RNA-binding protein DDX3 mediates posttranscriptional regulation of androgen receptor: A mechanism of castration resistance.

Prostate cancer (CaP) driven by androgen receptor (AR) is treated with androgen deprivation; however, therapy failure results in lethal castration-resistant prostate cancer (CRPC). AR-low/negative (ARL/-) CRPC subtypes have recently been characterized and cannot be targeted by hormonal therapies, resulting in poor prognosis. RNA-binding protein (RBP)/helicase DDX3 (DEAD-box helicase 3 X-linked) is a key component of stress granules (SG) and is postulated to affect protein translation. Here, we investigated DDX3-mediated posttranscriptional regulation of AR mRNA (messenger RNA) in CRPC. Using patient samples and preclinical models, we objectively quantified DDX3 and AR expression in ARL/- CRPC. We utilized CRPC models to identify DDX3:AR mRNA complexes by RNA immunoprecipitation, assess the effects of DDX3 gain/loss-of-function on AR expression and signaling, and address clinical implications of targeting DDX3 by assessing sensitivity to AR-signaling inhibitors (ARSI) in CRPC xenografts in vivo. ARL/- CRPC expressed abundant AR mRNA despite diminished levels of AR protein. DDX3 protein was highly expressed in ARL/- CRPC, where it bound to AR mRNA. Consistent with a repressive regulatory role, DDX3 localized to cytoplasmic puncta with SG marker PABP1 in CRPC. While induction of DDX3-nucleated SGs resulted in decreased AR protein expression, inhibiting DDX3 was sufficient to restore 1) AR protein expression, 2) AR signaling, and 3) sensitivity to ARSI in vitro and in vivo. Our findings implicate the RBP protein DDX3 as a mechanism of posttranscriptional regulation for AR in CRPC. Clinically, DDX3 may be targetable for sensitizing ARL/- CRPC to AR-directed therapies.

Expression, Localization, and Function of the Nucleolar Protein BOP1 in Prostate Cancer Progression.

Differentiating between indolent and aggressive prostate cancers (CaP) is important to decrease overtreatment and increase survival for men with the aggressive disease. Nucleolar prominence is a histologic hallmark of CaP; however, the expression, localization, and functional significance of specific nucleolar proteins have not been investigated thoroughly. The nucleolar protein block of proliferation 1 (BOP1) is associated with multiple cancers but has not been implicated in CaP thus far. Meta-analysis of publicly available data showed increased BOP1 expression in metastatic CaP and recurrent CaP, and was inversely associated with overall survival. Multiplexed immunohistochemistry was used to analyze expression and localization of BOP1 and nucleolar protein 56 in human tissue samples from various stages of CaP progression. Here, increased BOP1 expression was observed at later stages of CaP progression, coinciding with a localization change from nuclear to cytoplasmic. In patient samples, cytoplasmic BOP1 was also inversely associated with overall survival. In models of prostate cancer progression, BOP1 expression showed expression and localization similar to that in human patient samples. The functional significance of BOP1 in metastatic CaP was assessed by genetic knockdown, where BOP1 knockdown resulted in decreased proliferation and motility compared with control. Taken together, these data suggest prognostic significance of BOP1 expression and localization in CaP progression and provide a foundation for further investigation into the functional role of nucleolar proteins in advanced CaP.

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia.

BACKGROUND: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. METHODS: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1ß and TGF-ß1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. RESULTS: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-ß1 stimulated OPN secretion by NHPrE-1 cells and both IL-1ß and TGF-ß1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. CONCLUSIONS: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

Expression and Localization of DDX3 in Prostate Cancer Progression and Metastasis.

Survival rates decrease significantly when localized prostate cancer (CaP) becomes metastatic, emphasizing the need for improved targeted therapies. DDX3, an RNA helicase, has widespread functions in RNA regulation, in both the nucleus and cytoplasm. Although DDX3 has been implicated as a prognostic marker for many cancers, including primary CaP, its expression, localization, and function in metastatic CaP have not been investigated. Analysis of metadata and cell line models found increased DDX3 expression in metastatic versus primary CaP and benign prostate. Quantification of DDX3 expression in 320 human prostate samples, representing different stages of CaP progression, revealed an increase in epithelial whole cell, cytoplasmic, and nuclear DDX3 in primary CaP compared with benign prostate. In metastatic tissues, cytoplasmic DDX3 remained highly expressed, whereas nuclear DDX3 significantly decreased compared with primary CaP, suggesting a potential role for cytoplasmic DDX3 in metastatic CaP. Genetic and pharmacologic loss of function for DDX3 in metastatic CaP produced a significant decrease in cell viability, proliferation, and motility but did not affect apoptosis. The data suggest that cytoplasmic DDX3 is highly expressed in metastatic CaP and that inhibition of DDX3 affects metastatic growth by decreasing proliferation and motility. These findings introduce a novel role for cytoplasmic DDX3 in CaP progression and provide a foundation for clinically targeting DDX3 in metastatic CaP.

Modeling human prostate cancer progression in vitro.

Detailed mechanisms involved in prostate cancer (CaP) development and progression are not well understood. Current experimental models used to study CaP are not well suited to address this issue. Previously, we have described the hormonal progression of non-tumorigenic human prostate epithelial cells (BPH1) into malignant cells via tissue recombination. Here, we describe a method to derive human cell lines from distinct stages of CaP that parallel cellular, genetic and epigenetic changes found in patients with cancers. This BPH1-derived Cancer Progression (BCaP) model represents different stages of cancer. Using diverse analytical strategies, we show that the BCaP model reproduces molecular characteristics of CaP in human patients. Furthermore, we demonstrate that BCaP cells have altered gene expression of shared pathways with human and transgenic mouse CaP data, as well as, increasing genomic instability with TMPRSS2-ERG fusion in advanced tumor cells. Together, these cell lines represent a unique model of human CaP progression providing a novel tool that will allow the discovery and experimental validation of mechanisms regulating human CaP development and progression. This BPH1-derived Cancer Progression (BCaP) model represents different stages of cancer. The BCaP model reproduces molecular characteristics of prostate cancer. The cells have altered gene expression with TMPRSS2-ERG fusion representing a unique model for prostate cancer progression.

Incidence of androgen receptor and androgen receptor variant 7 coexpression in prostate cancer.

BACKGROUND: Prostate cancer (PRCA) is an androgen-driven disease, where androgens act through the androgen receptor (AR) to induce proliferation and survival of tumor cells. Recently, AR splice variant 7 (ARv7) has been implicated in advanced stages of PRCA and clinical recurrence. With the widespread use of AR-targeted therapies, there has been a rising interest in the expression of full-length AR and ARv7 in PRCA progression and how these receptors, both independently and together, contribute to adverse clinicopathologic outcomes. METHODS: Despite a multitude of studies measuring the expression levels of AR and ARv7 in PRCA progression, the results have been inconsistent and sometimes contradictory due to technical and analytical discrepancies. To circumvent these inconsistencies, we used an automated multiplexed immunostaining platform for full-length AR and ARv7 in human PRCA samples and objectively quantified expression changes with machine learning-based software. With this technology, we can assess receptor prevalence both independently, and coexpressed, within specific tissue and cellular compartments. RESULTS: Full-length AR and ARv7 expression increased in epithelial nuclei of metastatic samples compared to benign. Interestingly, a population of cells with undetectable AR persisted through all stages of PRCA progression. Coexpression analyses showed an increase of the double-positive (AR+ /ARv7+ ) population in metastases compared to benign, and an increase of the double-negative population in PRCA samples compared to benign. Importantly, analysis of clinicopathologic outcomes associated with AR/ARv7 coexpression showed a significant decrease in the double-positive population with higher Gleason score (GS), as well as in samples with recurrence in under 5 years. Conversely, the double-negative population was significantly increased in samples with higher GS and in samples with recurrence in under 5 years. CONCLUSIONS: Changes in AR and ARv7 coexpression may have prognostic value in PRCA progression and recurrence. A better understanding of the prevalence and clinicopathologic outcomes associated with changes in these receptors' coexpression may provide a foundation for improved diagnosis and therapy for men with PRCA.

Androgen hormone action in prostatic carcinogenesis: stromal androgen receptors mediate prostate cancer progression, malignant transformation and metastasis.

It has been postulated that prostatic carcinogenesis is androgen dependent and that androgens mediate their effects primarily through epithelial cells; however, definitive proof of androgen hormone action in prostate cancer (PRCA) progression is lacking. Here we demonstrate through genetic loss of function experiments that PRCA progression is androgen dependent and that androgen dependency occurs via prostatic stromal androgen receptors (AR) but not epithelial AR. Utilizing tissue recombination models of prostatic carcinogenesis, loss of AR function was evaluated by surgical castration or genetic deletion. Loss of AR function prevented prostatic carcinogenesis, malignant transformation and metastasis. Tissue-specific evaluation of androgen hormone action demonstrated that epithelial AR was not necessary for PRCA progression, whereas stromal AR was essential for PRCA progression, malignant transformation and metastasis. Stromal AR was not necessary for prostatic maintenance, suggesting that the lack of cancer progression due to stromal AR deletion was not related to altered prostatic homeostasis. Gene expression analysis identified numerous androgen-regulated stromal factors. Four candidate stromal AR-regulated genes were secreted growth factors: fibroblast growth factors-2, -7, -10 and hepatocyte growth factor which were significantly affected by androgens and anti-androgens in stromal cells grown in vitro. These data support the concept that androgens are necessary for PRCA progression and that the androgen-regulated stromal microenvironment is essential to carcinogenesis, malignant transformation and metastasis and may serve as a potential target in the prevention of PRCA.

White's operation: the history of 19th century attempts to treat prostate disease with castration.

To understand the roots of 19th century hormonal treatments for BPH in the career of J. William White, a prominent surgeon scientist at the University of Pennsylvania. We reviewed primary and secondary literature available in PUBMED, the University of Pennsylvania Archives, and internet resources. In 1893, Dr. White presented a series of experiments demonstrating atrophy of the canine prostate following castration and advocated for this procedure in men suffering from prostatic hypertrophy. This approach was adopted by many of White's contemporaries. In 1895, White presented findings from 111 patients and reported improvement of urinary symptoms in three quarters of these patients. Improvements in surgical techniques for prostatectomy have predominantly eliminated castration as a clinical procedure for BPH treatment. These early experiments demonstrated the critical dependence of the prostate on testicular androgens and were the basis for subsequent hormonal therapies for BPH. In conclusion, the bold experiments of late 19th century surgeons paved the way for our contemporary understanding of the important role of sex steroid hormones in BPH.

Fetal bisphenol A and ethinylestradiol exposure alters male rat urogenital tract morphology at birth: Confirmation of prior low-dose findings in CLARITY-BPA.

Bisphenol A (BPA) is a contaminant in virtually all Americans. To examine BPA's adverse effects, the FDA-NCTR, NIEHS, and 14 groups of academic scientists formed a consortium: CLARITY-BPA. The purpose of our study was to investigate the effects of a wide range of doses of BPA on fetal development of the NCTR CD-SD male rat urogenital sinus (UGS). Pregnant rats were administered BPA or positive control ethinylestradiol (EE2) daily, via oral gavage, from gestational day 6 through parturition. Tissues were collected on postnatal day 1 and the UGS was analyzed using computer-assisted 3-D reconstruction. Importantly, only low doses of BPA, as well as EE2, significantly changed birth weight and UGS morphology, including an increased size of the colliculus and decreased size of the urethra, consistent with prior reported BPA and EE2 effects. Our findings provide further evidence that BPA mediates nonmonotonic developmental effects on the fetal urogenital sinus.

Prostate enlargement and altered urinary function are part of the aging process.

Prostate disease incidence, both benign and malignant, directly correlates with age. Men under 40 years of age are rarely diagnosed with benign or malignant prostate disease, while 90% of men over the age of 80 have histological evidence of benign disease (benign prostatic hyperplasia; BPH). Although rodent models have been invaluable in the study of disease progression and treatment efficacy, the effect of age is often not considered. In examining aged (24-month-old) mice, we observed changes within the lower urinary tract that is typically associated with lower urinary tract dysfunction (LUTD) similar to models of BPH. In this study, we identify LUTD using functional testing as well as various imaging technologies. We also characterize the histological differences within the lower urinary tract between young (2-month-old) and aged mice including proliferation, stromal remodeling, and collagen deposition. Additionally, we examined serum steroid hormone levels, as steroid changes drive LUTD in mice and are known to change with age. We conclude that, with age, changes in prostate function, consistent with LUTD, are a consequence. Therapeutic targeting of endocrine and prostatic factors including smooth muscle function, prostate growth and fibrosis are likely to reestablish normal urinary function.

Tissue-specific quantification and localization of androgen and estrogen receptors in prostate cancer.

Androgens and estrogens, working together, promote prostate cancer (PRCA) initiation and progression, with androgens acting via androgen receptor (AR) and estrogens acting primarily through estrogen receptor α (ERα). While the interplay between these steroid hormones has been established, the interaction between steroid hormone receptors in prostatic disease remains unstudied. The goal of this study was to objectively determine the incidence, stage specificity, and tissue/cell type specificity of AR and ERα expression, both independently and simultaneously, during the progression of PRCA. Using multiplexed immunohistochemistry and multispectral imaging analysis, AR, ERα, and smooth muscle α-actin expression was detected and quantitated in benign prostate tissue (BPT), high-grade prostatic intraepithelial neoplasia (HGPIN), PRCA, and metastasis (MET) from patient specimens (n=340). Epithelial AR expression was significantly increased in HGPIN, PRCA, and MET compared with BPT, whereas ERα expression in epithelial and stromal cells was highest in HGPIN. With analysis of AR and ERα coexpression, we identified a unique population of double-positive (AR+/ERα+) cells that increased in HGPIN specimens in both the stroma and the epithelium. Double-negative (AR-/ERα-) cells significantly decreased across PRCA progression, from 65% in BPT to 30% in MET. Preliminary analysis of this AR+/ERα+ population indicates potential cell type specificity in smooth muscle α-actin-negative stromal cells. This study demonstrates stage-, tissue-, and cell type-specific AR and ERα expression changes during PRCA progression, both independently and coexpressed. A more complete understanding of steroid hormones and their receptors in the initiation and progression of prostatic disease may elucidate improved strategies for PRCA prevention or therapy.

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Expression and colocalization of ß-catenin and lymphoid enhancing factor-1 in prostate cancer progression.

The purpose of this study was to objectively investigate ß-catenin and LEF1 abundance, subcellular localization, and colocalization across benign and staged prostate cancer (PCa) specimens. A tissue microarray containing tumor-adjacent histologically benign prostate tissue (BPT; n = 48 patients), high-grade prostatic intraepithelial neoplasia (HGPIN; n = 25), localized PCa (n = 42), aggressive PCa (n = 31), and metastases (n = 22) was stained using multiplexed immunohistochemistry with antibodies toward E-cadherin, ß-catenin, and LEF1. Multispectral imaging was used for quantitation, and protein expression and colocalization was evaluated across PCa progression. Stromal nuclear ß-catenin abundance was greater in HGPIN and PCa compared with BPT (P < .05 for both), and epithelial nuclear ß-catenin abundance was lower in metastatic PCa than in BPT (P < .05 for both). Epithelial and stromal nuclear LEF1 abundance was greater in HGPIN compared with BPT, whereas epithelial nuclear LEF1 was also greater in metastases. The proportion of epithelial and stromal nuclear double-positive ß-catenin(+)/LEF1(+) cells was greater in HGPIN compared with BPT. In addition, the proportion of epithelial ß-catenin(+)/LEF1(+) cells was greater in localized PCa and metastases compared with BPT. A significant amount of stromal cells were positive for LEF1 but not ß-catenin. ß-Catenin and LEF1 abundance were negatively correlated in the epithelium (P < .0001) but not the stroma (P > .05). We conclude that ß-catenin and LEF1 colocalization is increased in HGPIN and metastasis relative to BPT, suggesting a role for ß-catenin/LEF1-mediated transcription in both malignant transformation and metastasis of PCa. Furthermore, our results suggest that LEF1 abundance alone is not a reliable readout for ß-catenin activity in prostate tissues.

In Utero and Lactational TCDD Exposure Increases Susceptibility to Lower Urinary Tract Dysfunction in Adulthood.

Benign prostatic hyperplasia, prostate cancer, and changes in the ratio of circulating testosterone and estradiol often occur concurrently in aging men and can lead to lower urinary tract (LUT) dysfunction. To explore the possibility of a fetal basis for the development of LUT dysfunction in adulthood, Tg(CMV-cre);Nkx3-1(+/-);Pten(fl/+) mice, which are genetically predisposed to prostate neoplasia, were exposedin uteroand during lactation to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 µg/kg po) or corn oil vehicle (5 ml/kg) after a single maternal dose on 13 days post coitus, and subsequently were aged without further manipulation, or at 8 weeks of age were exposed to exogenous 17 ß-estradiol (2.5 mg) and testosterone (25 mg) (T+E2) via slow release subcutaneous implants.In uteroand lactational (IUL) TCDD exposure in the absence of exogenous hormone treatment reduced voiding pressure in adult mice, but otherwise had little effect on mouse LUT anatomy or function. By comparison, IUL TCDD exposure followed by exogenous hormone treatment increased relative kidney, bladder, dorsolateral prostate, and seminal vesicle weights, hydronephrosis incidence, and prostate epithelial cell proliferation, thickened prostate periductal smooth muscle, and altered prostate and bladder collagen fiber distribution. We propose a 2-hit model whereby IUL TCDD exposure sensitizes mice to exogenous-hormone-induced urinary tract dysfunction later in life.

Endometriotic haptoglobin binds to peritoneal macrophages and alters their function in women with endometriosis.

OBJECTIVE: To evaluate the effects of endometriotic haptoglobin on peritoneal macrophage function. DESIGN: Prospective laboratory study. SETTING: School of medicine. PATIENT(S): Twenty-three women with and without endometriosis. INTERVENTION(S): Peritoneal macrophages cultured without or with haptoglobin. MAIN OUTCOME MEASURE(S): Peritoneal macrophage haptoglobin immunoreactivity, adhesion, and interleukin-6 (IL-6) production. RESULT(S): In vivo, significantly more peritoneal macrophages from women with endometriosis bound haptoglobin and exhibited reduced adhesion compared to women without endometriosis. In vitro, haptoglobin treatment significantly decreased peritoneal macrophage adherence only in women without endometriosis; this effect was not seen in women with endometriosis, probably owing to in vivo haptoglobin saturation. Conversely, haptoglobin treatment robustly increased IL-6 production only by macrophages from women with endometriosis, suggesting differential immune response in these women. CONCLUSION(S): Endometriotic lesions synthesize and secrete a unique form of haptoglobin (endometriosis protein-I) that is up-regulated by IL-6. This study shows that haptoglobin adheres to peritoneal macrophages; decreases adhesion, which may influence phagocytic function; and up-regulates IL-6 production. Hence, a feed-forward loop is proposed whereby endometriotic lesion haptoglobin decreases macrophage phagocytic function while increasing IL-6 production, which in turn increases endometriotic haptoglobin and promotes establishment of endometriosis.

Testosterone and 17ß-estradiol induce glandular prostatic growth, bladder outlet obstruction, and voiding dysfunction in male mice.

Benign prostatic hyperplasia (BPH) and bladder outlet obstruction (BOO) are common in older men and can contribute to lower urinary tract symptoms that significantly impact quality of life. Few existing models of BOO and BPH use physiological levels of hormones associated with disease progression in humans in a genetically manipulable organism. We present a model of BPH and BOO induced in mice with testosterone (T) and 17ß-estradiol (E(2)). Male mice were surgically implanted with slow-releasing sc pellets containing 25 mg T and 2.5 mg E(2) (T+E(2)). After 2 and 4 months of hormone treatment, we evaluated voiding patterns and examined the gross morphology and histology of the bladder, urethra, and prostate. Mice treated with T+E(2) developed significantly larger bladders than untreated mice, consistent with BOO. Some mice treated with T+E(2) had complications in the form of bladder hypertrophy, diverticula, calculi, and eventual decompensation with hydronephrosis. Hormone treatment caused a significant decrease in the size of the urethral lumen, increased prostate mass, and increased number of prostatic ducts associated with the prostatic urethra, compared with untreated mice. Voiding dysfunction was observed in mice treated with T+E(2), who exhibited droplet voiding pattern with significantly decreased void mass, shorter void duration, and fewer sustained voids. The constellation of lower urinary tract abnormalities, including BOO, enlarged prostates, and voiding dysfunction seen in male mice treated with T+E(2) is consistent with BPH in men. This model is suitable for better understanding molecular mechanisms and for developing novel strategies to address BPH and BOO.

Steroid hormones stimulate human prostate cancer progression and metastasis.

Tissue recombinants (TRs) composed of mouse urogenital mesenchyme (mUGM) plus an immortalized nontumorigenic human prostatic epithelial cell line (BPH-1) were grown under the kidney capsule of male athymic nude mice under different hormonal conditions. The objectives were to determine temporal plasma concentrations of testosterone (T) and estradiol-17beta (E2) that elicit progression of nontumorigenic human prostatic epithelial cells in vivo. Second, to determine whether mUGM+BPH-1 TRs in [T+E2]-treated hosts could progress to metastases. Control mouse hosts received no exogenous hormonal support, whereas treated mice received Silastic implants containing T and E2 for 1-4 months. Plasma from hormonally treated mice contained significantly higher (p < 0.01) concentrations of T at 1 month (11.7 vs. 0.9 ng/ml). Plasma levels of E2 in steroid implanted mice were significantly higher (p < 0.05) at 2 months (104.5 vs. 25.6 ng/l) and 4 months (122.8 vs. 19.2 pg/ml). Wet weights of mUGM+BPH-1 TRs from [T+E2]-implanted mice were significantly larger (p < 0.001) than those from untreated hosts. Untreated mUGM+BPH-1 TRs contained a well organized differentiated epithelium surrounded by smooth muscle stroma similar to developing prostate. In [T+E2]-implanted mice, mUGM+BPH-1 TRs formed carcinomas that contained a fibrous connective tissue stroma permeating the tumor; smooth muscle when present was associated with vasculature. Renal lymph nodes collected from [T+E2]-treated mice, but not untreated mice, contained metastatic carcinoma cells. Moreover, metastases could be observed at distant sites including lung and liver. Epithelial cells isolated from untreated mUGM+BPH-1 TRs exhibited benign histology and formed small nontumorigenic grafts when subsequently transplanted into athymic nude mice. In contrast, epithelial cells isolated from mUGM+BPH-1 tumors of [T+E2]-treated hosts formed large tumors that grew independent of stromal and hormonal support and developed lymph node metastases. We conclude that [T+E2]-treatment promotes prostatic cancer progression in mUGM+BPH-1 TRs. Use of mUGM in this system will allow future studies to utilize the power of mouse genetics to identify paracrine factors involved in human prostatic carcinogenesis.

A novel model of polyhydramnios: amniotic fluid volume is increased in aquaporin 1 knockout mice.

OBJECTIVE: To test the hypothesis that amniotic fluid volume is increased in aquaporin 1 knockout mice. STUDY DESIGN: Transgenic mice deficient in aquaporin 1 protein were generated by targeted gene disruption, as described previously. After a cesarean section was performed, intact, individual gestational sacs were removed from the uterus and weighed. Amniotic fluid volume, osmolality, and fetal and placental weights were determined. Data were analyzed by a 1-way analysis of variance for ranks; Dunn's post hoc test was used to analyze significant trends. RESULTS: Analysis of 16 litters showed 35 wild-type, 52 heterozygote, and 33 aquaporin 1 knockout mice. The knockout mice had a greater volume of amniotic fluid and lower amniotic fluid osmolality than their wild-type and heterozygote counterparts. There were no significant differences in fetal or placental weights among the groups. CONCLUSIONS: Aquaporin 1 null fetuses produce a greater volume of more dilute amniotic fluid. Our findings show that aquaporin 1 water channels in fetal membranes may contribute to amniotic fluid volume regulation. We speculate that idiopathic polyhydramnios may be associated with a deficiency of aquaporin 1 channels in human fetal membranes. Transgenic aquaporin 1 knockout mice provide a unique animal of polyhydramnios.

Expression and localization of aquaporin 1 and 3 in human fetal membranes.

OBJECTIVES: Aquaporins are a family of water-selective channels that facilitate fluid movement across cell membranes. Specifically, aquaporin 1 (AQP1) and aquaporin 3 (AQP3) have been found to be important in osmotic water movement across membranes. Our goal in this study was (1) to determine whether AQP1 or AQP3 messenger RNA are expressed in the chorioamniotic membrane and, if present, (2) to determine the precise membrane location of these aquaporins. STUDY DESIGN: Placentas were collected from women with intact membranes not in labor who underwent elective cesarean sections at term (37-40 weeks). The membranes (amnion and chorion) directly overlying the placenta were sampled as well as the free-floating reflected membranes. RNA and protein were isolated from the amnion and chorion. Reverse transcriptase-polymerase chain reaction, Western analysis, and immunohistochemistry were used to determine expression and localization of AQP1 and AQP3. RESULTS: AQP1 messenger RNA was found in amnion and chorion from both membrane locations. Western analysis also yielded positive results for amnion and chorion from both locations. Immunohistochemical localization of AQP1 showed it to be present on the apical aspect of the chorionic plate amnion. AQP3 protein was not found in the fetal membranes. CONCLUSIONS: AQP1 is present in the fetal membranes. AQP1 may play a role in water movement from the amniotic cavity across the placenta into the fetal circulation. Further studies are needed to clarify our understanding of the role of fetal membrane aquaporins in amniotic fluid homeostasis.