression is localized to the stroma of benign tissues, with expression also being detected in malignant epithelia. These data are consistent with previously reported data examining the influence of ER in the prostate. The predominant localization of ER to stroma implies that estrogen action on the epithelium primarily involves paracrine events, whereas in malignancy, the switch to aromatase expression in epithelia suggests that autocrine or intracrine events may elicit estrogen-mediated responses. The continued expression of ER and its role in PCa requires additional investigation. Alternately, estrogen produced in the epithelium may act via nonreceptor-mediated events or through other steroid receptors like the androgen receptor. In the latter instance, it is noteworthy that mutations in the androgen receptor detected in LNCaP cells (53) and in tumors (54) permit its activation by estrogenic compounds.
Our results confirm local aromatase gene expression in benign prostate, further supporting a role for estrogen in this organ. We have also demonstrated an induction of expression with malignancy that is similar to that occurring in breast cancer, and a level of aromatase activity in tumor cells that is within the range reported in breast tumors. The key differences between prostate and breast tumors with regard to aromatase expression and regulation need to be further examined if potential therapeutic targets are to be identified. These data provide a basis for the development of novel tissue-specific inhibitors, or selective aromatase modulators, as new possibilities for the treatment of prostate cancer.
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Acknowledgments |
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Footnotes |
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Abbreviations: BPH, Benign prostatic hyperplasia; d, deoxy; DNase, deoxyribonuclease; ER, estrogen receptor; LCM, laser capture microdissection; PCa, prostate cancer; PrEC, prostate epithelial cell (line); SDS, sodium dodecyl sulfate; TBST, Tris-buffered saline with Tween 20.
Received May 30, 2003.
Accepted February 1, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Reductase isozymes and aromatase are differentially expressed and active in the androgen-independent human prostate cancer cell lines DU145 and PC3. Prostate 41:224–232[CrossRef][Medline]
-reductase isozymes and aromatase in human prostate cancer cells and in benign prostate hyperplastic tissue. Prostate 34:283–291[CrossRef][Medline]
and -ß, and progesterone receptor mRNA expression in various prostatic lobes and microdissected normal and dysplastic epithelial tissues of the Noble rats. Endocrinology 139:424–427 mediates effects of estrogen on prostatic epithelium. Dev Biol 229:432–442[CrossRef][Medline]
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