A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential.

Neuroendocrine (NE) cells may be involved not only in growth and differentiation of the normal prostate but also in carcinogenesis and progression of prostate adenocarcinoma (Pca), including development of androgen resistance. However, the exact pathophysiology of NE cells in Pca remains poorly understood. Here we describe a transgenic model of Pca with progressive NE differentiation. Seven lines of transgenic mice with the rat prostate-specific large probasin promoter linked to the SV40-large T antigen (Tag) that develop prostatic neoplasia have been established. In this study, one of the seven lines (12T-10) was characterized by examination of 52 mice aged from 2-12 months. With advancing age, low-grade prostatic intraepithelial neoplasia, high-grade prostatic intraepithelial neoplasia, microinvasion, invasive carcinoma, and poorly or undifferentiated carcinoma with NE differentiation appeared in the prostates in sequential order. Whereas Tag is expressed uniformly in prostate epithelium, only an increasing subset of cells in prostatic intraepithelial neoplasia showed NE differentiation by chromogranin immunostaining. Frankly invasive carcinoma developing subsequently showed occasional definitive glandular differentiation (adenocarcinoma) and particularly undifferentiated carcinoma with NE histological features similar to those observed in NE carcinomas in humans. The NE carcinomas occurred in the dorsolateral and ventral lobes and were generally androgen receptor negative. Twenty-one of 32 (66%) mice aged > or = 6 months and 15 of 17 (88%) mice aged > or = 9 months developed metastatic tumors, as confirmed by histology and/or Tag immunohistochemistry. Metastases occurred at the later time points, with metastasis to regional lymph nodes, liver, and lung being particularly common. Metastases showed histological features of NE differentiation, as confirmed by chromogranin immunostaining and electron microscopy. An athymic nude mouse that received a s.c. implant of a primary NE tumor developed Tag-positive metastatic tumors with similar NE differentiation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified identical protein profiles between the primary NE tumor and lesions in the extraprostatic organs. Hence, in the 12T-10 large probasin promoter-Tag mouse, high-grade prostatic intraepithelial neoplasia develops progressively greater NE differentiation and progresses to invasive adenocarcinoma and NE carcinoma, with a high percentage of metastases. The predictable progression through these stages will allow testing of therapeutic interventions as well as possible further delineation of the role of NE cells in Pca progression.

A small composite probasin promoter confers high levels of prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo.

Transient transfection studies have shown that the probasin (PB) promoter confers androgen selectivity over other steroid hormones, and transgenic animal studies have demonstrated that the PB promoter will target androgen, but not glucocorticoid, regulation in a prostate-specific manner. Previous PB promoters either targeted low levels of transgene expression or became too large to be conveniently used. The goal was to design a PB promoter that would be small, yet target high levels of prostate-specific transgene expression. Thus, a composite probasin promoter (ARR2PB) coupled to the bacterial chloramphenicol acetyltransferase reporter (ARR2PBCAT) was generated and tested in prostatic and nonprostatic cell lines and in a transgenic mouse model. In PC-3, LNCaP, and DU145 prostate cancer cell lines, the ARR2PB promoter gave basal expression and was induced in response to androgen and glucocorticoid treatment after cotransfection with the respective steroid receptor. Basal expression of ARR2PBCAT in the nonprostatic COS-1, MCF-7, ZR-75-1, and PANC-1 cell lines was very low; however, CAT activity could be induced in response to androgens and glucocorticoids when cells were cotransfected with either the AR or GR. In contrast to the transfection studies, ARR2PBCAT transgene expression remained highly specific for prostatic epithelium in transgenic mice. CAT activity decreased after castration, and could be induced by androgens and, in addition, glucocorticoids. This demonstrates that the necessary sequences required to target prostate-specific epithelial expression are contained within the composite ARR2PB minimal promoter, and that high transgene expression can now be regulated by both androgens and glucocorticoids. The ARR2PB promoter represents a novel glucocorticoid inducible promoter that can be used for the generation of transgenic mouse models and in viral gene therapy vectors for the treatment of prostate cancer in humans.

A 5-kilobase pair promoter fragment of the murine epididymal retinoic acid-binding protein gene drives the tissue-specific, cell-specific, and androgen-regulated expression of a foreign gene in the epididymis of transgenic mice.

The murine epididymis synthesizes and secretes a retinoic acid-binding protein (mE-RABP) that belongs to the lipocalin superfamily. The gene encoding mE-RABP is specifically expressed in the mouse mid/distal caput epididymidis under androgen control. In transgenic mice, a 5-kilobase pair (kb) promoter fragment, but not a 0.6-kb fragment, of the mE-RABP gene driving the chloramphenicol acetyltransferase (CAT) reporter gene restricted high level of transgene expression to the caput epididymidis. No transgene expression was detected in any other male or female tissues. Immunolocalization of the CAT protein and in situ hybridization of the corresponding CAT mRNA indicated that transgene expression occurred in the principal cells of the mid/distal caput epididymidis, thereby mimicking the spatial endogenous mE-RABP gene expression. Transgene and mE-RABP gene expression was detected from 30 days and progressively increased until 60 days of age. Castration, efferent duct ligation, and hormone replacement studies demonstrated that transgene expression was specifically regulated by androgen but not by any other testicular factors. Altogether, our results demonstrate that the 5-kb promoter fragment of the mE-RABP gene contains all of the information required for the hormonal regulation and the spatial and temporal expression of the mE-RABP gene in the epididymis.

Inhibins, activins, and follistatins: expression of mRNAs and cellular localization in tissues from men with benign prostatic hyperplasia.

BACKGROUND: The transforming growth factor beta (TGF beta) superfamily of growth factors includes activins and inhibins, which have been shown to be present in the rat ventral prostate, and human prostate tumor cell lines, although their localization in benign prostatic hyperplasia (BPH) tissue is currently unknown. METHODS: BPH tissues were obtained at surgery, and the mRNA expression for the inhibin alpha, beta A, beta B subunits, the putative activin beta C subunit, the activin type II receptor (ActRII), and the activin binding protein, follistatin, was determined by reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis. Antibodies specific for alpha, beta A, beta B, activin A, and follistatin were used to determine the localization of these proteins in BPH tissue specimens. RESULTS: Southern blot analysis confirmed that mRNA for ActRII, beta C subunit, and follistatin was present in all biopsy samples assayed. However, alpha, beta A, and beta B subunit mRNA expression was variable between patient samples. Immunohistochemistry demonstrated the predominant localization of beta A, beta B, and activin A proteins to the epithelium of BPH tissues. No immunoreactivity for the inhibin alpha subunit was detected; follistatin immunoreactivity was localized to the fibroblastic stroma. CONCLUSIONS: The compartmentalization of activin subunit proteins to the epithelium, and of follistatin to the stroma, suggests that a paracrine interaction occurs between the activin ligands and follistatin-binding proteins in BPH tissue.

Expression and localization of activin subunits and follistatins in tissues from men with high grade prostate cancer.

Activins are growth and differentiation factors that have growth inhibitory effects on LNCaP and DU145, but not PC3, human prostate tumor cell lines. Activin-binding proteins, follistatins, block the inhibitory actions of exogenously added activins on LNCaP and DU145 tumor cell lines. Based on these in vitro observations using human prostate tumor cell lines, the aims of this study were to determine whether activins and follistatins are expressed in the human prostate in tissues from men with high grade prostate cancer. The expression and cellular localization of these proteins in malignant and nonmalignant regions of these tissues were compared to determine whether any changes occur with progression to malignancy. The results demonstrate that activins and follistatins are synthesized in tissues from men with high grade prostate cancer, and that messenger ribonucleic acid (mRNA) and protein for the activin beta A- and beta B-subunits and follistatin is expressed and localized to poorly differentiated tumor cells. In the nonmalignant regions, activin beta A and beta B subunit mRNA and proteins are predominantly localized to the epithelium. Follistatin mRNA was expressed in the basal epithelial cells and in the fibroblastic stroma; however, the localization of follistatin proteins using two specific antisera demonstrated a difference between the follistatin isoforms expressed in basal cells and the stroma. In the progression to malignancy, the colocalization of follistatin and activins to the tumor cells in vivo implies that resistance to the growth inhibitory effects of activin may be conferred by follistatins.

Growth inhibitory response to activin A and B by human prostate tumour cell lines, LNCaP and DU145.

Activins are growth and differentiation factors which have been shown to have proliferative and antiproliferative actions in many tissues. In addition, they have been implicated in tumourigenesis in reproductive tissues. Although activin and inhibin are present in rat ventral prostate, inhibin beta, but not alpha, subunit proteins have been detected in the human prostate epithelial tumour cell lines LNCaP, DU145 and PC3. With this absence of capacity to produce inhibins, the aims of this study were to determine the effect of activin A and B and follistatin on DNA synthesis by these human prostate tumour cell lines. The results demonstrate a differential response to exogenously added activin A and B on DNA synthesis in vitro by the tumour cell lines. The inhibitory effects were observed on LNCaP cells in the absence or presence of stimulation with 1 nM 5 alpha-dihydrotestosterone and on the androgen-independent DU145 cells, but not the PC3 cells. Activin A caused a dose-dependent inhibition of DNA synthesis and proliferation by LNCaP and androgen-independent DU145 cells which was maximal at 8 ng/ml. The effect of exogenously added activin A was completely reversed by follistatin, but not by inhibin A. The addition of human recombinant FS 288 alone (400 ng/ml) did not have any effect on DNA synthesis, whereas inhibin A alone (400 ng/ml) caused a significant inhibition of DNA synthesis. The capacity of all three cell lines to produce activins and follistatins was demonstrated by the expression of the mRNAs and confirmed by the localisation of immunoreactivity for these ligands to the cytoplasm of the tumour cells. The growth inhibitory response to activins A and B by LNCaP and DU145 cells, and the ability of follistatin to block these effects, suggest that the autocrine interactions between activins and follistatins have a role in the regulation of LNCaP and DU145 prostate tumour cell growth.