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Endocrinology Vol. 143, No. 6 2093-2105
Copyright © 2002 by The Endocrine Society

CANCER

Gene Expression Profiling of Testosterone and Estradiol-17ß-Induced Prostatic Dysplasia in Noble Rats and Response to the Antiestrogen ICI 182,780

Christopher J. Thompson, Neville N. C. Tam, Jennifer M. Joyce, Irwin Leav and Shuk-mei Ho

Department of Surgery-Division of Urology (C.J.T., N.N.C.T., J.M.J., I.L., S.-M.H.), Department of Cell Biology, and Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655; Department of Pathology (I.L.), School of Veterinary Medicine, Tufts University, Grafton, Massachusetts 01536; and Department of Pathology (I.L.), University of Massachusetts Medical School, Worcester, Massachusetts 01655

Address all correspondence and requests for reprints to: Dr. Shuk-Mei Ho, Room S4-746, Division of Urology, Department of Surgery, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655. E-mail: . Shuk-mei.Ho{at}umassmed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that 1) treatment of Noble rats for 16 wk with testosterone (T) and estradiol-17ß (E2) led to 100% incidence of dorsolateral prostate (DLP) dysplasia and hyperprolactinemia and 2) blockade of PRL release with bromocriptine cotreatment significantly lowered the incidence of DLP dysplasia. In the current study, we sought to determine whether E2 exerts direct effects, independent of PRL, in this model system. The pure antiestrogen ICI 182,780 (ICI), reported to have no effect on PRL release in female rats, was administered biweekly to T + E2-treated rats at 3 mg/kg body weight. ICI cotreatment completely prevented DLP dysplasia development but it also blocked hyperprolactinemia in the dual hormone-treated rats. Gene profiling with an 1185 gene rat cDNA array identified {approx}100 genes displaying >=3-fold changes in rat lateral prostates (LPs) following T + E2 treatment. Significantly more genes were up-regulated (77) than down-regulated (14), reflecting cellular/molecular changes associated with enhanced cell proliferation, DNA damage, heightened protein and RNA synthesis, increased energy metabolism, and activation of several proto-oncogenes and intracellular signaling pathways. Post hoc analyses, using quantitative real-time RT-PCR, corroborated differential expression of eight genes, exhibiting three different patterns of altered expression. Genes encoding the early growth response protein 1 and metalloendopeptidase meprin ß-subunit were similarly altered in T + E2- and T + E2 + ICI-treated animals when compared with untreated controls. In contrast, transcripts of fos-related antigen-2, growth arrest and DNA damage-inducible protein-45, and signal transducer and activator of transcription-3 were significantly increased in the LPs of T + E2-treated animals, but the increases were reversed by cotreatment with ICI. Differential expression of fos-related antigen-2 and growth arrest and DNA damage-inducible protein-45 were further confirmed at the protein level by immunohistochemistry. Lastly, levels of A-RAF, VIP-1 receptor, and calpastatin mRNA were distinctly lessen in rat LPs under T + E2 influence, but rebound with ICI cotreatment. In conclusion, our findings further implicated pituitary PRL in the induction of dysplasia in rat LP. Gene profiling provided clues that molecular events related to enhancement of cell proliferation, DNA damage, and activation of proto-oncogenes and transforming factors may be causally linked to the genesis of LP dysplasia in this rat model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WHILE THE PROSTATE is considered the prototypic androgen-dependent gland, estrogens also play a role in its growth and maintenance (1). In vitro studies have demonstrated direct mitogenic effects of estrogens on normal prostatic tissues (2) and on LNCaP, a human prostate cancer cell line (3). In vivo, estrogen has been shown to potentiate androgen-mediated prostate growth (4). Neonatal/perinatal exposure to estrogen has been reported to predispose the adult gland to prostatic inflammation (5) and hyperplasia/dysplasia (6, 7).

Epidemiological evidence has implicated androgens and estrogens as playing possible roles in the genesis of human prostate cancer (8, 9, 10). In aggregate, these studies have shown that prostate cancer, largely a disease of older men, develops in a hormonal milieu where circulating levels of estrogens predominate over declining levels of androgens. Moreover, African-American men, who have the highest incidence of prostate cancer in the world, have elevated levels of estrogens at early ages (11) and higher estrogen exposure in utero (12). In contrast, Japanese males, who have a low incidence of the disease, have low serum estradiol-17ß (E2) values when compared with age-matched Caucasians (13).

Experimental support for the theory that sex steroids play a role in the genesis of prostatic carcinoma comes from data obtained in Noble (NBL) rats (14). While NBL rats typically have a very low incidence of spontaneous prostatic carcinoma, we (15) and others (16, 17) have demonstrated induction of dysplasia and adenocarcinoma in the dorsolateral prostates (DLPs), but not in the ventral prostates, of rats chronically exposed to testosterone (T) and E2. Following 16 wk of combined T + E2 treatment, DLP dysplasia developed in 100% of the treated animals. The DLP lesion morphologically resembled prostatic intraepithelial neoplasia or PIN, the putative precursor of human prostate cancer (18). A number of cellular and molecular alterations were notable in the dysplastic DLP when compared with the normal prostatic lobe. These included a marked increase in epithelial cell proliferation (15), activation of the TGF-{alpha}/epidermal growth factor (EGF) receptor (EGFR) autocrine loop in dysplastic foci (19), modest enhancement of H- and K-ras proto-oncogene expression (20), up-regulation of metallothionein-I (21), changes in the MAPK/MKP-1 signaling (22), as well as evidence of oxidative stress-related DNA damage (23).

An important consequence of chronic T + E2 treatment of NBL rats is the dramatic elevation of serum PRL. Estrogen stimulates release of PRL from the pituitary, either through direct action on the lactotrophs or indirect action on the factors governing its release (24). In the prostate, PRL has been shown to potentiate the action of androgen (25) and exert direct, androgen-independent growth stimulatory effects on the prostate (2, 26). In this regard, striking enlargement of the prostate has been observed in transgenic mice overexpressing the PRL gene (27) and in rats with induced hyperprolactinemia (28). We have used the dopamine agonist bromocriptine to inhibit PRL oversecretion from the pituitaries of T + E2-treated NBL rats and observed blockade of hyperprolactinemia and a significant decrease in incidence of DLP dysplasia (29). However, it remains to be defined whether estrogen exerts direct trophic effects, independent of PRL induction, on the rat DLP and thereby participates in the genesis of dysplasia in the DLPs of NBL rats. To address this question, we used ICI 182,780 (ICI) to antagonize the action of estrogen at the tissue level in T + E2-treated rats. ICI is a steroidal, pure antiestrogen (30) that prevents transactivation from the estrogen response element (ERE) by both ER-{alpha} and -ß (31, 32). Importantly, in numerous experimental models and clinical trials, it has been demonstrated to be devoid of any estrogen agonist activity (33). ICI is also unique in that it is thought not to cross the blood brain barrier (34). Specifically, ICI blocked cyclical vaginal cornification without effecting body weight, serum LH, FSH, or PRL concentrations in female rats (30, 34).

In the current study, a cDNA microarray with 1185 known genes was used to profile gene expression changes associated with development of dysplasia in the lateral prostates (LPs) of T + E2-treated rats. Post hoc analyses of RNA samples isolated from LPs of untreated, T + E2-treated and T + E2 + ICI-treated rats with quantitative real-time RT-PCR corroborated significant changes in the expression of genes encoding early growth response protein 1 (EGR1), metalloendopeptidase meprin ß-subunit (meprin ß), growth arrest and DNA damage-inducible protein-45 (GADD45), fos-related antigen-2 (FRA2), A-RAF and VIP-1 receptor (VIP1R), signal transducers and activators of transcription-3 (STAT3) and calpastatin. Immunohistochemical studies demonstrated that expression of GADD45 and FRA2 was enhanced in the LP epithelium of T + E2-treated rats but the enhancement was blocked by ICI cotreatment. Thus, gene expression profiling has proven useful in the rapid identification of genes whose expression is altered by T + E2 treatment, either directly or indirectly via induction of hyperprolactinemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Treatment of animals, tissues collection and processing, and histological detection of dysplasia
The animal usage and care protocols were approved by the Institutional Animal Care Committees at Tufts University and at the University of Massachusetts Medical School, in compliance with NIH guidelines. Male NBL rats (5–6 wk old) were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Rats were housed at the university’s animal facility on a 12-h light/12-h dark cycle and allowed access to food and water ad libitum. At 11–12 wk of age (280–300 g), rats were separated into three groups. Thirteen animals were implanted sc with hormone-filled capsules made from SILASTIC brand silicon tubing (Dow Corning Corp., Midland, MI; 1.0-mm inner diameter x 2.2-mm outer diameter) packed with T or E2 (Sigma, St. Louis, MO). Each animal received two 2-cm long T capsules and one 1-cm long E2 capsule (for details see Refs. 15 and 35). Capsules were replaced after 8 wk. Control rats (n = 5) are untreated animals received empty capsules at the same schedule. ICI was a gift of Dr. B. M. Vose (Zeneca Pharmaceuticals, Cheshire, UK). It was dissolved in absolute ethanol and diluted in peanut oil (Sigma) to a final concentration of 3 mg/ml. The final volume of ethanol never exceeded 1%. One group (n = 6) of T + E2-implanted rats received ICI sc in the flank twice per week at a dose of 3 mg/kg according to (34). At the end of a 16-wk treatment period, all three groups of animals were killed by an overdose of Isoflurane (Ohmeda Caribe Inc., Guayama, PR) followed by decapitation. Trunk blood was collected from each animal for serum PRL analyses. Prostates were excised and separated into dorsal, lateral and ventral lobes. One lobe each from dorsal, lateral, and ventral prostate was fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 4- to 6-µm sections. Incidence of dysplasia was determined histologically in multiple-stepped sections through each prostatic lobe (at least 20 sections were examined for development of dysplasia by IL). The remaining portion of each prostatic lobe was snap-frozen for RNA preparation.

RIA analysis of serum PRL levels
Serum PRL was determined as previously described (29, 36) using the NIDKK rat PRL RIA kit provided by the National Hormone and Pituitary program (Dr. A. F. Parlow, Director).

RNA isolation and cDNA synthesis via RT
All reagents were purchased from Perkin-Elmer Corp. (Foster City, CA) unless stated otherwise. Total RNA was isolated from frozen tissues using STAT-60 reagent (Tel-Test, Inc., Friendswood, TX) according to manufacturer’s instructions with the following modifications: after isolation, total RNA was incubated for 1 h at 37 C, in a reaction mixture containing ribonuclease inhibitor (Perkin-Elmer Corp., Foster City, CA), dithiothreitol, 5x transcription buffer, and RQ1 ribonuclease-free deoxyribonuclease (Promega Corp., Madison, WI). One microgram of total RNA was reverse transcribed for 65 min at 42 C in a 60 µl reaction including 5 mM MgCl2, 1x GeneAmp PCR buffer II (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 1 mM each dNTP random hexamers, 60 U ribonuclease inhibitor, and 150 U MuLV reverse transcriptase. The RT reaction was terminated by heating to 95 C for 5 min. One microliter cDNA was used for subsequent PCRs.

cDNA microarray analysis, image quantification, and data analysis
The Atlas Rat 1.2 cDNA Expression Arrays (CLONTECH Laboratories, Inc., Palo Alto, CA) carrying 1185 rat cDNAs were used for global gene expression profiling. Total RNA was prepared from LPs of untreated and T + E2-treated rats followed by deoxyribonuclease treatment as described in the preceding paragraph. Each total RNA sample was checked for integrity and DNA contamination by measurement of optical density and size-fractionation of 18S and 28S rRNA on a denaturing gel capable of detecting RNA degradation. cDNA probes for the microarray were prepared according to manufacturer’s instructions using 3 µg total RNA and [{alpha}-32P]deoxy-ATP (>2,500 Ci/mmol, 10 µCi/µl) (NEN Life Science Products, Boston, MA). Approximately 0.6 x 106 cpm of each probe were hybridized to the membrane microarray overnight with continuous agitation at 68 C. Following washing, the membranes were covered in plastic wrap and exposed to a low-energy PhosphorImager Screen (Molecular Dynamics, Inc., Sunnyvale, CA) for 7 d at room temperature. The hybridization signals on the screens were read by PhosphorImager scanning using Storm 830 PhosphorImager (Molecular Dynamics, Inc.). Following exposure to PhosphorImager screens, membranes were stripped by boiling for 10 min and hybridized with a new sets of probes prepared from untreated, T + E2, and T + E2 + ICI animals. Stripping led to considerable loss of sensitivity. Signal intensities of the spots were quantified using a Kodak 1-D Image Analysis Software (Eastman Kodak Co., Rochester, NY) and exported to Microsoft Corp. Excel. Background intensity was normalized by subtracting an offset value calculating by taking the median of 24 negative genes. Labeling efficiency was normalized by calculating a ratio for each signal intensity against the mean value for three housekeeping genes: hypoxanthine-guanine phosphoribosyltransferase, glyceraldehyde 3-phosphate dehydrogenase, and tubulin {alpha} 1.

The following sets of genes were identified by comparison of the various datasets: 1) genes that were expressed only in the T + E2-treated LPs; 2) genes that were differentially expressed by 3-fold or greater between the T + E2-treated LP samples and the untreated LP samples; 3) genes that were up- or under-expressed in the T + E2-treated LPs when compared with the untreated LP but differential expression was reversed by cotreatment with ICI; and 4) genes demonstrating unique expression in the T + E2 + ICI-treated animals. Genes were then ranked for level of over- or under-expression. Genes that demonstrated approximately 3-fold or greater differential expression between untreated and T + E2 treated LPs and known to be associated with tumorigenesis, tumor suppression, growth, signal transduction or transcriptional regulation were selected for post hoc confirmation and further studies.

Real time quantitative RT-PCR
Confirmation of differentially expressed transcripts was performed using the iCycler IQ Real Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA) on cDNA obtained from the LPs of five untreated, seven T + E2-treated, and six T + E2 + ICI-treated rats. Total RNA was obtained from each LP and treated with deoxyribonuclease to remove DNA contamination. The integrity and quality of each total RNA samples were carefully checked as described above before RT into cDNA.

One microliter cDNA obtained by reversed transcription was amplified in a 25 µl reaction mix containing 1x SYBR Green PCR Master Mix (Perkin-Elmer Corp.) and 0.4 mM each primer. Following a 6-min Taq activation step at 95 C, reactions were subjected to 40 cycles of 30 sec denaturation at 94 C, 30 sec annealing, and 30 sec extension at 72 C. Primers were purchased from MWG Biotech, Inc. (High Point, NC). Primer pairs were chosen to minimize primer dimerization and to generate an amplicon between 150 and 350 bp. The following primer sets and annealing temperature were used to generate amplicons from the following cDNAs: GAPDH, 54 C, sense 5'-GTCGGTGTCAACGGATTTG-3' and antisense 5'-AGCTTCCCATTCTCAGCC-3'; EGR1, 59 C, sense 5'-GGGCGGCAGCAACAGCG-3' and antisense 5'-CCGGGTAGTTTGGCTGGGAT-3'; FRA2, 52 C, sense 5'-CGGGAACTTTGACACCTC-3' and antisense 5'-CGGATAGGGGTTGGA-3'; Meprin ß, 52 C, sense 5'-CGACTACACCTCGGTAATG-3' and antisense 5'-GTCACTGAAGTCCATTCG-3'; GADD45, 52 C, sense 5'-CAGAAGATCGAAAGGATGG-3' and antisense 5'-CAGAGCCACGTCCCGGTCGT-3'; Calpastatin, 56 C, sense 5'-AGAACTCGATGATGCCTTGG-3' and antisense 5'-TGGCTTCTCTGGTTTGTCCT-3'; STAT3, 56 C, sense 5'-CAGGTAGTGCTGCCCCTTAC-3' and antisense 5'-CACTCCGAGGTCAGATCCAT-3'; A-Raf, 55 C, sense 5'-ACAGGCTCTTTTGGCACTGT-3' and antisense 5'-CTGTGTGATGATGGCAAACC-3'; VIP1R, 56 C, sense 5'-GTGAAGACCGGCTACACCAT-3' and antisense 5'-GTCTATCTCCCCGCTGTTGA-3'. Optical data were collected during the annealing step of each cycle. Following PCR, reaction products were melted for 3 min at 95 C, then the temperature was lowered to 50 C in 0.5 C increments, 10 sec per increment. Optical data were collected over the duration of the temperature drop, with a dramatic increase in fluorescence occurring when the strands reanneal. This was done to ensure that only one PCR product was amplified per reaction. Relative expression of the RT-PCR products was determined using the {Delta}{Delta}Ct method (37). This method calculates relative expression using the equation: Fold induction = 2-[{Delta}{Delta}Ct], where Ct= the threshold cycle, i.e. the cycle number at which the sample’s relative fluorescence rises above the background fluorescence and {Delta}{Delta}Ct = [Ct gene of interest (unknown sample) - Ct GAPDH (unknown sample)] - [Ct gene of interest (calibrator sample) - Ct GAPDH (calibrator sample)]. One of the control samples was chosen as the calibrator sample and used in each PCR. Each sample was run in triplicate and the mean Ct was used in the {Delta}{Delta}Ct equation. GAPDH was chosen for normalization because this gene showed consistent expression relative to other housekeeping genes among the three treatment groups in our array experiments.

Statistical analyses
Statistical significance of expression level differences between treatment groups was determined using Systat software (student version 6.0.1) (SPSS, Inc., Chicago, IL) to perform one-way ANOVA with Tukey post hoc analyses. A P value of less than 0.05 was taken as statistically different between the two groups.

Immunohistochemistry
For immunohistochemical analyses, paraffin embedded tissues were cut into 5- to 6-µm sections. Tissue sections were deparaffinized overnight at 60 C and rehydrated through xylenes and graded alcohols. Endogenous peroxidase activity was quenched and antigen retrieval was performed by boiling sections for 2 x 5 min in citrate buffer, pH 6.0. Following antigen retrieval, the sections were pre-incubated with 5% normal goat serum in PBS for 30 min. Sections were then incubated overnight at 4 C with the primary antibody diluted in blocking solution. Antibodies for FRA2 (1:400) and GADD45 (1:300), along with their respective blocking peptides, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). This was followed by incubation with horseradish peroxidase-conjugated secondary antibody, goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) 1:200. Immunoreactivity was visualized with the Vectastain Elite ABC kit (Vector Laboratories) and DAB chromagen (Sigma).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Blockade of T + E2-induced dysplasia and hyperprolactinemia with ICI cotreatment
None of the animals treated with T + E2 + ICI developed dysplasia in their DLPs (Table 1Go and Fig. 1Go, E and F) compared with a 100% incidence of DLP lesions found in rats given T + E2 alone (Table 1Go and Fig. 1Go, C and D). Similarly, none of the untreated control animals developed this lesion (Table 1Go and Fig. 1Go, A and B). Data from the untreated and T + E2-treated groups are consistent with our previous findings using more than 100 rats in various studies (15, 19, 29, 35). As was the case with bromocriptine, ICI also inhibited the characteristic inflammation seen in the LPs of the T + E2-treated animals (Fig. 1CGo) that has been attributed to PRL action in the rat gland (38).


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  		Table 1. Blockade of T + E2 generated dysplasia with ICI 182,780

 


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  		Figure 1. Blockade of dysplasia induction in LP by ICI 182,780. Representative H&E-stained sections of rat lateral prostates with and without treatment. A, An acinus from an untreated intact NBL rat. Note the long fronds of acinar lining cells that project into the lumen (arrow). Magnification, x25. B, Higher magnification of the acinus illustrated in A. Note the orderly orientation of epithelial cells along the basement membrane of the acinus and the projecting fronds of connective tissue. Also, note the presence of apical vacuoles in most epithelial cells which if a feature of differentiated cells of the LP (arrows). Magnification, x132. C, Low-power micrograph of dysplasia in the LP of an intact NBL rat treated for 16 wk with T + E2 as described in Materials and Methods. Note the loss of acinar lining cell projections in most glands. Epithelial cells appeared piled up within affected glands (large arrows). Also note areas prostatic inflammation (small arrows). Magnification, x25. D, Higher magnification of the acinus shown in C. Epithelial cell crowding and piling up are evident. Epithelial cell nuclei in several dysplastic glands appear enlarged (arrows). Compare the appearance of these dysplastic glands with normal controls shown in A and B. Magnification, x132. E and F, Low- and high-power magnification of LP from an intact NBL rat treated for 16 wk with T + E2 + ICI. Note that the glands appear morphologically identical to the normal acini of untreated rats depicted in A and B. Magnification, x25 and x132.

 
Serum PRL analyses revealed that ICI was very effective in preventing the estrogen-induced hyperprolactinemia (serum PRL in T + E2-treated rats = 341 ± 50 ng/ml) (Fig. 2Go). The mean serum PRL levels of 15 ± 1.4 ng/ml found in T + E2 + ICI animals was actually lower than the mean PRL levels of 32 ± 10 ng/ml seen in T + E2-treated animals supplemented with bromocriptine (29) and those found in untreated controls (22 ± 4 ng/ml). Thus, ICI treatment effectively blocked the hyperprolactinemia induced by the combined treatment of rats with T + E2.



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  		Figure 2. Serum PRL levels after treatment with T + E2. Following the treatment period, NBL rats were killed and serum PRL levels were measured by RIA. Results are the mean serum PRL concentrations from five to six animals per treatment group. Error bars represent SEM.

 
Investigation of differential gene expression through cDNA microarray analyses
The CLONTECH Atlas 1.2 Rat cDNA Expression microarray containing 1185 known genes was used to detect gene expression changes in rat LP that are associated with T + E2-treatment of NBL rats. The LPs were used for these experiments because this lobe exhibited the most extensive dysplastic changes (15, 17, 35). Microarray analyses revealed 23 genes that were expressed in the LPs of T + E2-treated animals but were not detected in the untreated controls (Table 2Go). Three of these genes, maspin (a tumor-suppressing serpin), erbB2 proto-oncogene, and FRA2, belonged to the family of oncogenes/tumor suppressors. Three others were immune system proteins (myxovirus resistance protein 1, natural killer cell protease 4, interferon-induced GTP-binding protein mx-2 and -3), probably expressed by inflammatory cells that had infiltrated the LP in response to hyperprolactinemia (38). Additionally, 54 genes demonstrated 3-fold or greater enhanced expression in the T + E2-treated LPs when compared with the untreated LPs (Table 3Go). Interestingly, four additional proto-oncogenes (junD proto-oncogene, K-RAS 2B proto-oncogene, proto-oncogene c-crk and A-RAF proto-oncogene) were identified to be in this group. The gene encoding STAT-3 recently showed to promote prostate cancer progression (39), was also found to be up-regulated. Thus, expression levels of seven proto-oncogenes/tumor promoting molecules were found to be induced/enhanced following T + E2 treatment of NBL rats. Other classes of genes that demonstrated significant induced/enhanced expression by T + E2 were 1) genes encoding cell-cycle proteins or proliferation markers (G1/S-specific cyclin D1, presenilin 1, and proliferation cell nuclear antigen); 2) a gene encoding the GADD45; 3) genes involved in the synthesis, degradation and turn-over of proteins and RNAs; 4) genes regulating energy metabolism; 5) genes encoding metabolic enzymes; 6) genes controlling intracellular signaling; and 7) genes implicated in intracellular communication (Tables 2Go and 3Go). In summary, the LPs of T + E2-treated animals displayed a gene expression pattern that was consistent with a highly proliferative and anabolic tissue, with heightened energy metabolism and activation of a plethora of proto-oncogenes and signal transduction pathways. There were 14 genes that showed a 3-fold or greater reduction in expression in the LPs of T + E2-treated rats when compared with untreated LPs (Table 4Go). Due to the small number of genes in this category, we were not able to discern distinct functional clusters among them.


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  		Table 2. Genes whose expression was induced by T + E2 in the LPs of NBL rats

 

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  		Table 3. Genes whose expression was up-regulated by T + E2 treatment in LPs of NBL rats

 

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  		Table 4. Genes down-regulated in T + E2-treated animals

 
Interestingly, although ICI cotreatment largely reversed the expression changes observed with T + E2 treatment, this antiestrogen also produced gene expression changes unique to the LPs of ICI-exposed animals. Eight genes showed a 3-fold or greater down-regulation and one (fibroblast growth factor receptor-activating protein 1) demonstrated a 3-fold increase in expression exclusively in the T + E2 + ICI treatment group (Table 5Go).


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  		Table 5. Genes whose expression was exclusively affected by ICI cotreatment in the LPs of NBL rats

 
Confirmation of differential gene expression by real time RT-PCR
A total of 10 differentially expressed genes detected by microarray analyses were selected for post hoc confirmation analyses of expression levels by quantitative real-time RT-PCR. These genes were chosen because they have functions that are associated with cell proliferation and/or tumorigenesis. Total RNA was isolated from the LPs of individual animals in the three groups (untreated, T + E2-treated, and T + E2 + ICI-treated), deoxyribonuclease I-treated, reversed transcribed into cDNA and subjected to real-time PCR analyses. This method uses an intercalating dye to measure the production of double stranded nucleic acids, and data are collected optically as the reaction progresses, eliminating the need for cycle optimization and ensuring that samples are compared while amplification is in the linear range. Eight genes were confirmed to be differentially expressed, whereas two did not show significant differences in expression levels among the three treatment groups. The eight genes exhibited three distinct patterns of differential expression. The first pattern was represented by the genes encoding the EGR1 and meprin ß. These genes showed similar expression changes in both the T + E2-treated and T + E2 + ICI-treated LPs when compared with levels expressed in the untreated controls (Fig. 3Go), with EGR1 showed significant overexpression and meprin ß significant underexpression in the two T + E2 groups. The second pattern was represented by genes encoding GADD45, FRA2, and STAT3 (Fig. 4Go). Higher levels of transcripts for these genes were noted in the T + E2-treated LPs when compared with levels found in untreated LPs, with the T + E2-induced up-regulation blocked in each case by cotreatment with ICI. The third pattern was represented by genes encoding A-RAF, calpastatin, and VIP1R (Fig. 5Go). Expression levels of these genes were significantly reduced in the T + E2-treated LPs compared with untreated controls and ICI cotreatment was able to reverse the T + E2-induced declines in gene expression. These results were surprising for calpastatin and A-RAF, which had been indicated by the microarray analyses to be expressed at a higher level in the LPs of T + E2-treated animals when compared with untreated controls. Both genes were expressed at low levels in untreated and T + E2-treated samples. The unconfirmed genes probably represent false positives of microarray analyses, possibly due to low expression levels of these genes and/or high variability in expression levels among tissues from different animals. These findings clearly illustrate the significance of using quantitative real time RT-PCR as post hoc analyses for confirmation. Among all the T + E2 up-regulated genes, FRA2 showed the highest degree of enhanced expression, with a mean relative expression level 14-fold higher than control (Fig. 4BGo). Expression of GADD45 (Fig. 4AGo) and STAT3 (Fig. 4CGo) were significantly elevated in the T + E2-treated LP and ICI cotreatment was effective in reversing the hormone-induced up-regulation. Nevertheless, the difference in STAT3 mRNA levels between the T + E2-treated and T + E2 + ICI-treated LPs was found to be statistically not significant. Among the T + E2-down-regulated genes, calpastatin expression was significantly diminished in the T + E2-treated LPs compared with levels in the untreated LP (Fig. 5AGo). However, although ICI cotreatment appeared to reverse the T + E2-induced diminution, levels of calpastatin in the T + E2 + ICI-treated LPs was found to be statistically not different from those in the T + E2-LPs. In contrast, A-RAF and VIP1R expression were both significantly diminished in the T + E2-treated LPs when compared with levels measured in untreated LPs and in the ICI cotreated LPs (Fig. 5Go, B and C).



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  		Figure 3. Genes demonstrating similar expression patterns in T + E2 and T + E2 + ICI animals. Following real-time RT-PCR, relative mean expression for each treatment group was calculated using the {Delta}{Delta}Ct method (see Materials and Methods). Expression in expressed relative to one control sample referred to as the calibrator sample. n = 6 for control and T + E2 and 5 for T + E2 + ICI. Error bars represent SEM. *, P < 0.05 compared with control. A, EGR1. B, Meprin ß.

 


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  		Figure 4. Genes demonstrating elevated expression in T + E2 animals vs. control and T + E2 + ICI. Real-time RT-PCR showing increased expression in T + E2 treatment group compared with the control and T + E2 + ICI treatment groups. n = 6 for control and T + E2 and 5 for T + E2 + ICI. Error bars represent SEM. *, P < 0.01 compared with untreated control. A, GADD45. Note: n = 5 for T + E2. B, FRA2. C, STAT3.

 


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  		Figure 5. Genes demonstrating diminished expression in T + E2 animals vs. control and T + E2 + ICI. Real-time RT-PCR showing diminished expression in T + E2 treatment group compared with the control and T + E2 + ICI treatment groups. n = 6 for control and T + E2 and 5 for T + E2 + ICI. Error bars represent SEM. *, P < 0.05 compared with untreated control. **, P < 0.01 compared with untreated control. A, Calpastatin. Note: n = 3 for T + E2. B, A-Raf. Note: n = 4 for T + E2 and T + E2 + ICI. C, VIP1R.

 
Immunohistochemical confirmation of protein expression
To determine whether changes in gene expression at the transcript level were accompanied by parallel changes at the protein level, immunohistochemistry for FRA2 and GADD45 were performed (Fig. 6Go). FRA2 was expressed at a very low level in the cytoplasm of epithelial cells in the LPs of untreated animals (Fig. 6AGo). In contrast, treatment with T + E2 produced strong immunostaining for FRA2 in both the epithelial cytoplasm and nuclei (Fig. 6BGo). Nuclear staining appeared stronger in areas of dysplasia when compared with glands that were lesion free. Coadministration of ICI and T + E2 effectively negated nuclear staining (Fig. 6CGo). Expression of GADD45 protein was similar to that of FRA2. GADD45 immunostaining was not apparent in LPs obtained from untreated animals (Fig. 6DGo). T + E2 induced marked increases in cytoplasmic immunoreactivity in epithelial cells of LPs (Fig. 6EGo), whereas ICI cotreatment effectively negated increased expression of GADD45 (Fig. 6FGo). Interestingly, unlike FRA2, there was no apparent nuclear staining of GADD45 in LP sections from any of the three groups.



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  		Figure 6. Immunohistochemistry for FRA2 and GADD45. Genes shown to be up-regulated in the T + E2 animals via real-time RT-PCR were confirmed by immunohistochemistry. A–C, FRA2. D–F, GADD45. A and D, Untreated. B and E, T + E2. C and F, T + E2 + ICI. A, Note faint staining of cytoplasm. Magnification, x100. B, Note strong staining of epithelial nuclei (arrows). Inset, Section incubated with primary antibody preadsorbed with 10x (wt/wt) immunizing peptide. Magnification, x100. C, Note lack of nuclear staining as seen in untreated control (A). Magnification, x100. D, Magnification, x100. E, Note strong epithelial staining in areas of dysplasia. Inset, Section incubated with primary antibody preadsorbed with 10x (wt/wt) immunizing peptide. Magnification, x100. F, Note lack of staining similar to untreated control (D). Magnification, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
When planning the present study we had made the assumption, based on findings in female rats (30, 34), that ICI would not affect pituitary hormone levels and therefore only act locally, at the prostate level, as an antiestrogen. Subsequent administration of ICI to T + E2-treated NBL rats not only blocked the development of dysplasia in the DLP but also prevented the increase in serum PRL that accompanies the dual hormone treatment (35). In a previous study, we demonstrated that blockade of hyperprolactinemia in T + E2-treated NBL rats, via bromocriptine administration, effectively reduced incidence of dysplasia and inflammation in their DLPs (29). Results from our recent studies with NBL rat prostate organ cultures clearly established a direct action of PRL, but not E2, in stimulating prostatic epithelial cell proliferation (Thompson, C. J., C. Reily, I. Leav, and S.-M. Ho, unpublished). Taken together, these findings indicated that the inhibitory action of ICI on DLP dysplasia development is likely mediated primarily by its action in blocking the T + E2-induced hyperprolactinemia. However, the fact that our microarray data detected unique gene expression changes induced exclusively by the ICI cotreatment suggested that this antiestrogen might have direct local action in the prostate. Whether the local antiestrogenic action contributes significantly to the inhibition of DLP dysplasia remained to be determined. Significantly, cDNA microarray combined with post hoc quantitative analyses revealed gene expression changes associated with DNA damage, activation of proto-oncogenes/transforming factors, and enhancement of cell proliferation coincident with development of dysplasia in the LPs of T + E2-treated NBL rats. Our finding, therefore, provides new insights into the molecular mechanisms of hormone-induced tumorigenesis in this tissue.

cDNA microarray analysis is an effective tool for gene expression studies due to its ability to simultaneously monitor the expression pattern of a wide spectrum of genes. In the current study, it revealed many differences in LP gene expression between the three experimental groups. The major differences in gene expression occurred between the T + E2-treated group and the untreated controls. The T + E2-exposed LPs appeared to be more metabolically active than untreated LPs, with up-regulation of four genes classified to be involved in energy metabolism. They also displayed up-regulation of seven genes encoding ribosomal proteins and other genes that are involved in RNA processing, turnover and transport. Similarly, a large number of genes related to protein synthesis, trafficking, turnover, import and secretion were found to be up-regulated in the T + E2 treated LPs. Our microarray analyses also revealed enhanced expression of a large number of genes that encode proteins regulating intracellular signaling pathways. Most strikingly, these analyses detected the up-regulation of a total of 6 oncogenes/proto-oncogenes (erbB2 proto-oncogene, FRA2, junD proto-oncogene, K-RAS 2B proto-oncogene, proto-oncogene c-crk and A-RAF proto-oncogene) and a tumor suppressor (maspin) in rat LP following the dual hormone treatment. Activation of proto-oncogenes or overexpression of their protein products is a common cause of neoplastic transformation and cancer progression. However, the c-Crk oncogene, known to regulate cell migration (40), has not been linked to prostate carcinogenesis. HER-2/erbB-2 activation was observed during prostate cancer progression from androgen-dependent to androgen-independent state (41). A recent report identified FRA2/JunD complex as a transcriptional factor for the osteocalcin promoter in PC-3 cells that might be involved in establishment of bone metastasis (42). K-ras activation is a rare event in prostate cancers of North American men but quite commonly found in prostate cancers of Japanese patients (43). Lack of maspin protein expression in prostate cancer specimens was associated with increased risk of local recurrence and/or systemic tumor progression (44). In conclusion, the enhanced expression of these proto-oncogenes in the dysplastic LPs of NBL rats raises the possibility that activation of their regulatory pathways may predispose the tissue to early neoplastic transformation, a phenomenon worthy of future investigation in rat and human prostates. Three immune system proteins (myxovirus resistance protein 1, natural killer cell protease 4, interferon-induced GTP-binding protein mx-2 and –3) also demonstrated increased expression in the T + E2-treated LPs. These molecules are likely derived from inflammatory cells that infiltrated rat LP under the influence of PRL stimulation (38). Additionally, three genes, encoding cell-cycle proteins G1/S-specific cyclin D1, presenilin 1, and proliferation cell nuclear antigen, showed enhanced expression in T + E2-treated LPs. This finding is in accord with our early finding that a 7-fold increase in mitotic activity was noted in DLP epithelium following 16 wk of dual-hormone treatment (15).

In addition to identification of new molecular events, our microarray data have corroborated our previous findings on several cellular/molecular changes seen in the dysplastic prostates of the NBL rats (19, 20, 21, 22). Our array data did not detect any changes in TGF{alpha}/EGFR transcript levels in rat LPs following T + E2 treatment. This finding was in agreement with our earlier observation (19) that TGF{alpha}/EGFR mRNA levels in the dorsolateral prostates (DLPs) did not change significantly following T + E2 treatment. However, protein products of these two genes did show remarkable increases in the dysplastic foci of the dual hormone-treated DLPs. Metallothionein-1 was not represented on the array used in this experiment; thus, no comparison could be made. With regard to H- and K-ras proto-oncogene mRNA expression, we previously found a 50% and 60% increase, respectively, in the DLPs of T + E2-treated rats by Northern blot analyses (20). Here, array analyses detected a 2-fold increase in H-ras expression (not shown in Table 3Go) and a 4-fold augmentation in K-ras expression (Table 3Go) in the LPs of T + E2-treated rats. Thus, microarray data appeared to be in agreement with previous findings. As was previously showed (22), there were notable differences in the expression of several MAPKs and associated genes, but none of these genes demonstrated a 3-fold or greater changes in expression and therefore were not listed in Table 3Go. Most interestingly, one of our past studies reported the induction of oxidative stress and DNA damage in rat DLPs by T + E2 treatment (23). In our current study, microarray/post hoc analyses detected a gene linked to oxidative stress and related DNA damage (GADD45) that was up-regulated in the dysplastic LPs. This finding is consistent with our hypothesis that either direct or indirect (inflammation-mediated) oxidative damage of cellular DNA might be an important factor in the pathogenesis of prostatic dysplasia in the NBL rat model.

Several of the genes identified to be differentially expressed among our experimental groups, but not chosen for further analysis, are genes that have been shown to undergo changes in two rat prostate carcinogenesis models (Table 6Go). The first model is similar to that employed in this study except that a very high dose of T + E2 was used to treated the animals (45). The second model employs a transgenic rat engineered to express SV40 T antigen in its prostate (46). In both cases, the CLONTECH Atlas Rat cDNA Expression Arrays were used. Interestingly, several genes including TRPM-2, MMP7, proto-oncogene c-crk, transducin ß-1, microsomal glutathione S-transferase, and meprin ß were identified to be up- or down-regulated in one or both models as well as in our T + E2-treated LPs. Disregarding differences in rat strains and method of dysplasia/tumor induction, collectively, these findings suggest that these genes could be commonly involved in the pathogenesis of dysplasia/cancer in the rat prostate.


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  		Table 6. Differentially expressed genes in agreement with previously reported array data from other rat prostate cancer models

 
Of the eight differentially expressed LP genes that we have chosen for post hoc analyses by quantitative real-time RT-PCR, two genes show same direction of change in the two T + E2-treated groups. EGR1 was up-regulated by the T + E2-treatment and failed to respond to ICI cotreatment. Similarly, meprin ß was down-regulated in both T + E2- and T + E2 + ICI-treated groups. The expression pattern of these two genes suggests that they may be regulated by androgen and may not be tightly associated with the pathogenesis of prostatic dysplasia. Interestingly, EGR1 is a member of the early growth response family of nuclear transcription factors (47), and it has been shown to be up-regulated in human prostate cancers (48). Meprin ß is a membrane-bound metalloendopeptidase (49) that has been reported to be down-regulated in a previous study involving treatment of NBL rats with pharmacological doses of T + E2 (45). Neither gene has been documented to be regulated by androgens, although EGR1 transcription has been shown to be regulated by estrogen (50), and there is a putative ERE in the promoter of meprin ß (49).

With regard to the development of prostatic dysplasia, clearly the most informative genes were those that were up- or down-regulated in the T + E2-treated LPs but returned to control expression levels following cotreatment with ICI. Post hoc analyses confirmed up-regulation of FRA2 and GADD45, at both transcript and protein levels, in the LPs of T + E2-treated rats and their enhanced expression was reversed by cotreatment with ICI. The most dramatic expression change at the transcript level was seen in FRA2, a fos-family AP-1 transcription factor. FRA2 is more typically associated with cellular differentiation than with neoplasia development. Although broadly classified as proto-oncogenes, FRA2 does not possess the C-terminal transactivating domain that gives the other fos-family members the ability to transform rodent fibroblasts (51, 52). FRA2 is thought to regulate transcription by forming heterodimers with JunD (42, 53). Interestingly, in our microarray analyses, a 6-fold up-regulation of JunD was seen in the T + E2-treated LPs when compared with untreated LPs although JunD has not been chosen for post hoc analysis. FRA2 protein, despite its lack of transforming ability, is abundantly expressed in ras- and src-transformed murine and chicken fibroblasts, in neoplastic thyroid cells and in highly malignant mouse adenocarcinoma cells (52). cAMP has been shown to increase FRA2 expression, suggesting that the observed increase in FRA2 expression in the T + E2-treated LP could be mediated by estrogen, which has been shown to increase levels of cAMP in some cell types (54, 55). Action of FRA2 in the prostate has not been characterized, and the importance of its overexpression in the T + E2-treated LPs is not clear. However, the strong localization of the protein in cell nuclei of dysplastic lesions suggests that the gene is regulating transcription of products that are significant to the development of these lesions.

GADD45 also showed altered expression at both the transcript and protein levels in the T + E2-treated, but not the T + E2 + ICI-treated LPs. Unlike FRA2, increased transcription of this gene might not be directly linked to hormonal stimulation, but rather caused by DNA damage induced by the hormone-treatment regimen. GADD45 is known to play a role in growth arrest (56, 57) and DNA repair (58). Elevation in its expression is induced by DNA damage in both a p53-dependent (59) and -independent (60) manner. Interestingly, it has been reported that increased GADD45 expression in response to UV irradiation can be blocked by antioxidants, indicating that oxidative stress plays a role in up-regulation of the gene (61). In this regard, we have previously shown that T + E2-treatment of NBL rats, in addition to induction of dysplasia, leads to DNA damage and lipid peroxidation in the DLPs but not the ventral prostates of treated rats (23). Hence, the up-regulation of GADD45 in T + E2-treated LPs is in accord with this finding. Further, the PRL-induced inflammatory response in the T + E2-treated LPs (38) may also indirectly contribute to oxidative stress in this prostatic lobe. Our immunohistological data demonstrated higher levels of GADD45 protein expression in dysplastic lesions relative to adjacent normal acini, but they also revealed heterogeneity in GADD45 expression in normal acini. This pattern of expression may reflect focal elevation in GADD45 expression in response to local oxidative DNA damage associated with dysplasia induction.

We confirmed differential expression of four other genes at the mRNA level in the T + E2 group that was reversed by cotreatment with ICI. None of these genes have been shown previously to be transcriptionally regulated by PRL or estrogen. STAT3 has been identified as a transducer of the PRL receptor (62) and recently shown to mediate cytokine-induced proliferation in prostate cancer cell lines (39). It is up-regulated in the T + E2-treated LPs but levels remain low in the ICI cotreated LPs. The remaining three genes were down regulated in the T + E2-treated LPs when compared with untreated and the T + E2 + ICI-treated LPs. Calpastatin inhibits the protease calpain, which has been shown to play a role in the degradation of tumor suppressors such as BRCA1 (63) and retinoblastoma protein family member p107 (64). The MAPK cascade member, A-Raf, has been implicated in growth inhibition of prostate cancer cells (65). Type 1 VIPR has been identified as a marker of prostatic epithelial differentiation (66). Although the relationship between T + E2-induced down-regulation of these genes and prostatic dysplasia development remains unclear, these data suggest potential functional links that warrant future investigation.

In summary, we first determined that the antiestrogen ICI has profound effects in abolishing dysplasia development in LPs of T + E2-treated NBL rats. Importantly, this antiestrogen also blocks the E2-induced hyperprolactinemia. Together, with our past results from a bromocriptine study (29), the current findings strongly implicate PRL as a major factor in the induction of prostatic dysplasia in the NBL rats. Using cDNA microarray and post hoc confirmation analyses, we identified a plethora of genes whose expression was altered by T + E2 treatment and negated when ICI was coadministered with the two sex steroids. Among them, gene clusters with functions related to increased cell proliferation, DNA damage, proto-oncogene activation, and intra/intercellular signaling are likely involved in the pathogenesis of this prostatic lesion.


   Acknowledgments
 
We wish to thank Dr. Jim Voogt and Dr. Young-soo Lee at the University of Kansas Medical Center for performing serum PRL RIA. We also wish to thank Dr. B. M. Vose (Zeneca Pharmaceuticals, Cheshire, UK) for providing ICI 182,780 for this study.


   Footnotes
 
Research was supported, in part, by NIH Grants CA-15776, CA-62269, and AG-13965 (awarded to S.-M.H.). Part of this work was conducted by C.J.T. and S.M.H. at the Biology Department, Tufts University (Medford, MA).

Abbreviations: DLP, Dorsolateral prostate; EGF, epidermal growth factor; EGFR, EGF receptor; EGR1, early growth response protein 1; ERE, estrogen response element; FRA2, fos-related antigen-2; GADD45, growth arrest and DNA damage-inducible protein-45; ICI, pure antiestrogen ICI 182,780; LP, lateral prostate; meprin ß, metalloendopeptidase meprin ß-subunit; NBL, Noble rats; STAT3, signal transducers and activators of transcription-3; VIP1R, VIP-1 receptor.

Received December 6, 2001.

Accepted for publication February 13, 2002.


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 Materials and Methods
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