Conditional activation of FGFR1 in the prostate epithelium induces angiogenesis with concomitant differential regulation of Ang-1 and Ang-2.

The expression of fibroblast growth factor receptor (FGFR)-1 correlates with angiogenesis and is associated with prostate cancer (CaP) progression. To more precisely define the molecular mechanisms whereby FGFR1 causes angiogenesis in the prostate we exploited a transgenic mouse model, JOCK-1, in which activation of a conditional FGFR1 allele in the prostate epithelium caused rapid angiogenesis and progressive hyperplasia. By labeling the vasculature in vivo and applying a novel method to measure the vasculature in three dimensions, we were able to observe a significant increase in vascular volume 1 week after FGFR1 activation. Although vessel volume and branching both continued to increase throughout a 6-week period of FGFR1 activation, importantly, we discovered that continued activation of FGFR1 was not required to maintain the new vasculature. Exploring the molecular mediators of the angiogenic phenotype, we observed consistent upregulation of HIF-1alpha, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2), whereas expression of Ang-1 was lost. Further analysis revealed that loss of Ang-1 expression occurred in the basal epithelium, whereas the increase in Ang-2 expression occurred in the luminal epithelium. Reporter assays confirmed that the Ang-2 promoter was regulated by FGFR1 signaling and a small molecule inhibitor of FGFR activity, PD173074, could abrogate this response. These findings establish a method to follow spontaneous angiogenesis in a conditional autochthonous system, implicate the angiopoietins as downstream effectors of FGFR1 activation in vivo, and suggest that therapies targeting FGFR1 could be used to inhibit neovascularization during initiation and progression of CaP.

The insulin-like growth factor axis and prostate cancer: lessons from the transgenic adenocarcinoma of mouse prostate (TRAMP) model.

We have characterized the temporal expression of the insulin-like growth factor (IGF) axis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model as prostate cancer progression in this model closely mimics that observed in the human disease, and the model provides samples representing the earliest stages of prostate cancer that are clinically the most difficult to obtain. We report that prostate-specific IGF-I mRNA expression increased during prostate cancer progression in TRAMP mice and was elevated in the accompanying metastatic lesions, whereas prostatic IGF-I mRNA remained at nontransgenic levels in androgen-independent disease. Expression of IGF-II mRNA, however, was reduced in primary prostate cancer, metastatic lesions, and androgen-independent disease. Expression of type-1 IGF receptor (IGF1R) mRNA, encoding the cognate receptor for both IGF-I and IGF-II, as well as type-2 IGF receptor (IGF2R) mRNA was not found to be altered during primary prostate cancer progression in intact TRAMP mice but was dramatically reduced in metastatic lesions and in androgen-independent disease. Similar to reports from clinical disease, serum IGF-I levels were observed to increase precociously in TRAMP mice early in disease progression but remained at nontransgenic levels after castration. Elevated serum levels of IGF-binding protein 2 were observed to correlate with advanced prostate cancer in the TRAMP model. Together these observations implicate IGF-I as an important factor during the initiation and progression of primary prostate cancer and provide evidence that there is a strong selection against expression of IGF1R and IGF2R in metastatic and androgen-independent disease.

Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP).

Latent prostate tumors are commonly found with similar frequency in many countries and ethnic groups. In contrast, aggressive prostate cancer (PC) is significantly less prevalent among Asian men, where the intake of soy products is very high. High consumption of foods containing soy results in high plasma, urine, and prostatic fluid concentrations of phytoestrogens, including genistein. The objective of the present study was to test the hypothesis that dietary genistein might prevent PC progression in a transgenic mouse model of PC (TRAMP). By 28-30 weeks of age, all TRAMP mice in the study had developed prostate tumors, with about half of them displaying well-differentiated prostatic adenocarcinoma (WD, score 4), and the other half divided between moderately differentiated (MD, score 5) and poorly differentiated adenocarcinoma (PD, score 6). Two lines of evidence supported the possibility that prostates with PD may represent a more advanced stage of PC in TRAMP mice: (a) the weight of prostates with PD was two orders of magnitude higher than that of prostates with WD or MD; and (b) expression of androgen receptor transcripts was altered in PD as compared with WD and MD. To test the potential of genistein to prevent the incidence of mice with PD, starting at 5-6 weeks of age, transgenic males were fed a phytoestrogen-free diet (AIN-76A) containing 0, 100, 250, or 500 mg of genistein per kg AIN-76A (25, 10, 17, and 7 mice/group, respectively). Mice were on the diet until they were 28-30 weeks of age. Each mouse was weighed once a week throughout the study. At necropsy, selected organs were weighed and prepared for histopathological evaluation. Serum levels of genistein in mice on diet containing 0, 250, or 500 mg of genistein per kg AIN-76A were 52.4 +/- 32.7, 138.9 +/- 69.6, and 397.3 +/- 104.9 nM, respectively, comparable with those found in Asian men on regular soy diet (276 nM). Using body and organ weights as indicators, dietary genistein had no toxic effect on TRAMP mice. The percentage of transgenic males that developed PD was reduced in a dose-dependent manner by dietary genistein.

Angiogenesis and prostate cancer: identification of a molecular progression switch.

To elucidate the sequence of molecular events intricate with angiogenesis and the initiation and progression prostate cancer, the temporal and spatial expression patterns of platelet endothelial cell adhesion molecule-1 (PECAM1/CD31), hypoxia-induced factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and the cognate receptors VEGFR1 and VEGFR2 were characterized. Immunohistochemical and in situ analyses of prostate tissue specimens derived from the spontaneous autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) model identified a distinct early angiogenic switch consistent with the expression of PECAM-1, HIF-1alpha, and VEGFR1 and the recruitment of new vasculature to lesions representative of high-grade prostatic epithelial neoplasia (PIN). During progression of prostate cancer, the intraductal microvessel density (IMVD) was also observed to increase as a function of tumor grade. Immunoblot and in situ analyses further demonstrated a distinct late angiogenic switch consistent with decreased expression of VEGFR1, increased expression of VEGFR2, and the transition from a differentiated adenocarcinoma to a more poorly differentiated state. Analysis of clinical prostate cancer specimens validated the predictions of the TRAMP model. This resolution of prostate cancer-associated angiogenesis into distinct early and late molecular events establishes the basis for a "progression-switch" model to explain how the targets of antiangiogenic therapy might change as a function of tumor progression.

Chemoprevention of prostate carcinogenesis by alpha-difluoromethylornithine in TRAMP mice.

Development of effective chemopreventive agents for human consumption requires conclusive evidence of their efficacy in animal models that have relevance to human diseases. Transgenic adenocarcinoma mouse prostate (TRAMP) is an excellent model of prostate cancer that mimics progressive forms of human disease inasmuch as 100% of males develop histological PIN by 8-12 weeks of age that progress to adenocarcinoma with distant site metastases by 24-28 weeks of age. In these animals, ornithine decarboxylase (ODC) activity (>3-fold) as well as protein expression (>4-fold) was found to be markedly higher in the dorsolateral prostate as compared with the nontransgenic littermates, suggesting their suitability to determine the chemopreventive effect of alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, against prostate cancer. Using male TRAMP mice, we studied the effect of oral consumption of DFMO on development of prostate carcinogenesis and surrogate end point biomarkers related to prostate cancer progression. In two independent experiments, each consisting of 8 animals on test, the cumulative incidence of prostatic cancer development at 28 weeks of age in 16 untreated TRAMP mice was 100% (16 of 16), whereas 94% (15 of 16) and 69% (11 of 16) of the animals exhibited distant site metastases to lymph nodes and lungs, respectively. Oral consumption of 1% DFMO (w/v) in the drinking water to TRAMP mice from 8 to 28 weeks of age resulted in a significant decrease in (a) weight (59%) and volume (66%) of prostate, (b) genitourinary weight (63%), and (c) ODC enzyme activity (52%) in the dorsolateral prostate. Importantly, in none of the DFMO-fed TRAMP mice were any distant metastases to lymph node and lungs observed. Furthermore, DFMO treatment resulted in the marked reduction in the protein expression of proliferation cell nuclear antigen, ODC, and probasin in the dorsolateral prostate. The protein expression of antimetastases markers, i.e., E-cadherin and alpha- and beta-catenin, was found to be restored in DFMO-fed animals as compared with the non-DFMO-fed mice. These chemopreventive effects of DFMO were further confirmed by immunohistochemical analysis of the dorsolateral prostate. Histological analysis of the dorsolateral prostate of DFMO-fed animals displayed marginal epithelial stratification, a small number of cribriform structures, elongated hyperchromatic epithelial nuclei, and a significant increase in apoptotic index. Non-DFMO-fed animals, on the other hand, displayed extensive epithelial stratification with profound cribriform structures accompanied with marked thickening, remodeling, and hypercellularity of the fibromuscular stroma. In nontransgenic littermates fed with DFMO, no significant alterations in the above parameters were evident. These data demonstrate that ODC represents a promising and rational target for chemoprevention of human prostate cancer and that TRAMP mice are excellent models for screening of novel drugs and chemopreventive regimens for potential human use.

Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

To develop a syngeneic transplantable system to study immunotherapeutic approaches for the treatment of prostate cancer, three cell lines were established from a heterogeneous 32 week tumor of the transgenic adenocarcinoma mouse prostate (TRAMP) model. TRAMP is a transgenic line of C57BL/6 mice harboring a construct comprised of the minimal -426/+28 rat probasin promoter driving prostate-specific epithelial expression of the SV40 large T antigen. TRAMP males develop histological prostatic intraepithelial neoplasia by 8-12 weeks of age that progress to adenocarcinoma with distant metastases by 24-30 weeks of age. The three cell lines (TRAMP-C1, TRAMP-C2, and TRAMP-C3) express cytokeratin, E-cadherin, and androgen receptor by immunohistochemical analysis and do not appear to have a mutated p53. Although TRAMP-C1 and TRAMP-C2 are tumorigenic when grafted into syngeneic C57BL/6 hosts, TRAMP-C3 grows readily in vitro but does not form tumors. The T antigen oncoprotein is not expressed by the cell lines in vitro or in vivo. The rationale for establishing multiple cell lines was to isolate cells representing various stages of cellular transformation and progression to androgen-independent metastatic disease that could be manipulated in vitro and, in combination with the TRAMP model, provide a system to investigate therapeutic interventions, such as immunotherapy prior to clinical trials.

Metastatic prostate cancer in a transgenic mouse.

We have previously reported the development of a transgenic mouse model for prostate cancer derived from PB-Tag transgenic line 8247, henceforth designated the TRAMP (transgenic adenocarcinoma mouse prostate) model. We now describe the temporal and spatial consequences of transgene expression and report the identification and characterization of metastatic disease in the TRAMP model. TRAMP mice characteristically express the T antigen oncoprotein by 8 weeks of age and develop distinct pathology in the epithelium of the dorsolateral prostate by 10 weeks of age. Distant site metastases can be detected as early as 12 weeks of age. The common sites of metastases are the periaortic lymph nodes and lungs, with occasional metastases to the kidney, adrenal gland, and bone. By 28 weeks of age, 100% harbor metastatic prostate cancer in the lymph nodes or lungs. We have also demonstrated the loss of normal E-cadherin expression, as observed in human prostate cancer, as primary tumors become less differentiated and metastasize. The TRAMP model provides a consistent source of primary and metastatic tumors for histopathobiological and molecular analysis to further define the earliest molecular events involved in the genesis, progression, and metastasis of prostate cancer.

Androgen-independent prostate cancer progression in the TRAMP model.

We previously established the autochthonous transgenic adenocarcinoma mouse prostate (TRAMP) model to facilitate characterization of molecular mechanisms involved in the initiation and progression of prostate cancer. TRAMP mice display high grade prostatic intraepithelial neoplasia or well-differentiated prostate cancer by 10-12 weeks of age. To test the hypothesis that molecular events leading to androgen independence and metastasis can occur early in the natural history of prostate cancer yet remain silent until selective pressures such as androgen deprivation are applied, we have examined the consequences of castration on the initiation and progression to metastatic prostate cancer in TRAMP mice. Cohorts were castrated at 12 weeks of age and sacrificed at 18 (T12/18) or 24 (T12/24) weeks of age, and the development of primary cancer and metastatic disease was compared to noncastrated (T18 and T24) controls. Median T12/18 and T12/24 genitourinary (GU) weight was significantly less than T18 and T24, respectively. In addition, T12/24 GU weight was significantly greater than T12/18. Histological prostate tumors developed in 3 of 7 T12/18 and 8 of 10 T12/24 mice. All tumors that developed in castrated mice were poorly differentiated in contrast to 27% in noncastrated controls. Although castration significantly decreased GU tumor burden, overall progression to poorly differentiated and metastatic disease was not ultimately delayed. These results demonstrate that prostate cancer in the TRAMP model is heterogeneous with respect to androgen dependence as early as 12 weeks of age; therefore, early androgen ablation may have a variable impact on progression in an individual mouse. Further analysis of this prostate cancer model to identify specific molecular mechanisms that determine androgen sensitivity may facilitate future initiation of appropriate individualized hormonal therapy for the management of human prostate cancer.

Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade.

We have previously shown that antibodies to CTLA-4, an inhibitory receptor on T cells, can be effective at inducing regression of transplantable murine tumors. In this study, we demonstrate that an effective immune response against primary prostate tumors in transgenic (TRAMP) mice can be elicited using a strategy that combines CTLA-4 blockade and an irradiated tumor cell vaccine. Treatment of TRAMP mice at 14 weeks of age resulted in a significant reduction in tumor incidence (15% versus control, 75%), as assessed 2 months after treatment. Histopathological analysis revealed that treated mice had a lower tumor grade with significant accumulation of inflammatory cells in interductal spaces when treated with anti-CTLA-4 and a granulocyte-macrophage colony-stimulating factor-expressing vaccine. Vaccination of nontransgenic mice with this regimen resulted in marked prostatitis accompanied by destruction of epithelium, indicating that the immune response was, at least in part, directed against normal prostate antigens. These findings demonstrate that this combinatorial treatment can elicit a potent antiprostate response and suggest potential of this approach for treatment of prostate cancer.

E-7869 (R-flurbiprofen) inhibits progression of prostate cancer in the TRAMP mouse.

E-7869 (R-flurbiprofen) is a single enantiomer of a racemic nonsteroidal anti-inflammatory drug. E-7869 does not inhibit either cyclooxygenase-1 or cyclooxygenase-2. We used the transgenic adenocarcinoma mouse prostate (TRAMP) mouse, a prostate cancer model, to evaluate the effect of this drug on prostate cancer progression. Sixty 12-week-old male TRAMP mice were placed randomly into five groups. The animals were treated by daily oral gavage with vehicle (1% carboxymethylcellulose) or E-7869 for 18-weeks. During the course of the study, two diets were used. Three groups (vehicle, 15-mg/kg, and 20-mg/kg drug treatments) received a Teklad diet containing 2.4% saturated fat [a high saturated fat (HSF) diet], and two groups (vehicle and 20 mg/kg drug treatment) received an AIN-93G diet containing 1.05% saturated fat [a low saturated fat (LSF) diet]. At necropsy, the urogenital system and periaortic lymph nodes were removed and weighed. The prostate lobes, seminal vesicles, lungs, and periaortic lymph nodes were preserved and sectioned for histological evaluation. The lung and periaortic lymph nodes were graded as to the presence (+) or absence (-) of metastasis; the urogenital tissues were graded on a 1-6 scale for degree of neoplasia/carcinoma. For both diets, the urogenital wet weights and lymph node wet weights in the 20-mg/kg treatment groups were significantly lower as compared to vehicle control groups. In addition, treatment with 20 mg/kg E-7869 in the LSF diet group resulted in a significantly lower primary tumor incidence (P < 0.05) and reduced incidence of metastasis. In this treatment group, the reduced incidence of metastasis was not statistically significant because the LSF diet itself resulted in a remarkably lower incidence of metastasis in the vehicle control group (10% LSF versus 40% HSF). Treatment with 20 mg/kg E-7869 on the HSF diet resulted in a significantly lower incidence of metastasis (P < 0.05) and a reduction in the primary tumor incidence. These results suggest that E-7869 is a promising chemopreventive and treatment for human prostate cancer.

Role of RhoA activation in the growth and morphology of a murine prostate tumor cell line.

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

A role for phospholipase C-gamma-mediated signaling in tumor cell invasion.

The invasive and metastatic transformation of cancers often results in death. However, the mechanisms that promote this transformation remain unclear. Two closely related receptors, the epidermal growth factor receptor (EGFR) and ErbB2, are overexpressed in a significant percentage of breast and prostate carcinomas, among others, with this up-regulated signaling correlating with tumor progression. Previous studies in our laboratory have demonstrated that an EGFR-phospholipase C (PLC)gamma-mediated motility-associated signaling pathway is rate-limiting for tumor cell invasion in vitro and in vivo in one model of prostate carcinoma. Therefore, we investigated whether this PLCgamma signaling pathway also was rate-limiting for invasion in other tumor cell lines and types and whether this EGFR activity is subsumed by the closely related ErbB2. We determined the effects of PLCgamma signal abrogation by pharmacological (U73122) and molecular (expression of the dominant-negative PLCz) means on the in vitro invasiveness of tumor cells. Inhibition of PLCgamma signaling concomitantly decreased invasiveness of de novo-occurring transgenic adenocarcinoma mouse prostate (TRAMP) lines and the human breast cancer cell lines MDA-468 and MDA-231; these lines present up-regulated EGFR signaling. Because the prostate and breast cancer lines usually present autocrine stimulatory loops involving EGFR, we also examined transgenic adenocarcinoma mouse prostate C1 and MDA-468 treated with the EGFR-specific kinase inhibitor PD153035 to determine whether invasiveness is dependent on EGFR signaling. PD153035 reduced invasiveness to levels similar to those seen with U73122, suggesting that the autocrine EGFR stimulatory loop is functioning to promote invasiveness. To determine whether this signaling pathway also promotes invasiveness of ErbB2-overexpressing tumors, we examined the human breast carcinoma line MDA-361; again, U73122 inhibition of PLCgamma decreased invasiveness. In all situations, the inhibition of PLCgamma signaling did not decrease mitogenic signaling. Thus, the motility-associated PLCgamma signaling pathway is a generalizable rate-limiting step for tumor cell progression.

Collocation of androgen receptor gene mutations in prostate cancer.

Consistent with both the development of the normal prostate gland and prostate tumorigenesis being dependent on testicular androgens, targeting the androgen-signaling axis (i.e., androgen ablation therapy) remains the predominant treatment regime for patients with metastatic prostate cancer. Although there is a very good initial response to androgen ablation, these treatments are essentially palliative. Recent evidence suggests that treatment failure may not result from a loss of androgen signaling but, rather, from the acquisition of genetic changes that lead to aberrant activation of the androgen-signaling axis. A consistent finding is that androgen receptor (AR) gene mutations, present in metastatic prostate cancer and in human prostate cancer cell lines as well as in xenograft and other animal models, result in decreased specificity of ligand-binding and inappropriate receptor activation by estrogens, progestins, adrenal androgens, glucocorticoids and/or AR antagonists. Because a significant proportion of missense mutations in the AR gene reported in prostate cancer collocate to the signature sequence and AF-2, two discrete regions of the ligand-binding domain critical for androgen signaling, we recently proposed that collocation of mutations identified in prostate cancer would identify additional regions of the AR important in receptor function. This approach led to the identification of a four-amino acid region at the boundary of the hinge and ligand-binding domains of the receptor that forms half of a potential protein-protein binding site. AR gene mutations have also been identified that collocate to areas in the DNA-binding domain, to the NH(2)-terminal transactivation domain, and to the hinge region in prostate tumors. In nearly every case, missense mutations in the AR gene identified in prostate cancer that collocate to discrete regions of the receptor contribute to altered androgen signaling and provide a potential mechanism to explain the reemergence of tumor growth during the course of hormone ablation therapies.

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice.

An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.

Mutations at the boundary of the hinge and ligand binding domain of the androgen receptor confer increased transactivation function.

The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.

Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids.

A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.

Targeted expression of des(1-3) human insulin-like growth factor I in transgenic mice influences mammary gland development and IGF-binding protein expression.

To test the hypothesis that insulin-like growth factor I (IGF-I) regulates mammary gland development and lactation, the expression of both human (h) IGF-I and des(1-3)hIGF-I was targeted to the mammary gland in transgenic mice using a novel exon replacement strategy and the rat whey acidic protein (rWAP) gene regulatory sequences. Both transgenes expressed a 0.7-kilobase messenger RNA (mRNA). The abundance of WAP-IGE-I and WAP-DES mRNA on day 10 of lactation ranged from 0.2-1.0% and 0.2-13% of the endogenous mouse WAP mRNA, respectively. For WAP-DES mice, transgene expression was greatest from midpregnancy throughout lactation. Western blot analysis showed the presence of correctly processed hIGF-I in milk from these transgenic mice. This hIGF-I was capable of stimulating protein synthesis in cultured rat L6 myoblasts. Ligand blotting indicated changes in mammary gland secretion of IGFBP in response to WAP-DES expression. Histological analysis of mammary tissue from mice overexpressing des(1-3)hIGF-I showed incomplete mammary involution, ductile hypertrophy, and loss of secretory lobules associated with increased deposition of collagen. These changes are believed to occur through autocrine and paracrine effects of des(1-3)-hIGF-I on both epithelial and stromal cells.

Transgenic models for prostate cancer research.

The ability to introduce novel or specifically altered genes into the germ line of mice and directly perturb gene expression in a specific tissue can facilitate characterization of the molecular mechanisms governing transformation of differentiating tissue within the context of an intact developing animal. Transgenics provide a powerful and remarkably flexible system that can be used to study the cooperation between proto-oncogenes, tumor suppressor genes, and other epigenetic factors in the development of cancer.

Models of metastatic prostate cancer: a transgenic perspective.

PURPOSE: Transgenic mouse models are proving to be invaluable in our effort to understand the molecular basis of metastatic prostate cancer (CaP). We review and discuss how current animal models have contributed to our understanding of the metastatic cascade and how transgenic technology is being used to develop the next generation of mouse models. Our goal is to provide a review of the recent advances and provide a framework for further studies. MATERIALS AND METHODS: We performed a MEDLINE search of the literature on CaP metastasis transgenic and animal models. RESULTS: We present a summary of the characteristics of nine different animal models of CaP. Each model is unique and provides valuable insight into the molecular mechanisms governing the progression of CaP. Our experience with transgenic models and all the new data from the literature predicts that we will be able to develop genetically engineered mice that accurately mimic the heterogeneity, androgen-independent growth, and metastatic spread seen in clinical disease. CONCLUSION: In order to elucidate the molecular mechanisms of CaP metastasis, it will be necessary to compare gene and protein expression patterns and biochemical analyses of clinical metastatic disease with data obtained from current models. We will also need to refine our ability to engineer and characterize genetic perturbation models. This type of integrative and iterative approach should facilitate better understanding of the molecular biology of CaP metastases.