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Molecular Biology, Pathobiology, and Genetics |
1 Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio and 2 Department of Urology, University of Illinois at Chicago, Chicago, Illinois
Requests for reprints: Gail S. Prins, Department of Urology, University of Illinois at Chicago, 820 South Wood Street, MC 955, Chicago, IL 60612. Phone: 312-413-9766; Fax: 312-996-1291; E-mail: gprins{at}uic.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostate gland development, which occurs during fetal life in humans and the perinatal period in rodents, is exquisitely sensitive to estrogen imprinting. The in utero estrogen environment of African-American mothers has been suggested to affect the elevated prostate cancer risk of their offspring because they have higher estradiol levels during pregnancy when compared with their Caucasian counterparts (8, 9). In rodent models, brief perinatal exposure to pharmacologic doses of natural or synthetic estrogens permanently alters prostate growth and differentiation (10–12) and results in precancerous lesions and tumors with aging (13). However, although perinatal exposures to environmentally relevant doses of bisphenol A or estradiol have been shown to augment prostatic size (14), they have not, as yet, been shown to induce pathologic prostatic lesions. Thus, it remains unclear whether low-dose exposures to estradiol or environmental estrogens can influence prostate cancer risk.
Because early exposure to low-dose estrogen augments estrogen responsiveness of adult female reproductive organs (15, 16), we asked whether analogous circumstances exist in the prostate. This is particularly relevant because relative estradiol levels increase in the aging male, partly due to increased body fat content and aromatase activity, at a time when prostate cancer incidence increases (17). Furthermore, estrogens have been associated with increased prostate cancer risk in men (18), whereas, in the Noble rat model, prolonged adult exposure to conjoint estradiol and testosterone drives prostatic carcinogenesis (19). In this context, we established a carcinogenesis model that involved neonatal exposure to high- or low-dose estradiol or low-dose bisphenol A followed by adult exposure to elevated but nonpharmacologic testosterone plus estradiol in the Sprague-Dawley rat, a strain less sensitive to hormone-induced prostate carcinogenesis. Our goal was to determine if neonatal low-dose exposures to estradiol or bisphenol A might increase cancer susceptibility as a result of adult exposure to elevated estradiol. We herein present the first evidence that indeed low-dose as well as high-dose estrogenic exposures predispose to neoplastic prostatic lesions in the aging male.
We next sought to determine the molecular underpinnings by which developmental estrogenic exposures can imprint or transform the prostate long after the initial hormone exposure. One distinct possibility is through permanent epigenetic modifications of the genome by DNA methylation at CpG-rich regions (CpG islands), which can silence (hypermethylation) or activate (hypomethylation) gene transcription (20). Once established in somatic cells, CpG methylation patterns within the genome are stable and heritable through subsequent cell divisions, except during early embryonic development and tumorigenesis. Importantly, alterations in DNA methylation have been shown to contribute to both cancer initiation and promotion (20, 21). Furthermore, previous studies have revealed an association between aberrant CpG methylation of specific genes in the reproductive tract and neonatal exposures to phytoestrogens, diethylstilbestrol, and the environmental toxicants vinclozolin and methoxychlor (22–25). To determine whether neonatal estrogenic exposures imprint the prostate gland via this epigenetic modification, we did methylation-sensitive restriction fingerprinting (MSRF) followed by specific methylation analysis to identify and characterize candidate genes as methylation targets in prostate glands exposed to our two-hit model. We herein present evidence for altered methylation patterns of several candidate genes and characterize phosphodiesterase type 4 variant 4 (PDE4D4), the enzyme involved in cyclic AMP (cAMP) degradation, as an estrogen-imprinted gene directly associated with preneoplastic prostatic lesions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hormonal treatment regime, consisting of newborn rats briefly exposed to estrogens followed by prolonged adult exposure to elevated estradiol with appropriate controls, resulted in a total of eight animal groups. Newborn pups were assigned to one of four neonatal treatment groups with 20 to 30 pups per group: (a) controls given tocopherol-stripped corn oil vehicle alone (ICN Biomedicals, Inc., Aurora, OH), (b) high-dose 17ß-estradiol 3-benzoate (EB) at 25 µg/pup (or 2,500 µg EB/kg body weight), (c) low-dose EB at 0.001 µg EB/pup (or 0.1 µg EB/kg body weight), or (c) bisphenol A at 0.1 µg/pup (or 10 µg/kg body weight). All steroids were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). The high-dose EB was chosen based on our published data of an estrogenized phenotype with adult-onset prostatic intraepithelial neoplasia (PIN; ref. 13). The low-dose EB was chosen because this dose delayed puberty and permanently affected other reproductive structures in our dose-response study (26). An environmentally relevant dose of bisphenol A was chosen based on a predicted exposure range from leached bisphenol A in the environment (27). To avoid litter effects, male pups within each litter were randomly assigned to a treatment and toe clipped for permanent identification. Treatments were given on postnatal days 1, 3, and 5 by s.c. injections in the nape of the neck. The pups were weaned at postnatal day 21, and siblings were housed three per cage until postnatal day 90 and individually thereafter. At postnatal day 90, half of the rats from each treatment group were given implants of Silastic capsules (inside diameter 1.5 mm, outer diameter 2.0 mm; Dow Corning, Midland, MI) packed with estradiol (one 1-cm tube) and testosterone (two 2-cm tubes) for 16 weeks (replaced after 8 weeks), whereas the remaining half were given empty tubes. The testosterone capsules were necessary to maintain physiologic levels of testosterone because estradiol treatment alone results in hypothalamic-testicular feedback inhibition of endogenous testosterone secretion with resultant prostatic involution. These testosterone plus estradiol capsule lengths result in 
75 pg/mL serum estradiol and 3 ng/mL serum testosterone (28) and produce PIN in the dorsolateral prostates at 100% incidence in Noble rats (29) but only 33% incidence in Sprague-Dawley rats (30). At 28 weeks of age, the animals were sacrificed by decapitation, and prostate glands were quickly removed and microdissected into ventral, lateral, and dorsal lobes. Half of each lobe was snap frozen and stored in liquid nitrogen for subsequent methylation analysis, whereas the contralateral lobe was fixed in 10% buffered formalin overnight and stored in 70% ethanol for histopathologic diagnosis. In addition to the above animals sacrificed on day 200, rats from the four neonatal treatment groups were sacrificed, and prostates were removed on postnatal days 10 and 90 (n = 5-7 per group) for DNA methylation analysis.
Histopathology. Fixed prostatic tissues were processed, paraffin embedded, and sectioned along the longitudinal axis at three levels of the tissue block (10 sections per lobe). The sections were coded to prevent reader bias and stained with H&E. Each lobe was scored in a blinded fashion for epithelial and stromal hyperplasia, inflammation, and the presence of PIN and other notable pathology (adenoma, metaplasia, basement membrane breakdown, microinvasion, etc). PIN lesions were characterized by the presence of nuclear atypia (enlarged and elongated nuclei, hyperchromasia, and prominent nucleoli) with or without aberrant cellular piling and ductal formation (31). PIN lesions were graded on a scale of 0 to 3 (0, no atypia; 1, low-grade PIN; 2, focal high-grade PIN; and 3, extensive high-grade PIN). For PIN lesions, the incidence and the mean PIN score per treatment group were determined. Incidence was analyzed by 
2, and PIN scores were analyzed by ANOVA after square root transformation of the data followed by Fisher's exact test with significance accepted at P < 0.05.
Immunohistochemistry and in situ apoptosis labeling. Proliferation was measured by immunohistochemistry using a polyclonal Ki-67 primary antibody (1:2,500; Novocastra, Newcastle upon Tyne, United Kingdom). For apoptosis assessment, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining was used with ApopTag peroxidase in situ apoptosis detection kit (Chemicon International, Temecula, CA). To calculate the proliferation and apoptotic indices, multiple representative areas of each lobe were captured with a color digital AxioCam camera on an Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY). Positive and negative Ki-67-stained or TUNEL-labeled epithelial cells were counted using Zeiss Image version 3.0 (Carl Zeiss), with an average of 1,000 cells counted per slide. Data were analyzed by ANOVA and post hoc Bonferroni tests, and P < 0.05 was considered significant.
Methylation-sensitive restriction fingerprinting. MSRF was done as described (32), with minor modifications. In brief, 1 µg genomic DNA extracted from tissues with the DNeasy Tissue kit (Qiagen, Valencia, CA) was digested with MseI alone or double digested with BstUI and MseI (New England Biolabs, Beverly, MA). Digested DNA was amplified by PCR using 2 µCi [
-32P] dCTP (3,000 Ci/mmol; NEN, Boston, MA) with various combinations of paired arbitrary primers chosen from the following: Bs7, 5'-GAGGTGCGCG; Bs11, 5'-GAGAGGCGCG; Bs17, 5'-GGGGACGCGA; PCG1, 5'-AAGGAAGACG; and PCG4, 5'-TCCTTCCTCG. PCR products were separated on 6% nondenaturing polyacrylamide gels, which were dried and exposed to Kodak MS film (Kodak, New Haven, CT) to visualize the labeled bands. Candidate bands displaying the appropriate differential methylation status among "control" and "comparative" samples were cut, reamplified, and cloned directly into pCR2.1 vector (Invitrogen, Carlsbad, CA) for sequencing. The sequence obtained was aligned with database from Genbank and RefSeq using BLAST, expressed sequence tag homology (National Center for Biotechnology Information), and BLAT search (University of California Genome Research, Santa Cruz, CA).
5'-Rapid amplification of cDNA ends. The first-strand cDNA of PDE4D4 was amplified using a reverse specific primer (5'-AAAGACGAGGGCCAGGACAT-3') and the GeneRacer 5' Primer (Invitrogen). Nested PCR was done, and products were subcloned into pCR4-TOPO vector (Invitrogen). At least 10 clones were chosen and sequenced.
Bisulfite genomic sequencing. Genomic DNA (200 ng) from rat prostate tissue samples or rat cell lines was modified with sodium bisulfite using CpGenome DNA Modification kit (Chemicon International) and used in nested PCR for bisulfite sequencing. Primers for amplifying the PDE4D4 gene promoter/exon 1 region in completely converted DNA were designed with Primer3 and MethPrimer (33). First PCR was done using forward (5'-AGTGGTTTTGGAGAAGTTAGAGTTTA-3') and reverse (5'-CCAAAACATCCTAAATTTCTTCAAA-3') primers. Nested PCR was done with forward (5'-TTATTGTTGTGAAGAGTAGATTTTGTG-3') and reverse (5'-ATCCTAAATTTCTTCAAACCTAACC-3') primers. Both PCRs were done at 94°C for 9 minutes, 40 cycles of denaturing (94°C for 30 seconds), annealing (56°C for 1 minute), and extension (72°C for 1 minute) followed by a 12-minute final extension. The PCR product was gel purified and cloned into pCR2.1 vector. Six clones were picked from each sample for sequencing (Macrogen, Rockville, MD), and at least three sets of samples from each group were used. The DNA methylation data from sequencing were analyzed by BiQ Analyzer (34).
Methylation-specific PCR. PCR was done on bisulfite-treated DNA samples (40 ng) using primers specific for methylated (5'-GGTACGAGTAGTATTATTAGTATTCGTTTC-3' and 5'-CACGACAATACAAATAACGCTCCGT-3') or unmethylated (5'-GGTATGAGTAGTATTATTAGTATTTGTTTT-3' and 5'-CACAACAATACAAATAACACTCCAT-3') DNA. Forty PCR cycles were done with the following conditions: denature at 94°C for 30 seconds, anneal at 58°C for 1 minute, and extension at 72°C for 1 minute followed by 12-minute final extension. PCR products were separated on 2% agarose gel and visualized with ethidium bromide.
Real-time reverse transcription-PCR. Total RNA was isolated and reverse transcribed, and PDE4D4 expression was quantitated by a fluorogenic method with 2x SYBR Green Master Mix using an iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) as described previously (35). Primers specific for PDE4D4 (AF031373) were designed in the exon/exon spanning region and were as follows: PDE4D4, 5'-ACGAGCAGCACCACC AGTA-3' (forward) and 5'-CTTGAGGCGTAGCGACCAC-3' (reverse). PDE4D4 mRNA levels were normalized to RPL19 and the postnatal day 10 oil-treated control value was arbitrarily assigned an abundance value of 1. All data groups were analyzed by ANOVA followed by post hoc Bonferroni tests.
Prostate cell cultures and demethylation assay. Normal prostate epithelial NbE-1 cells and tumorigenic AIT cells were established from the Noble rat and immortalized as described previously (36). Cell cultures were treated with 0.5 or 1 µmol/L 5-aza-2'-deoxycytidine (5-Aza-dC; Sigma-Aldrich) for 8 days. Drugs were replenished every 4 days, and equivalent concentrations of DMSO were added in replicate control samples. At the end of the treatment, DNA and RNA were extracted from the cells and subjected to bisulfite genomic sequencing to determine the methylation status of the 5'-flanking region of the PDE4D4 gene and real-time reverse transcription-PCR (RT-PCR) to quantitate PDE4D4 gene expression.
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Results |
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The prostatic tissues were assessed for alterations in epithelial cell proliferation and apoptosis, which are normally low in the adult prostate gland. Low rates of proliferation and apoptosis were consistently observed in all areas of the treated prostates, except for those exposed neonatally to high-dose EB or bisphenol A (Fig. 2A-B ). Neonatal high EB treatment alone or with adult hormones increased basal proliferation rates throughout the tissue, with a higher rate observed in high-grade PIN regions (Fig. 2B, inset). Bisphenol A exposure followed by adult hormones also significantly increased proliferation in regions with high-grade PIN (Fig. 2A and B, inset). Similarly, low basal rates of apoptosis were detected throughout the prostate tissues, except for regions of high-grade PIN in the animals exposed neonatally to high-dose EB or bisphenol A with adult hormones (Fig. 2C-D). This provides support for the hypothesis that developmental estrogenic exposures initiate or activate precancerous pathways, resulting in an imbalance in cell proliferation and apoptosis that may contribute to prostatic pathology with aging. Taken together, the present experimental paradigm suggests that early low-dose estrogen exposures predispose the prostate to PIN with aging and that environmentally relevant doses of bisphenol A during development increase prostatic susceptibility to carcinogenesis following additional adult insults.
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), phosphokinase C pathway (PLCß3), cAMP pathways (PDE4D4 and HPCAL1), and neural or cardiac development (CARXI and CARK).
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Direct association of DNA methylation at CpGs 49 to 56 and its resultant effect on PDE4D4 gene expression were shown by using methylation-specific PCR and real-time RT-PCR. There were no differences in DNA methylation or gene expression between treatment groups at day 10 (Fig. 4A-B ). However, as the animals aged, CpGs 49 to 56 became entirely methylated in oil-control prostates, whereas neonatal high EB-treated, low EB-treated, and bisphenol A–treated prostates possessed completely unmethylated sequences (Fig. 4A). Importantly, these differential methylation patterns were inversely correlated to PDE4D4 gene expression (Fig. 4B). PDE4D4 message levels in prostates exposed neonatally to estradiol or bisphenol A were markedly higher at day 90 than control tissues and remained elevated with aging. We thus conclude that the prostatic PDE4D4 gene is normally silenced with aging through promoter hypermethylation but remains expressed in neonatally estrogenized prostates by virtue of hypomethylation at CpGs 49 to 56. Notably, this phenomenon was observed in all neonatal high- and low-dose EB and low-dose bisphenol A groups before the "second hit" of hormones and before adult-onset PIN lesions. Thus PDE4D4 may have potential as a marker for prostate cancer risk assessment.
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Discussion |
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Although the mechanism(s) by which developmental exposures to endogenous and environmental estrogens alter the carcinogenic potential of the prostate have not been fully clarified, the present findings support the hypothesis, initially proposed by McLachlan (43), that altered epigenetic memory by endocrine disruptors may play a critical role. In this study, we have provided direct evidence in support of this premise. Our data show that several genes exhibit methylation changes in response to the neonatal estrogen treatments, many of which are permanent. It is noteworthy that several of these genes encode signaling pathway proteins that are involved in cell cycle and/or apoptosis, suggesting that neonatal estrogen exposures may perturb proliferation/apoptosis equilibrium through epigenetic gene (de)regulation. It is also interesting that overlapping as well as unique methylation alterations were observed for high- and low-dose estrogen and bisphenol A. This suggests two important points. First, common prostatic genes may be epigenetically imprinted by different estrogenic compounds and doses, suggesting common pathways that predispose to prostate carcinogenesis with aging. Second, unique candidate genes specific to the neonatal estrogenic exposure and/or dose may mediate the subtle differences in phenotypes that were observed following the separate neonatal exposures.
The epigenetic regulation of gene expression by neonatal estrogen exposure was confirmed by detailed analysis of the PDE4D4 gene. The estradiol and bisphenol A–initiated alterations in PDE4D4 gene methylation occurred at a CpG island that spans the promoter/exon 1 region, a site typically involved in epigenetic regulation. Importantly, the degree of methylation at this site was inversely related to PDE4D4 gene expression in the prostate tissues. Thus, the PDE4D4 promoter undergoes gradual hypermethylation with aging in normal prostates, resulting in PDE4D4 gene repression in the adult gland. In contrast, it remains hypomethylated in animals briefly exposed to neonatal estradiol or bisphenol A, thus engendering persistent PDE4D4 overexpression throughout life. This pattern of PDE4D4 methylation and transcriptional regulation was also observed in normal and malignant prostate epithelial cells where normal NbE-1 cells with hypermethylated PDE4D4 gene had low gene expression, whereas tumorigenic AIT cells had hypomethylation at the CpG island and elevated PDE4D4 expression. Taken together, these findings suggest the potential involvement of epigenetically mediated PDE4D4 dysregulation in prostate epithelial cell transformation.
At present, it is premature to suggest that PDE4D4 dysregulation is a direct mediator of the prostatic dysgenesis as a result of early exposures to low- and high-dose estradiol or low-dose bisphenol A, particularly because the phenotypic response to the hormonal agents has specific differences widely, whereas the PDE4D4 methylation and expression alterations are quite similar. Nonetheless, PDE4 is a promising lead candidate that deserves further discussion. PDE4 is a member of a large family of intracellular PDE enzymes involved in cyclic nucleotide monophosphate breakdown, and it specifically degrades cAMP (44). There are multiple downstream signals for cAMP in the cell, including activation of protein kinase A with resultant phosphorylation of cAMP-responsive element binding protein, which regulates transcription of genes involved in cell growth and differentiation (45). PDE4D has been shown to regulate cAMP levels in hormone-targeted cells, and the PDE4D4 variant, which localizes to the cytoskeletal structures, is itself activated by hormones (46). Sustained expression of PDE4D4 by hypomethylation could thus result in decreased intracellular cAMP in specific subcellular locations, creating a potential for aberrant cell signaling and potentially neoplastic transformation. In this regard, recent studies have shown a tight association between PDE4 expression and cancer cell proliferation, including glioma cells (47), osteosarcomas (48), and chronic lymphocytic leukemia (49). Importantly, PDE4 is currently being pursued as a possible chemotherapeutic target (50).
In addition to providing insight into the molecular underpinnings of estrogen imprinting, the methylated candidate genes identified herein have potential to serve as molecular markers for risk assessment of prostate disease due to early environmental exposures. PDE4D4 shows particular promise in this regard because alterations in both gene methylation and expression were apparent before adult hormonal exposures and, importantly, before the onset of histopathologic changes in the prostate gland. This suggests that subtle alterations in gene expression may be more sensitive indicators of underlying pathology than the histologic alterations that occur when the disease is further progressed. Future studies are planned to develop a panel of methylated genes that may be used as markers for prostatic disease following early bisphenol A exposures.
In summary, we have shown that a range of estrogenic exposures during the developmental critical period, from environmentally relevant bisphenol A exposure to low-dose and pharmacologic estradiol exposures, results in an increased incidence and susceptibility to neoplastic prostatic lesions in the aging male, which may provide a fetal basis for this adult disease. Furthermore, the present findings provide evidence that developmental exposure to environmental endocrine disruptors (bisphenol A) and natural estrogens impacts the prostate epigenome during early life, which suggests an epigenetic basis for estrogen imprinting of the prostate gland. Methylation patterns and/or expression of candidate genes, such as PDE4D4, may serve as early biomarkers of prostate malignancy due to developmental exposure to endocrine disruptors or the in utero estrogenic environment.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the Department of Surgery of the University of Massachusetts (Worcester, MA), David Hepps, MD, and Lynn Birch for technical contributions.
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Footnotes |
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S-M. Ho and W-Y. Tang contributed equally to this work.
Received 2/ 9/06. Revised 3/20/06. Accepted 3/29/06.
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, P < 0.01 versus bisphenol A (BPA) alone. C, incidence of PIN lesions across treatment groups at day 200. LGPIN, low-grade PIN; HGPIN, high-grade PIN. D, representative H&E sections from dorsal prostates of the eight treatment groups. Bar, 50 µm.
, unmethylated; 
, methylated. Boxed region, potential CpG sites epigenetically altered by bisphenol A. C, percentage methylation at each CpG site within the 5'-CpG island of PDE4D4 in the separate treatment groups at days 10, 90, and 200 with or without adult testosterone + estradiol. The percentage methylation at each site was averaged from three individual sample sets. 
, oil control; 
, high-dose estradiol; 
, low-dose estradiol; x, bisphenol A.
, P < 0.05 versus control NbE-1 cells.