Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance.

PURPOSE: Latent grade group ≥2 prostate cancer can impact the performance of active surveillance protocols. To date, molecular biomarkers for active surveillance have relied solely on RNA or protein. We trained and independently validated multimodal (mRNA abundance, DNA methylation, and/or DNA copy number) biomarkers that more accurately separate grade group 1 from grade group ≥2 cancers. MATERIALS AND METHODS: Low- and intermediate-risk prostate cancer patients were assigned to training (n=333) and validation (n=202) cohorts. We profiled the abundance of 342 mRNAs, 100 DNA copy number alteration loci, and 14 hypermethylation sites at 2 locations per tumor. Using the training cohort with cross-validation, we evaluated methods for training classifiers of pathological grade group ≥2 in centrally reviewed radical prostatectomies. We trained 2 distinct classifiers, PRONTO-e and PRONTO-m, and validated them in an independent radical prostatectomy cohort. RESULTS: PRONTO-e comprises 353 mRNA and copy number alteration features. PRONTO-m includes 94 clinical, mRNAs, copy number alterations, and methylation features at 14 and 12 loci, respectively. In independent validation, PRONTO-e and PRONTO-m predicted grade group ≥2 with respective true-positive rates of 0.81 and 0.76, and false-positive rates of 0.43 and 0.26. Both classifiers were resistant to sampling error and identified more upgrading cases than a well-validated presurgical risk calculator, CAPRA (Cancer of the Prostate Risk Assessment; P < .001). CONCLUSIONS: Two grade group classifiers with superior accuracy were developed by incorporating RNA and DNA features and validated in an independent cohort. Upon further validation in biopsy samples, classifiers with these performance characteristics could refine selection of men for active surveillance, extending their treatment-free survival and intervals between surveillance.

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.

Increased expression of class III beta-tubulin in castration-resistant human prostate cancer.

BACKGROUND: Class III beta-tubulin (betaIII-tubulin) is expressed in tissues of neuronal lineage and also in several human malignancies, including non-small-cell lung carcinoma, breast and ovarian cancer. Overexpression of betaIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies. At present, betaIII-tubulin expression remains largely uncharacterised in prostate cancer. METHODS: In this report, we evaluated the expression of betaIII-tubulin in 138 different human prostate tumour specimens by immunohistochemistry from patients with hormone-treated or hormone-untreated prostate cancer. betaIII-tubulin expression was also examined in various prostatic cancer cell lines including in androgen-sensitive human prostate cancer cells, LNCaP, grown in androgen-depleted medium in 2D cultures or as tumour xenografts when the host mouse was castrated. RESULTS: Whereas moderate-to-strong betaIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of betaIII-tubulin. These findings were supported by in vitro and in vivo settings. CONCLUSION: Our data indicate that betaIII-tubulin expression is augmented in prostate cancer by androgen ablation and that the expression of this beta-tubulin isoform is associated with the progression of prostate cancer to the castration-resistant state, a stage largely responsible for mortality from prostate cancer.

Anti-prostate cancer activity of a beta-carboline alkaloid enriched extract from Rauwolfia vomitoria.

The tropical shrub, Rauwolfia vomitoria, is a medicinal plant used traditionally to treat a variety of ailments. A bioactive beta-carboline alkaloid, alstonine, present in this extract was previously shown to have anti-cancer activity against cancer cell lines. This study considers the potential anti-prostate cancer activity of this extract in vitro and in vivo. Rauwolfia vomitoria extract standardized for beta-carboline alkaloids was tested for ability to influence the growth and survival of the human LNCaP prostate cancer cell line. A WST-1 assay was used to measure cell growth, and cell cycle analyses were conducted with flow cytometry. Western blot detection of PARP cleavage and accumulation of cells containing sub-genomic DNA indicated induction of apoptosis. Pathway specific microarray analyses were utilized to identify the effect of Rauwolfia extract on the expression of 225 genes. Mice xenografted with LNCaP cells were treated with the extract or placebo control, and tumor growth was measured for 5 weeks. The effects of the extract on xenografted tumor cell proliferation and apoptosis were measured by in situ BrdU incorporation and TUNEL staining. Rauwolfia extract decreased in vitro cell growth in a dose-dependent manner (p<0.001) and induced the accumulation of G1 phase cells. PARP cleavage demonstrated that apoptosis was induced only at the highest concentration tested (500 microg/ml) which was confirmed by detection of cells containing sub-genomic DNA. The expression of genes associated with DNA damage signaling pathway was up-regulated by Rauwolfia treatment, including that of GADD153 and MDG. The expression of a few cell cycle genes (p21, cyclin D1 and E2F1) was also modulated. These alterations were confirmed by RT-PCR. Tumor volumes were decreased by 60%, 70% and 58% in the groups fed the 75, 37.5 or 7.5 mg/kg Rauwolfia, respectively (Kruskal-Wallis test, p<0.001). The Rauwolfia vomitoria extract significantly suppressed the growth and cell cycle progression of LNCaP cells, in vitro and in vivo.

Complex regulation of human androgen receptor expression by Wnt signaling in prostate cancer cells.

beta-Catenin, a component of the Wnt signaling pathway, is a coactivator of human androgen receptor (hAR) transcriptional activity. Here, we show that Wnt signaling also influences androgen-mediated signaling through its ability to regulate hAR mRNA and protein in prostate cancer (PCa) cells. Three functional LEF-1/TCF binding sites lie within the promoter of the hAR gene as shown by CHIP assays that captured beta-catenin-bound chromatin from Wnt-activated LNCaP cells. Chimeric reporter vectors that use the hAR gene promoter to drive luciferase expression confirmed that these LEF-1/TCF binding elements are able to confer robust upregulation of luciferase expression when stimulated by Wnt-1 or by transfection with beta-catenin and that dominant-negative TCF or mutations within the dominant TCF-binding element abrogated the response. Semi-quantitative and real time RT-PCR assays confirmed that Wnt activation upregulates hAR mRNA in PCa cells. In contrast, hAR protein expression was strongly suppressed by Wnt activation. The reduction of hAR protein is consistent with evidence that Wnt signaling increased phosphorylation of Akt and its downstream target, MDM2 that promotes degradation of hAR protein through a proteasomal pathway. These data indicate that the hAR gene is a direct target of LEF-1/TCF transcriptional regulation in PCa cells but also show that the expression of the hAR protein is suppressed by a degradation pathway regulated by cross-talk of Wnt to Akt that is likely mediated by Wnt-directed degradation of the B regulatory subunit of protein phosphatase, PP2A.

Reduction of wild type p53 function confers a hormone resistant phenotype on LNCaP prostate cancer cells.

BACKGROUND: The protein encoded by the p53 gene is required for some forms of apoptosis and loss or mutations in this gene are found with increased frequency in advanced and hormone resistant human prostate cancers. In order to better appreciate whether reduction of wildtype p53 function in prostate cancer cells might contribute to the development of therapeutic-resistance by these cells, we created stable variants of the androgen-responsive, wild type p53-expressing human prostate cancer cell line, LNCaP, by transfection with expression vectors designed to reduce expression or function of wildtype p53 in them. These cells were then tested for their ability to form tumors in castrated male nude mice. METHODS: A conditional eukaryotic expression vector (under tetracycline regulation) expressing antisense p53 cDNA was constructed and either directly transfected into LNCaP cells or tranduced into these cells using recombinant retroviruses containing the vector. Stably transfected/transduced cells (LNCaP/Asp53) were evaluated by Western blot analysis for the ability of doxycycline to reduce p53 protein expression and for their ability to form tumors in castrated male nude mice treated or untreated with doxycycline. Additionally, we derived an LNCaP subline (LNCaP/DD) stably expressing a dominant-negative form of p53 and tested these cells for their ability to form tumors in castrated male nude mice. RESULTS: LNCaP/Asp53 cells showed reduced expression of p53 protein when cultured in a medium containing doxycycline and tested sublines were able to efficiently form tumors in castrated male nude mice only when the mice were treated with doxycycline. LNCaP/DD cells were readily able to form tumors in castrated male nude mice whereas parental LNCaP cells or control-transfected LNCaP cells were not. CONCLUSION: Loss of wildtype p53 function can contribute to the phenotype of hormone resistance of prostate cancer cells.

Therapeutic potential of curcumin in human prostate cancer. III. Curcumin inhibits proliferation, induces apoptosis, and inhibits angiogenesis of LNCaP prostate cancer cells in vivo.

BACKGROUND: Earlier work from our laboratory highlighted the therapeutic potential of curcumin (turmeric), used as a dietary ingredient and as a natural anti-inflammatory agent in India and other Southeast Asian countries. This agent was shown to decrease the proliferative potential and induce the apoptosis potential of both androgen-dependent and androgen-independent prostate cancer cells in vitro, largely by modulating the apoptosis suppressor proteins and by interfering with the growth factor receptor signaling pathways as exemplified by the EGF-receptor. To extend these observations made in vitro and to study the efficacy of this potential anti-cancer agent in vivo, the growth of LNCaP cells as heterotopically implanted tumors in nude mice was followed. METHODS: The androgen-dependent LNCaP prostate cancer cells were grown, mixed with Matrigel and injected subcutaneously into nude mice. Experimental group received a synthetic diet containing 2% curcumin for up to 6 weeks. At the end point, sections taken from the excised tumors were evaluated for pathology, cell proliferation, apoptosis, and vascularity. RESULTS: Curcumin causes a marked decrease in the extent of cell proliferation as measured by the BrdU incorporation assay and a significant increase in the extent of apoptosis as measured by an in situ cell death assay. Moreover, a significant decrease in the microvessel density as measured by the CD31 antigen staining was also seen. CONCLUSIONS: Curcumin could be a potentially therapeutic anti-cancer agent, as it significantly inhibits prostate cancer growth, as exemplified by LNCaP in vivo, and has the potential to prevent the progression of this cancer to its hormone refractory state.

Biomarker analysis demonstrates a hypoxic environment in the castrated rat ventral prostate gland.

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.

Hormone-refractory prostate cancer: a multi-step and multi-event process.

Since the pioneering studies of Huggins in 1941, it has been known that prostate cancer cells, like certain normal epithelial cells, can chronically depend on a critical level of androgenic stimulation for their continuous growth and survival. The entire issue of the development of resistance to androgen ablation therapy for metastatic prostate cancer is based on the fact that a portion of cells can survive without androgen stimulation. The cell mechanism of androgen independent status is unclear. For some authors, a portion of the cells present within a patient with a prostate cancer before therapy is naturally androgen independent (selection hypothesis). However, this hypothesis does not consider gene alteration during prostate cancer natural history and probably hormone-refractory prostate cancer (HRPC) is due to a multi-step and multi-event process. In this literature review, different cell pathways that lead to HRPC are described.Prostate Cancer and Prostatic Diseases (2001) 4, 204-212.

Reduction of endothelial and smooth muscle density in the corpora cavernosa of the streptozotocin induced diabetic rat.

PURPOSE: Erectile dysfunction is one of the most prevalent complications of diabetes in males. Because adequate vascular perfusion is needed for appropriate erectile tissue function a likely reason for the high incidence of this complication in diabetics is a pathological change associated with the disease in vascularization of erectile tissues. We investigate whether chronic diabetes may induce changes in vascularization of the corpora cavernosa using a computerized image analysis system to quantify changes in the smooth muscle and endothelial cell content of the corpora cavernosa of diabetic rats induced by streptozotocin 6 months previously, and compare these changes to those associated with aging. MATERIALS AND METHODS: We studied 3 groups of rats, including 10-week-old untreated controls, diabetic rats treated with streptozotocin for 6 months starting at age 10 weeks and 18-month-old rats (aged). Penile shafts from these groups were excised, fixed, sectioned and immunostained with anti-smooth muscle actin to identify smooth muscle cells and anti-CD31 to identify endothelial cells. Computerized image analysis was used to quantify the percent area within the corpora cavernosa occupied by smooth muscle cells or endothelial cells, and the data were compared among the groups. RESULTS: We identified a highly significant decrease in the percentage of smooth muscle and endothelial cells within the cavernosa areas of diabetic rats compared to control or aged rats. Mean cavernous smooth muscle cell content was 15.28 +/- 2.54% in control rats and 9.83 +/- 1.21% in diabetic rats (p = 0.0001). Likewise, cavernous endothelial cell content was 6.93 +/- 0.86% in the control group and 4.01 +/- 1.08% in the diabetic group (p = 0. 0001). However, no statistical difference of smooth muscle or endothelial cell content was found between control and aged rats. CONCLUSIONS: Using the streptozotocin treated rat as a model for diabetes, we showed that smooth muscle and endothelial cell density is significantly decreased in diabetic corpora cavernosa but not in normal aged rats. This observation is a further step toward the understanding of the pathomechanisms for diabetic related erectile dysfunction.

The effects of androgen deprivation on the prostate gland: cell death mediated by vascular regression.

Androgenic steroids are required to maintain the prostate gland in the adult state. Consistent with this requirement, androgen deprivation therapies typically induce a drastic regression of mature prostate tissue that is accompanied by the extensive loss of prostate cells through the programmed cell death process referred to as apoptosis. Whereas, in the past, the loss of prostate cells associated with androgen deprivation has generally been perceived to be a direct response of the androgen receptor-expressing prostate cells to an androgen-depleted environment, more recent studies of the prostate regression process suggest that it might instead be initiated by an indirect response of the prostatic parenchyma to an ischemic/hypoxic environment caused by a drastic reduction of blood flow to the tissue that occurs when androgens are withdrawn. This article reviews evidence that the prostatic vascular system is a primary target of androgen action and other evidence suggesting that the regression of the prostate parenchyma occurs secondarily to the regression of the prostate vascular system through cell death mediated by tissue ischemia/hypoxia.

Herbal therapy PC-SPES: in vitro effects and evaluation of its efficacy in 69 patients with prostate cancer.

PURPOSE: We investigate the potential use of the phytotherapeutic PC-SPES to treat human prostate cancer, and evaluate its in vivo and in vitro activity, and clinical efficacy. MATERIALS AND METHODS: PC-SPES was evaluated for its ability to induce apoptosis on prostate cancer cell lines LNCaP, PC3 and DU145. The effect of oral PC-SPES on growth of PC3 tumors present in male immunodeficient mice was studied. A total of 30 male nude mice were divided in 5 groups. In groups 1 control and 2 full dose therapy was started the same day of the tumor injection. In groups 3 control, 4 half dose and 5 full dose PC-SPES therapy was initiated 1 week after tumor injection. A total of 69 patients with prostate cancer were treated with 3 capsules of 320 mg. PC-SPES daily. Serum prostate specific antigen (PSA) responses and side effects were evaluated. RESULTS: All of the cultured prostate cancer cell lines had a significant dose dependent induction of apoptosis following exposure to an alcoholic PC-SPES extract. Immunodeficient mice xenografted with the PC3 cell line had reduced tumor volume compared with sham treated controls when they were treated with a PC-SPES extract from the time of tumor cell implantation (931 +/- 89 versus 1,424 +/- 685 mm.3, p not significant) but not when the treatment was begun 1 week after tumor cell implantation. The testis, prostate, bladder and seminal vesicles of the treated mice were significantly reduced in weight compared with the sham treated animals. Of the patients with prostate cancer 82% had decreased serum PSA 2 months, 78% 6 months and 88% 12 months after treatment with PC-SPES. Side effects in the treated patient population included nipple tenderness in 42% and phlebitis requiring heparinization in 2%. CONCLUSIONS: An extract of the phytotherapeutic agent PC-SPES proved to be active in inducing apoptosis of hormone sensitive and insensitive prostate cancer cells in vitro, and in suppressing the growth rate of a hormone insensitive prostate cancer cell line in vivo. The overwhelming majority of patients with prostate cancer treated with the agent experienced a decrease in serum PSA but also demonstrated a side effect profile comparable to estrogen treatment.

Blood-based RT-PCR assays of MN/CA9 or PSMA: clinical application in renal cancer patients.

OBJECTIVES: To report a survey of blood-based RNAs obtained from common groups of control and renal cancer patients for expression of both MN/CA9 and prostate-specific membrane antigen (PSMA) messenger RNAs. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) assays for MN/CA9 and PSMA were performed on RNAs extracted from 81 blood samples (59 patients with renal cancer, 7 with benign tumors, and 15 control volunteers). The results of these assays were statistically analyzed to determine whether a positive result (individually or combined) correlates with any tumor characteristics. RESULTS: Neither MN/CA9 nor PSMA amplification products were detected in the RNAs from peripheral blood samples of the 15 control volunteers and from the 7 patients with benign renal tumor (sensitivity 100%). MN/CA9 alone was detected in 11 (19%) of 59 samples and PSMA alone in 12 (20%) of 59 samples from patients with renal cancer. PSMA positivity was significantly correlated with vascular invasion of the primary tumor. Expression of one or both of these molecular tumor markers was detected in 21 (36%) of 59 renal cancer patients. When combined, the results of the MN/CA9 and PSMA RT-PCR tests were found to be highly associated with vascular invasion in nephrectomy specimens (sensitivity 67%, specificity 77%, odds ratio = 6.89, P = 0.002). CONCLUSIONS: Combination of RT-PCR assays for MN/CA9 and PSMA provides a sensitive blood test for molecular detection of clear cell carcinoma of the kidney and its potential for vascular invasion. Further testing of this assay will be required to evaluate its efficacy in the diagnosis, screening, and follow-up of patients with kidney cancer.

Vascular response of the rabbit bladder to short term partial outlet obstruction.

Partial bladder outlet obstruction of the rabbit bladder results in a rapid increase in mass characterized by remodeling of the bladder wall. In this study we investigated the effect of partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium, and on blood flow using a fluorescent microsphere technique. Transverse sections of bladder wall were examined after 0 (unobstructed), 1, 3, 5, 7, and 14 days of obstruction. The microvasculature of obstructed rabbit bladder mucosa and detrusor smooth muscle apparently increased relative to augmentation of these compartments, while new vessels appeared in the thickening serosa. These vascular changes correlated with results showing that, at 1 week after obstruction, blood flow (ml/min/g tissue) to the mucosa and detrusor was unchanged. Thickening of the serosa, apparent after 1 day of obstruction, began before its vascularization. Then, 1 week post-obstruction, there was significant microvessel formation in the transition region between the detrusor smooth muscle and the increasing serosa; after 2 weeks, the entire serosa was vascularized. The vascularization of the muscle-serosal transition region and then the remaining serosa apparently precedes fibroblast differentiation, providing blood supply and thus metabolic support for this process. All obstructed rabbit bladders in this study were in a state of compensated function based on their weights. Our working hypothesis is that blood flow per unit tissue mass is normal in compensated obstructed bladders, thus allowing for normal contractile function and cellular metabolism. The results of this study indicate the presence of an augmented microvasculature in compensated obstructed rabbit bladders that provides adequate blood perfusion for normal function.

Is microvascular invasion on radical prostatectomy specimens a useful predictor of PSA recurrence for prostate cancer patients?

OBJECTIVE: Angiogenesis is believed to play an important role in tumor progression and metastasis. The goal of this study was to investigate the clinical utility of vascular invasion in prostate cancer patients treated by radical prostatectomy as a predictor of PSA recurrence. METHODS: Between 1993 and 1998, 241 patients underwent radical prostatectomy at our institution and had routine analysis of vascular and/or lymphatic invasion (V/LI). V/LI was correlated with preoperative parameters including digital rectal examination (DRE), Gleason score (GS) on biopsy and serum PSA, with the pathological findings and with biochemical recurrence. RESULTS: V/LI incidence was 12.4% (30 of 241 patients). Of the 30 patients with V/LI, 28 (93%) had GS > or =7 (67%) had a pT3 disease and 7 had SV invasion (23%). V/LI was not associated with DRE and GS on prostate needle biopsy. However, V/LI was correlated with the worst pathological findings including pT3 disease, seminal invasion, positive surgical margins and GS on prostate specimen > or = 7. Biochemical recurrence-free survival was 92.5% for the patients without V/LI as compared to 30.1% for patients with V/LI on prostate specimen examination (p = 0.0001). Multivariate analysis showed that preoperative serum PSA, Stage and V/LI were independent predictors of PSA recurrence. Patients with pT2 disease without V/LI had a biochemical recurrence-free survival of 99 vs. 31% in patients with V/LI (p = 0.0001). CONCLUSION: This study demonstrated that V/LI is strongly associated with biochemical recurrence after radical prostatectomy. The routine analysis of V/LI should be considered as a routine evaluation of the radical prostatectomy specimen.

Prostatic neoplasia in transgenic mice with prostate-directed overexpression of the c-myc oncoprotein.

BACKGROUND: Promoter elements within the 5' DNA region of the rat C(3)1 gene have been shown to direct prostate-specific expression of gene products when they are fused through recombinant DNA procedures and used to produce transgenic mice. In order to test the in vivo effects of chronic overexpression of the mouse c-myc protooncogene on the prostate glands of transgenic mice, we created several lines of C(3)1-c-myc transgenic mice and then examined the phenotype of males with this genetic alteration. METHODS: The modified promoter and 5' region of the rat C(3)1 gene was fused to the coding region of the mouse c-myc gene using recombinant DNA techniques. This DNA was used to create three different founder lines of transgenic mice. Tissues from males and females heterozygous for the transgene were examined for expression of the recombinant mouse c-myc mRNA by an RNase protection assay. Prostates from males were examined for expression of recombinant c-myc mRNA by in situ hybridization. Thin sections of fixed ventral prostates from males were analyzed by microscopy for histological abnormalities. RESULTS: Three different lines of transgenic mice were obtained from these procedures. These mice demonstrated expression of recombinant mouse c-myc mRNA in the testis and ventral prostates of males and in the uterus of females. In situ hybridization demonstrated that the epithelial cells were the source of recombinant c-myc expression in the ventral prostates of the transgenic lines. Microscopic analysis of the ventral prostates from these mice demonstrated abnormalities in epithelial cell morphology seemingly typical of an intraepithelial neoplasia-like phenotype. However, none of the males of any of the lines developed overt prostatic adenocarcinoma over their lifetimes. CONCLUSIONS: Chronic overexpression of c-myc in the ventral prostate epithelial cells of C3(1)-c-myc transgenic mice leads to the development of epithelial cell abnormalities similar to those seen in low-grade prostatic intraepithelial neoplasia in humans. These abnormalities were not found to progress to adenocarcinoma over the lifetimes of the transgenic mice, suggesting the need for additional oncogenic changes in the pathway to prostatic adenocarcinomas. Furthermore, our cumulative experience with the use of the C3(1) gene promoter in the generation of transgenic mice suggests that the probasin promoter element provides a much more specific and effective means to target transgenes to the prostate glands of mice.

Expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia.

OBJECTIVES: Cellular senescence is a unique cellular response pathway thought to be closely associated with the aging process. The senescent phenotype is characterized by the loss of a cell's ability to respond to proliferative and apoptotic stimuli even while normal metabolic activity and vitality is maintained. Recently, a novel biomarker, senescent-associated beta-galactosidase (SA-beta-gal), was found to identify cells with the senescent phenotype. In the present study, we examined whether human prostatic epithelial cells adopt a senescence-associated phenotype after prolonged culture and analyzed a series of human benign prostatic hyperplasia (BPH) specimens to determine whether the cellular senescence process might be a factor in the development of BPH. METHODS: A primary culture of epithelial cells was established from the normal tissue of the peripheral zone of a radical prostatectomy specimen and was serially passaged until senescence. Forty-three human prostate specimens were obtained subsequent to radical prostatectomy or transrectal ultrasound-guided biopsy. The cultured cells and tissue specimens were histochemically stained to reveal the expression of SA-beta-gal, the cellular senescence biomarker. RESULTS: As has been reported for other types of cultured cells, human prostatic epithelial cells demonstrated widespread expression of the cellular senescence marker, SA-beta-gal, on prolonged culture. In our survey of hypertrophied human prostate tissues, 17 specimens (40%) of the 43 analyzed demonstrated positive staining for SA-beta-gal. In these tissues, SA-beta-gal expression was noted only in the epithelial cells. No statistical correlation (P = 0.42) between the chronologic age of the patient donor and SA-beta-gal expression was found. However, a high prostate weight (greater than 55 g) was found to correlate strongly with the expression of the SA-beta-gal biomarker (P = 0. 0001). CONCLUSIONS: Cultured prostatic epithelial cells expressed SA-beta-gal on reaching replicative senescence in vitro. The survey of human BPH specimens for the senescent marker showed that prostatic epithelial cells in patients with BPH with more advanced enlargement of the prostate (greater than 55 g prostate weight) expressed SA-beta-gal, and the prostates from patients with BPH that weighed less than 55 g tended to lack senescent epithelial cells. On the basis of these results, we propose that the accumulation of senescent epithelial cells may play a role in the development of the prostatic enlargement associated with BPH.

Biomarkers of renal cell carcinoma. Past and future considerations.

Renal cancer includes several distinct entities with a range of biologic and clinical behaviors from relatively indolent to extremely aggressive tumors. Although conventional prognostic factors such as stage and grade are quite useful, other clinical, laboratory, and pathologic findings are now believed to have additional predictive values. This article reviews the literature on the potential utility of biomarkers in renal cell carcinoma. To date, only a few biomarkers, such as Ki-67, appeared to be potentially useful for monitoring renal cancer patients. New biomarkers including MN/CA9 and circulating cell detection require further and extensive studies to assess their potential clinical utility.

Vascular endothelial growth factor-A expression in the rat ventral prostate gland and the early effects of castration.

BACKGROUND: Blood flow to the rat ventral prostate gland is drastically reduced during the very early period after castration, and this reduction coincides with the appearance of striking degenerative changes within the prostatic vascular system. These early effects on the prostate vascular system are likely to be important for the subsequent regression of the ventral prostate that occurs in response to castration. Since the endothelial cells of the ventral prostate do not express androgen receptor protein (AR), we proposed that these early effects might be indirectly mediated by changes in the local expression of vascular regulatory factors. In order to evaluate whether vascular endothelial growth factor-A (VEGF-A) might be among the primary mediators of these effects, we measured expression of VEGF-A mRNA and protein in the rat ventral prostate gland prior to and within the first 3 days after castration. METHODS: Ventral prostate tissues were obtained from control (unoperated) rats, sham-operated rats, or rats at sequential daily intervals (1-3 days) after castration. A quantitative RNase protection assay and a comparative RT-PCR assay were used to evaluate the extent to which the expression of VEGF-A mRNA in the ventral prostate was affected by castration. In situ immunohistochemistry, using an anti-VEGF-A antibody, was performed to localize VEGF-A protein in the various cells of the tissue. Western blot analysis and a quantitative ELISA assay using anti-VEGF-A antibodies were performed to determine how VEGF-A protein expression in the rat ventral prostate was affected by castration. RESULTS: Results of VEGF-A mRNA analysis in the rat ventral prostate gland during the first 3 days after castration showed a biphasic change characterized by a transient reduction of VEGF-A mRNA expression (by approximately 50%) on the second day after castration that was restored to higher than control levels by the third day after castration. Immunohistochemical analysis for VEGF-A in control and castrated ventral prostates showed that the prostatic epithelial and smooth muscle cells were the major source of VEGF-A expression in this tissue. Quantitative analysis of VEGF-A protein expression by Western blot and ELISA methods confirmed a biphasic change in the expression of the polypeptide that correlated well with the results of the mRNA analyses. CONCLUSIONS: VEGF-A expression in the ventral prostate gland of the Sprague-Dawley rat is downregulated on the second day after castration but returns to control levels by the third day after castration. Since critical changes in the ventral prostate vascular system are already evident by 1 day after castration, we believe that these findings indicate that VEGF-A is not likely to be the critical or sole mediator of the early effects of castration on the vascular system of the rat ventral prostate gland.