Intravesical treatment of advanced urothelial bladder cancers with oncolytic HSV-1 co-regulated by differentially expressed microRNAs.

Urothelial bladder cancer is the most common malignancy of the urinary tract. Although most cases are initially diagnosed as non-muscle-invasive, more than 80% of patients will develop recurrent or metastatic tumors. No effective therapy exists currently for late-stage metastatic tumors. By intravesical application, local administration of oncolytic Herpes Simplex virus (oHSV-1) can provide a promising new therapy for this disease. However, its inherent neurotoxicity has been a perceived limitation for such application. In this study, we present a novel microRNA-regulatory approach to reduce HSV-1-induced neurotoxicity by suppressing viral replication in neurons while maintaining oncolytic selectivity toward urothelial tumors. Specifically, we designed a recombinant virus that utilizes differentially expressed endogenous microR143 (non-cancerous, ubiquitous) and microR124 (neural-specific) to regulate expression of ICP-4, a gene essential for HSV-1 replication. We found that expression of ICP-4 must be controlled by a combination of both miR143 and miR124 to achieve the most effective attenuation in HSV-1-induced toxicity while retaining maximal oncolytic capacity. These results suggest that interaction between miR143 and miR124 may be required to successfully regulate HSV-1 replication. Our resent study is the first proof-in-principle that miRNA combination can be exploited to fine-tune the replication of HSV-1 to treat human cancers.

[Oncolytic vesicular stomatitis viruses as intravesical agents against non-muscle-invasive bladder cancer]. / Intravesikale Therapie nicht muskelinvasiver Blasentumoren mit onkolytischen Vesikular-Stomatitisviren.

Patients with high-risk bladder cancer who do not respond to bacillus Calmette-Guerin (BCG) immunotherapy represent a significant therapeutic challenge. The addition of interferon to BCG has recently evolved as a second-line treatment option; however, many high-grade tumors are nonresponsive to interferon. Thus, replication-competent oncolytic vesicular stomatitis viruses (VSV) that selectively target interferon-refractory tumors are promising intravesical agents. In vitro, wild-type VSV as well as a mutant variant (AV3) that has an impaired ability to shut down innate immunity preferentially killed undifferentiated, interferon-nonresponsive bladder cancer cells. Testing of these viruses in an orthotopic murine model of high-grade bladder cancer, which we have recently validated, revealed that both AV3 and wild-type VSV significantly inhibited orthotopic tumor growth. Despite the use of immunocompromised nude mice, there was no evidence of toxicity. In conclusion, VSV instillation therapy demonstrated strong antitumor activity and safety in an orthotopic model of high-risk disease. These findings provide preclinical proof-of-principle for the intravesical use of VSV, especially in interferon-refractory patients.

An HSV-1 amplicon system for prostate-specific expression of ICP4 to complement oncolytic viral replication for in vitro and in vivo treatment of prostate cancer cells.

The aim of the present study was to determine whether a prostate-specific amplicon, containing a probasin-derived promoter (ARR(2)PB) upstream of an essential Herpes simplex virus-1 (HSV-1) viral gene, infected-cell polypeptide 4 (ICP4), could complement an HSV-1 helper virus with this gene deleted (ICP4-) and cause lytic replication specifically in prostate cancer cells. Two amplicon constructs, CMV-ICP4 and ARR(2)PB-ICP4, were packaged by a replication-deficient ICP4- helper virus. The amplicon viruses could complement ICP4- helper viruses to efficiently replicate and cause cell lysis in prostate cancer cells. Intratumoral injection of LNCaP human prostate cancer xenografts with either amplicon/helper virus resulted in >75% reduction in tumor volume and serum prostate specific antigen (PSA). Histological and Q-PCR (quantitative PCR) analyses indicated that the toxicity in nontumor tissues was much lower with ARR(2)PB-ICP4 than with CMV-ICP4 amplicon/helper virus. In conclusion, a replication-deficient HSV-1 virus could be complemented by an amplicon virus to restore its oncolytic activity in a tissue-specific and low toxicity fashion, illustrating that this approach could be a potentially useful strategy for developing an oncolytic viral therapy for prostate cancer.

Neuropilin-1 is upregulated in the adaptive response of prostate tumors to androgen-targeted therapies and is prognostic of metastatic progression and patient mortality.

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.

Targeting and killing of prostate cancer cells using lentiviral constructs containing a sequence recognized by translation factor eIF4E and a prostate-specific promoter.

To develop a gene therapy that would selectively kill prostate cancer cells while sparing normal cells, we have constructed lentiviral vectors that contain a therapeutic gene with a short DNA sequence in the 5'-untranslated region (UTR) that is recognized by the translation initiation factor, eIF4E, which is often overexpressed in malignant cells. Infection of cancer (LNCaP, PC-3M, DU145, and MCF-7 cells) and noncancer cell lines (BPH-1, 267-B1, Plat-E, and Huvec-c cells) with lentivirus having a CMV-promoter and EGFP reporter resulted in high levels of EGFP expression in all cells, whereas, inclusion of the eIF4E UTR recognition sequence restricted high expression to cancer cells and Plat-E cells, which also express substantial levels of eIF4E. Infection of the cells with lentiviral vectors having this UTR in front of the HSV thymidine kinase suicide gene resulted in differential sensitivity to the killing effects of ganciclovir, with at least 100-fold more drug required to kill noncancer cells than cancer cells. Furthermore, in experiments where the CMV promoter was replaced by the prostate-specific ARR(2)PB promoter, the killing effects of ganciclovir were restricted to prostate cancer cells and not seen in nonprostate cancer cells. Our results indicate that combined translational regulation, by incorporation of an eIF4E-UTR recognition sequence into a therapeutic gene, together with transcriptional regulation with a prostate-specific promoter, may provide a means to selectively destroy prostate cancer cells while sparing normal prostate cells.

Gene expression during the early phases of regression of the androgen-dependent Shionogi mouse mammary carcinoma.

The effects of castration on gene expression were measured in the androgen-dependent Shionogi mouse mammary carcinoma. From 0 to 144 h after castration, polyadenylated RNA from the tumors was analyzed by Northern blotting for the expression of genes associated with cell maintenance (beta-actin and alpha-tubulin), cell growth and differentiation (c-fos and c-myc), and cell stress and death (heat shock 70 and TRPM-2). During the first 48-72 h after castration, the tumor continued to increase its mass but thereafter (72-144 h) began to regress. Throughout, the concentration of polyadenylated RNA recovered and the expression of both beta-actin and alpha-tubulin remained relatively constant, implying that in the surviving cells there are no major decreases in RNA synthesis. By comparison, c-myc exhibited a small increase in relative expression during the entire period examined, whereas c-fos displayed a transient peak at 12 h after castration, suggesting that this gene is acutely sensitive to androgen withdrawal. Heat shock protein 70 displayed a pattern similar to that of c-fos; however, the transient rise in the level of heat shock protein 70 expression could be induced by sham operations alone. The concentration of the transcripts encoding TRPM-2 rose only when tumor regression was most evident (72-144 h after castration). Thus, gene expression in the Shionogi carcinoma following castration can be grouped according to those genes which show little or no response, those which are acutely sensitive, and those which show a late effect and are more closely affiliated with cell death.

Acquisition of chemoresistant phenotype by overexpression of the antiapoptotic gene testosterone-repressed prostate message-2 in prostate cancer xenograft models.

Testosterone-repressed prostate message-2 (TRPM-2) expression is highly up-regulated in normal and malignant prostate cells after androgen withdrawal. Although recent studies have suggested a protective role of TRPM-2 expression against apoptosis in several experimental models, the functional role of TRPM-2 in chemotherapy-induced apoptosis remains undefined. Here, we demonstrated that overexpression of TRPM-2 in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to paclitaxel treatment than control LNCaP cells, with a 20-fold higher IC50 through the inhibition of apoptotic cell death. In mice bearing TRPM-2-overexpressing LNCaP tumors, tumor volume and serum prostate-specific antigen increased two to three times faster after castration and paclitaxel treatment compared with mice bearing control tumors. We then tested the efficacy of combined treatment with antisense TRPM-2 oligodeoxynucleotide (ODN) and paclitaxel in the mouse androgen-dependent Shionogi tumor model. Antisense TRPM-2 ODN treatment significantly enhanced paclitaxel chemosensitivity of Shionogi tumor cells in a dose-dependent manner, reducing the IC50 by 75%. Combined treatment of Shionogi cells with 500 nM antisense TRPM-2 ODN and 10 nM paclitaxel-induced apoptosis, either agent alone did not. Adjuvant administration of antisense TRPM-2 ODN and polymeric micellar paclitaxel after castration resulted in reduced TRPM-2 levels in vivo and a significant delay of emergence of androgen-independent recurrent Shionogi tumors compared with administration of either agent alone. Furthermore, combined treatment of mice bearing androgen-independent recurrent Shionogi tumors with antisense TRPM-2 ODN and micellar paclitaxel inhibited tumor growth compared with treatment with either agent alone. Collectively, these findings demonstrate that TRPM-2 overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis, and that antisense TRPM-2 ODN may be useful in enhancing the effects of cytotoxic chemotherapy in hormone-refractory prostate cancer.

Testosterone-repressed prostate message-2 is an antiapoptotic gene involved in progression to androgen independence in prostate cancer.

Although initially reported as an androgen-repressed gene in the rat prostate, the functional role of testosterone-repressed prostate message-2 (TRPM-2) in apoptosis remains undefined. Inhibition of castration-induced apoptosis by calcium channel blocker treatment in androgen-dependent Shionogi tumors resulted in the prevention of TRPM-2 gene up-regulation, suggesting that TRPM-2 is not directly androgen-repressed, but is regulated by apoptotic stimuli. The overexpression of the TRPM-2 gene in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to androgen ablation in vivo. We then tested the efficacy of antisense TRPM-2 oligodeoxynucleotide (ODN) therapy in the Shionogi tumor model and demonstrated that the systemic administration of antisense TRPM-2 ODNs in mice bearing Shionogi tumors after castration resulted in a more rapid onset of apoptosis and time to complete regression, as well as a significant delay of emergence of androgen-independent recurrent tumors compared to control ODN treatment. Collectively, these findings illustrate that TRPM-2 is an antiapoptotic rather than an androgen-repressed gene that confers resistance to androgen ablation and thereby helps accelerate the progression to androgen independence.

Intestinal uptake of hexoses and fatty acids in tumor-bearing rats.

An in vitro technique was used to examine the rate of uptake of varying concentrations of glucose, galactose, and a homologous series of saturated fatty acids into the jejunum of control rats and Noble rats bearing prostatic tumor variants of primary human prostatic adenocarcinoma 52. Both groups of animals were healthy, eating and gaining weight normally. The uptake of acetic and butyric acid was reduced in tumor-bearing rats, but the uptake of four medium chain-length fatty acids was unchanged. The similar value of the incremental change in free energy in the control and tumor-bearing animals indicates similar passive permeability properties of the jejunum in the two groups. The uptake of varying concentrations of glucose and galactose was similar in the control and in the tumor-bearing animals. It is concluded that there are only subtle changes in nutrient uptake in tumor-bearing rats that are eating and gaining weight normally. It is suggested that any alteration in nutrient absorption which might occur in the cachectic tumor-bearing animal is probably due to alterations in food intake or body weight, rather than due to a direct effect of the tumor on the intestine.

A metastatic and androgen-sensitive human prostate cancer model using intraprostatic inoculation of LNCaP cells in SCID mice.

Several metastasizing murine and human animal models for prostate cancer are available. However, these models are androgen-independent and lack differentiated features such as androgen receptor and androgen-regulated gene expression like prostate-specific antigen (PSA). The objective of this study was to develop a metastasizing prostate cancer model with differentiated features using the human LNCaP cell line. Athymic and SCID mice were injected either s.c. or intraprostatically with 1 x 10(6) LNCaP cells. Changes in serum and tumor PSA mRNA levels were determined before and after castration to assess time to androgen-independent progression. Local tumor and metastatic growth was assessed at sacrifice after 12 weeks. Reverse transcription-PCR (RT-PCR) was used to detect circulating LNCaP cells. LNCaP tumor incidence after s.c. injection was 100% (65 of 65) in SCID mice and 80% in athymic mice. No lymph node or distant metastases were observed with s.c. tumors, and RT-PCR for PSA transcripts was negative. Primary tumor incidence after intraprostatic injection was 89% (39 of 44) in SCID mice and 60% in athymic mice. In 10 SCID mice with primary tumors followed for 12 weeks, retroperitoneal or mediastinal lymph node metastases were found in 100%, and microscopic pulmonary metastases were identified in 40%. RT-PCR for PSA transcripts was positive in 3 of 10 mice tested. Serum PSA levels in mice with s.c. and intraprostatic tumors decreased by 65% to nadir levels at 7 and 4 days after castration, respectively. Serum PSA and LNCaP tumor PSA mRNA levels increased to precastration levels earlier in SCID mice with intraprostatic tumors compared to those with s.c. tumors. Intraprostatic injection of LNCaP cells in SCID mice provides a useful animal model to investigate mechanisms of metastasis and to evaluate therapies targeted toward inhibiting the metastatic cascade.

Effects of androgen withdrawal on the stem cell composition of the Shionogi carcinoma.

The parent Shionogi mouse mammary carcinoma is androgen dependent but cells that survive hormone withdrawal progress and give rise to an androgen-independent tumor. To determine whether renewed growth might be attributed to the persistence or partial recovery of an androgenic stimulus, we compared the amount of dihydrotestosterone and nuclear androgen receptor in parent and recurrent tumors. The whole tissue concentration of dihydrotestosterone in the parent tumor before castration was 1.40 +/- 0.46 (SE) as compared with 0.22 +/- 0.10 pmol/mg of DNA in the recurrent tumor. The initial concentration of nuclear androgen receptor in the parent was 0.65 +/- 0.12 pmol/mg of DNA; this was reduced to zero within 24 h after castration. Also in keeping with the androgen independence, no receptor was detected in the nuclear fraction of the recurrent carcinoma. In an attempt to relate malignant potential to nonhormonal factors associated with progression, we compared the proportions of androgen-dependent and -independent tumorigenic (stem) cells in parent and recurrent tumors using an in vivo limiting dilution assay. The difference observed, i.e., one stem cell per 4000 tumor cells in the parent versus one stem cell per 200 tumor cells in the recurrent carcinoma, was consistent with a marked enrichment of stem cells in the latter. The proportion of androgen-independent stem cells was also determined by assaying tumor takes in female hosts. The difference, i.e., one stem cell per 370,000 tumor cells in the parent versus one stem cell per 800 tumor cells in the recurrent carcinoma, demonstrated a striking 500-fold increase in androgen-independent stem cells resulting from androgen withdrawal. Unexpectedly, no enrichment of androgen-independent stem cells was evident in regressing parent tumors; rather, the proportion of such cells was very small, i.e., one androgen-independent stem cell per 2,200,000 regressing parent cells. This finding implies that the androgen-independent state of cells which survive androgen withdrawal may result from the ability of a small number of initially androgen-dependent stem cells to adapt to an altered hormone environment.

Variability of androgen-related phenotypes in the Shionogi mammary carcinoma during growth, involution, recurrence, and progression to hormonal independence.

Several parameters of androgen action were measured in hormone-dependent Shionogi carcinoma cells during phases of growth, regression, and recurrence. In the parental C1 line under steady state conditions, dihydrotestosterone is localized exclusively in the nucleus while testosterone is confined almost entirely to the cytoplasm. After castration, the concentration of testosterone declines more rapidly than that of dihydrotestosterone. Spontaneous recurrent growth is not accompanied by significant elevation of the whole-tissue concentration of either androgen. Neither are changes observed in the concentration of cytoplasmic receptor or in the rate of uptake of androgens into the nucleus. However, relapse is associated with the appearance of a glucose-6-phosphate dehydrogenase double-enzyme phenotype and a loss of responsiveness to androgen withdrawal. The autonomous C3 variant line which is devoid of androgen-related markers is characterized by a deficiency of androgen retention by whole tissue and possibly a permeability defect of the plasma membrane. This variant tends to express a glucose-6-phosphate dehydrogenase double-enzyme phenotype. In contrast, the autonomous C4 variant line retains the ability to concentrate modest levels of testosterone in whole tissue and high levels of dihydrotestosterone in the nucleus. Although the number of nuclear binding sites is the same as that observed in the parental C1 line, the concentration of cytoplasmic receptor and the rate of nuclear uptake of androgens are relatively decreased. Expression of a glucose-6-phosphate dehydrogenase double-enzyme phenotype is less frequent than in the autonomous C3 variant line. The above results suggest that a recurrent tumor may contain hormone-sensitive cells which resume growth in an androgen-depleted environment. They also imply that progression from the androgen-dependent to the autonomous condition involves the selection and outgrowth of hormone-insensitive cells of variable phenotype.

Studies on the regulation of the concentration of androgens and androgen receptors in nuclei of prostatic cells.

Experiments were performed to assess the effect of intracellular androgen metabolism and the availability of cytoplasmic receptors on the concentration of androgens and androgen receptors in nuclei of prostatic cells. It was found that androgens are incorporated into the nucleus by a regulated, selective process which appears to limit the type and amount of androgen transported across the nuclear membrane. The metabolic conversion of testosterone to dihydrotestosterone which takes place in cytoplasm does not reduce transport and, very likely, affects only the ratio of testosterone and dihydrotestosterone transferred into the nucleus. In vivo, when the intranuclear concentration of androgens approaches 250 nM (8 pmol per mg DNA), an apparent concentration ceiling is reached even in the presence of a downward concentration gradient that would be expected to promote further transport across the nuclear membrane. This finding strongly suggests that in vivo the nuclear membrane acts as a barrier to the passage of androgens and, therefore, mitigates against the possibility that passive diffusion is an important mechanism of afferent transport of androgens into the nucleus. The ability of the nucleus to concentrate testosterone and dihydrotestosterone was clearly demonstrated in vivo when cytoplasmic concentrations of androgens of approximately 20 nM were accompanied by intranuclear concentrations in the vicinity of 250 nM. Since the measured concentration of testosterone and dihydrotestosterone in prostate of several species fall within the 5-20 nM range, it is evident that androgen concentrations in the nucleus as high as 250 nM may be typical of the physiological steady state. At the latter concentration the nucleus contains 60 000 androgen molecules: in approximate terms one third of this total is bound to a large molecular weight component of the nucleus, one third is bound to a 3.3 S receptor and one third is free or loosely bound. Since 60 000 androgen molecules and 20 000 receptor molecules appear in the nucleus before transport stops, it seems that the quantity of 4.4 S cytoplasmic receptor estimated at 174 plus or minus 24 pmol per mg protein (equivalent to about 8000 molecules per cell) is insufficient to account for the total influx of androgens and androgen receptors into the nucleus. Thus, although these results support the view that cytoplasmic receptors and the capacity to transport androgens are closely linked phenotypic markers of intracellular steroid hormone action, they suggest that the control of androgen concentration in the nucleus is achieved in a more intricate fashion than simply through a dependence on the presumed translocation of 4.4 S androgen-receptor complex into the nucleus.

Plasminogen activator in prostatic tumors of Nb rats.

Using a fluorometric assay, nonspecific proteolytic activity and plasminogen activator were measured in transplantable tumors of the dorsal prostate of Nb rats. Nonspecific proteolytic activity in prostatic tumors did not differ significantly from that measured in normal dorsal prostate, whereas plasminogen activator activity, undetectable in the latter tissue, was readily measurable in the tumors. Furthermore, plasminogen activator activity in prostatic tumors characterized by hormone-insensitive growth was 8-fold higher than in tumors characterized by androgen-stimulated growth. In both typess of tumors, the plasminogen activator activity per mg protein was highest in the lysosomal fractions. The results indicate that plasminogen activator may be a useful marker for discriminating between androgen-stimulated and autonomous prostatic tumors.

Nuclear binding of androgens and acid phosphatase activity in prostatic tumors of Nb rats.

A transplantable prostatic adenocarcinoma derived from the dorsal lobe of the prostate gland of an Nb rat was analyzed for the concentration of nuclear androgen-binding sites and the presence of acid phosphatase activity. When extracts of nuclei from normal prostatic tissue were labelled with [1,2-3H]dihydrotestosterone in the absence and presence of competitor, two types of specific binding were observed: type 1 was characterized by an association constant (Ka) of 6 x 10(7) M-1 and involved a molecule that was excluded from Sephadex G-200; type 2 was characterized by a Ka of 3 x 10(8) M-1 and depended on a binding component that was retained by Sephadex G-200. Nuclei from androgen-stimulated tumors contained reduced concentrations of both androgen-binding components, whereas nuclei from autonomous tumors had only a trace amount of type 1 sites and were entirely devoid of type 2 sites. In all tumors the acid phosphatase activity per mg of protein was markedly elevated. Relative to normal, the activity of this enzyme was 140% and 350% higher in androgen-stimulated and autonomous tumors, respectively. These findings indicate that prostatic tumors are characterized by a decrease in nuclear androgen-binding, and an increase in specific activity of acid phosphatase, and also that such changes are more pronounced in autonomous than in androgen-stimulated tumors.

Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene.

The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the rat androgen receptor [glutathione-S-transferase (GST)-AR1] and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT. In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements. Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.

Cortisol alters gene expression during involution of the rat ventral prostate.

The ability of high doses of cortisol to retard the involution process in the rat ventral prostate was related to alterations in the pattern of gene expression. Poly(A)+ RNA preparations from the prostates of noncastrated, castrated, and castrated rats injected daily for 7 days with cortisol were compared by Northern blot hybridizations for the relative expression of genes associated with cell differentiation and maintenance (the C1 prostatic steroid binding protein gene and alpha-tubulin), with cell death (TRPM-2, hsp 70, and c-fos), and with hormone regulation (the androgen and glucocorticoid receptors). As anticipated, the concentration of C1 mRNA in the prostate fell to less than 4% of that in the noncastrated controls within 4 days after castration and was nearly undetectable after 7 days. This decline was retarded by cortisol treatment of 7-day castrated animals which sustained the level of C1 transcripts at approximately 50% of control. While the pattern of expression of alpha-tubulin indicated some minor fluctuations, with the highest level occurring 7 days after castration, the prostates of the cortisol-treated group had essentially the same concentration of this mRNA as the noncastrates. Cortisol also modified the expression of genes associated with prostatic cell death. The large increase in prostatic TRPM-2 mRNA, seen 7 days after castration, was reduced by over 80% after treatment with the glucocorticoid. Although not as abundantly expressed as TRPM-2, the castration-induced levels of transcripts for both hsp 70 and the protooncogene c-fos were substantially reduced by cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinants of DNA sequence specificity of the androgen, progesterone, and glucocorticoid receptors: evidence for differential steroid receptor response elements.

While androgen, progesterone, and glucocorticoid receptors perform distinct physiological functions by regulating unique sets of genes, in vitro they can transactivate a common high-affinity DNA-binding target. Naturally occurring steroid response elements display nucleotide divergence that lowers binding affinity in comparison to the optimal binding element, but enhances receptor-type specificity. We investigated the role of nucleotide deviations within the DNA-binding site for contribution to steroid receptor specificity. We hypothesized that receptor specificity drives the evolution of binding site sequence, rather than strictly receptor-binding affinity. Receptor-selective targets can evolve by some nucleotides selected on the basis of additional bond energy, and others may be selected by differential tolerance to discourage binding from inappropriate receptors. To identify receptor-specific binding sites, we mimicked these dual selection pressures in a receptor-competitive environment in which DNA binding sites for the androgen or progesterone receptors were selected in the presence of the glucocorticoid receptor. These analyses also demonstrated that steroid receptors strongly select nucleotides in the spacer and flanking regions of the half-site and do so in an asymmetric fashion, indicating that steroid receptors interact with DNA in an allosteric manner that affects the transcriptional activation potential.

Overexpression of insulin-like growth factor binding protein-5 helps accelerate progression to androgen-independence in the human prostate LNCaP tumor model through activation of phosphatidylinositol 3'-kinase pathway.

Although insulin-like growth factor (IGF) binding protein-5 (IGFBP-5) is highly up-regulated in normal and malignant prostate tissues after androgen withdrawal, its functional role in castration-induced apoptosis and androgen-independent progression remains undefined. To analyze the functional significance of IGFBP-5 overexpression in IGF-I-mediated mitogenesis and progression to androgen-independence, IGFBP-5-overexpressing human androgen-dependent LNCaP prostate cancer cells were generated by stable transfection. The growth rates of IGFBP-5-transfected LNCaP cells were significantly faster, compared with either the parental or vector-only transfected LNCaP cells in both the presence and absence ofdihydrotestosterone. IGFBP-5-induced increases in LNCaP cell proliferation occurs through both IGF-I-dependent and -independent pathways, with corresponding increases in the cyclin D1 messenger RNA expression and the fraction of cells in S + G2/M phases of the cell cycle. Changes in Akt/protein kinase B, a downstream component of phosphatidylinositol 3'-kinase (PI3K) pathway, in the LNCaP sublines also paralleled changes in their growth rates. Although treatment with a PI3K inhibitor induced apoptosis in both control and IGFBP-5-overexpressing LNCaP cells, this PI3K inhibitor-induced apoptosis was prevented by exogenous IGF-I treatment only in IGFBP-5 transfectants, suggesting that IGFBP-5 overexpression can potentiate the antiapoptotic effects of IGF-I. Furthermore, tumor growth and serum prostate-specific antigen levels increased several fold faster in mice bearing IGFBP-5-transfected LNCaP tumors after castration, despite having similar tumor incidence and tumor growth rates with controls when grown in intact mice before castration. Collectively, these data suggest that IGFBP-5 overexpression in prostate cancer cells after castration is an adaptive cell survival mechanism that helps potentiate the antiapoptotic and mitogenic effects of IGF-I, thereby accelerating progression to androgen independence through activation of the PI3K-Akt/ protein kinase B signaling pathway.

Kinetic parameters of 5 alpha-reductase activity in stroma and epithelium of normal, hyperplastic, and carcinomatous human prostates.

To study the influence of 5 alpha-reductase on the concentration of dihydrotestosterone in prostatic tissue, we measured the activity of this enzyme in stroma and epithelium from 15 normal, 50 hyperplastic, and 20 carcinomatous prostates. Maximum velocity (Vmax) and Km parameters based on the Lineweaver-Burk and Eadie-Hofstee transformations of the Michaelis-Menten equation were related to the stromal and epithelial concentrations of dihydrotestosterone. On the basis of relative Vmax values, there was 6-15 times more 5 alpha-reductase activity in stroma than in epithelium regardless of the histology of the prostate. Stromal enzyme activity also was unique in having a 2- to 5-fold larger mean Km value and greater resistance to competitive and noncompetitive inhibition. Despite the enrichment of 5 alpha-reductase activity in stroma, the dihydrotestosterone concentrations in the stromal and epithelial fractions were very similar. In addition, similar concentrations were found in the stromal fractions of hyperplastic and carcinomatous tissues, notwithstanding a 4-fold difference in the mean Vmax values. This anomaly occurred in association with a large disparity in mean Km values, i.e. 68.3 +/- 1.6 (+/- SE) nmol/L in hyperplasia vs. 23.0 +/- 2.9 nmol/L in carcinoma. The dissociation between parameters of 5 alpha-reductase activity and tissue dihydrotestosterone concentrations was apparent to some extent in benign prostatic hyperplasia, in which the lowest stromal androgen concentrations were found in prostates with the largest Vmax and Km values; also, a rise in stromal Km was almost invariably associated with a proportional increase in Vmax (correlation coefficient = 0.95). These data strongly suggest that the stromal and epithelial forms of 5 alpha-reductase are separate isoenzymes, and that the excess of 5 alpha-reductase in stroma does not promote accumulation of an abnormal amount of dihydrotestosterone. They also imply that both the augmentation of 5 alpha-reductase activity in hyperplastic stroma and the condition of benign hyperplasia of the prostate are mutual consequences of a primary increase in Km.