Bio

Academic Appointments


Honors & Awards


  • Cardura International Competitive Award, Pfizer (2000/2001)
  • Prostate Cancer Research Award, Prostate Cancer Foundation (2003, 2008)
  • Prostate Cancer Research Award, CaP CURE (1993,1995,1996,1997,1999,2000)

Professional Education


  • B.S., Stanford University, Biology (1974)
  • Ph.D., University of Colorado, biology (1979)

Research & Scholarship

Current Research and Scholarly Interests


The overall goal of my research is to develop realistic experimental models of benign and malignant prostatic diseases. Both benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are major medical problems, causing significant morbidity and mortality. My lab has developed techniques to establish primary cultures of epithelial and stromal cells from normal, benign or malignant human prostatic tissues, and we have used these cell cultures to investigate many aspects of the molecular and cellular biology of the prostate. More recently, we have established “tissue slice cultures” (TSCs), which are live, precision-cut thin sections of tissues that can be maintained in culture for several days. TSCs are perhaps the most representative experimental model of the human prostate available, containing almost all of the cells typically present in the body and maintaining essential epithelial-stromal interactions as well as differentiated cells, which are typically lost in mono-culture. TSCs can also be established as grafts under the renal capsule of mice, providing an in vivo model of the benign and malignant prostate. In addition to our cell and tissue models, our research takes advantage of the archival patient materials available in the Department of Urology. These include tissues from a well-characterized series of radical prostatectomy specimens and a serum bank. Using these diverse models and resources, we carry out studies related to diagnosis, prognosis and treatment of PCa. Currently, some of our projects include: 1) investigating the association of a splice-variant of the androgen receptor with aggressive PCa, 2) studying cell surface proteoglycans as novel and specific biomarkers of PCa, 3) searching for serum autoantibodies or proteins that could be used to diagnose PCa, 4) creating induced pluripotent stem (iPS) cells from PCa cells as a novel model to characterize cancer-related methylation, 5) determining the role of monoamine oxidase A (MAOA) in normal prostate differentiation and as a therapeutic target, 6) using TSCs to screen phage display libraries to discover PCa-specific cell surface molecules for cancer-specific targeting, 7) developing hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging for rapid evaluation of therapeutic response, 8) creating additional cell culture models of metastatic PCa, and 9) testing the efficacy of a novel organic arsenical compound against PCa. These projects are funded in part by the NIH, the DoD, and the Prostate Cancer Foundation.

Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate. Laboratory investigation; a journal of technical methods and pathology Maund, S. L., Nolley, R., Peehl, D. M. 2014; 94 (2): 208-221

    Abstract

    Few preclinical models accurately depict normal human prostate tissue or primary prostate cancer (PCa). In vitro systems typically lack complex cellular interactions among structured prostatic epithelia and a stromal microenvironment, and genetic and molecular fidelity are concerns in both in vitro and in vivo models. 'Tissue slice cultures' (TSCs) provide realistic preclinical models of diverse tissues and organs, but have not been fully developed or widely utilized for prostate studies. Problems encountered include degeneration of differentiated secretory cells, basal cell hyperplasia, and poor survival of PCa. Here, we optimized, characterized, and applied a TSC model of primary human PCa and benign prostate tissue that overcomes many deficiencies of current in vitro models. Tissue cores from fresh prostatectomy specimens were precision-cut at 300 μm and incubated in a rotary culture apparatus. The ability of varied culture conditions to faithfully maintain benign and cancer cell and tissue structure and function over time was evaluated by immunohistological and biochemical assays. After optimization of the culture system, molecular and cellular responses to androgen ablation and to piperlongumine (PL), purported to specifically reduce androgen signaling in PCa, were investigated. Optimized culture conditions successfully maintained the structural and functional fidelity of both benign and PCa TSCs for 5 days. TSCs exhibited androgen dependence, appropriately undergoing ductal degeneration, reduced proliferation, and decreased prostate-specific antigen expression upon androgen ablation. Further, TSCs revealed cancer-specific reduction of androgen receptor and increased apoptosis upon treatment with PL, validating data from cell lines. We demonstrate a TSC model that authentically recapitulates the structural, cellular, and genetic characteristics of the benign and malignant human prostate, androgen dependence of the native tissue, and cancer-specific response to a potentially new therapeutic for PCa. The work described herein provides a basis for advancing the experimental utility of the TSC model.

    View details for DOI 10.1038/labinvest.2013.141

    View details for PubMedID 24296879

  • Recent advances in the development of animal and cell culture models for prostate cancer research A minireview. Urologic oncology Bosland, M. C., Chung, L. W., Greenberg, N. M., Ho, S. M., Isaacs, J. T., Lane, K., Peehl, D. M., Thompson, T. C., van Steenbrugge, G. J., van Weerden, W. M. ; 2 (4): 99

    View details for PubMedID 21224146

  • Transcription Factor KLLN Inhibits Tumor Growth by AR Suppression, Induces Apoptosis by TP53/TP73 Stimulation in Prostate Carcinomas, and Correlates With Cellular Differentiation JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Wang, Y., Radhakrishnan, D., He, X., Peehl, D. M., Eng, C. 2013; 98 (3): E586-E594

    Abstract

    KLLN is a newly identified gene with unknown function and shares a bidirectional promoter with PTEN.The objective of the study was to analyze the relationship between KILLIN (KLLN) expression and prostate cancer and the potential tumor suppressive effect.We conducted an in silico analysis to compare KLLN expression in normal prostate and matched primary carcinoma tissues. We subsequently used immunohistochemistry to examine KLLN expression and association with Gleason grade and score in 109 prostatectomy samples. KLLN's tumor-suppressive effect was studied in androgen-dependent and androgen-independent cell models.Patients were diagnosed with peripheral zone prostate carcinomas without metastasis at the time of prostatectomy. Each patient's primary tumor comprised at least 2 tumoral regions with different Gleason grades.KLLN expression decreased from normal prostate tissue to primary carcinomas (P < .0001). The loss of epithelial and stromal KLLN expression is associated with poor differentiation and high Gleason scores (P < .0001), consistent with our in vitro observation that KLLN inhibits tumor cell proliferation and invasiveness. KLLN decreases prostate-specific antigen levels and suppresses androgen-mediated cell growth by inhibiting androgen receptor (AR) transcription. As an androgen receptor-regulated target, KLLN also functions as a transcriptional activator, directly promoting the expression of TP53 and TP73, with consequent elevated apoptosis, regardless of AR status.Our observations suggest that KLLN is a transcription factor directly regulating AR, TP53, and TP73 expression, with a role in prostate carcinogenesis. Loss of KLLN associates with high Gleason scores, suggesting that KLLN might be used as a potential prognostic marker for risk management and as a novel therapy target for advanced prostate carcinomas.

    View details for DOI 10.1210/jc.2012-3490

    View details for Web of Science ID 000316417200022

    View details for PubMedID 23386643

  • Establishment and serial passage of cell cultures derived from LuCaP xenografts. The Prostate Young, S. R., Saar, M., Santos, J., Nguyen, H. M., Vessella, R. L., Peehl, D. M. 2013

    Abstract

    BACKGROUND: LuCaP serially transplantable xenografts derived from primary and metastatic human prostate cancer encompass the molecular and cellular heterogeneity of the disease and are an invaluable resource for in vivo preclinical studies. A limitation of this model, however, has been the inability to establish and passage cell cultures derived from the xenografts. Here, we describe a novel spheroid culture system that supports long-term growth of LuCaP cells in vitro. METHODS: Xenografts were minced and digested with collagenase. Tissue dissociation was terminated while the majority of cells remained as clusters rather than single cells. The cell clusters were suspended in StemPro medium supplemented with R1881 and Y-27632, a Rho kinase inhibitor, and placed in ultralow attachment dishes for spheroid culture. Serial passage was achieved by partial digestion to small clusters with trypsin/EDTA in the presence of Y-27632. Cell viability, growth and phenotype were monitored with LIVE/DEAD®, MTS, qRT-PCR, and immunocytochemical assays. RESULTS: Cells from six LuCaP xenografts formed proliferating spheroids that were serially passaged a minimum of three times and cryopreserved. Two of the cell lines, LuCaP 136 and LuCaP 147, were further passaged and characterized. Both expressed biomarkers characteristic of the xenografts of origin, were determined to be of independent origin by STR fingerprinting, and were free of mycoplasma. LuCaP 147 formed tumors similar to the original xenograft when injected into mice. CONCLUSIONS: The ability to culture LuCaP cells affords new opportunities for fast, cheap, and efficient preclinical studies and extends the value of the LuCaP xenograft models. Prostate 9999: XX-XX, 2013. © 2013 Wiley Periodicals, Inc.

    View details for PubMedID 23740600

  • Induced pluripotency of human prostatic epithelial cells. PloS one Zhao, H., Sun, N., Young, S. R., Nolley, R., Santos, J., Wu, J. C., Peehl, D. M. 2013; 8 (5)

    Abstract

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.

    View details for DOI 10.1371/journal.pone.0064503

    View details for PubMedID 23717621

  • Transcript Levels of Androgen Receptor Variant AR-V1 or AR-V7 Do Not Predict Recurrence in Patients with Prostate Cancer at Indeterminate Risk for Progression JOURNAL OF UROLOGY Zhao, H., Coram, M. A., Nolley, R., Reese, S. W., Young, S. R., Peehl, D. M. 2012; 188 (6): 2158-2164

    Abstract

    AR-V7, a ligand independent splice variant of androgen receptor, may support the growth of castration resistant prostate cancer and have prognostic value. Another variant, AR-V1, interferes with AR-V7 activity. We investigated whether AR-V7 or V1 expression would predict biochemical recurrence in men at indeterminate (about 50%) risk for progression following radical prostatectomy.AR-V7 and V1 transcripts in a mixed grade cohort of 53 men in whom cancer contained 30% to 70% Gleason grade 4/5 and in a grade 3 only cohort of 52 were measured using a branched chain DNA assay. Spearman rank correlations of the transcripts, and histomorphological and clinical variables were determined. AR-V7 and V1 levels were assessed as determinants of recurrence in the mixed grade cohort by logistic regression and survival analysis. The impact of TMPRSS2-ERG gene fusion on prognosis was also evaluated.Neither AR-V7 nor V1 levels in grade 3 or 4/5 cancer in the mixed grade cohort were associated with recurrence or time to recurrence. However, AR-V7 and V1 inversely correlated with serum prostate specific antigen and positively correlated with age. The AR-V1 level in grade 3 cancer in the grade 3 only cohort was higher than in grade 3 or grade 4/5 components of mixed grade cancer. TMPRSS2-ERG fusion was not associated with AR-V7, AR-V1 or recurrence but it was associated with the percent of grade 4/5 cancer.The AR-V1 or V7 transcript level does not predict recurrence in patients with high grade prostate cancer at indeterminate risk for progression. Grade 3 cancer in mixed grade tumors may differ from 100% grade 3 cancer, at least in AR-V1 expression.

    View details for DOI 10.1016/j.juro.2012.08.014

    View details for Web of Science ID 000311581400035

    View details for PubMedID 23088973

  • DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer GENOME RESEARCH Kobayashi, Y., Absher, D. M., Gulzar, Z. G., Young, S. R., McKenney, J. K., Peehl, D. M., Brooks, J. D., Myers, R. M., Sherlock, G. 2011; 21 (7): 1017-1027

    Abstract

    Candidate gene-based studies have identified a handful of aberrant CpG DNA methylation events in prostate cancer. However, DNA methylation profiles have not been compared on a large scale between prostate tumor and normal prostate, and the mechanisms behind these alterations are unknown. In this study, we quantitatively profiled 95 primary prostate tumors and 86 benign adjacent prostate tissue samples for their DNA methylation levels at 26,333 CpGs representing 14,104 gene promoters by using the Illumina HumanMethylation27 platform. A 2-class Significance Analysis of this data set revealed 5912 CpG sites with increased DNA methylation and 2151 CpG sites with decreased DNA methylation in tumors (FDR < 0.8%). Prediction Analysis of this data set identified 87 CpGs that are the most predictive diagnostic methylation biomarkers of prostate cancer. By integrating available clinical follow-up data, we also identified 69 prognostic DNA methylation alterations that correlate with biochemical recurrence of the tumor. To identify the mechanisms responsible for these genome-wide DNA methylation alterations, we measured the gene expression levels of several DNA methyltransferases (DNMTs) and their interacting proteins by TaqMan qPCR and observed increased expression of DNMT3A2, DNMT3B, and EZH2 in tumors. Subsequent transient transfection assays in cultured primary prostate cells revealed that DNMT3B1 and DNMT3B2 overexpression resulted in increased methylation of a substantial subset of CpG sites that showed tumor-specific increased methylation.

    View details for DOI 10.1101/gr.119487.110

    View details for Web of Science ID 000292298000003

    View details for PubMedID 21521786

  • Serum Mac-2BP Does Not Distinguish Men With High Grade, Large Volume Prostate Cancer From Men With Benign Prostatic Hyperplasia PROSTATE Peehl, D. M., Chen, Z., Nolley, R. 2011; 71 (1): 26-31

    Abstract

    Mac-2 binding protein (Mac-2BP) is a secreted protein that has been used as a serum prognostic marker for several types of cancers. A previous study showed that serum Mac-2BP was significantly higher (?2-fold) in men with prostate cancer compared to healthy men. We investigated whether serum Mac-2BP could distinguish men with high grade, large volume prostate cancer from men with benign prostatic hyperplasia (BPH).A commercially available ELISA kit was used to measure Mac-2BP in paired pre- and post-prostatectomy sera from 10 men with high grade, large volume prostate cancer, in pre-operative sera from 50 untreated men with high grade, large volume prostate cancer, and in sera from 50 men with clinical symptoms of BPH and biopsy-negative for prostate cancer. Results were analyzed by Student's t-test and receiver operating characteristic (ROC) curves.Levels of Mac-2BP did not decrease in post-prostatectomy sera, and Mac-2BP values were not significantly different in the sera of men with prostate cancer versus those with BPH.Serum Mac-2BP does not appear to originate in the prostate and it is unlikely that Mac-2BP can be used for the differential diagnosis of prostate cancer versus BPH.

    View details for DOI 10.1002/pros.21218

    View details for Web of Science ID 000285849000003

    View details for PubMedID 20583127

  • Anti-tumor and radiosensitization activities of the iron chelator HDp44mT are mediated by effects on intracellular redox status CANCER LETTERS Tian, J., Peehl, D. M., Zheng, W., Knox, S. J. 2010; 298 (2): 231-237

    Abstract

    A novel iron chelator, HDp44mT, has been reported to have potent anti-proliferative effects on cancer cells; however, the underlying mechanism of action is not well understood. In this study, we characterized the cytotoxic effect of HDp44mT in a chemo- and radio-resistant cell line (PC-3) of prostatic cancer origin. The activity of HDp44mT at nM concentrations was dependent on the intracellular GSH and atmospheric O(2) concentration, rather than iron deprivation. HDp44mT also radiosensitized PC-3 cells in a GSH-dependent manner. Interestingly, this radiosensitizing effect was observed under aerobic and, to a larger extent, hypoxic conditions, suggesting its potential utility as a radiosensitizer for some radioresistant tumors.

    View details for DOI 10.1016/j.canlet.2010.07.010

    View details for Web of Science ID 000284300000011

    View details for PubMedID 20678860

  • Targeting monoamine oxidase A in advanced prostate cancer JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY Flamand, V., Zhao, H., Peehl, D. M. 2010; 136 (11): 1761-1771

    Abstract

    Inhibitors of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades neurotransmitters including serotonin and norepinephrine, are commonly used to treat neurological conditions including depression. Recently, we and others identified high expression of MAOA in normal basal prostatic epithelium and high-grade primary prostate cancer (PCa). In contrast, MAOA is low in normal secretory prostatic epithelium and low-grade PCa. An irreversible inhibitor of MAOA, clorgyline, induced secretory differentiation in primary cultures of normal basal epithelial cells and high-grade PCa. Furthermore, clorgyline inhibited several oncogenic pathways in PCa cells, suggesting clinical value of MAOA inhibitors as a pro-differentiation and anti-oncogenic therapy for high-risk PCa. Here, we extended our studies to a model of advanced PCa, VCaP cells, which were derived from castration-resistant metastatic PCa and express a high level of MAOA.Growth of VCaP cells in the presence or absence of clorgyline was evaluated in vitro and in vivo. Gene expression changes in response to clorgyline were determined by microarray and validated by quantitative real-time polymerase chain reaction.Treatment with clorgyline in vitro inhibited growth and altered the transcriptional pattern of VCaP cells in a manner consistent with the pro-differentiation and anti-oncogenic effects seen in treated primary PCa cells. Src, beta-catenin, and MAPK oncogenic pathways, implicated in androgen-independent growth and metastasis, were significantly downregulated. Clorgyline treatment of mice bearing VCaP xenografts slowed tumor growth and induced transcriptome changes similar to those noted in vitro.Our results support the possibility that anti-depressant drugs that target MAOA might find a new application in treating PCa.

    View details for DOI 10.1007/s00432-010-0835-6

    View details for Web of Science ID 000282183700016

    View details for PubMedID 20204405

  • Metalloporphyrin Synergizes with Ascorbic Acid to Inhibit Cancer Cell Growth Through Fenton Chemistry CANCER BIOTHERAPY AND RADIOPHARMACEUTICALS Tian, J., Peehl, D. M., Knox, S. J. 2010; 25 (4): 439-448

    Abstract

    Ascorbic acid (AA) has been reported to inhibit tumor cell growth through the generation of extracellular hydrogen peroxide (H(2)O(2)). However, the clinical utility of AA has been limited by relatively low potency and in vivo efficacy. This study reports that the metalloporphyrin, Mn(III) tetrakis(N-methylpyridinium-2-yl)porphyrin(5+) (MnTMPyP), has a potent synergistic cytotoxic effect when combined with AA in a variety of cancer cell lines. In the presence of MnTMPyP, the concentration of AA required to inhibit cancer cell growth was markedly reduced. In vitro (cell-free) experiments demonstrated that AA alone enhanced the Fenton reaction that produces cytotoxic hydroxyl radical (HO(*)); however, this reaction was limited by the low rate by which AA generates H(2)O(2) (Fenton reaction substrate) from O(2). MnTMPyP catalyzed H(2)O(2) generation through the AA-facilitated Mn(II <--> III)TMPyP redox cycle and thereby markedly potentiated the Fenton reaction. Accordingly, MnTMPyP and AA resulted in increased cellular levels of H(2)O(2) and HO(*) in cancer cells, which mediate the synergistic cytotoxicity of this combined treatment. This effect was inhibited by cellular enzymes that metabolize H(2)O(2), such as catalase and glutathione peroxidase, suggesting that selective killing of cancer cells deficient in such enzymes can be achieved in vivo.

    View details for DOI 10.1089/cbr.2009.0756

    View details for Web of Science ID 000281255100008

    View details for PubMedID 20735206

  • Tissue Slice Grafts An in Vivo Model of Human Prostate Androgen Signaling AMERICAN JOURNAL OF PATHOLOGY Zhao, H., Nolley, R., Chen, Z., Peehl, D. M. 2010; 177 (1): 229-239

    Abstract

    We developed a tissue slice graft (TSG) model by implanting thin, precision-cut tissue slices derived from fresh primary prostatic adenocarcinomas under the renal capsule of immunodeficient mice. This new in vivo model not only allows analysis of approximately all of the cell types present in prostate cancer within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Both benign and cancer tissues displayed characteristic histology and expression of cell-type specific markers for up to 3 months. Moreover, androgen-regulated protein expression diminished in TSGs after androgen ablation of the host and was restored after androgen repletion. Finally, many normal secretory epithelial cells and cancer cells in TSGs remained viable 2 months after androgen ablation, consistent with similar observations in postprostatectomy specimens following neoadjuvant androgen ablation. Among these were putative Nkx3.1(+) stem cells. Our novel TSG model has the appropriate characteristics to serve as a useful tool to model all stages of disease, including normal tissue, premalignant lesions, well-differentiated cancer, and poorly differentiated cancer.

    View details for DOI 10.2353/ajpath.2010.090821

    View details for Web of Science ID 000279805100025

    View details for PubMedID 20472887

  • Methods for Metabolic Evaluation of Prostate Cancer Cells Using Proton and C-13 HR-MAS Spectroscopy and [3-C-13] Pyruvate as a Metabolic Substrate MAGNETIC RESONANCE IN MEDICINE Levin, Y. S., Albers, M. J., Butler, T. N., Spielman, D., Peehl, D. M., Kurhanewicz, J. 2009; 62 (5): 1091-1098

    Abstract

    Prostate cancer has been shown to undergo unique metabolic changes associated with neoplastic transformation, with associated changes in citrate, alanine, and lactate concentrations. (13)C high resolution-magic angle spinning (HR-MAS) spectroscopy provides an opportunity to simultaneously investigate the metabolic pathways implicated in these changes by using (13)C-labeled substrates as metabolic probes. In this work, a method to reproducibly interrogate metabolism in prostate cancer cells in primary culture was developed using HR-MAS spectroscopy. Optimization of cell culture protocols, labeling parameters, harvesting, storage, and transfer was performed. Using [3-(13)C] pyruvate as a metabolic probe, (1)H and (13)C HR-MAS spectroscopy was used to quantify the net amount and fractional enrichment of several labeled metabolites that evolved in multiple cell samples from each of five different prostate cancers. Average enrichment across all cancers was 32.4 +/- 5.4% for [3-(13)C] alanine, 24.5 +/- 5.4% for [4-(13)C] glutamate, 9.1 +/- 2.5% for [3-(13)C] glutamate, 25.2 +/- 5.7% for [3-(13)C] aspartate, and 4.2 +/- 1.0% for [3-(13)C] lactate. Cell samples from the same parent population demonstrated reproducible fractional enrichments of alanine, glutamate, and aspartate to within 12%, 10%, and 10%, respectively. Furthermore, the cells produced a significant amount of [4-(13)C] glutamate, which supports the bioenergetic theory for prostate cancer. These methods will allow further characterization of metabolic properties of prostate cancer cells in the future. Magn Reson Med, 2009. (c) 2009 Wiley-Liss, Inc.

    View details for DOI 10.1002/mrm.22120

    View details for Web of Science ID 000271431200001

    View details for PubMedID 19780158

  • Anti-oncogenic and pro-differentiation effects of clorgyline, a monoamine oxidase A inhibitor, on high grade prostate cancer cells BMC MEDICAL GENOMICS Zhao, H., Flamand, V., Peehl, D. M. 2009; 2

    Abstract

    Monoamine oxidase A (MAO-A), a mitochondrial enzyme that degrades monoamines including neurotransmitters, is highly expressed in basal cells of the normal human prostatic epithelium and in poorly differentiated (Gleason grades 4 and 5), aggressive prostate cancer (PCa). Clorgyline, an MAO-A inhibitor, induces secretory differentiation of normal prostate cells. We examined the effects of clorgyline on the transcriptional program of epithelial cells cultured from high grade PCa (E-CA).We systematically assessed gene expression changes induced by clorgyline in E-CA cells using high-density oligonucleotide microarrays. Genes differentially expressed in treated and control cells were identified by Significance Analysis of Microarrays. Expression of genes of interest was validated by quantitative real-time polymerase chain reaction.The expression of 156 genes was significantly increased by clorgyline at all time points over the time course of 6 - 96 hr identified by Significance Analysis of Microarrays (SAM). The list is enriched with genes repressed in 7 of 12 oncogenic pathway signatures compiled from the literature. In addition, genes downregulated >or= 2-fold by clorgyline were significantly enriched with those upregulated by key oncogenes including beta-catenin and ERBB2, indicating an anti-oncogenic effect of clorgyline. Another striking effect of clorgyline was the induction of androgen receptor (AR) and classic AR target genes such as prostate-specific antigen together with other secretory epithelial cell-specific genes, suggesting that clorgyline promotes differentiation of cancer cells. Moreover, clorgyline downregulated EZH2, a critical component of the Polycomb Group (PcG) complex that represses the expression of differentiation-related genes. Indeed, many genes in the PcG repression signature that predicts PCa outcome were upregulated by clorgyline, suggesting that the differentiation-promoting effect of clorgyline may be mediated by its downregulation of EZH2.Our results suggest that inhibitors of MAO-A, already in clinical use to treat depression, may have potential application as therapeutic PCa drugs by inhibiting oncogenic pathway activity and promoting differentiation.

    View details for DOI 10.1186/1755-8794-2-55

    View details for Web of Science ID 000272814900001

    View details for PubMedID 19691856

  • Tumor-Promoting Phenotype of CD90(hi) Prostate Cancer-Associated Fibroblasts PROSTATE Zhao, H., Peehl, D. M. 2009; 69 (9): 991-1000

    Abstract

    Cancer-associated stroma contributes to the malignant behavior of adenocarcinomas of the prostate and other organs. CD90 is a marker of mesenchymal stem cells (MSCs) and its expression is higher in prostate cancer stroma compared to normal tissue. Cultured prostate cancer-associated fibroblasts (CAFs) expressing high versus low levels of CD90 were analyzed for an MSC-like or tumor-promoting phenotype.CD90(hi) and CD90(lo) cells were collected by fluorescence-activated cell sorting (FACS). Expression of genes associated with MSCs and/or tumor-promoting activities was measured by quantitative polymerase chain reaction (qPCR). Effects of stromal cell co-culture or conditioned media were tested on BPH-1 epithelial cells.The pattern of gene expression did not support the hypothesis that CD90(hi) cells were MSCs. However, CD90(hi) cells expressed higher levels of many genes associated with tumor promotion, including cytokines, angiogenic factors, hedgehog signaling components, and transforming growth factor (TGF)-beta. Co-culture or conditioned medium from CD90(hi) cells increased CXCR4 expression in BPH-1 cells, at least in part due to TGF-beta, and protected BPH-1 cells from apoptosis.Our results suggest that the elevated expression of CD90 previously observed in the cancer-associated stroma of the human prostate is biologically significant. Although our results do not support the idea that CD90(hi) cells cultured from the cancer stroma are MSCs, our findings suggest that the phenotype of these cells is more tumor-promoting than that of cells expressing low CD90.

    View details for DOI 10.1002/pros.20946

    View details for Web of Science ID 000266470700010

    View details for PubMedID 19267366

  • Inhibition of prostaglandin synthesis and actions by genistein in human prostate cancer cells and by soy isoflavones in prostate cancer patients INTERNATIONAL JOURNAL OF CANCER Swami, S., Krishnan, A. V., Moreno, J., Bhattacharya, R. S., Gardner, C., Brooks, J. D., Peehl, D. M., Feldman, D. 2009; 124 (9): 2050-2059

    Abstract

    Soy and its constituent isoflavone genistein inhibit the development and progression of prostate cancer (PCa). Our study in both cultured cells and PCa patients reveals a novel pathway for the actions of genistein, namely the inhibition of the synthesis and biological actions of prostaglandins (PGs), known stimulators of PCa growth. In the cell culture experiments, genistein decreased cyclooxygenase-2 (COX-2) mRNA and protein expression in both human PCa cell lines (LNCaP and PC-3) and primary prostate epithelial cells and increased 15-hydroxyprostaglandin dehydrogenase (15-PGDH) mRNA levels in primary prostate cells. As a result genistein significantly reduced the secretion of PGE(2) by these cells. EP4 and FP PG receptor mRNA were also reduced by genistein, providing an additional mechanism for the suppression of PG biological effects. Further, the growth stimulatory effects of both exogenous PGs and endogenous PGs derived from precursor arachidonic acid were attenuated by genistein. We also performed a pilot randomised double blind clinical study in which placebo or soy isoflavone supplements were given to PCa patients in the neo-adjuvant setting for 2 weeks before prostatectomy. Gene expression changes were measured in the prostatectomy specimens. In PCa patients ingesting isoflavones, we observed significant decreases in prostate COX-2 mRNA and increases in p21 mRNA. There were significant correlations between COX-2 mRNA suppression, p21 mRNA stimulation and serum isoflavone levels. We propose that the inhibition of the PG pathway contributes to the beneficial effect of soy isoflavones in PCa chemoprevention and/or treatment.

    View details for DOI 10.1002/ijc.24161

    View details for Web of Science ID 000264647600007

    View details for PubMedID 19127598

  • The Significance of Monoamine Oxidase-A Expression in High Grade Prostate Cancer JOURNAL OF UROLOGY Peehl, D. M., Coram, M., Khine, H., Reese, S., Nolley, R., Zhao, H. 2008; 180 (5): 2206-2211

    Abstract

    Gleason grade 4/5 prostate cancer is a determinant for recurrence following radical prostatectomy. Monoamine oxidase-A is over expressed in grade 4/5 compared to grade 3 cancer. Monoamine oxidase-A is also expressed by normal basal cells and in vitro studies suggest that its function is to repress secretory differentiation. Therefore, monoamine oxidase-A in grade 4/5 cancer might reflect dedifferentiation to a basal cell-like phenotype. We investigated whether monoamine oxidase-A expression correlates with another basal cell protein, CD44, in high grade cancer and whether either is associated with an aggressive phenotype.A total of 133 grade 4/5 archival cancers from a cohort previously used to evaluate the prognostic significance of histomorphological variables were scored for monoamine oxidase-A and CD44 immunohistochemical labeling. Spearman rank correlations of the proteins, and histomorphological and clinical variables were determined. The univariate and multivariate value of each variable as a determinant of biochemical recurrence was assessed by logistic regression.Monoamine oxidase-A expression correlated with CD44. Neither was prognostic for biochemical recurrence. However, monoamine oxidase-A expression positively correlated with preoperative serum prostate specific antigen and the percent of grade 4/5 cancer.Concurrent expression of monoamine oxidase-A and CD44 suggests that grade 4/5 cancer may be basal cell-like in nature, despite the absence of other classic basal cell biomarkers such as cytokeratins 5 and 14, and p63. The correlation of monoamine oxidase-A expression with prostate specific antigen and the percent of grade 4/5 cancer suggests that monoamine oxidase-A may contribute to growth of high grade cancer and that antidepressant drugs that target monoamine oxidase-A may have applications in treating prostate cancer.

    View details for DOI 10.1016/j.juro.2008.07.019

    View details for Web of Science ID 000260102000083

    View details for PubMedID 18804811

  • Inhibition of monoamine oxidase A promotes secretory differentiation in basal prostatic epithelial cells DIFFERENTIATION Zhao, H., Nolley, R., Chen, Z., Reese, S. W., Peehl, D. M. 2008; 76 (7): 820-830

    Abstract

    Monoamine oxidase A (MAO-A) expression is associated with high-grade prostate cancer. Immunohistochemistry showed that MAO-A is also expressed in the basal epithelial cells of normal prostate glands. Using cultured primary prostatic epithelial cells as a model, we showed that MAO-A prevents basal epithelial cells from differentiating into secretory cells. Under differentiation-promoting conditions, clorgyline, an irreversible MAO-A inhibitor, induced secretory cell-like morphology and repressed expression of cytokeratin 14, a basal cell marker. More importantly, clorgyline induced mRNA and protein expression of androgen receptor (AR), a hallmark of secretory epithelial cells. In clorgyline-treated cells, androgen induced luciferase activity controlled by the promoter of prostate-specific antigen, an AR target gene, in a dose-dependent manner. This activity was blocked by the AR antagonist Casodex, showing that AR is functional. In turn, androgen decreased MAO-A expression in clorgyline-treated, secretory-like cells. Our results demonstrated that cultured basal epithelial cells have the potential to differentiate into secretory cells, and that inhibition of MAO-A is a key factor in promoting this process. Increased expression of MAO-A in high-grade prostate cancer may be an important contributor to its de-differentiated phenotype, raising the possibility that MAO-A inhibition may restore differentiation and reverse the aggressive behavior of high-grade cancer.

    View details for DOI 10.1111/j.1432-0436.2007.00263.x

    View details for Web of Science ID 000258581400007

    View details for PubMedID 18248494

  • Centrosomal PKC beta II and pericentrin are critical for human prostate cancer growth and angiogenesis CANCER RESEARCH Kim, J., Choi, Y., Vallentin, A., Hunrichs, B. S., Hellerstein, M. K., Peehl, D. M., Mochly-Rosen, D. 2008; 68 (16): 6831-6839

    Abstract

    Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth, but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCbetaII was activated during increased angiogenesis and that PKCbetaII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCbetaII-selective inhibitor, betaIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCbetaII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCbetaII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be up-regulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is up-regulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCbetaII inhibitor such as betaIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.

    View details for DOI 10.1158/0008-5472.CAN-07-6195

    View details for Web of Science ID 000258548200042

    View details for PubMedID 18701509

  • Calcitriol as a chemopreventive and therapeutic agent in prostate cancer: Role of anti-inflammatory activity JOURNAL OF BONE AND MINERAL RESEARCH Krishnan, A. V., Moreno, J., Nonn, L., Swami, S., Peehl, D. M., Feldman, D. 2007; 22: V74-V80

    Abstract

    Calcitriol, the hormonally active form of vitamin D, inhibits the growth and development of several cancers. Inflammation has been implicated in the development and progression of many cancers, including prostate cancer (PCa). Recent research from our laboratory suggests that calcitriol exhibits anti-inflammatory actions that may contribute to its inhibitory effects in PCa. We found that calcitriol inhibits the synthesis and actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: (1) inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (2) induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs, and (3) decreasing the expression of prostaglandin E and prostaglandin F PG receptors, which are the mediators of PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Acting on a separate anti-inflammatory pathway, calcitriol induces the expression of mitogen-activated protein kinase phosphatase 5 (MKP5), a member of a family of phosphatases that are negative regulators of MAP kinases, causing the selective dephosphorylation and inactivation of the stress-activated protein kinase p38. Because p38 activation may be both procarcinogenic and promote inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, may contribute to the chemopreventive activity of calcitriol. We conclude that calcitriol exerts several anti-inflammatory actions in prostate cells, which contribute to its potential as a chemopreventive and therapeutic agent in PCa.

    View details for DOI 10.1359/JBMR.07S213

    View details for Web of Science ID 000253417900016

    View details for PubMedID 18290727

  • Vitamin D inhibition of the prostaglandin pathway as therapy for prostate cancer NUTRITION REVIEWS Feldman, D., Krishnan, A., Moreno, J., Swami, S., Peehl, D. M., Srinivas, S. 2007; 65 (8): S113-S115

    View details for Web of Science ID 000248995500012

    View details for PubMedID 17867384

  • Potentiation of the growth-inhibitory effects of vitamin D in prostate cancer by genistein NUTRITION REVIEWS Krishnan, A. V., Swami, S., Moreno, J., Bhattacharyya, R. B., Peehl, D. M., Feldman, D. 2007; 65 (8): S121-S123

    View details for Web of Science ID 000248995500015

    View details for PubMedID 17867387

  • Chemopreventive anti-inflammatory activities of curcumin and other phytochemicals mediated by MAP kinase phosphatase-5 in prostate cells CARCINOGENESIS Nonn, L., Duong, D., Peehl, D. M. 2007; 28 (6): 1188-1196

    Abstract

    As inflammation emerges as a risk factor for prostate cancer (PCa), there is potential for chemoprevention by anti-inflammatory agents. Dietary phytochemicals have been shown to have chemopreventive properties which may include anti-inflammatory activities. In this study, we demonstrate a role for mitogen-activated protein kinase phosphatase-5 (MKP5) in mediating anti-inflammatory activities of the phytochemicals curcumin, resveratrol and [6]-gingerol. We utilized the cytokines tumor necrosis factor-alpha (TNFalpha) and interleukin (IL)-1beta to increase p38-dependent nuclear factor kappa-B (NFkappaB) activation and expression of pro-inflammatory genes cyclooxygenase-2 (COX-2), IL-6 and IL-8 in normal prostatic epithelial cells. MKP5 over-expression decreased cytokine-induced NFkappaB activation, COX-2, IL-6 and IL-8 in normal prostatic epithelial cells, suggesting potent anti-inflammatory activity of MKP5. Pretreatment of cells with a p38 inhibitor mimicked the results observed with MKP5 over-expression, further implicating p38 inhibition as the main activity of MKP5. Curcumin, the phytochemical found in turmeric, up-regulated MKP5, subsequently decreasing cytokine-induced p38-dependent pro-inflammatory changes in normal prostatic epithelial cells. Resveratrol and [6]-gingerol, phytochemicals present in red wine and ginger, respectively, also up-regulated MKP5 in normal prostate epithelial cells. Moreover, we found that PCa cell lines DU 145, PC-3, LNCaP and LAPC-4 retained the ability to up-regulate MKP5 following curcumin, resveratrol and [6]-gingerol exposure, suggesting utility of these phytochemicals in PCa treatment. In summary, our findings show direct anti-inflammatory activity of MKP5 in prostate cells and suggest that up-regulation of MKP5 by phytochemicals may contribute to their chemopreventive actions by decreasing prostatic inflammation.

    View details for DOI 10.1093/carcin/bgl241

    View details for Web of Science ID 000247999700008

    View details for PubMedID 17151092

  • Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Krishnan, A. V., Moreno, J., Nonn, L., Malloy, P., Swami, S., Peng, L., Peehl, D. M., Feldman, D. 2007; 103 (3-5): 694-702

    Abstract

    Calcitriol, the hormonally active form of Vitamin D, inhibits the growth and development of many cancers through multiple mechanisms. Our recent research supports the contributory role of several new and diverse pathways that add to the mechanisms already established as playing a role in the actions of calcitriol to inhibit the development and progression of prostate cancer (PCa). Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in the inhibition of PCa cell growth by increasing the expression of the cell cycle inhibitor p21. Calcitriol inhibits the prostaglandin (PG) pathway by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, the inhibition of the PG pathway may add to the ability of calcitriol to prevent and inhibit PCa development and growth. The combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of a family of phosphatases that are negative regulators of MAP kinases. Calcitriol induces MKP5 expression in prostate cells leading to the selective dephosphorylation and inactivation of the stress-activated kinase p38. Since p38 activation is pro-carcinogenic and is a mediator of inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, contributes to the chemopreventive activity of calcitriol in PCa. Mullerian Inhibiting Substance (MIS) has been evaluated for its inhibitory effects in cancers of the reproductive tissues and is in development as an anti-cancer drug. Calcitriol induces MIS expression in prostate cells revealing yet another mechanism contributing to the anti-cancer activity of calcitriol in PCa. Thus, we conclude that calcitriol regulates myriad pathways that contribute to the potential chemopreventive and therapeutic utility of calcitriol in PCa.

    View details for DOI 10.1016/j.jsbmb.2006.12.051

    View details for Web of Science ID 000245826800094

    View details for PubMedID 17229571

  • Distinctive gene expression of prostatic stromal cells cultured from diseased versus normal tissues JOURNAL OF CELLULAR PHYSIOLOGY Zhao, H., Ramos, C. F., Brooks, J. D., Peehl, D. M. 2007; 210 (1): 111-121

    Abstract

    To obtain a comprehensive view of the transcriptional programs in prostatic stromal cells of different histological/pathological origin, we profiled 18 adult human stromal cell cultures from normal transition zone (TZ), normal peripheral zone (PZ), benign prostatic hyperplasia (BPH), and prostate cancer (CA) using cDNA microarrays. A hierarchical clustering analysis of 714 named unique genes whose expression varied at least threefold from the overall mean abundance in at least three samples in all 18 samples demonstrated that cells of different origin displayed distinct gene expression profiles. Many of the differentially expressed genes are involved in biological processes known to be important in the development of prostatic diseases including cell proliferation and apoptosis, cell adhesion, and immune response. Significance Analysis of Microarrays (SAM) analysis identified genes that showed differential expression with statistical significance including 24 genes between cells from TZ versus BPH, 34 between BPH versus CA, and 101 between PZ versus CA. S100A4 and SULF1, the most up- and downregulated genes in BPH versus TZ, respectively, showed expression at the protein level consistent with microarray analysis. In addition, sulfatase assay showed that BPH cells have lower SULF1 activity compared to TZ cells. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed differential expression of ENPP2/autotoxin and six other genes between PZ versus CA, as well as differential expression of six genes between BPH versus CA. Our results support the hypothesis that prostatic stromal cells of different origin have unique transcriptional programs and point towards genes involved in actions of stromal cells in BPH and CA.

    View details for DOI 10.1002/jcp.20828

    View details for Web of Science ID 000242568200012

    View details for PubMedID 17044071

  • Calcitriol and genistein actions to inhibit the prostaglandin pathway: Potential combination therapy to treat prostate cancer JOURNAL OF NUTRITION Swami, S., Krishnan, A. V., Moreno, J., Bhattacharyya, R. B., Peehl, D. M., Feldman, D. 2007; 137 (1): 205S-210S

    Abstract

    We present an overview of the prostaglandin (PG) pathway as a novel target for the treatment of prostate cancer (PCa) using a combination of calcitriol and genistein, both of which have known antiproliferative properties. Calcitriol inhibits the PG pathway in PCa cells in 3 separate ways: by decreasing cyclooxygenase-2 (COX-2) expression, stimulating 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression, and decreasing EP (PGE2) and FP (PGF(2alpha)) receptors. These actions of calcitriol result in reduced levels of biologically active PGE2, leading ultimately to growth inhibition of the PCa cells. We also demonstrate the advantages of using calcitriol in combination with genistein for the treatment of PCa. Genistein, a major component of soy, is a potent inhibitor of the activity of CYP24, the enzyme that initiates the degradation of calcitriol. This leads to increased half-life of bioactive calcitriol, thereby enhancing all of calcitriol's actions including those on the PG pathway. In addition to inhibiting CYP24 enzyme activity, genistein has its own independent actions on the PG pathway in PCa cells. Like calcitriol it inhibits COX-2 expression and activity, leading to decreased synthesis of PGE2. It also inhibits the EP and FP receptors, thereby reducing the biological function of PGE2. Thus, the combination of calcitriol and genistein acts additively to inhibit the PG pathway. Both calcitriol and genistein are relatively safe and have little toxicity associated with their intake. We postulate that the combination of calcitriol and genistein is an attractive therapeutic option for the treatment of PCa.

    View details for Web of Science ID 000243330800009

    View details for PubMedID 17182827

  • Transcript profiling of the androgen signal in normal prostate, benign prostatic hyperplasia, and prostate cancer ENDOCRINOLOGY Bauman, D. R., Steckelbroeck, S., Peehl, D. M., Penning, T. M. 2006; 147 (12): 5806-5816

    Abstract

    Human prostate adenocarcinoma (CaP) and benign prostatic hyperplasia (BPH) have epithelial and stromal cell origins, respectively. To determine whether the androgen signal is processed differently in these cell types the expression of transcripts for enzymes that control ligand access to the androgen receptor (AR) were measured. Transcripts for type 2 5alpha-reductase, ketosteroid reductases [aldo-keto reductase (AKR)1C1-AKR1C4], the major oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) retinol dehydrogenase (RODH)-like 3alpha-HSD (RL-HSD) and nuclear receptors [AR, estrogen receptor (ER)alpha, and ERbeta] were determined in whole human prostate and in cultures of primary epithelial cells (PEC) and primary stromal cells (PSC) from normal prostate, CaP and BPH by real-time RT-PCR. Normal PEC (n=14) had higher levels of AKR1C1 (10-fold, P<0.001), AKR1C2 (115-fold, P<0.001) and AKR1C3 (6-fold, P<0.001) than normal PSC (n=15), suggesting that reductive androgen metabolism occurs. By contrast, normal PSC had higher levels of AR (8-fold, P<0.001) and RL-HSD (21-fold, P<0.001) than normal PEC, suggesting that 3alpha-androstanediol is converted to 5alpha-dihydrotestosterone to activate AR. In CaP PEC (n=14), no significant changes in transcript levels vs. normal PEC were observed. In BPH PSC (n=21) transcripts for AR (2-fold, P<0.001), AKR1C1 (4-fold, P<0.001), AKR1C2 (10-fold P<0.001), AKR1C3 (4-fold, P<0.001) and RL-HSD (3-fold, P<0.003) were elevated to increase androgen response. Differences in the AR:ERbeta transcript ratios (eight in normal PEC vs. 280 in normal PSC) were maintained in PEC and PSC in diseased prostate. These data suggest that CaP may be more responsive to an ERbeta agonist and BPH may be more responsive to androgen ablation.

    View details for DOI 10.1210/en.2006-0627

    View details for Web of Science ID 000242047200034

    View details for PubMedID 16959841

  • Mechanisms of vitamin D-mediated growth inhibition in prostate cancer cells: Inhibition of the prostaglandin pathway ANTICANCER RESEARCH Moreno, J., Krishnan, A. V., Peehl, D. M., Feldman, D. 2006; 26 (4A): 2525-2530

    Abstract

    Calcitriol (1,25-dihydroxyvitamin D3), the active form of vitamin D, promotes growth inhibition and differentiation in prostate cancer (PCa) cells. To unravel the molecular pathways of calcitriol actions, cDNA microarray analysis was used to identify novel calcitriol target genes including two that play key roles in the metabolism of prostaglandins (PGs), known stimulators of PCa growth and progression. Calcitriol significantly decreases the expression of the PG synthesizing cyclooxygenase-2 (COX-2) gene, while increasing that of PG inactivating 15-prostaglandin dehydrogenase (15-PGDH). Calcitriol also inhibits the expression of the PG receptors EP2 and FP. It reduces the levels of biologically active PGs and inhibits PG actions in PCa cells, thereby decreasing the proliferative stimulus of PGs. We postulate that the regulation of the PG pathway contributes to the growth inhibitory actions of calcitriol. We also propose that calcitriol can be combined with non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit COX enzyme activity, as a potential therapeutic strategy in PCa.

    View details for Web of Science ID 000241391500003

    View details for PubMedID 16886660

  • Inhibition of p38 by vitamin D reduces interleukin-6 production in normal prostate cells via mitogen-activated protein kinase phosphatase 5: Implications for prostate cancer prevention by vitamin D CANCER RESEARCH Nonn, L., Peng, L. H., Feldman, D., Peehl, D. M. 2006; 66 (8): 4516-4524

    Abstract

    Although numerous studies have implicated vitamin D in preventing prostate cancer, the underlying mechanism(s) remains unclear. Using normal human prostatic epithelial cells, we examined the role of mitogen-activated protein kinase phosphatase 5 (MKP5) in mediating cancer preventive activities of vitamin D. Up-regulation of MKP5 mRNA by 1,25-dihydroxyvitamin-D3 (1,25D) was dependent on the vitamin D receptor. We also identified a putative positive vitamin D response element within the MKP5 promoter that associated with the vitamin D receptor following 1,25D treatment. MKP5 dephosphorylates/inactivates the stress-activated protein kinase p38. Treatment of prostate cells with 1,25D inhibited p38 phosphorylation, and MKP5 small interfering RNA blocked this effect. Activation of p38 and downstream production of interleukin 6 (IL-6) are proinflammatory. Inflammation and IL-6 overexpression have been implicated in the initiation and progression of prostate cancer. 1,25D pretreatment inhibited both UV- and tumor necrosis factor alpha-stimulated IL-6 production in normal cells via p38 inhibition. Consistent with inhibition of p38, 1,25D decreased UV-stimulated IL-6 mRNA stabilization. The ability of 1,25D to up-regulate MKP5 was maintained in primary prostatic adenocarcinoma cells but was absent in metastases-derived prostate cancer cell lines. The inability of 1,25D to regulate MKP5 in the metastasis-derived cancer cells suggests there may be selective pressure to eliminate key tumor suppressor functions of vitamin D during cancer progression. These studies reveal MKP5 as a mediator of p38 inactivation and decreased IL-6 expression by 1,25D in primary prostatic cultures of normal and adenocarcinoma cells, implicating decreased prostatic inflammation as a potential mechanism for prostate cancer prevention by 1,25D.

    View details for DOI 10.1158/0008-5472.CAN-05-3796

    View details for Web of Science ID 000236843200071

    View details for PubMedID 16618780

  • Tumor-selective killing by selenite in patient-matched pairs of normal and malignant prostate cells PROSTATE Husbeck, B., Nonn, L., Peehl, D. M., Knox, S. J. 2006; 66 (2): 218-225

    Abstract

    Selenium compounds have been shown to induce apoptosis in a variety of human prostate cancer cell lines. However, the effects of selenium have yet to be examined in normal and malignant cells derived from the same individual. Selenite metabolism consumes glutathione (GSH) and produces superoxide. The generation of reactive oxygen species is an important mechanism in selenite-induced apoptosis.Three patient-matched pairs of primary prostatic epithelial cell cultures from normal and cancer were evaluated for their response to selenite. Apoptosis was measured and the differential response of normal and cancer cells was correlated with the expression of bcl-2, bax, GSH, and manganese superoxide dismutase (MnSOD).The cancer-derived cells were significantly more sensitive to selenite-induced apoptosis than the corresponding normal cells. Tumor-selective killing was not observed in cells treated with selenomethionine. The ratio of bcl-2:bax was decreased in the cancer-derived cells treated with selenite. Total GSH concentrations were similar in paired normal and cancer cells. Therefore, differences in GSH content do not appear to play a role in tumor-selective killing by selenite. Superoxide is a by-product of selenite metabolism and normal cells showed increased MnSOD expression and SOD activity compared to the cancer-derived cells. Prostate cancer cells treated with the MnSOD mimetic, MnTMPyP, were protected against the cytotoxic effects of selenite.Higher MnSOD expression in normal cells may play an important role in eliminating superoxide radicals produced as a result of selenite metabolism and contribute to the tumor-selective killing by selenite in prostate cancer.

    View details for DOI 10.1002/pros.20337

    View details for Web of Science ID 000234838300011

    View details for PubMedID 16173037

  • Identification of the major oxidative 3 alpha-hydroxysteroid dehydrogenase in human prostate that converts 5 alpha-androstane-3 alpha,17 beta-diol to 5 alpha-dihydrotestosterone: A potential therapeutic target for androgen-dependent disease MOLECULAR ENDOCRINOLOGY Bauman, D. R., Steckelbroeck, S., Williams, M. V., Peehl, D. M., Penning, T. M. 2006; 20 (2): 444-458

    Abstract

    Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.

    View details for DOI 10.1210/me.2005-0287

    View details for Web of Science ID 000235129600018

    View details for PubMedID 16179381

  • Comparative gene and protein expression in primary cultures of epithelial cells from benign prostatic hyperplasia and prostate cancer CANCER LETTERS Rose, A., Xu, Y., Chen, Z. X., Fan, Z. B., Stamey, T. A., McNeal, J. E., Caldwell, M., Peehl, D. M. 2005; 227 (2): 213-222

    Abstract

    Primary cultures are widely used to investigate the disease-specific biology of prostate cancer and benign prostatic hyperplasia (BPH). To identify genes differentially expressed between epithelial cells cultured from adenocarcinomas versus BPH tissues, we used probe array technology. Gene expression profiles were evaluated on Affymetrix Human Cancer G110 Array Chips containing approximately 1900 cancer-related genes. After defined statistical analysis, genes that were over-expressed in cancer cultures were identified. Protein expression of four of the differentially expressed genes was measured in immunoblots, and the expression of two other genes was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). While no gene or protein was consistently over-expressed in all cancer versus BPH cell cultures, cytokeratin 16 protein was highly elevated in several of the cancer cultures, suggesting that a hyperproliferative phenotype may be characteristic of prostate cancer cells.

    View details for DOI 10.1016/j.canlet.2005.01.037

    View details for Web of Science ID 000231845600014

    View details for PubMedID 16112424

  • Genistein potentiates the growth inhibitory effects of 1,25-dihydroxyvitamin D-3 in DU145 human prostate cancer cells: Role of the direct inhibition of CYP24 enzyme activity MOLECULAR AND CELLULAR ENDOCRINOLOGY Swami, S., Krishnan, A. V., Peehl, D. M., Feldman, D. 2005; 241 (1-2): 49-61

    Abstract

    In a search for improved therapies for prostate cancer, we investigated the effect of genistein in combination with 1alpha-25-dihydroxyvitamin D3 [1,25(OH)2D3], on the growth of DU145 human prostate cancer cells. DU145 cells were very resistant to the growth inhibitory action of 1,25(OH)2D3 or genistein when administered individually. However, the combination caused a significant growth inhibition seen at lower concentrations of both agents. 1,25(OH)2D3 induces the expression of the CYP24 gene, which codes for the enzyme that initiates the catabolism of 1,25(OH)2D3. We showed for the first time that genistein at low doses (50-100 nM) directly inhibited CYP24 at the enzyme level. Addition of genistein to mitochondrial preparations inhibited CYP24 enzyme activity in a noncompetitive manner. CYP24 inhibition by genistein increased the half-life of 1,25(OH)2D3 thereby augmenting the homologous up-regulation of the vitamin D receptor (VDR) both at the mRNA and protein levels. Genistein co-treatment enhanced 1,25(OH)2D3-mediated transactivation of the vitamin D responsive reporters OC-Luc and OP-Luc transfected into DU145 cells. Consistent with the growth inhibition due to the combination treatment, significant changes in the expression of genes involved in growth arrest and apoptosis were seen. We conclude that genistein potentiates the antiproliferative actions of 1,25(OH)2D3 in DU145 cells by two mechanisms: (i) an increase in the half-life of 1,25(OH)2D3 due to the direct inhibition of CYP24 enzyme activity and (ii) an amplification of the homologous up-regulation of VDR. Together these two effects lead to a substantial enhancement of the cellular responses to the growth inhibitory and pro-apoptotic signaling by 1,25(OH)2D3.

    View details for DOI 10.1016/j.mce.2005.05.001

    View details for Web of Science ID 000232439700006

    View details for PubMedID 15955619

  • Regulation of prostaglandin metabolism by calcitriol attenuates growth stimulation in prostate cancer cells CANCER RESEARCH Moreno, J., Krishnan, A. V., Swami, S., Nonn, L., Peehl, D. M., Feldman, D. 2005; 65 (17): 7917-7925

    Abstract

    Calcitriol exhibits antiproliferative and pro-differentiation effects in prostate cancer. Our goal is to further define the mechanisms underlying these actions. We studied established human prostate cancer cell lines and primary prostatic epithelial cells and showed that calcitriol regulated the expression of genes involved in the metabolism of prostaglandins (PGs), known stimulators of prostate cell growth. Calcitriol significantly repressed the mRNA and protein expression of prostaglandin endoperoxide synthase/cyclooxygenase-2 (COX-2), the key PG synthesis enzyme. Calcitriol also up-regulated the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme initiating PG catabolism. This dual action was associated with decreased prostaglandin E2 secretion into the conditioned media of prostate cancer cells exposed to calcitriol. Calcitriol also repressed the mRNA expression of the PG receptors EP2 and FP, providing a potential additional mechanism of suppression of the biological activity of PGs. Calcitriol treatment attenuated PG-mediated functional responses, including the stimulation of prostate cancer cell growth. The combination of calcitriol with nonsteroidal anti-inflammatory drugs (NSAIDs) synergistically acted to achieve significant prostate cancer cell growth inhibition at approximately 2 to 10 times lower concentrations of the drugs than when used alone. In conclusion, the regulation of PG metabolism and biological actions constitutes a novel pathway of calcitriol action that may contribute to its antiproliferative effects in prostate cells. We propose that a combination of calcitriol and nonselective NSAIDs might be a useful chemopreventive and/or therapeutic strategy in men with prostate cancer, as it would allow the use of lower concentrations of both drugs, thereby reducing their toxic side effects.

    View details for DOI 10.1158/0008-5472.CAN-05-1435

    View details for Web of Science ID 000231659500052

    View details for PubMedID 16140963

  • Molecular targets of Doxazosin in human prostatic stromal cells PROSTATE Zhao, H. J., Lai, F., Nonn, L., Brooks, J. D., Peehl, D. M. 2005; 62 (4): 400-410

    Abstract

    We used cDNA microarray analysis to obtain insights into the mechanisms of action of doxazosin, an alpha(1)-adrenergic receptor antagonist used to treat benign prostatic hyperplasia (BPH).Hierarchical clustering analysis and significance analysis of microarray (SAM) were performed to identify genes differentially expressed between untreated stromal cells cultured from normal tissue and BPH, and changes in gene expression induced by doxazosin. Transcript levels of selected genes were validated by real-time reverse-transcription polymerase chain reaction (RT-PCR).Hierarchical clustering analyses separated untreated normal and BPH cells. Sixty-seven genes whose expression varied at least twofold after doxazosin treatment in both normal and BPH cells were identified, as were 93 genes differentially regulated in normal versus BPH cells. Molecular targets consistent with tumor necrosis factor (TNF)-alpha-related activity were identified.Normal versus BPH stromal cells differ in global gene transcription. Doxazosin induced gene expression changes relevant to proliferation/apoptosis, immune defense, cell-cell signaling/signal transduction, and transcriptional regulation.

    View details for DOI 10.1002/pros.20161

    View details for Web of Science ID 000226991500011

    View details for PubMedID 15378519

  • Primary cell cultures as models of prostate cancer development ENDOCRINE-RELATED CANCER Peehl, D. M. 2005; 12 (1): 19-47

    Abstract

    This review focuses on primary cultures of human prostatic epithelial cells and their applications as models of normal and malignant biological behavior. Current abilities to culture cells from normal tissues, from premalignant dysplastic lesions (prostatic intraepithelial neoplasia), from primary adenocarcinomas, and from metastases are described. Evidence for representation of the interrelated cells of the normal prostatic epithelium--stem cells, basal epithelial cells, secretory epithelial cells, transit amplifying cells and neuroendocrine cells--in primary cultures is presented. Comparisons between normal and cancer-derived primary cultures are made regarding biological activities relevant to carcinogenesis, such as proliferation, apoptosis, differentiation, senescence, adhesion, migration, invasion, steroid hormone metabolism, other metabolic pathways and angiogenesis. Analyses of tumor suppressor activity, differential gene expression and cytogenetics in primary cultures have revealed changes relevant to prostate cancer progression. Preclinical studies with primary cultures have provided information useful for designing new strategies for chemoprevention, chemotherapy, cytotoxin therapy, radiation therapy, gene therapy and imaging. While the behavior of normal primary cultures is often used as a basis for comparison with established, immortal prostate cancer cell lines, the most informative studies are performed with donor-matched pairs of normal and malignant primary cultures, grown under identical conditions. Challenges that remain to be addressed if the full potential of primary cultures as a model system is to be realized include isolation, culture and characterization of stem cells, improved methodology to induce or maintain a fully differentiated, androgen-responsive phenotype, and identification of cell surface antigens or other markers with which to purify pure populations of live cancer or premalignant cells apart from non-malignant epithelial cells prior to culture.

    View details for DOI 10.1677/erc.1.00795

    View details for Web of Science ID 000228049800002

    View details for PubMedID 15788637

  • Redox modulation of human prostate carcinoma cells by selenite increases radiation-induced cell killing FREE RADICAL BIOLOGY AND MEDICINE Husbeck, B., Peehl, D. M., Knox, S. J. 2005; 38 (1): 50-57

    Abstract

    Although selenium compounds have been extensively studied as chemopreventative agents for prostate cancer, little is known about the potential use of selenium compounds for chemotherapy. We have shown that selenite inhibits cell growth and induces apoptosis in androgen-dependent LAPC-4 prostate cancer cells. LAPC-4 cells were more sensitive to selenite-induced apoptosis than primary cultures of normal prostate cells. Selenite-induced apoptosis in LAPC-4 cells correlated with a decrease in the Bcl-2:Bax expression ratio. Selenite-induced oxidative stress and apoptosis are dependent upon its reaction with reduced GSH. LAPC-4 cells treated with selenite showed decreased levels of total GSH and increased concentrations of GSSG. Thus, selenite altered the intracellular redox status toward an oxidative state by decreasing the ratio of GSH:GSSG. Because increased levels of Bcl-2 and GSH are associated with radioresistance, we examined the ability of selenite to sensitize prostate cancer cells to gamma-irradiation. Both LAPC-4 and androgen-independent DU 145 cells pretreated with selenite showed increased sensitivity to gamma-irradiation as measured by clonogenic survival assays. Importantly, selenite-induced radiosensitization was observed in combination with a clinically relevant dose of 2 Gy. These data suggest that altering the redox environment of prostate cancer cells with selenite increases the apoptotic potential and sensitizes them to radiation-induced cell killing.

    View details for DOI 10.1016/j.freeradbiomed.2004.09.022

    View details for Web of Science ID 000226172900006

    View details for PubMedID 15589371

  • Interaction of nuclear receptor ligands with the vitamin D signaling pathway in prostate cancer JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Peehl, D. M., Feldman, D. 2004; 92 (4): 307-315

    Abstract

    A number of hormonal ligands and/or the nuclear receptors that mediate their actions have been targeted for prostate cancer therapy. Androgens, the ligands for the androgen receptor (AR), are critical for the growth of prostate cancer. Inhibition of androgen production has been the mainstay of treatment for advanced prostate cancer for decades. Other more recently tested targets include retinoid receptors (RAR and RXR), glucocorticoid receptors (GR), estrogen receptors (ER) and peroxisome proliferator-activated receptors (PPAR). Calcitriol, acting through the Vitamin D receptor (VDR), has many tumor suppressive activities in the prostate, including inhibition of proliferation, induction of apoptosis and/or differentiation, and reduction of cellular invasion. Because of these properties, calcitriol and its less hypercalcemic analogs are being evaluated as agents to prevent or treat prostate cancer. Androgens, retinoids, glucocorticoids, estrogens and agonists of PPAR directly or indirectly impact Vitamin D signaling pathways, and vice versa. In order to design the most effective strategies to use calcitriol to prevent or treat prostate cancer, the interactions of other nuclear receptors and their ligands with the Vitamin D signaling pathway need to be considered.

    View details for DOI 10.1016/j.jsbmb.2004.10.006

    View details for Web of Science ID 000227022500010

    View details for PubMedID 15663994

  • Molecular and cellular pathogenesis of benign prostatic hyperplasia JOURNAL OF UROLOGY Lee, K. L., Peehl, D. M. 2004; 172 (5): 1784-1791

    Abstract

    Symptomatic benign prostatic hyperplasia (BPH) is one of the most common ailments seen by the urologist. Significant advances have occurred in medical and surgical therapy, and in the understanding of the biology of this disease. However, the basic science literature is often conflicting and confusing, without a unified voice. We report the current state of knowledge of the molecular and cellular basis of BPH.We compiled and interpreted basic science studies relevant to BPH pathogenesis.Cellular alterations that include changes in proliferation, differentiation, apoptosis and senescence in the epithelium and stroma are implicated in BPH pathogenesis. Molecular analyses have yielded numerous candidate genes important in disease progression. Differential expression of cytokines and growth factors in BPH tissue suggests roles for inflammation and hypoxia. Through the use of cell culture models the complex regulatory mechanisms of growth control in BPH are becoming defined.The scientific endeavor has resulted in great strides in our understanding of BPH on a molecular and cellular level. It is hopeful that basic science and translational research will improve treatment and prevention strategies for this common disease of elderly men.

    View details for DOI 10.1097/01.ju.0000133655.71782.14

    View details for Web of Science ID 000224463600005

    View details for PubMedID 15540721

  • Molecular activity of 1,25-dihydroxyvitamin D-3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Peehl, D. M., Shinghal, R., Nonn, L., Seto, E., Krishnan, A. V., Brooks, J. D., Feldman, D. 2004; 92 (3): 131-141

    Abstract

    1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] exerts anti-proliferative, differentiating and apoptotic effects on prostatic cells. These activities, in addition to epidemiologic findings that link Vitamin D to prostate cancer risk, support the use of 1,25(OH)(2)D(3) for prevention or therapy of prostate cancer. The molecular mechanisms by which 1,25(OH)(2)D(3) exerts antitumor effects on prostatic cells are not well-defined. In addition, there is heterogeneity among the responses of various prostate cell lines and primary cultures to 1,25(OH)(2)D(3) with regard to growth inhibition, differentiation and apoptosis. To understand the basis of these differential responses and to develop a better model of Vitamin D action in the prostate, we performed cDNA microarray analyses of primary cultures of normal and malignant human prostatic epithelial cells, treated with 50 nM of 1,25(OH)(2)D(3) for 6 and 24 h. CYP24 (25-hydroxyvitamin D(3)-24-hydroxylase) was the most highly upregulated gene. Significant and early upregulation of dual specificity phosphatase 10 (DUSP10), validated in five additional primary cultures, points to inhibition of members of the mitogen-activated protein kinase (MAPK) superfamily as a key event mediating activity of 1,25(OH)(2)D(3) in prostatic epithelial cells. The functions of other regulated genes suggest protection by 1,25(OH)(2)D(3) from oxidative stress. Overall, these results provide new insights into the molecular basis of antitumor activities of Vitamin D in prostate cells.

    View details for DOI 10.1016/j.jsbmb.2004.07.003

    View details for Web of Science ID 000226009000003

    View details for PubMedID 15555907

  • Mechanisms of decreased Vitamin D 1 alpha-hydroxylase activity in prostate cancer cells MOLECULAR AND CELLULAR ENDOCRINOLOGY Ma, J. F., Nonn, L., Campbell, M. J., Hewison, M., Feldman, D., Peehl, D. M. 2004; 221 (1-2): 67-74

    Abstract

    Vitamin D 1alpha-hydroxylase (1alpha(OH)ase), which converts the circulating prohormone 25-hydroxyvitamin-D(3) (25(OH)D(3)) to the active 1alpha-25-dihydroxyvitamin-D(3) (1,25(OH)(2)D(3)), is present in normal prostatic epithelium. However, prostate cancer cells, both primary cultured cells and cell lines, have greatly decreased activity of 1alpha(OH)ase and are therefore resistant to the tumor suppressor activity of circulating 25(OH)D(3). We quantitated 1alpha(OH)ase mRNA and protein levels to investigate mechanism(s) responsible for decreased 1alpha(OH)ase enzymatic activity in prostate cancer. Prostate cancer cell lines had low 1alpha(OH)ase mRNA levels. Primary prostate cell cultures derived from normal and cancer tissues had equivalent levels of 1alpha(OH)ase RNA and protein. Equivalent 1alpha(OH)ase protein levels were observed in prostate tissue sections containing normal and malignant cells. The protein levels of hsc70, whose homolog intracellular Vitamin D binding protein (IDBP-1) facilitates delivery of 25(OH)D(3) to 1alpha(OH)ase in monkey cells, were equivalent in the normal and cancer cells. Equivalent activity in normal and cancer cells of Vitamin D 24-hydroxylase, a mitochondrial enzyme that also uses 25(OH)D(3) as a substrate, further ruled out lack of access to substrate as a basis for low activity of 1alpha(OH)ase in cancer cells. We conclude that diminished 1alpha(OH)ase activity in prostate cancer cell lines is through decreased gene expression, whereas decreased activity in primary cultures and tissues is post-translational.

    View details for DOI 10.1016/j.mce.2004.03.003

    View details for Web of Science ID 000222854100007

    View details for PubMedID 15223133

  • Analysis of vitamin D-regulated gene expression in LNCaP human prostate cancer cells using cDNA microarrays PROSTATE Krishnan, A. V., Shinghal, R., Raghavachari, N., Brooks, J. D., Peehl, D. M., Feldman, D. 2004; 59 (3): 243-251

    Abstract

    1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] exerts growth inhibitory, pro-differentiating, and pro-apoptotic effects on prostate cells. To better understand the molecular mechanisms underlying these actions, we employed cDNA microarrays to study 1,25(OH)2D3-regulated gene expression in the LNCaP human prostate cancer cells.mRNA isolated from LNCaP cells treated with vehicle or 50 nM 1,25(OH)2D3 for various lengths of time were hybridized to microarrays carrying approximately 23,000 genes. Some of the putative target genes revealed by the microarray analysis were verified by real-time PCR assays.1,25(OH)2D3 most substantially increased the expression of the insulin-like growth factor binding protein-3 (IGFBP-3) gene. Our analysis also revealed several novel 1,25(OH)2D3-responsive genes. Interestingly, some of the key genes regulated by 1,25(OH)2D3 are also androgen-responsive genes. 1,25(OH)2D3 also down-regulated genes that mediate androgen catabolism.The putative 1,25(OH)2D3 target genes appear to be involved in a variety of cellular functions including growth regulation, differentiation, membrane transport, cell-cell and cell-matrix interactions, DNA repair, and inhibition of metastasis. The up-regulation of IGFBP-3 gene has been shown to be crucial in 1,25(OH)2D3-mediated inhibition of LNCaP cell growth. 1,25(OH)2D3 regulation of androgen-responsive genes as well as genes involved in androgen catabolism suggests that there are interactions between 1,25(OH)2D3 and androgen signaling pathways in LNCaP cells. Further studies on the role of these genes and others in mediating the anti-cancer effects of 1,25(OH)2D3 may lead to better approaches to the prevention and treatment of prostate cancer.

    View details for DOI 10.1002/pros.20006

    View details for Web of Science ID 000220910600003

    View details for PubMedID 15042599

  • Are primary cultures realistic models of prostate cancer? JOURNAL OF CELLULAR BIOCHEMISTRY Peehl, D. M. 2004; 91 (1): 185-195

    Abstract

    Primary cultures fill a unique niche among the repertoire of in vitro model systems available to investigate the biology of the normal and malignant human prostate. This review summarizes some of the properties of primary cultures, with special emphasis on two questions: are primary cultures from adenocarcinomas really comprised of cancer rather than normal cells, and do primary cultures faithfully retain characteristics of cells of origin?

    View details for DOI 10.1002/jcb.10691

    View details for Web of Science ID 000187714700016

    View details for PubMedID 14689590

  • Pathways mediating the growth-inhibitory actions of vitamin D in prostate cancer JOURNAL OF NUTRITION Peehl, D. M., Krishnan, A. V., Feldman, D. 2003; 133 (7): 2461S-2469S

    Abstract

    Vitamin D is emerging as an important dietary factor that affects the incidence and progression of many malignancies including prostate cancer. The active form of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)], inhibits the growth and stimulates the differentiation of prostate cancer cells. We have studied primary cultures of normal and cancer-derived prostatic epithelial cells as well as established human prostate cancer cell lines to elucidate the molecular pathways of 1,25(OH)(2)D(3) actions. These pathways are varied and appear to be cell specific. In LNCaP cells, 1,25(OH)(2)D(3) mainly causes growth arrest through the induction of insulin-like growth factor binding protein-3 and also stimulates apoptosis to a much smaller extent. We have used cDNA-microarray analyses to identify additional genes that are regulated by 1,25(OH)(2)D(3) and to raise novel therapeutic targets for use in the chemoprevention or treatment of prostate cancer. Less calcemic analogs of 1,25(OH)(2)D(3) that have more antiproliferative activity are being developed that will be more useful clinically. In target cells, 1,25(OH)(2)D(3) induces 24-hydroxylase, the enzyme that catalyzes its self inactivation. Cotreatment with 24-hydroxylase inhibitors enhances the antiproliferative activity of 1,25(OH)(2)D(3). The combination of other anticancer agents such as retinoids with vitamin D offers another promising therapeutic approach. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate-specific antigen increase in prostate cancer patients, which demonstrates proof of the concept that vitamin D or its analogs are clinically effective. Our research is directed at understanding the mechanisms of vitamin D action in prostate cells with the goal of developing chemoprevention and treatment strategies to improve prostate cancer therapy.

    View details for Web of Science ID 000184163200012

    View details for PubMedID 12840225

  • Primary culture model of peroxisome proliferator-activated receptor gamma activity in prostate cancer cells JOURNAL OF CELLULAR PHYSIOLOGY Xu, Y., Iyengar, S., Roberts, R. L., Shappell, S. B., Peehl, D. M. 2003; 196 (1): 131-143

    Abstract

    BRL 49653 (rosiglitazone) is a thiazolidinedione anti-diabetic drug that activates the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma). Pilot clinical trials have shown evidence of therapeutic activity of PPARgamma agonists against prostate cancer. To more effectively use PPARgamma ligands to treat this common and generally chemo-resistant type of cancer, it will be necessary to better understand the nature of PPARgamma activity in prostate cancer cells. Tumor suppressor effects of activation of PPARgamma may include suppression of growth and/or induction of differentiation or apoptosis. We investigated responses of primary cultures of human prostatic cancer cells to BRL 49653. PPARgamma was expressed in all of the cell strains examined. BRL 49653 caused dose- and time-dependent growth inhibition that was associated with increased expression of the transcription repressor, transforming growth factor beta-stimulated clone 22 (TSC-22), and markedly increased expression of the secretory differentiation-associated gene adipophilin. Adipocyte-type fatty acid binding protein (aFABP), neutrophil gelatinase-associated lipocalin (NGAL), glycerol kinase (GyK), and beta-catenin, which are regulated by PPARgamma ligands in certain other types of cells, were not regulated by BRL 49653 in prostate cells. Upregulation of adipophilin coincided with morphological changes and the appearance of cytoplasmic vacuoles with ultrastructural features of secondary lysosomes. These results extend previous studies with established cancer cell lines and show that PPARgamma agonists can inhibit proliferation and modulate expression of secretory-associated genes in primary cultures of prostate cancer cells, further warranting consideration of these agents as pro-differentiating chemotherapeutic or chemoprevention agents for the treatment of prostate cancer.

    View details for DOI 10.1002/jcp.10281

    View details for Web of Science ID 000183361700015

    View details for PubMedID 12767049

  • Human type 3 3 alpha-hydroxysteroid dehydrogenase (Aldo-Keto reductase 1C2) and androgen metabolism in prostate cells ENDOCRINOLOGY Rizner, T. L., Lin, H. K., Peehl, D. M., Steckelbroeck, S., Bauman, D. R., Penning, T. M. 2003; 144 (7): 2922-2932

    Abstract

    Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto-, and 20-ketosteroid reductases or as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidases. These AKRs can convert potent sex hormones (androgens, estrogens, and progestins) into their cognate inactive metabolites or vice versa. By controlling local ligand concentration AKRs may regulate steroid hormone action at the prereceptor level. AKR1C2 is expressed in prostate, and in vitro it will catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent oxidation of 3alpha-androstanediol (3alpha-diol) to 5alpha-dihydrotestosterone (5alpha-DHT). This reaction is potently inhibited by reduced NAD phosphate (NADPH), indicating that the NAD(+): NADPH ratio in cells will determine whether AKR1C2 makes 5alpha-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5alpha-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity, which eliminated 3alpha-diol as androsterone. PC-3 cells expressed retinol dehydrogenase/3alpha-HSD and 11-cis-retinol dehydrogenase, but these endogenous enzymes did not oxidize 3alpha-diol to 5alpha-DHT. In stable LNCaP-AKR1C2 transfectants, AKR1C2 did not alter androgen metabolism due to a high rate of glucuronidation. In primary cultures of epithelial cells, high levels of AKR1C2 transcripts were detected in prostate cancer, but not in cells from normal prostate. Thus, in prostate cells AKR1C2 acts as a 3-ketosteroid reductase to eliminate 5alpha-DHT and prevents activation of the androgen receptor. AKR1C2 does not act as an oxidase due to either potent product inhibition by NADPH or because it cannot surmount the oxidative 17beta-HSD present. Neither AKR1C2, retinol dehydrogenase/3alpha-HSD nor 11-cis-retinol dehydrogenase is a source of 5alpha-DHT in PC-3 cells.

    View details for DOI 10.1210/en.2002-0032

    View details for Web of Science ID 000183547800024

    View details for PubMedID 12810547

  • The role of vitamin D and retinoids in controlling prostate cancer progression ENDOCRINE-RELATED CANCER Peehl, D. M., Feldman, D. 2003; 10 (2): 131-140

    Abstract

    Prostate cancer is a leading cause of cancer-related deaths in many countries. Premalignant lesions and invasive cancer occur more frequently in the prostate than in any organ other than the skin. Yet, the incidence of clinically detected prostate cancer is much lower than the histopathological incidence. The slow growth of prostate cancer and the low incidence of clinically manifest disease in some geographical locations or racial/ethnic groups suggest that prostate cancer can be controlled, perhaps by dietary factors. Vitamin D and retinoids have emerged as leading candidates both to prevent and to treat prostate cancer. Many of the activities of these compounds, established from epidemiological studies, research with cell culture and animal models, and clinical trials, are consistent with tumor suppressor effects. However, retinoids may have additional tumor enhancer properties that balance or negate anti-cancer activity. This perhaps explains the overall lack of protective effects of vitamin A compounds against prostate cancer found in epidemiological studies, and the minimal efficacy of retinoids in clinical trials to treat prostate cancer. While current efforts focus on developing strategies to use vitamin D compounds to control prostate cancer, the possibility exists that prostate cancer cells may become resistant to tumor suppressor effects of vitamin D. Analyses of experimental model systems show that prostate cancer cells become less sensitive to vitamin D through loss of receptors or signaling molecules that mediate vitamin D's actions, or through changes in metabolic enzymes that synthesize or degrade vitamin D compounds. The potential promise of exploiting vitamin D to control prostate cancer is tempered by the possibility that prostate cancer, perhaps even at early stages, may develop mechanisms to escape tumor suppressor activities of vitamin D and/or retinoids.

    View details for Web of Science ID 000184151400003

    View details for PubMedID 12790775

  • Leptomycin B stabilizes and activates p53 in primary prostatic epithelial cells and induces apoptosis in the LNCaP cell line PROSTATE Lecane, P. S., Kiviharju, T. M., Sellers, R. G., Peehl, D. M. 2003; 54 (4): 258-267

    Abstract

    Previous studies showed that primary cultures of normal and malignant human prostatic epithelial cells are defective in their ability to upregulate the tumor suppressor protein p53 in response to DNA damage. This dysfunctional regulation of p53 may be relevant to both the high incidence of prostate cancer and its resistance to chemotherapy. Leptomycin B (LMB) has recently been found to increase the protein level and transcriptional activity of p53 by interfering with nucleocytoplasmic export and subsequent degradation by the proteasome. We investigated the ability of LMB to activate p53 in prostatic epithelial cells.Primary cultures and the cell lines LNCaP and DU 145 were treated with LMB. p53 protein was evaluated in Western blots and by immunocytochemistry. Induction of downstream targets of p53 was evaluated in Western and Northern blots. Growth inhibition, cell cycle arrest, and apoptosis in response to LMB were measured in clonal growth assays, by flow cytometry, and by Hoescht/propidium iodide staining, respectively.Treatment of prostatic epithelial cells with LMB led to post-translational stabilization of p53, activation of downstream target genes, and induction of cell cycle arrest in primary cultures and apoptosis in LNCaP (with wild-type p53) but not DU 145 (with mutant p53) cells.p53 in primary cultures of normal and malignant prostate cells, although dysfunctional in that it is not responsive to DNA damage, is activated by LMB. The ability of LMB to stabilize p53 and induce expression of p53-responsive growth inhibitory genes may be a useful lead in the development of chemopreventive or therapeutic small molecules that can modulate p53 function in prostatic epithelial cells.

    View details for DOI 10.1002/pros.10197

    View details for Web of Science ID 000181056800002

    View details for PubMedID 12539224

  • Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression JOURNAL OF CELLULAR BIOCHEMISTRY Krishnan, A. V., Peehl, D. M., Feldman, D. 2003; 88 (2): 363-371

    Abstract

    Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued.

    View details for DOI 10.1002/jcb.10334

    View details for Web of Science ID 000180479100024

    View details for PubMedID 12520538

  • Growth of prostatic epithelial and stromal cells in vitro. Methods in molecular medicine Peehl, D. M. 2003; 81: 41-57

    View details for PubMedID 12725113

  • Vitamin D receptor start codon polymorphism (FokI) and prostate cancer progression CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Xu, Y., Shibata, A., McNeal, J. E., Stamey, T. A., Feldman, D., Peehl, D. M. 2003; 12 (1): 23-27

    Abstract

    Vitamin D plays an important role in cell growth and differentiation and is proposed to protect against cancer initiation and/or progression. The vitamin D receptor (VDR) has a thymine/cytosine (T/C) polymorphism located in the first of two potential start (ATG) codons that can be detected by a RFLP using the endonuclease FokI. The C variant, which lacks the first ATG, results in a shorter VDR and is referred to as the F allele. The T variant (f allele) initiates at the first ATG. We examined the association of the VDR FokI genotype with histopathological characteristics and prognosis of prostate cancer among 191 mostly Caucasian subjects who had undergone radical prostatectomy between 1984 and 1992. The frequencies of the FF, Ff, and ff genotypes were 41%, 38%, and 21%, respectively. Subjects with the ff genotype had a lower mean percentage of Gleason grade 4/5 cancer (30.3%) than subjects with the FF or Ff genotypes (42.8% and 43.8%, respectively; P = 0.015 by t test for ff versus FF + Ff). The data suggest that the presence of an F allele increased the risk of being diagnosed with more aggressive cancer because higher percentage of Gleason grade 4/5 is associated with worse prognosis. The age-adjusted risk of prostate-specific antigen failure was lower for the ff genotype than for the FF genotype by Cox proportional hazards analysis but did not achieve statistical significance (hazard ratio = 0.76; 95% confidence interval, 0.44-1.32). This risk reduction disappeared after further adjustment for percentage of Gleason grade 4/5, cancer volume, and preoperative serum prostate-specific antigen level (hazard ratio = 1.03; 95% confidence interval, 0.58-1.85). In conclusion, the ff genotype was associated with less aggressive histopathological findings than Ff or FF genotypes. Additional studies with a larger sample size and investigation of the functional significance of the FokI polymorphism in prostate cancer cells are warranted.

    View details for Web of Science ID 000180536800005

    View details for PubMedID 12540499

  • The role of vitamin D in prostate cancer VITAMIN D ANALOGS IN CANCER PREVENTION AND THERAPY Krishnan, A. V., Peehl, D. M., Feldman, D. 2003; 164: 205-221

    Abstract

    Prostate cancer (PCa) cells harbor receptors for vitamin D (VDR) as well as androgens (AR). 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] increases AR expression and enhances androgen actions linking the two receptor systems. 1,25(OH)2D3 exhibits antiproliferative activity in both AR-positive and AR-negative PCa cells. Less calcemic analogs of 1,25(OH)2D3, with more antiproliferative activity, are being developed and will be more useful clinically. The mechanisms underlying differential analog activity are being investigated. In target cells, 1,25(OH)2D3 induces 24-hydroxylase, the enzyme that catalyzes its self-inactivation. Co-treatment with 24-hydroxylase inhibitors enhances the antiproliferative activity of calcitriol. Primary cultures of normal or cancer-derived prostatic epithelial cells express 1alpha-hydroxylase, the enzyme that catalyzes the synthesis of 1,25(OH)2D3, the levels being much lower in the cancer-derived cells and in PCa cell lines. This finding raises the possibility of using 25-hydroxyvitamin D3 [25(OH)D3] as a chemopreventive agent in PCa. In LNCaP human PCa cells, 1,25(OH)2D3 and its analogs exert antiproliferative activity predominantly by cell cycle arrest, but also induce apoptosis, although to a much lesser degree. Growth arrest is mediated by induction of IGF binding protein-3 (IGFBP-3), which in turn increases the expression of the cell cycle inhibitor p21, leading to growth arrest. Other actions of 1,25(OH)2D3 in PCa cells include promotion of pro-differentiation effects and inhibition of tumor cell invasion, metastasis and angiogenesis. Combination therapy with retinoids, other anticancer agents or 24-hydroxylase inhibitors augments the inhibitory activity of 1,25(OH)2D3 in PCa and provides another effective approach in PCa treatment. Small clinical trials have shown that 1,25(OH)2D3 can slow the rate of prostate specific antigen (PSA) rise in PCa patients, demonstrating proof of concept that 1,25(OH)2D3 or its analogs will be clinically effective in PCa therapy. Current research involves further investigation of the role of 1,25(OH)2D3 and its analogs for the therapy or chemoprevention of PCa.

    View details for Web of Science ID 000184907200015

    View details for PubMedID 12899524

  • Preclinical activity of ketoconazole in combination with calcitriol or the vitamin D analogue EB 1089 in prostate cancer cells JOURNAL OF UROLOGY Peehl, D. M., Seto, E., Hsu, J. Y., Feldman, D. 2002; 168 (4): 1583-1588

    Abstract

    Ketoconazole is a general inhibitor of P450 enzymes, of which some are necessary for androgen biosynthesis and the metabolism of vitamin D compounds. We tested the growth inhibitory activity of ketoconazole combined with 1,25-dihydroxyvitamin D3 (calcitriol) and with the vitamin D analogue EB 1089 in a preclinical model of prostate cancer.Clonal assays with primary cultures of human prostatic cancer cells were performed to test anti-proliferative effects of ketoconazole alone or in combination with calcitriol or EB 1089. Enzyme substrate reactions were done to determine whether the ability of ketoconazole to potentiate the activity of calcitriol or EB 1089 was due to the inhibition of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the enzyme that initiates conversion of active vitamin D compounds to inactive products.Ketoconazole, calcitriol and EB 1089 each inhibited the growth of prostatic cancer cells. In combination 0.1 microg./ml. ketoconazole potentiated growth inhibitory activity of calcitriol 50-fold and EB 1089 10-fold. Induction of 24-hydroxylase by calcitriol or EB 1089 was partially blocked by this level of ketoconazole.Combination therapy with ketoconazole and calcitriol or EB 1089 may enhance antitumor activities of vitamin D compounds for prostate cancer and alleviate side effects of vitamin D deficiency that are likely associated with ketoconazole therapy.

    View details for DOI 10.1097/01.ju.0000030158.18335.84

    View details for Web of Science ID 000178187800086

    View details for PubMedID 12352462

  • Polymorphisms in the androgen receptor and type II 5 alpha-reductase genes and prostate cancer prognosis PROSTATE Shibata, A., Garcia, M. I., Cheng, I., Stamey, T. A., McNeal, J. E., Brooks, J. D., Henderson, S., Yemoto, C. E., Peehl, D. M. 2002; 52 (4): 269-278

    Abstract

    Cytosine-adenine-guanine repeat length of the androgen receptor gene and the A49T and V89L polymorphisms of the 5 alpha-reductase (SRD5A2) gene have been associated with prostate cancer.We investigated the relationship of the three genetic polymorphisms to tumor grade among 211 men who had undergone radical prostatectomy. Subjects had prostate cancer <3 cm(3) with a percentage of cancer represented by Gleason grade 4 or 5 (% Gleason grade 4/5) of either > or = 20% or < or = 5%. We also examined the association between those genetic markers and prostate specific antigen (PSA) failure among 112 subjects with > or = 20% Gleason grade 4/5.In cross-sectional analysis, none of the polymorphisms was a significant predictor of % Gleason grade 4/5. In longitudinal analysis, the LL genotype at the V89L site was associated with statistically significant four- to sixfold increase in PSA failure risk after adjustment for clinicopathologic variables.We observed poorer prognosis among men with the LL genotype at codon 89 of the SRD5A2 gene. Lack of consistency between studies must be resolved before clinical utility of this marker is established.

    View details for DOI 10.1002/pros.10119

    View details for Web of Science ID 000177541200003

    View details for PubMedID 12210487

  • Antiproliferative and proapoptotic activities of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells CLINICAL CANCER RESEARCH Kiviharju, T. M., Lecane, P. S., Sellers, R. G., Peehl, D. M. 2002; 8 (8): 2666-2674

    Abstract

    Interest in exploiting traditional medicines for prevention or treatment of cancer is increasing. Extracts from the herb Tripterygium wilfordii hook F have been used in China for centuries to treat immune-related disorders. Recently it was reported that triptolide (PG490), a purified compound from Tripterygium, possessed antitumor properties and induced apoptosis by p53-independent mechanisms in a variety of malignant cell lines. This property of triptolide attracted our attention because we have found that primary cultures of human prostatic epithelial cells derived from normal tissues and adenocarcinomas are in general extremely resistant to apoptosis. Furthermore, the function of wild-type p53 is impaired in these cells such that drugs that require p53 activity to induce cell death are ineffective. Therefore, the properties of triptolide and the recent approval of its water-soluble form (PG490-88) for entry into Phase I clinical trials suggested that this drug was a promising candidate to test for antitumor activity against prostate cells. Experiments presented here demonstrated that triptolide had dose-dependent effects on both normal and cancer-derived primary cultures of human prostatic epithelial cells. Low concentrations of triptolide inhibited cell proliferation and induced a senescence-like phenotype. Higher concentrations of triptolide induced apoptosis that was unexpectedly associated with nuclear accumulation of p53. Paradoxically, levels of the p53 target genes, p21(WAF1/CIP1) and hdm-2, were reduced, as was bcl-2. Our preclinical studies suggest that triptolide might be an effective preventive as well as therapeutic agent against prostate cancer and that triptolide may activate a functional p53 pathway in prostate cells.

    View details for Web of Science ID 000177520700029

    View details for PubMedID 12171899

  • Silencing of pi-class glutathione S-transferase in MDA PCa 2a and MDA PCa 2b cells PROSTATE Vidanes, G. M., Paton, V., Wallen, E., Peehl, D. M., Navone, N., Brooks, J. D. 2002; 51 (4): 225-230

    Abstract

    Loss of expression of the glutathione S-transferase-pi (GSTP1) is the most common genetic alteration described in human prostate cancer, occurring in virtually all tumors regardless of grade or stage. Of the available human prostate cancer cell lines, only LNCaP mirrors this phenotype. We investigated whether the prostate cancer cell lines MDA PCa 2a and MDA PCa 2b share this phenotype.GSTP1 protein and mRNA levels were assessed in the MDA PCa 2a and MDA PCa 2b cell lines by Western and Northern blot. DNA methylation was evaluated by Southern blot analysis of genomic DNA digested with the methylation-sensitive restriction enzymes BssHII, NotI, and SacII. Re-expression of GSTP1 was determined by RT-PCR following treatment with 5-azacytidine, a DNA methyltransferase inhibitor, and/or the histone deacetylase inhibitor trichostatin A (TSA).Like all human prostatic carcinomas in vivo, both the MDA PCa 2a and 2b cell lines lack protein and mRNA expression of GSTP1. This lack of expression is associated with methylation in the GSTP1 gene promoter. Treatment with the methyltransferase inhibitor 5-azacytidine resulted in re-expression of GSTP1. By itself, TSA did not result in re-expression of GSTP1, nor did it augment expression induced by 5-azacytidine.MDA PCa 2a and 2b appear to be useful models of human prostatic carcinoma in that they lack expression of GSTP1 due to gene silencing via promoter methylation. Inhibition of histone acetylation does not appear to affect GSTP1 expression.

    View details for DOI 10.1002/pros.10093

    View details for Web of Science ID 000175909900001

    View details for PubMedID 11987150

  • A glucocorticoid-responsive mutant androgen receptor exhibits unique ligand specificity: Therapeutic implications for androgen-independent prostate cancer ENDOCRINOLOGY Krishnan, A. V., Zhao, X. Y., Swami, S., Brive, L., Peehl, D. M., Ely, K. R., Feldman, D. 2002; 143 (5): 1889-1900

    Abstract

    The cortisol/cortisone-responsive AR (AR(ccr)) has two mutations (L701H and T877A) that were found in the MDA PCa human prostate cancer cell lines established from a castrated patient whose metastatic tumor exhibited androgen-independent growth. Cortisol and cortisone bind to the AR(ccr) with high affinity. In the present study, we characterized the structural determinants for ligand binding to the AR(ccr). Our data revealed that many of the C17, C19, and C21 circulating steroids, at concentrations that are found in vivo, functioned as effective activators of the AR(ccr) but had little or no activity via the wild-type AR or GRalpha. Among the synthetic glucocorticoids tested, dexamethasone activated both GRalpha and AR(ccr), whereas triamcinolone was selective for GRalpha. In MDA PCa 2b cells, growth and prostate-specific antigen production were stimulated by potent AR(ccr) agonists such as cortisol or 9alpha-fluorocortisol but not by triamcinolone (which did not bind to or activate the AR(ccr)). Of the potential antagonists tested, bicalutamide (casodex) and GR antagonist RU38486 showed inhibitory activity. We postulate that corticosteroids provide a growth advantage to prostate cancer cells harboring the promiscuous AR(ccr) in androgen-ablated patients and contribute to their transition to androgen-independence. We predict that triamcinolone, a commonly prescribed glucocorticoid, would be a successful therapeutic agent for men with this form of cancer, perhaps in conjunction with the antagonist casodex. We hypothesize that triamcinolone administration would inhibit the hypothalamic-pituitary-adrenal axis, thus suppressing endogenous corticosteroids, which stimulate tumor growth. Triamcinolone, by itself, would not activate the AR(ccr) or promote tumor growth but would provide glucocorticoid activity essential for survival.

    View details for Web of Science ID 000175170500037

    View details for PubMedID 11956172

  • Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line CELL DEATH AND DIFFERENTIATION Armstrong, J. S., Steinauer, K. K., Hornung, B., Irish, J. M., Lecane, P., Birrell, G. W., Peehl, D. M., Knox, S. J. 2002; 9 (3): 252-263

    Abstract

    The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.

    View details for DOI 10.1038/sj.cdd.4400959

    View details for Web of Science ID 000174032600006

    View details for PubMedID 11859408

  • Genetic polymorphisms in the androgen receptor and type II 5 alpha-reductase genes in prostate enlargement JOURNAL OF UROLOGY Shibata, A., Stamey, T. A., McNeal, J. E., Cheng, I., Peehl, D. M. 2001; 166 (4): 1560-1564

    Abstract

    We examined the association of androgen receptor gene cytosine-adenine-guanine (CAG) repeat length and the 2 single nucleotide polymorphisms A49T and V89L in the type II 5 alpha-reductase gene with prostate enlargement measured as the weight of the surgically removed prostate.A total of 68 men with a prostate weighing 80 gm. or greater were compared with 197 controls with a prostate weighing less than 80 gm. These men had undergone radical prostatectomy between 1992 and 1996. DNA was extracted from archival prostate tissue uninvolved with cancer and genotyped for 3 polymorphic markers. The effects of genetic variants and clinicopathological variables on prostate enlargement risk were estimated by logistic regression.The age adjusted odds ratio estimate of prostate enlargement risk in men with 23 or greater versus 20 or fewer CAG repeats was 0.41 (95% confidence interval 0.19 to 0.89). This risk reduction was consistently found when an alternative prostate enlargement definition and subject restriction were used. No consistent association with prostate enlargement risk was observed for A49T or V89L polymorphisms.Our finding further supports the hypothesis that the shorter CAG repeat length of the androgen receptor gene is related to prostate enlargement.

    View details for Web of Science ID 000170950100102

    View details for PubMedID 11547131

  • Rationale for combination ketoconazole/ vitamin D treatment of prostate cancer. Urology Peehl, D. M., Seto, E., Feldman, D. 2001; 58 (2): 123-126

    Abstract

    The high rate of progression of prostate cancer after androgen deprivation therapy mandates that new strategies be developed. Adjuvant therapy combined with androgen deprivation may slow or prevent progression. Ketoconazole plus calcitriol therapy is an example of 1 such a combination with a mechanistic basis for synergistic activity. Ketoconazole is commonly used as a second-line androgen deprivation therapy. This imidazole derivative is an inhibitor of P-450 enzymes, including those involved in steroidogenesis. Other P-450 enzymes that are inhibited by ketoconazole include 1alpha-hydroxylase and 24-hydroxylase, which metabolize vitamin D. Growth inhibition of prostate cancer cells by vitamin D depends on levels of the active metabolite, 1,25-dihydroxyvitamin D(3) (calcitriol). The enzyme 24-hydroxylase converts calcitriol to less active products. The inhibition of 24-hydroxylase by ketoconazole maintains the magnitude and duration of response to calcitriol. Combined ketoconazole/calcitriol therapy might therefore potentiate the antitumor activity of calcitriol. Because androgen-independent prostate cancer cells often remain responsive to growth inhibition by calcitriol, it is also possible that calcitriol would slow or prevent development of androgen-independent cancer growth. Another consideration is that ketoconazole blocks 1alpha-hydroxylase activity, which is the key enzyme that creates calcitriol in the body. Therefore, patients receiving ketoconazole therapy are likely to be deficient in vitamin D. The detrimental consequences of vitamin D deficiency in these patients would also be alleviated by the addition of calcitriol to the therapeutic regimen.

    View details for PubMedID 11502466

  • Reduced 1 alpha-hydroxylase activity in human prostate cancer cells correlates with decreased susceptibility to 25-hydroxyvitamin D-3-induced growth inhibition CANCER RESEARCH Hsu, J. Y., Feldman, D., McNeal, J. E., Peehl, D. M. 2001; 61 (7): 2852-2856

    Abstract

    Evidence from epidemiological, molecular, and genetic studies suggests a role for vitamin D in the development and/or progression of prostate cancer. In experimental models and clinical trials, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was shown to exert antiproliferative, prodifferentiating, and antimetastatic/invasive effects on prostatic epithelial cells. Because the direct clinical application of 1,25(OH)2D3 is limited by the major side effect of hypercalcemia, we investigated the potential therapeutic utility of its less calcemic precursor, 25-hydroxyvitamin D3 [25(OH)D3], which is converted locally within the prostate to 1,25(OH)2D3 by 1alpha-hydroxylase. Quantification of 1alpha-hydroxylase activity in human prostatic epithelial cells by enzyme-substrate reaction analyses revealed a significantly decreased activity in cells derived from adenocarcinomas compared with cells derived from normal tissues or benign prostatic hyperplasia (BPH). In growth assays, we found that 25(OH)D3 inhibited growth of normal or BPH cells similarly to 1,25(OH)2D3. In contrast, in primary cultures of cancer cells and established cell lines, the antiproliferative action of 25(OH)D3 was significantly less pronounced than that of 1,25(OH)2D3. Our results indicate that growth inhibition by 25(OH)D3 depends on endogenous 1alpha-hydroxylase activity, and that this activity is deficient in prostate cancer cells. This finding has ramifications for both the prevention and therapy of prostate cancer with vitamin D compounds.

    View details for Web of Science ID 000167967100012

    View details for PubMedID 11306457

  • SMAD3 represses androgen receptor-mediated transcription CANCER RESEARCH Hayes, S. A., Zarnegar, M., Sharma, M., Yang, F. J., Peehl, D. M., ten Dijke, P., Sun, Z. J. 2001; 61 (5): 2112-2118

    Abstract

    The androgen-signaling pathway is important in the growth and progression of prostate cancer. Androgen ablation therapy, which may result in programmed cell death, is often used to treat advanced prostate cancer. The growth-promoting effects of androgen are mediated mostly through the androgen receptor (AR). Transforming growth factor beta (TGF-beta) plays critical roles in controlling prostate cell proliferation, differentiation, and apoptosis. Normal transcripts and proteins of TGF-beta receptors are frequently lost in prostate cancer cells, especially in advanced stages of the disease. However, the mechanisms by which TGF-beta inhibits proliferation and induces apoptosis in prostate cancer cells is not clear. We investigated the molecular mechanism by which TGF-beta inhibits transcriptional activation mediated by AR. Using transient transfection systems, we demonstrated that Smad3 specifically represses transcriptional activation mediated by AR on two natural androgen-responsive promoters. This repression is transmitted through TGF-beta signaling and can be regulated by other Smad proteins. A protein-protein interaction between AR and Smad3 was identified in vitro and in vivo, and the transcription activation domain of AR and the MH2 of Smad3 were identified as being responsible for binding. Additional functional experiments showed that the repression of AR by Smad3 is mediated solely through the MH2 domain. These results provide fresh insight for understanding the mechanism by which TGF-beta regulates the androgen-signaling pathway in prostate cancer cells.

    View details for Web of Science ID 000167568100056

    View details for PubMedID 11280774

  • Cultured stromal cells: An in vitro model of prostatic mesenchymal biology PROSTATE Peehl, D. M., Sellers, R. G. 2000; 45 (2): 115-123

    Abstract

    Initial efforts to develop in vitro models to study prostatic biology focused on the culture and characterization of epithelial cells. Recently, attention has turned towards inclusion of stromal cells in experimental systems.Improved methods to isolate and culture stromal cells have been developed. An array of markers are employed to characterize subtypes of stromal cells, with particular interest in smooth muscle differentiation.Defined, serum-free media are available for certain experimental applications. Conditions that promote smooth muscle differentiation have been identified. Investigators have characterized hormonal and peptide factors that regulate the growth of prostatic stromal cells, and have also described paracrine factors produced by stromal cells that influence epithelial biology.Prostatic stromal-cell cultures are now widely employed by a large number of investigators for a diverse array of experimental purposes. While further refinement is required to obtain model systems that fully mimic in vivo processes, the availability of stromal- and epithelial-cell cultures provides a valuable resource for studying normal prostatic biology as well as benign prostatic hyperplasia (BPH) and cancer.

    View details for Web of Science ID 000089614600005

    View details for PubMedID 11027410

  • Brefeldin A induces p53-independent apoptosis in primary cultures of human prostatic cancer cells JOURNAL OF UROLOGY Wallen, E., Sellers, R. G., Peehl, D. M. 2000; 164 (3): 836-841

    Abstract

    The objective of this study was to investigate growth-inhibitory and apoptotic activity of the experimental antitumor drug, brefeldin A (BFA), on primary cultures of human epithelial cells derived from prostatic adenocarcinomas.Clonal assays were performed to evaluate the effects of BFA on growth of prostatic cancer cell strains. Loss of cell viability in response to BFA was assessed by trypan blue exclusion. Induction of apoptosis by BFA was evaluated by morphologic criteria, electrophoretic assay of DNA fragmentation, and a cell death ELISA. Immunoblots were used to monitor p53 and pRB expression in response to BFA.BFA was growth-inhibitory at a half-maximal concentration of 5 ng./ml. (18 nM). Morphological manifestations of apoptosis were evident by 24 hours of treatment. Cell viability declined and the cell death ELISA indicated an 18-fold increase in apoptosis in BFA-treated versus untreated cells at 48 hours. DNA fragmentation was also seen at 48 hours. Levels of p53 were not altered by BFA, but pRB was maintained in a hypophosphorylated state by BFA treatment.BFA is a potent inducer of apoptosis in prostatic cancer cells via a p53-independent mechanism. Cells derived from low-grade as well as high-grade cancers responded similarly to BFA. Since p53-mediated pathways of apoptosis may frequently be abrogated in prostatic cancer cells, agents such as BFA that induce p53-independent cell death may be promising candidates for chemotherapeutic agents.

    View details for Web of Science ID 000088682100068

    View details for PubMedID 10953164

  • 1 alpha,25-dihydroxyvitamin D-3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms ENDOCRINOLOGY Zhao, X. Y., Peehl, D. M., Navone, N. M., Feldman, D. 2000; 141 (7): 2548-2556

    Abstract

    We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.

    View details for Web of Science ID 000088386200032

    View details for PubMedID 10875257

  • Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor NATURE MEDICINE Zhao, X. Y., Malloy, P. J., Krishnan, A. V., Swami, S., Navone, N. M., Peehl, D. M., Feldman, D. 2000; 6 (6): 703-706

    Abstract

    The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

    View details for Web of Science ID 000087438300044

    View details for PubMedID 10835690

  • Two mutations identified in the androgen receptor of the new human prostate cancer cell line MDA PCa 2a JOURNAL OF UROLOGY Zhao, X. Y., Boyle, B., Krishnan, A. V., Navone, N. M., Peehl, D. M., Feldman, D. 1999; 162 (6): 2192-2199

    Abstract

    We have characterized the androgen receptor (AR) in a new human prostate cancer cell line, MDA PCa 2a, that has recently been established from a bone metastasis of a patient whose cancer exhibited androgen-independent growth.Androgen responsiveness of these cells was assessed by measuring the effect of DHT and R1881 on cell growth and PSA secretion. Scatchard analysis was used to characterize the affinity and abundance of AR protein. Using a PCR based strategy, genomic DNA of the entire coding region of AR gene was sequenced to identify possible mutations.These cells express abundant AR (Nmax = 685 +/- 149 fmol./mg. protein), but the AR binding affinity (Kd) for DHT is only 25 nM, approximately 50-fold lower affinity than the mutated AR in LNCaP prostate cancer cells (Kd = 0.5 nM) or the wildtype AR in MCF-7 breast cancer cells (Kd = 0.4 nM). Two mutations, L701H and T877A, were identified in the ligand binding domain of the AR gene. Compared with LNCaP cells, the new cell line is significantly less responsive to DHT and R1881 as well as to other androgens such as testosterone, androstenedione, and DHEA. Similar to LNCaP cells, the ligand specificity of the AR in MDA PCa 2a cells appears to be relaxed and non-androgens such as progesterone and estradiol act as agonists although with less potency than in LNCaP cells. Interestingly, in the absence of androgens, the new cell line expresses 15-fold higher baseline levels of PSA than LNCaP.Two mutations were identified in the AR gene of the MDA PCa 2a cell line that are likely responsible for the decreased androgen sensitivity and altered ligand specificity observed in these cells. Thus, this new cell line with partial androgen responsiveness and PSA expression can serve as a functionally relevant model system of bone metastatic prostate cancer, and can be used to investigate the role of AR mutations in prostate cancer and its progression to androgen independence.

    View details for Web of Science ID 000083639500119

    View details for PubMedID 10569618

  • Altered growth regulation and loss of response to retinoic acid accompany tumorigenic transformation of prostatic cells ANTICANCER RESEARCH Peehl, D. M., Sellers, R. G., Arnstein, P., Kung, H. F., Rhim, J. S. 1999; 19 (5B): 3857-3864

    Abstract

    In vitro models of human prostatic carcinogenesis are increasingly available and include representatives of normal, immortal, tumorigenic and metastatic phenotypes. In this study, growth regulation of immortal, but non-tumorigenic, human papillomavirus-transformed prostatic epithelial cells was compared to that of their tumorigenic variants. These variants were created either by exposure to a carcinogen or by passage through mice. In all cases, tumorigenic cells retained responsiveness to a potent mitogen, epidermal growth factor, and to a potent growth inhibitory factor, 1,25-dihydroxyvitamin D3. Responses to other growth regulatory factors were altered. One set of transformants, CA-HPV-10 and its tumorigenic variants 5019 and 5019IIc, lost their requirement for insulin-like growth factor. Another set, RWPE-1 and its tumorigenic variant 129Nu5002-1 Tu, became unresponsive to growth inhibition by transforming growth factor-beta. The only alteration uniquely correlated with the tumorigenic phenotype was loss of response to retinoic acid. This factor, which inhibits growth of normal and immortal but non-tumorigenic prostatic epithelial cells, had no effect on tumorigenic 129Nu5002-1 Tu cells. We previously reported that conversion of an SV40-immortalized prostatic epithelial cell line to tumorigenicity by introduction of the ras oncogene also resulted in loss of responsiveness to growth inhibitory activity of retinoic acid. 129Nu5002-1 Tu cells, which do not have an altered ras gene, gained the same phenotype. This suggests that loss of inhibition by retinoic acid may be a critical element in the tumorigenic conversion of prostatic epithelial cells.

    View details for Web of Science ID 000083973200008

    View details for PubMedID 10628324

  • Induction of androgen receptor by 1 alpha,25-dihydroxyvitamin D-3 and 9-cis retinoic acid in LNCaP human prostate cancer cells ENDOCRINOLOGY Zhao, X. Y., Ly, L. H., Peehl, D. M., Feldman, D. 1999; 140 (3): 1205-1212

    Abstract

    We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.

    View details for Web of Science ID 000078733700025

    View details for PubMedID 10067845

  • Defined medium for normal adult human prostatic stromal cells IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL Peehl, D. M., Sellers, R. G., Wong, S. T. 1998; 34 (7): 555-560

    Abstract

    Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10(3) cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn't stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.

    View details for Web of Science ID 000075277700011

    View details for PubMedID 9719415

  • Basic FGF, EGF, and PDGF modify TGF beta-induction of smooth muscle cell phenotype in human prostatic stromal cells PROSTATE Peehl, D. M., Sellers, R. G. 1998; 35 (2): 125-134

    Abstract

    We investigated the ability of a variety of growth factors to regulate the differentiation of prostatic fibroblasts into smooth muscle cells.Smooth muscle actin levels were monitored by immunoblot analysis and immunocytochemistry. Proliferation was measured in clonal growth assays and by cell counts.We determined that TGFbeta inhibited proliferation and induced smooth muscle differentiation of stromal cells derived from prostatic adenocarcinomas, as we previously reported for cells derived from the normal peripheral zone. Basic FGF, EGF, TGFalpha, and PDGF, but not IGF, retinoic acid, 1,25-dihydroxyvitamin D3, or androgen, attenuated induction of differentiation by TGFbeta, by a mechanism apparently unrelated to proliferation.Regulation of growth and differentiation occurs equivalently in prostatic stromal cells derived from adenocarcinomas and normal peripheral zone. TGFbeta is a potent inducer of the smooth muscle phenotype. Basic FGF, EGF and/or TGFalpha, and PDGF attenuate TGFbeta's activity, and promote a fibroblastic phenotype. Our studies provide an in vitro model system in which fibroblastic or smooth muscle cells can be promoted, maintained, and investigated in a defined manner. The results suggest that the ratio of fibroblasts to smooth muscle cells in the stroma reflects the relative levels of growth factors, which may be altered in diseased states.

    View details for Web of Science ID 000073180900006

    View details for PubMedID 9568676

  • Selection of human cervical epithelial cells that possess reduced apoptotic potential to low-oxygen conditions CANCER RESEARCH Kim, C. Y., Tsai, M. H., OSMANIAN, C., Graeber, T. G., Lee, J. E., Giffard, R. G., DiPaolo, J. A., Peehl, D. M., Giaccia, A. J. 1997; 57 (19): 4200-4204

    Abstract

    Since human papillomavirus (HPV) infection is strongly associated with cervical neoplasia and tumor hypoxia has prognostic significance in human cervical carcinomas, we examined the relationship between hypoxia and apoptosis in human cervical epithelial cells expressing high-risk HPV type 16 oncoproteins. In vitro, hypoxia stimulated both p53 induction and apoptosis in primary cervical epithelial cells infected with the HPV E6 and E7 genes but not in cervical fibroblasts infected with E6 and E7. Furthermore, cell lines derived from HPV-associated human cervical squamous cell carcinomas were substantially less sensitive to apoptosis induced by hypoxia, indicating that these cell lines have acquired additional genetic alterations that reduced their apoptotic sensitivity. Although the process of long-term cell culturing resulted in selection for subpopulations of HPV oncoprotein-expressing cervical epithelial cells with diminished apoptotic potential, the exposure of cells to hypoxia greatly accelerated the selection process. These results provide evidence for the role of hypoxia-mediated selection of cells with diminished apoptotic potential in the progression of human tumors and can in part explain why cervical tumors that possess low pO2 values are more aggressive.

    View details for Web of Science ID A1997XZ01100013

    View details for PubMedID 9331075

  • 1 alpha 25-dihydroxyvitamin D-3 actions in LNCaP human prostate cancer cells are androgen-dependent ENDOCRINOLOGY Zhao, X. Y., Ly, L. H., Peehl, D. M., Feldman, D. 1997; 138 (8): 3290-3298

    Abstract

    We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.

    View details for Web of Science ID A1997XL84200028

    View details for PubMedID 9231780

  • Loss of response to epidermal growth factor and retinoic acid accompanies the transformation of human prostatic epithelial cells to tumorigenicity with v-Ki-ras CARCINOGENESIS Peehl, D. M., Wong, S. T., Sellers, R. G., Jin, S., Rhim, J. S. 1997; 18 (8): 1643-1650

    Abstract

    Growth factor-independent proliferation and loss of response to differentiation factors are believed to be critical elements in carcinogenesis. We have developed an in vitro model of human prostatic carcinogenesis by the introduction of SV40 DNA into normal prostatic epithelial cells to create a transformed, immortal cell line, pRNS-1-1. This non-tumorigenic cell line responded similarly to normal prostatic epithelial cells to most growth- and differentiation-regulatory factors, with the notable exception of loss of response to the inhibitory factor 1,25-dihydroxyvitamin D3. In this study, we describe the introduction of the ras oncogene into pRNS-1-1 cells to create a tumorigenic cell line, pRNS-1-1/ras. In addition to an attenuated response to 1,25-dihydroxyvitamin D3, these cells also became unresponsive to retinoic acid and gained the ability to undergo clonal proliferation in the absence of epidermal growth factor (EGF). EGF-independent growth could not be linked to the production of autocrine transforming growth factor-alpha, but instead was likely due to sustained signaling by the ras oncogene, bypassing ligand-activation of the EGF receptor. Ligand-independent proliferation, coupled with the loss of response to the growth-inhibitory and differentiation agent retinoic acid, may be important elements in the conversion of human prostatic epithelial cells to tumorigenicity.

    View details for Web of Science ID A1997XQ87800030

    View details for PubMedID 9276642

  • Induction of smooth muscle cell phenotype in cultured human prostatic stromal cells EXPERIMENTAL CELL RESEARCH Peehl, D. M., Sellers, R. G. 1997; 232 (2): 208-215

    Abstract

    Stromal cells are key regulators of growth and differentiation in the adult human prostate. Alterations in the stroma are believed to initiate the development of benign prostatic hyperplasia, and stromal-epithelial interactions may have a role in malignant progression. The prostatic stroma is composed of two major cell types, smooth muscle cells and fibroblasts. Cell cultures from the prostatic stroma have been established by several investigators, but the phenotype of these cells has not been extensively characterized and it is not clear whether they are fibroblastic or smooth muscle-like. In this study, the response of stromal cells cultured from normal prostatic tissues to transforming growth factor-beta (TGF beta) was investigated. We confirmed a previous report that TGF beta inhibited the growth of prostatic stromal cells in serum-containing medium, and showed that inhibition also occurred in serum-free medium. Growth inhibition by TGF beta was irreversible after 24 to 72 h of exposure. In the absence of TGF beta, cells were fibroblastic and expressed vimentin and fibronectin but little alpha-smooth muscle actin. After 3 days of exposure to 1 ng/ml of TGF beta, the majority of cells expressed alpha-smooth muscle actin and desmin, as demonstrated by immunocytochemistry and immunoblot analysis. This effect was specific and alpha-smooth muscle actin was not induced by two other growth-inhibitory factors, retinoic acid or 1,25-dihydroxyvitamin D3. These results suggest that TGF beta is an important regulator of growth and differentiation of prostatic stromal cells and that a smooth muscle cell phenotype is promoted in the presence of TGF beta.

    View details for Web of Science ID A1997WX48900002

    View details for PubMedID 9168795

  • Parathyroid hormone-related protein is not an autocrine growth factor for normal prostatic epithelial cells PROSTATE Peehl, D. M., Edgar, M. G., Cramer, S. D., Deftos, L. J. 1997; 31 (1): 47-52

    Abstract

    Parathyroid hormone-related protein (PTHrP) is the primary factor responsible for humoral hypercalcemia of malignancy. The hypercalcemic actions of PTHrP occur via stimulation of renal distal tubular calcium reabsorption and increased osteoclastic bone resorption. These effects of PTHrP are thought to be mediated through a common parathyroid hormone (PTH)/PTHrP receptor. In addition to the well-established role of PTHrP in bone remodeling, PTHrP is believed to be an important mediator of cellular growth and differentiation in a number of nonbony tissues. We recently demonstrated abundant expression of PTHrP in normal and malignant human prostatic tissues, and in cultured prostatic epithelial cells.In vitro assays were used to test growth-regulatory activity of synthetic and endogenous PTHrP peptides on normal prostatic epithelial cells.No growth-regulatory activity could be demonstrated.PTHrP is not an autocrine growth factor for normal prostatic epithelial cells.

    View details for Web of Science ID A1997WT87500008

    View details for PubMedID 9108886

  • Prostate specific antigen cleaves parathyroid hormone-related protein in the PTH-like domain: Inactivation of PTHrP-stimulated cAMP accumulation in mouse osteoblasts JOURNAL OF UROLOGY Cramer, S. D., Chen, Z. X., Peehl, D. M. 1996; 156 (2): 526-531

    Abstract

    To determine whether parathyroid hormone-related protein (PTHrP) is a substrate of prostate-specific antigen (PSA) and how the biological activity of PTHrP may be altered by cleavage with PSA.Prostate-specific antigen cleavage of recombinant human PTHrP 1-141 was conducted in vitro at 37C and analyzed by SDS-PAGE. Five rounds of automated amino-terminal amino acid sequence analysis were performed on blotted PSA-cleaved PTHrP peptide fragments to determine the PSA cleavage sites. The mouse osteoblast cell line MC3T3-E1 was used to test whether PSA cleavage of PTHrP 1-141 altered its ability to stimulate cAMP production.Prostate-specific antigen was found to specifically cleave PTHrP 1-141 in a time- and dose-dependent manner. Cleavage of PTHrP 1-141 by PSA generated fragments on Coomassie-stained acrylamide gels that migrated with mobilities that corresponded to 19.5, 17, 15 and < 7 kd. The preferred PSA cleavage site of PTHrP 1-141 was determined to be at the carboxyl-terminus of phenylalanine 23, consistent with chymotryptic-like enzymatic activity of PSA. Cleavage of PTHrP by PSA completely abolished the ability of PTHrP to stimulate cAMP production.Cleavage of PTHrP 1-141 by PSA carboxyl-terminal to phenylalanine 23 represents a unique pattern of PTHrP processing that may be specific to the prostate. Prostate-specific antigen inactivation of the cAMP-inducing activity of PTHrP 1-141 demonstrates that PSA cleavage regulates the biological activity of PTHrP. These results have implications for the role of PTHrP in prostate cancer metastasis to bone and its subsequent regulation of bone remodeling. Study of the biological activities of the PSA-generated PTHrP peptides identified in this study merits further investigation.

    View details for Web of Science ID A1996UX15200074

    View details for PubMedID 8683730

  • Parathyroid hormone-related protein (PTHrP) is an epidermal growth factor-regulated secretory product of human prostatic epithelial cells PROSTATE Cramer, S. D., Peehl, D. M., Edgar, M. G., Wong, S. T., Deftos, L. J., Feldman, D. 1996; 29 (1): 20-29

    Abstract

    Parathyroid hormone-related protein (PTHrP) has previously been shown to be expressed in human prostatic tissue and in prostatic cancer cell lines. In the present study, PTHrP immunoreactivity was detected in the glandular epithelium of normal prostate and benign prostatic hyperplasia (BPH), as well as in prostatic adenocarcinoma (CaP). Epithelial cell cultures derived from normal, BPH, and CaP tissues were also stained by antibodies against PTHrP, and northern analysis revealed multiple transcripts of PTHrP in the cellular RNA. PTHrP (1-34) was measurable by radioimmunoassay (RIA) in media conditioned by the prostatic epithelial cell cultures, and PTHrP accumulated in conditioned media during a 72 hr time course. Addition of complete growth medium to starved cells resulted in increased PTHrP mRNA levels by 1 hr, with maximal stimulation at 8-24 hr. Several individual factors contained in the complete growth medium were tested for their ability to regulate PTHrP expression. Epidermal growth factor (EGF) was the major inducer of PTHrP expression, while cholera toxin, bovine pituitary extract, hydrocortisone, and insulin had minimal or no effect on PTHrP transcript levels. Since each of these factors is growth stimulatory, the unique ability of EGF to induce PTHrP is apparently unrelated to mitogenicity. 1,25-Dihydroxyvitamin D3[1,25(OH)2D3], an inhibitor of PTHrP expression in several other cell types, had no effect on steady-state levels of PTHrP mRNA expressed by epithelial cells in complete growth medium, although prostate cells have vitamin D receptors and are responsive to 1,25(OH)2D3 in other ways. Our results indicate that PTHrP expression is not confined to the neuroendocrine cells of the human prostate and that our culture system can be used as a model to investigate the role of PTHrP in the prostate.

    View details for Web of Science ID A1996UX88000003

    View details for PubMedID 8685051

  • Keratin 19 in the adult human prostate: Tissue and cell culture studies CELL AND TISSUE RESEARCH Peehl, D. M., Sellers, R. G., McNeal, J. E. 1996; 285 (1): 171-176

    Abstract

    Keratin 19 is found primarily in simple epithelia. In mammary epithelia, keratin 19 was localized to a subset of luminal cells, suggesting that keratin 19-negative cells may be the proliferative compartment of the secretory cell lineage. The structural and functional similarities of prostate and breast led us to examine keratin 19 expression in the prostate. Immunohistochemical studies revealed that keratin 19 expression was heterogeneous and frequently occurred in basal as well as in luminal cells of normal, dysplastic, and benign hyperplastic tissues. Keratin 19 was observed in cancer, but usually in a minority of cells. This was in dramatic contrast to invasive breast cancers, which are reportedly uniformly positive for keratin 19. Prostatic epithelial cells cultured from tissues of all histologies expressed keratin 19. In summary, keratin 19 does not obviously correlate with any epithelial cell lineage or phenotype in the prostate.

    View details for Web of Science ID A1996UW98300020

    View details for PubMedID 8766871

  • KGF and EGF differentially regulate the phenotype of prostatic epithelial cells GROWTH REGULATION Peehl, D. M., Wong, S. T., Rubin, J. S. 1996; 6 (1): 22-31

    Abstract

    Previous studies indicate that keratinocyte growth factor (KGF) acts as a paracrine factor in the prostatic epithelium and epidermal growth factor (EGF) acts as an autocrine factor. In serum-free medium, KGF or EGF promoted similar growth of human prostatic epithelial cells. Response to two growth-inhibitory factors (suramin and transforming growth factor-beta), and expression of keratins and prostate-specific antigen (PSA), were similar with either mitogen. However, colonies in medium with KGF were very compact with extensive intercellular bonds, whereas colonies with EGF consisted of widely-separated cells. Growth was decreased to a greater extent by deletion of growth factors from medium with KGF versus EGF, and retinoic acid was 10-fold more potent at inducing growth inhibition and differentiation-associated keratin with KGF compared with EGF. We conclude that regulation of growth and differentiation in the prostate might vary depending on the availability of KGF versus EGF.

    View details for Web of Science ID A1996UK51000004

    View details for PubMedID 8717447

  • Simian virus 40-, but not human papillomavirus-, transformation of prostatic epithelial cells results in loss of growth-inhibition by 1,25-dihydroxyvitamin D-3 INTERNATIONAL JOURNAL OF ONCOLOGY Gross, C., Skowronski, R. J., Plymate, S. R., Rhim, J. S., Peehl, D. M., Feldman, D. 1996; 8 (1): 41-47

    Abstract

    In addition to its well known calcemic actions, 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D] exhibits differentiating and antiproliferative effects in several types of cancer cells. 1,25(OH)(2)D receptors (VDR) as well as 1,25(OH)(2)D-mediated growth-inhibition have been demonstrated in human prostate cancer cell lines. In order to further develop model systems for the study of 1,25(OH)(2)D action and to elucidate the mechanism of growth-inhibition, we studied several human prostate cell lines immortalized with either simian virus 40 (SV40) or human papillomavirus type 18 (HPV). The SV40-transformed cell lines P69SV40-T and P153SV40-T were not growth-inhibited by 1,25(OH)(2)D at concentrations as high as 100 nM, whereas the HPV-transformed cells PZ-HPV-7 and CA-HPV-10 were growth-inhibited. All cell lines expressed VDR, and VDR mRNA was demonstrated by Northern blot analysis. All cells exhibited induction of 24-hydroxylase mRNA, a 1,25(OH)(2)D responsive gene, after 1,25(OH)(2)D treatment. In an attempt to understand the apparent dissociation of 1,25(OH)(2)D actions in the SV40-transformed cells, we turned to the human prostate cancer cell line DU 145. These cells, like the SV40-transformed cells, are not growth-inhibited but demonstrate induction of 24-hydroxylase mRNA after 1,25(OH)(2)D treatment. DU 145 cells contain a mutated retinoblastoma gene (Rb) which contributes to their uncontrolled growth, analogous to the disruption of Rb by SV40 and HPV. We compared DU,145 cells to DU 145 cells transfected with normal Rb (DU 145/Rb). Similar to DU 145, DU 145/Rb cells were not growth-inhibited by 1,25(OH)(2)D, while 24-hydroxylase mRNA was induced. These results suggest that divergent pathways mediate the growth-inhibitory effect of 1,25(OH)(2)D and its induction of 24-hydroxylase. It also appears that the antiproliferative effect of 1,25(OH)(2)D is mediated by an Rb-independent mechanism.

    View details for Web of Science ID A1996TM02400006

    View details for PubMedID 21544329

  • The role of insulin-like growth factors in prostate biology JOURNAL OF ANDROLOGY Peehl, D. M., Cohen, P., Rosenfeld, R. G. 1996; 17 (1): 2-4

    View details for Web of Science ID A1996UA10800002

    View details for PubMedID 8833734

  • KERATINOCYTE GROWTH-FACTOR - AN ANDROGEN-REGULATED MEDIATOR OF STROMAL EPITHELIAL INTERACTIONS IN THE PROSTATE WORLD JOURNAL OF UROLOGY Peehl, D. M., Rubin, J. S. 1995; 13 (5): 312-317

    Abstract

    Members of the fibroblast growth factor (FGF) family are important growth-regulatory elements. Of the FGFs, keratinocyte growth factor (KGF) appears to have unique properties that implicate it as a paracrine factor in the prostate. Two KGF transcripts (approximately to 2.4 and 5.0 kb) encode a protein of approximately 22 kDa. In contrast to several other members of the FGF family, KGF has a signal peptide and is actively secreted. Cellular response to KGF is mediated by a specific receptor that is transcribed from an alternately spliced variant of the FGF type 2 receptor (FGFR-2). KGF transcripts have been detected in prostatic tissues and in stromal cells cultured from rat and human prostates as well as in a variety of stromal cells derived from other organs. Prostatic epithelial cells and numerous other types of epithelial cells are targets of KGF's mitogenic activity. Several factors involved in wound healing regulate the expression of KGF, but androgen regulation of KGF is of greatest relevance to the role of KGF in the prostate. Current efforts to localize and manipulate KGF activity in vivo should reveal the significance of KGF expression and function in the prostate and in other organs.

    View details for Web of Science ID A1995TC93300008

    View details for PubMedID 8581003

  • THE INSULIN-LIKE GROWTH-FACTOR SYSTEM IN THE PROSTATE WORLD JOURNAL OF UROLOGY Peehl, D. M., Cohen, P., Rosenfeld, R. G. 1995; 13 (5): 306-311

    Abstract

    The insulin-like growth factor (IGF) system is involved in the regulation of cell growth. The system involves a network of molecules that includes the IGFs themselves (IGF-I and -II), IGF receptors (types I and II), IGF-binding proteins (IGFBP-1 through -6), and IGFBP proteases. Characterization of this complex system in the prostate has recently been initiated. Prostatic cell lines as well as primary cultures of prostatic epithelial and stromal cells have been analyzed for expression of IGFs, receptors, and IGFBPs. Prostatic epithelial cells and, quite likely, stromal cells as well respond to the mitogenic activity of IGFs via the type I IGF receptor. Prostatic stromal cells synthesize and secrete IGF-II; there is evidence that prostatic cell lines also synthesize IGFs, but this has not been confirmed in primary cultures of prostatic epithelial cells. Prostatic stromal and epithelial cells secrete a number of IGFBPs. The biological impact of some of these IGFBPs on the growth of prostatic cells has been examined, and proteolytic cleavage of IGFBP-3 by prostate-specific antigen (PSA) has been demonstrated. Aberrations in several elements of the IGF system have been observed in stromal cells derived from benign prostatic hyperplasia (BPH). The IGF system may therefore have a part in the etiology of BPH as well as in normal and malignant processes in the prostate.

    View details for Web of Science ID A1995TC93300007

    View details for PubMedID 8581002

  • Suramin, hydrocortisone, and retinoic acid modify inhibitory effects of 1,25-dihydroxyvitamin D(3) on prostatic epithelial cells. Urologic oncology Peehl, D. M., Wong, S. T., Cramer, S. D., Gross, C., Feldman, D. 1995; 1 (5): 188-194

    Abstract

    The proliferation of prostatic epithelial cells is regulated by the complex interplay of numerous growth-stimulatory and growth-inhibitory factors. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has recently been identified as a potent inhibitor of the growth of prostatic epithelial cells. Epidemiologic studies indicate that vitamin D deficiency may be a risk factor for the development of clinical prostate cancer, possibly due to increased growth and reduced differentiation of prostatic cells in an environment with decreased 1,25(OH)(2)D(3). The application of vitamin D or analogs in chemotherapy against prostate and other cancers is being explored by several investigators. In order to use vitamin D most efficaciously in a clinical setting, it may be beneficial to learn more about the interaction of 1,25(OH)(2)D(3) with other factors that regulate prostatic epithelial cellular growth. In this study, we examined the effect of the proliferative status of cultured cells on their ability to respond to 1,25(OH)(2)D(3), and found that minimally proliferative cells were equally as responsive to 1,25(OH)2D3 as actively dividing cells. We noted no apparent interaction of 1,25(OH)(2)D(3) with epidermal growth factor, insulin-like growth factor, cholera toxin, or transforming growth factor-?, but we did find synergistic inhibitory effects of 1,25(OH)(2)D(3) with suramin and retinoic acid. Perhaps most noteworthy was the dramatic increase in potency of 1,25(OH)(2)D(3) that occurred upon deletion of hydrocortisone from the culture medium. Our in vitro studies indicate that combination therapy of vitamin D analogs with suramin, vitamin A analogs, or anti-glucocorticoids might be considered for prostate cancer.

    View details for PubMedID 21224116

  • ACTIONS OF VITAMIN-D-3 ANALOGS ON HUMAN PROSTATE-CANCER CELL-LINES - COMPARISON WITH 1,25-DIHYDROXYVITAMIN-D-3 ENDOCRINOLOGY Skowronski, R. J., Peehl, D. M., Feldman, D. 1995; 136 (1): 20-26

    Abstract

    Data from epidemiological studies has suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. We have previously demonstrated the presence of vitamin D receptors (VDR) in three human prostate carcinoma cell lines (LNCaP, PC-3, and DU-145) as well as in primary cultures of stromal and epithelial cells derived from normal and malignant prostate tissues. We have also shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can elicit an antiproliferative action in these cells. In the present study we compared the biological actions of 1,25-(OH)2D3 to those of a series of natural vitamin D3 metabolites and several synthetic analogs of vitamin D3 known to exhibit less hypercalcemic activity in vivo. In ligand binding competition experiments, we demonstrated the following order of potency in displacing [3H]1,25-(OH)2D3 from VDR: EB-1089 > 1,25-(OH)2D3 > MC-903 > 1,24,25-(OH)3D3 > 22-oxacalcitriol (OCT) > 1 alpha,25-dihydroxy-16-enecholecalciferol (Ro24-2637) > 25-hydroxyvitamin D3, with EB-1089 being approximately 2-fold more potent than the native hormone. No competitive activity was found for 25-hydroxy-16,23-diene-cholecalciferol. When compared for ability to inhibit proliferation of LNCaP cells, MC-903, EB-1089, OCT, and Ro24-2637 exhibited 4-, 3-, and 2-fold greater inhibitory activity than 1,25-(OH)2D3. Interestingly, although OCT and Ro24-2637 exhibit, respectively, 10 and 14 times lower affinity for VDR than 1,25-(OH)2D3, both compounds inhibited the proliferation of LNCaP cells with a potency greater than that of the native hormone. The relative potency of vitamin D3 metabolites and analogs to inhibit cell proliferation correlated well with the ability of these compounds to stimulate prostate-specific antigen secretion by LNCaP cells as well as with their potency to induce the 25-hydroxyvitamin D3-24-hydroxylase messenger RNA transcript in PC-3 cells. In conclusion, these results demonstrate that synthetic analogs of vitamin D3, known to exhibit reduced calcemic activity, can elicit antiproliferative effects and other biological actions in LNCaP and PC-3 cell lines. It is noteworthy that although binding to VDR is critical for 1,25-(OH)2D3 action, the analog data indicate that additional factors significantly contribute to the magnitude of the biological response. Finally, the strong antiproliferative effects of several synthetic analogs known to exhibit less calcemic activity than 1,25-(OH)2D3 suggest that these compounds potentially may be useful as an additional therapeutic option for the treatment of prostate cancer.

    View details for Web of Science ID A1995QD58400004

    View details for PubMedID 7530193

  • LIPOFECTION-MEDIATED IMMORTALIZATION OF HUMAN PROSTATIC EPITHELIAL-CELLS OF NORMAL AND MALIGNANT ORIGIN USING HUMAN PAPILLOMAVIRUS TYPE-18 DNA CANCER RESEARCH Weijerman, P. C., Konig, J. J., Wong, S. T., Niesters, H. G., Peehl, D. M. 1994; 54 (21): 5579-5583

    Abstract

    Human papillomavirus (HPV) type 18 DNA was introduced into epithelial cell strains derived from normal and cancer tissues of human prostatectomy specimens by the lipofection transfection method. Two cell lines were established: PZ-HPV-7 (transfected normal cell) and CA-HPV-10 (transfected cancer-derived cell). These lines have been maintained for over 100 passages. Incorporation of HPV type 18 DNA was confirmed by polymerase chain reaction. Immunocytochemical analysis showed expression of keratins 5 and 8, similar to the cells of origin, and the early region 6 oncoprotein of HPV. PZ-HPV-7, derived from normal diploid cells, had a modal chromosome number of 46 in early passages but became tetraploid later. CA-HPV-10 cells were aneuploid, and some retained the double minute chromosomes that were noted in the cancer-derived cells of origin. The cell lines showed a typical transformed morphology and were nontumorigenic in nude mice. We conclude that human prostatic epithelial cells derived from both normal and cancer tissues have been successfully transformed to immortality with HPV type 18 DNA. The establishment of these cell lines provides an opportunity for further development of an in vitro model of carcinogenesis for prostate cancer.

    View details for Web of Science ID A1994PP19000018

    View details for PubMedID 7923200

  • KERATIN EXPRESSION - A MEASURE OF PHENOTYPIC MODULATION OF HUMAN PROSTATIC EPITHELIAL-CELLS BY GROWTH-INHIBITORY FACTORS CELL AND TISSUE RESEARCH Peehl, D. M., Leung, G. K., Wong, S. T. 1994; 277 (1): 11-18

    Abstract

    Expression of certain cytokeratins can be indicative of the state of differentiation of epithelial cells. The basal cells in the normal adult human prostatic epithelium are characterized by the expression of cytokeratins 5 and 14, whereas the secretory luminal cells contain cytokeratins 8 and 18. Cells cultured from the prostatic epithelium expressed cytokeratins 5, 8, and 18, and thus had features of both basal and luminal cells. Certain growth-inhibitory conditions altered keratin expression in conjunction with growth modulation. Deletion of peptide factors and hormones from the culture medium induced the expression of cytokeratins 1 and 10, associated with a squamous phenotype. These same squamous keratins were found in very dense, stratified cultures that were maintained at confluency in standard, complete medium for extended periods. Retinoic acid enhanced the expression of secretory luminal cell-associated cytokeratins 8 and 18 in semi-confluent cultures. Other growth inhibitory factors such as suramin, transforming growth factor-beta, and interferon-gamma had no effect on keratin expression. These observations indicate that the differentiation of prostatic epithelial cells can be directed toward alternate pathways, either squamous or secretory, by different growth-inhibitory conditions. However, not all growth inhibitory factors altered differentiation, demonstrating that growth inhibition in itself is not a sufficient inducer of differentiation.

    View details for Web of Science ID A1994NU28100002

    View details for PubMedID 7519968

  • CHARACTERIZATION OF ADULT HUMAN PROSTATIC EPITHELIAL-CELLS IMMORTALIZED BY POLYBRENE-INDUCED DNA TRANSFECTION WITH A PLASMID CONTAINING AN ORIGIN-DEFECTIVE SV40-GENOME INTERNATIONAL JOURNAL OF ONCOLOGY Lee, M. S., GARKOVENKO, E., Yun, J. S., Weijerman, P. C., Peehl, D. M., Chen, L. S., Rhim, J. S. 1994; 4 (4): 821-830

    Abstract

    Normal adult human prostatic epithelial cells were infected with an adenovirus 12-SV40 virus or transfected by polybrene-induced gene transfer with a plasmid (pRSV-T) containing the SV40 early region genes or with a plasmid (pRNS-1) containing an origin-defective SV40 genome and a plasmid carrying the neomycin resistance gene. Colonies of morphologically altered cells were isolated, cultured in a serum-free medium and characterized. These cells had extended lifespan in culture compared to normal adult human prostatic epithelial cells. Both Ad12-SV40-infected and pRSV-T-transfected cultures eventually underwent senescence. pRNS-1-transfected cells (pRNS-1-1), however, have now been grown for more than 50 passages. These cells contain the SV40 genome, express SV40 T-antigen, and are not tumorigenic in nude mice. They express cytokeratins 5 and 8, like the parent cells, and are pseudodiploid. Analysis of growth regulatory processes revealed that the growth of pRNS-1-1 cells was stimulated similarly to that of normal prostatic epithelial cells by epidermal growth factor, insulin-like growth factor, and pituitary extract. The response of pRNS-1 cells to a growth-inhibitory factor, retinoic acid, was also similar to that of normal cells. However, pRNS-1-1 cells were less responsive than normal cells to growth inhibition by transforming growth factor-beta, and had lost altogether the ability of normal cells to be inhibited by tumor necrosis factor-alpha and 1,25 (OH)2 vitamin D3. Therefore transformation appeared to alter growth-inhibitory but not growth - stimulatory mechanisms. These cells should be useful in elucidating the multistep mechanism of carcinogenesis of the prostate.

    View details for Web of Science ID A1994ND85600006

    View details for PubMedID 21566988

  • ANTIPROLIFERATIVE EFFECTS OF 1,25-DIHYDROXYVITAMIN-D(3) ON PRIMARY CULTURES OF HUMAN PROSTATIC CELLS CANCER RESEARCH Peehl, D. M., Skowronski, R. J., Leung, G. K., Wong, S. T., Stamey, T. A., Feldman, D. 1994; 54 (3): 805-810

    Abstract

    Cultures of adult human prostatic epithelial and fibroblastic cells were established from normal, benign hyperplastic, and malignant tissues. Vitamin D receptors were detected by ligand binding of [3H]1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in cytosolic extracts prepared from all types of cell cultures as well as from fresh prostatic tissues. Vitamin D receptor transcripts were demonstrated by Northern blot analysis. 1,25-(OH)2D3 inhibited the growth of epithelial cells with half-maximal inhibition at approximately 1 nM. The growth of fibroblasts was also inhibited by 1,25(OH)2D3 but to a lesser extent. This is consistent with the apparently lower level of vitamin D receptors in fibroblasts compared to epithelial cells determined by ligand binding and Northern analysis of RNA transcripts. The growth inhibition of epithelial cells by 1,25(OH)2D3 was irreversible even after a short 2-h exposure, but morphology and keratin expression were not appreciably altered by long-term exposure to the hormone. A physiological role for 1,25(OH)2D3 in the prostate is postulated, and the inhibitory effect of 1,25(OH)2D3 on cancer-derived prostate cells may provide a basis for new preventive or therapeutic strategies.

    View details for Web of Science ID A1994MU67700036

    View details for PubMedID 7508338

  • Human papilloma virus type 18 DNA immortalized cell lines from the human prostate epithelium. Progress in clinical and biological research Weijerman, P. C., ROMIJN, H. C., Peehl, D. M. 1994; 386: 67-69

    View details for PubMedID 7800701

  • VITAMIN-D AND PROSTATE-CANCER - 1,25-DIHYDROXYVITAMIN-D3 RECEPTORS AND ACTIONS IN HUMAN PROSTATE-CANCER CELL-LINES ENDOCRINOLOGY Skowronski, R. J., Peehl, D. M., Feldman, D. 1993; 132 (5): 1952-1960

    Abstract

    It has been suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. In this study three human prostate carcinoma cell lines, LNCaP, DU-145, and PC-3, were examined both for the presence of specific 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] receptors (VDRs) and also employed to study the effects of hormone on cell proliferation and differentiation. Ligand binding experiments demonstrated classical VDR in all three cell lines examined with an apparent dissociation constant of 7.5, 5.4, and 6.3 x 10(-11) M for LNCaP, DU-145, and PC-3 cells, respectively. Corresponding binding capacity for the three prostate carcinoma cell lines were 27, 31, and 78 fmol/mg protein, respectively. The presence of VDR in the three cell lines was also confirmed by immunocytochemistry. In addition, one major 4.6-kilobase messenger RNA transcript hybridizing with a specific human VDR complementary DNA probe was identified in all three cell lines. Interestingly, both DU-145 and PC-3 but not LNCaP cell lines exhibited 1,25(OH)2D3-stimulated induction of 24-hydroxylase messenger RNA employed as a marker of 1,25(OH)2D3 action. Physiological levels of 1,25(OH)2D3 dramatically inhibited proliferation of the LNCaP and PC-3 cell lines. However, in spite of the presence of high affinity VDR, proliferation of DU-145 cells was not inhibited by 1,25(OH)2D3 at the doses tested. Treatment with 1,25(OH)2D3 caused a dose-dependent stimulation of prostate-specific antigen secretion by LNCaP cells. In conclusion, these results demonstrate that these three human prostate carcinoma cell lines all possess specific VDR and that 1,25(OH)2D3 treatment can elicit both an antiproliferative and a differentiating action on these cancer cells. The findings lend support to the hypothesis that vitamin D might exert beneficial actions on prostate cancer risk.

    View details for Web of Science ID A1993KZ69900011

    View details for PubMedID 7682937

  • ELEVATED LEVELS OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-2 IN THE SERUM OF PROSTATE-CANCER PATIENTS JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Cohen, P., Peehl, D. M., Stamey, T. A., Wilson, K. F., Clemmons, D. R., Rosenfeld, R. G. 1993; 76 (4): 1031-1035

    Abstract

    We have previously documented the presence of specific insulin-like growth factor (IGF)-binding protein (IGFBPs) in seminal plasma and prostate epithelial cell-conditioned medium IGFBP-2 is the prevalent IGFBP in both fluids. To assess whether patients with prostate carcinoma have alterations in serum IGFP levels related to the production of IGFBPs by their tumors, we performed Western ligand blots (WLB) and IGFBP-2 RIA on serum samples from 32 patients with prostate carcinoma of various degrees of clinical severity and compared them to results in 16 healthy age-matched controls. We have also measured serum IGF-I and -II by RIA. The mean level of IGFBP-2 in the prostate cancer patients was 170% of control levels by WLB analysis and 195% of control levels by RIA (P < 0.01). The degree of elevation of IGFBP-2 was related to the stage of the tumor and the levels of the serum tumor marker, prostate-specific antigen. Serum IGFBP-3 levels determined by WLB and serum IGF-I and IGF-II levels measured by RIA after acid chromatography were not different among the subjects with cancer and the normal controls. We conclude that IGFBP-2, which is the main IGFBP produced by prostate epithelial cells, is elevated in the serum of patients with prostate carcinoma, and that the degree of this elevation is related to serum prostate-specific antigen levels and the stage of the tumor. We speculate that prostate-derived IGFBPs may be secreted by prostate tumors and could e of value in understanding the pathophysiology of prostatic tumor growth as well as provide potential diagnostic markers.

    View details for Web of Science ID A1993KW77300041

    View details for PubMedID 7682560

  • VITAMIN-A REGULATES PROLIFERATION AND DIFFERENTIATION OF HUMAN PROSTATIC EPITHELIAL-CELLS PROSTATE Peehl, D. M., Wong, S. T., Stamey, T. A. 1993; 23 (1): 69-78

    Abstract

    The response of cultured human prostatic epithelial cells to vitamin A was measured by clonal growth assay in serum-free medium. Retinoic acid at 3 nM or higher inhibited the proliferation of cell strains derived from normal, benign hyperplastic and malignant tissues, while lower levels (0.03 nM) were stimulatory. Reduced proliferation induced by retinoic acid was accompanied by a marked change in morphology, as intercellular adhesion decreased. In conjunction, the expression of keratins 8 and 18, associated with the differentiated luminal phenotype of prostatic epithelia, was increased. In post-confluent cultures, retinoic acid prevented the appearance of keratin 1, which accompanied the development of a squamous phenotype by cells maintained under these conditions. The findings of this study indicate a role for vitamin A as a modulator of the growth and differentiation of prostatic epithelial cells.

    View details for Web of Science ID A1993LQ79400006

    View details for PubMedID 7687781

  • PROSTATE-SPECIFIC ANTIGEN (PSA) IS AN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-3 PROTEASE FOUND IN SEMINAL PLASMA JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Cohen, P., GRAVES, H. C., Peehl, D. M., Kamarei, M., Giudice, L. C., Rosenfeld, R. G. 1992; 75 (4): 1046-1053

    Abstract

    Insulin-like growth factor binding protein-3 (IGFBP-3), the major serum carrier protein for the IGFs, is absent from Western ligand blots of seminal plasma, but is detectable by RIA. IGFBP-3 protease activity has recently been described in pregnancy serum. We investigated the possibility that seminal plasma contains an IGFBP-3 protease, by incubating seminal plasma with 125I-labeled human IGFBP-3. Seminal plasma was found to have potent IGFBP-3 protease activity with a cleavage pattern different from that of pregnancy serum. Prostate-specific antigen (PSA) is a serine protease found in semen. Autoradiographs measuring IGFBP-3 protease activity demonstrated that purified PSA cleaved IGFBP-3, yielding a cleavage pattern identical to that of seminal plasma. IGFBP-2 and -4 in seminal plasma were not degraded by PSA. Cleavage of IGFBP-3 by PSA resulted in a marked reduction in the binding affinity of the fragments to IGF-I, but not IGF-II. We speculate that PSA may serve to modulate IGF function within the reproductive system or in prostate cancer by altering IGF-IGFBP-3 interactions.

    View details for Web of Science ID A1992JT81500015

    View details for PubMedID 1383255

  • INSULIN-LIKE GROWTH-FACTORS (IGFS), IGF RECEPTORS, AND IGF-BINDING PROTEINS IN PRIMARY CULTURES OF PROSTATE EPITHELIAL-CELLS JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Cohen, P., Peehl, D. M., Lamson, G., Rosenfeld, R. G. 1991; 73 (2): 401-407

    Abstract

    Insulin-like growth factors (IGFs) are potent mitogens that bind with high affinity and specificity to IGF receptors and IGF-binding proteins (IGFBPs). We studied the roles of these three groups of proteins in prostate epithelial cells (PEC) in primary culture grown under serum-free conditions. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from PEC revealed an abundance of type 1 IGF receptors and no evidence of type 2 IGF receptors. Western ligand blots of conditioned media (CM) from PEC demonstrated the presence of two specific IGFBP bands similar to those previously demonstrated in seminal plasma, with approximate mol wt of 31 and 24 kDa. The 31-kDa band was immunoprecipitable with an antibody to IGFBP-2, and neither band could be deglycosylated with endoglycosidase-F. Northern blot analysis of poly(A)+ RNA prepared from PEC with cDNAs for hIGFBP-1, -2, and -3 documented the expression of mRNA for hIGFBP-2 only. Modifications of the serum-free conditions of PEC did not significantly alter the IGFBP profile of PEC CM. The ability of IGF-I, IGF-II, and insulin to stimulate clonal growth of PEC was examined. IGF-I stimulated PEC growth with an ED50 of 0.1 ng/mL. IGF-II and insulin, respectively, were 1 and 3 orders of magnitude less effective than IGF-I in stimulating the growth of PEC. Radioimmunoassayable IGF-I and IGF-II levels in PEC CM were below the assay detection levels. In conclusion, we suggest that IGFs are important growth stimulators of PEC in culture, that their actions are mediated through the type 1 IGF receptor, and that PEC produce hIGFBP-2 and a 24-kDa IGFBP which may modulate IGF action in these cells.

    View details for Web of Science ID A1991FY51100029

    View details for PubMedID 1713219

  • INVITRO PROTON SPECTROSCOPY OF NORMAL AND ABNORMAL PROSTATE MAGNETIC RESONANCE IN MEDICINE YACOE, M. E., Sommer, G., Peehl, D. 1991; 19 (2): 429-438

    Abstract

    Previous biochemical and 13C NMR spectroscopic data have suggested that the metabolism of citrate, a secretory product of normal prostate, may be interrupted in prostate cancer. In the present study in vitro 1H NMR spectroscopy was used to see if cell strains derived from prostate cancers could be reliably distinguished from those of normal prostate epithelium. High-resolution one-dimensional and two-dimensional J-resolved 1H NMR spectra as well as gas chromatography coupled with mass spectroscopy were used to study extracts of highly defined cell strains from normal peripheral zone, normal central zone, adenocarcinoma, and benign prostatic hyperplasia. Resonances assigned to citric acid and related metabolites were identified. Cell strains derived from prostate cancers tended to have smaller amounts of citrate than those from normal prostate epithelium. However, the differences were small and not statistically significant. The lack of statistically significant differences may reflect the variability present in both normal and abnormal cell strains and thus underscore the well-known difficulty in differentiating normal and cancerous tissues.

    View details for Web of Science ID A1991FQ24300033

    View details for PubMedID 1715505

  • CYTOSTATIC EFFECTS OF SURAMIN ON PROSTATE-CANCER CELLS CULTURED FROM PRIMARY TUMORS JOURNAL OF UROLOGY Peehl, D. M., Wong, S. T., Stamey, T. A. 1991; 145 (3): 624-630

    Abstract

    Suramin is currently undergoing clinical trials as a chemotherapeutic agent for prostate cancer. The effects of suramin on cultured human epithelial cells derived from normal, benign hyperplastic, and malignant prostate tissues were examined. In serum-free medium, suramin inhibited the clonal growth of prostate cells at a half-maximal dose of approximately 10 micrograms/ml. Growth inhibition by suramin was completely reversible even after 24 hours of exposure. In conjunction, suramin did not alter cellular phenotype with regard to expression of keratins and prostate-specific antigens. Although suramin is reportedly an antagonist of growth factor-mediated mitogenesis, ten-fold excesses of growth factors did not appreciably suppress the cytostatic activity of suramin. In comparison to the activities of other possible chemotherapeutic agents, suramin would appear suboptimal because its inhibitory effects are reversible and it does not induce a terminally differentiated cellular phenotype.

    View details for Web of Science ID A1991FA69900038

    View details for PubMedID 1705296

  • CULTURE OF PROSTATIC EPITHELIAL-CELLS FROM ULTRASOUND-GUIDED NEEDLE BIOPSIES PROSTATE Peehl, D. M., Wong, S. T., Terris, M. K., Stamey, T. A. 1991; 19 (2): 141-147

    Abstract

    A protocol which was developed for the culture of epithelial cells from radical prostatectomy specimens was slightly modified to permit the culture of cells from ultrasound-guided prostatic needle biopsies. The collagenase digestion step of the standard protocol was omitted, and biopsies were simply minced and allowed to attach to collagen-coated dishes in serum-free medium. Cell outgrowths from biopsies were free of fibroblasts, and expression of keratin, prostate specific antigen, and prostatic acid phosphatase was maintained in vitro. The establishment of a bank of frozen cells from primary cultures permitted repetitive studies with individual cell strains, which could be serially passaged and were capable of clonal growth. The ability to derive cultures from biopsies will facilitate the biological characterization of cells from primary prostate tumors of high malignant grade, which are not commonly available from radical prostatectomy specimens.

    View details for Web of Science ID A1991GW40600006

    View details for PubMedID 1717964

  • SEARCHING FOR SUPPRESSOR GENES IN PROSTATE-CANCER CANCER SURVEYS Peehl, D. M. 1991; 11: 25-34

    Abstract

    Prostate cancer poses many challenges for clinicians. Methods for early detection are not satisfactory, and in fact more efficient detection will lead to even further dilemmas. We know from necropsy studies that the occurrence of prostate cancer is much higher than the clinical incidence (Yatani et al, 1982). Obviously, only some of the prostate cancers detected early would progress to a health threatening state if left untreated--the question is, which ones? This is a question that must be addressed, because prostate cancer has the highest occurrence rate of any cancer among males in the US, and current approaches to eradicating early stage prostate cancers involve expensive surgical or irradiation techniques. Even for primary prostatic tumours of more advanced stages, current prognostic indicators are not ideal. What is the potential of specific suppressor genes lost in prostate cancer for identifying useful prognostic indicators? Even though numerous molecular events, including loss as well as gain of gene activity, have been delineated for colon cancer, no single genetic change has proven prognostic. Rather, increasing malignant potential of colon cancer seems to be correlated with an accumulation of several genetic events. On the other hand, a specific chromosomal abnormality, a deletion of chromosome 17p, does appear to be a prognostic indicator for distinguishing high grade from low grade transitional cell carcinoma (Olumi et al, 1990). Whether loss of chromosome 17p will prove to be a better prognostic marker than other parameters, however, is still unknown. At present, no consistent genetic changes have been associated with any stage of prostate cancer, so we are a long way from determining whether such changes will prove their hypothetical usefulness as new and improved prognostic indicators. The other clinical realm in which the identification of loss of suppressor genes in prostate cancer might prove a resource is therapy. Suppressor gene products obviously provide key regulatory functions, perhaps linked to differentiation, in normal cells. Restoration of these regulatory functions in cancer cells, either by directly supplying the lost regulatory element or by circumventing the missing element by other means, is a novel therapeutic approach that might offer hope even in metastatic disease. Realization of this dream will require much more knowledge of the precise nature of the suppressor gene products lost in cancer, and a detailed understanding of the hierarchy of regulation of growth and differentiation in normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1991HQ11500004

    View details for PubMedID 1668728

  • ANALYSIS OF SOMATIC-CELL HYBRIDS DERIVED FROM NORMAL HUMAN PROSTATIC EPITHELIAL-CELLS FUSED WITH HELA-CELLS PROSTATE Peehl, D. M., Wong, S. T., McNeal, J. E., Stamey, T. A. 1990; 17 (2): 123-136

    Abstract

    Somatic cell hybrids have been instrumental in the recognition of specific chromosomes containing genes capable of suppressing the malignant phenotype. As a first step towards the identification of possible suppressor genes in prostate cells, we created hybrids by fusing normal prostate cells with malignant HeLa cells. Similar to hybrids made with other combinations of normal and malignant cells, the normal phenotype was dominant and the malignant phenotype was suppressed. The phenotype of the nontumorigenic hybrids after injection into nude mice resembled that of normal keratinocyte X HeLa hybrids, and tiny, nonprogressive keratinized nodules were produced. One hybrid clone was tumorigenic, possible due to the loss of a normal suppressor gene, and displayed glandular as well as squamous elements. Further characterization of these hybrids should permit isolation of specific suppressor genes, as well as promote recognition of elements that regulate the glandular phenotype.

    View details for Web of Science ID A1990DZ96800005

    View details for PubMedID 1697966

  • CLONAL GROWTH OF HUMAN PROSTATIC EPITHELIAL-CELLS IS STIMULATED BY FIBROBLASTS PROSTATE Kabalin, J. N., Peehl, D. M., Stamey, T. A. 1989; 14 (3): 251-263

    Abstract

    The interaction of fibroblasts with adult human prostatic epithelial cells was studied in vitro. Stimulation of epithelial cell proliferation, measured by clonal growth assay, was demonstrated when prostatic epithelial cells were grown in coculture with fibroblasts. Epithelial growth in cocultures with fibroblasts was greater than could be obtained in isolated culture in an "optimized," serum-free medium previously described. Fibroblasts were able to compensate for the deletion of several growth factors from this "optimized" medium, including epidermal growth factor and insulin, but were notably unable to replace bovine pituitary extract. Epithelial growth stimulation identical to that achieved in coculture was produced by medium conditioned by fibroblasts. Similar results were obtained by using fibroblasts of adult human prostate origin, adult human skin, human fetal lung fibroblasts (IMR-90), and mouse 3T3 cells. No difference in response was demonstrated between prostatic epithelial cells derived from normal or malignant tissues. Fixed fibroblast monolayers and extracellular matrix prepared from fibroblast cultures failed to stimulate prostatic epithelial growth. These results suggest that a soluble growth factor is secreted by prostatic fibroblasts and other human fibroblasts of nonprostatic origin, as well as by embryonic mesenchymal cells from nonhuman species, which is capable of producing a marked proliferative response in vitro from adult human prostatic epithelium. This proliferative response could not be reproduced by the addition of a variety of known growth factors to prostatic epithelial cell cultures.

    View details for Web of Science ID A1989AA83000005

    View details for PubMedID 2499876

  • In vitro studies of human prostatic epithelial cells: attempts to identify distinguishing features of malignant cells. Growth factors Peehl, D. M., Wong, S. T., Bazinet, M., Stamey, T. A. 1989; 1 (3): 237-250

    Abstract

    Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-beta and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.

    View details for PubMedID 2698220

  • CLONAL GROWTH-CHARACTERISTICS OF ADULT HUMAN PROSTATIC EPITHELIAL-CELLS IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY Peehl, D. M., Wong, S. T., Stamey, T. A. 1988; 24 (6): 530-536

    Abstract

    The ability of human epithelial cells derived from adult prostatic tissues to undergo clonal growth in culture was examined. In a previously described serum-free culture system, such cells exhibited a density-dependent growth requirement. It was found that raising the level of one of the constituents of the culture medium, bovine pituitary extract, to 100 micrograms/ml permitted excellent clonal growth when as few as 100 cells were inoculated/60-mm2 dish. Raising the levels of supplements other than pituitary extract (cholera toxin, epidermal growth factor, hydrocortisone, or insulin) did not produce this result. The average colony-forming efficiency of cells derived from primary or early passage cultures was approximately 25%. When single cell suspensions were prepared from tissue isolates and directly analyzed for clonal growth, colony-forming efficiencies were approximately 5%, perhaps indicating the proportion of stem cells with proliferative potential in the original isolates. The colony-forming efficiency of a cell population derived from cancer tissue was not significantly different from those of populations derived from normal tissues.

    View details for Web of Science ID A1988P034100007

    View details for PubMedID 3391931

  • Oncogene expression. Progress in clinical and biological research Peehl, D. M. 1987; 239: 77-90

    View details for PubMedID 3309967

  • ONCOGENES - A REVIEW WITH RELEVANCE TO CANCERS OF THE UROGENITAL TRACT JOURNAL OF UROLOGY Peehl, D. M., Stamey, T. A. 1986; 135 (5): 897-904

    View details for Web of Science ID A1986C176400001

    View details for PubMedID 3007783

  • SERUM-FREE GROWTH OF ADULT HUMAN PROSTATIC EPITHELIAL-CELLS IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY Peehl, D. M., Stamey, T. A. 1986; 22 (2): 82-90

    Abstract

    Proliferation of adult human prostatic epithelial cells in serum-free medium occurs upon the addition of cholera toxin, epidermal growth factor, pituitary extract, and hydrocortisone to basal medium PFMR-4A. Insulin and selenium enhance proliferation and permit growth at lower cell densities. Reducing the level of calcium in the medium dramatically alters morphology and also seems to increase proliferation. Mortal strains of cells derived from normal central or peripheral zone, benign hyperplasia, or cancer respond similarly to growth factors and calcium, but two populations of cancer cells which have been long-lived and may be immortal lines behave differently. GKC-CA cells require serum proteins or high levels of pituitary extract for optimal growth, and neither GKC-CA cells or cells of another cancer line, WB-CA, proliferate well in medium containing reduced levels of calcium. These observations may, however, be a reflection of attachment phenomena rather than of growth responses per se. Growth of cells in serum-free medium has allowed definitive studies of the effects of androgens, and regardless of cell type no response to androgens of prostate epithelial cells under any experimental conditions has been seen.

    View details for Web of Science ID A1986A180200004

    View details for PubMedID 2419302

  • GROWTH-RESPONSES OF NORMAL, BENIGN HYPERPLASTIC, AND MALIGNANT HUMAN PROSTATIC EPITHELIAL-CELLS INVITRO TO CHOLERA-TOXIN, PITUITARY EXTRACT, AND HYDROCORTISONE PROSTATE Peehl, D. M., Stamey, T. A. 1986; 8 (1): 51-61

    Abstract

    Normal, benign hyperplastic (BPH), and malignant prostatic epithelial cells from adult humans can be serially passaged in medium PFMR-4 supplemented with 20% whole serum, cholera toxin, and epidermal growth factor (EGF). Improved growth of these cells has been achieved by optimizing the concentrations of the components of the basal medium. In the new modified medium, PFMR-4A, the only supplements required are 5% dialyzed serum, cholera toxin, EGF, pituitary extract, and hydrocortisone. Comparative studies of the growth responses of prostatic cells derived from normal central zone, normal peripheral zone, BPH nodules, and adenocarcinomas did not reveal any qualitative differences; all respond positively to the addition of cholera toxin, pituitary extract, and hydrocortisone to the culture medium. Cells of one cancer strain, however, were much less density-dependent for growth than were the other strains of normal, BPH, or malignant cells.

    View details for Web of Science ID A1986AZB3700005

    View details for PubMedID 2418431

  • Serial propagation of adult human prostatic epithelial cells with cholera toxin. In vitro Peehl, D. M., Stamey, T. A. 1984; 20 (12): 981-986

    Abstract

    Reproducible subculture of adult human prostatic epithelial cells from normal, benign hyperplastic and malignant tissue has been achieved. Cholera toxin is the key component in the culture system, but use of an optimal basal medium (PFMR-4) supplemented with a high level of serum in collagen-coated dishes also improves growth and serial propagation.

    View details for PubMedID 6085065

Conference Proceedings


  • Vitamin D and prostate cancer risk Peehl, D. M. ELSEVIER SCIENCE BV. 1999: 392-394

    Abstract

    Prostate cancer is a progressive, multistep disease which presents many stages for intervention. Microscopic cancer is found in the prostate beginning by age 30 in about 20% of men, and the incidence increases steadily so that by the time a man is 90 years old, he has almost a 100% chance of having cancer in his prostate. Independent, multiple foci of cancer are present in the majority of prostate specimens, and the incidence of premalignant lesions is even higher than that of cancer. Yet, despite the high incidence of microscopic cancer, only 8% of men in the US present with clinically significant disease during their lifetime. Furthermore, only 3% of men in the US die of prostate cancer. In no other human cancer is there such disparity between the high incidence of microscopic malignancy and the relatively low death rate. Thus, there are many windows of opportunity for control of prostate cancer. Evidence from diverse areas of study - epidemiologic, molecular, genetic, cellular, animal models, and clinical trials - suggests that vitamin D may be an effective preventive agent against prostate cancer.

    View details for Web of Science ID 000080400800009

    View details for PubMedID 10325494

  • Human papillomavirus detection by polymerase chain reaction in benign and malignant prostate tissue is dependent on the primer set utilized Terris, M. K., Peehl, D. M. CAHNERS PUBL CO. 1997: 150-156

    Abstract

    Prior investigations evaluating the presence of human papillomavirus (HPV) in prostatic tissue by polymerase chain reaction (PCR) technology have yielded detection rates of 0% to 100%. Contamination by viral DNA from prostatic urethral colonization or less than optimal laboratory conditions have been suggested to explain this discrepancy. In addition, the various investigations have differed in the specific oligonucleotide primers utilized for amplification and, therefore, have searched for different segments of the viral genome. The objective of this study is to address these differences.Forty-one archival radical prostatectomy specimens were evaluated, identifying areas of normal and abnormal histology. Meticulous technique was used during tissue acquisition, histologic confirmation, DNA isolation, PCR amplification, polyacrylamide gel electrophoresis, and staining. Primers for a 126- and 99-base pair (bp) fragment of the E6 portion of HPV 16 as well as a consensus primer for the L1 portion of the papillomavirus genome were utilized.Of the normal prostatic tissues, 13.5% (5/37) contained the 126-bp E6 viral DNA as did 33.3% (7/21) of benign prostatic hyperplasia (BPH) samples, 25% (5/20) of dysplasia, 18.2% (2/11) of Gleason grades 1 and 2 adenocarcinoma, 25.9% (7/27) of Gleason grade 3 adenocarcinoma, and 6.7% (1/15) of Gleason grade 4 adenocarcinoma. Sections from the urethras of the prostatectomy specimens contained viral DNA in 31.7% (13/41). Viral detection was variable among different specimens in the same patient. With amplification for the 99-bp fragment of HPV 16, 1 of 37 normal (2.7%), 2 of 21 BPH (9.5%), 1 of 20 dysplasia (5.0%), and 2 of 53 cancer (3.8%) specimens revealed HPV DNA. In none of the specimens was DNA amplified using primers for a 450-bp fragment of the L1 portion of HPV.Previously published discrepancies in HPV detection may be solely due to the differences in primer sets utilized.

    View details for Web of Science ID A1997XJ07600032

    View details for PubMedID 9218040

  • Cellular biology of prostatic growth factors Peehl, D. M. WILEY-BLACKWELL. 1996: 74-78

    Abstract

    The etiology of prostate cancer or of benign prostatic hyperplasia (BPH) is essentially not understood. It is becoming clear, however, that major determinants of the malignant or hyperplastic phenotype are various growth-stimulatory or -inhibitory factors and their receptors, whose inappropriate expression or loss disrupts normal regulation of cell proliferation and differentiation. Cell culture has been a versatile tool for studying the expression and interaction of growth factors in prostatic cells. Immunohistochemistry and in situ hybridization have provided a view of growth factor expression coupled with histopathology. The eventual definition of autocrine, paracrine, and endocrine pathways of growth regulation in the human prostate will facilitate the design of new preventative, diagnostic, and therapeutic strategies.

    View details for Web of Science ID A1996UB91100014

    View details for PubMedID 8630234

  • Vitamin D and prostate cancer Feldman, D., Skowronski, R. J., Peehl, D. M. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1995: 53-63

    Abstract

    Our findings demonstrate the presence of VDR in various human prostate cancer cell lines and in primary cultures derived from normal, BPH and prostate cancer. In addition, 1,25-D induced several bioresponses in these cells including growth inhibition and PSA stimulation. Based on examples in many different malignant cells as well as our data in prostate cells, that vitamin D is anti-proliferative and promotes cellular maturation, it seem clear that vitamin D must be viewed as an important cellular modulator of growth and differentiation if addition to its classical role as regulator of calcium homeostasis. In this respect, vitamin D has the potential to have beneficial actions on various malignancies including prostate cancer. Its ultimate role in prostate cancer remains to be determined, but 1,25-D may prove useful in chemoprevention and/or differentiation therapy. We believe the data currently available provide the basis for an optimistic view on the possible use of vitamin D to treat prostate cancer in patients and that further investigation is clearly warranted to better define its potential therapeutic utility.

    View details for Web of Science ID A1995BD36X00005

    View details for PubMedID 7645428

  • ONCOGENES IN PROSTATE-CANCER - AN UPDATE Peehl, D. M. WILEY-LISS. 1993: 1159-1164

    Abstract

    Oncogenes have been implicated in the carcinogenic development of many diverse types of human malignancies. For some cancers, the expression of specific oncogenes has been shown to have diagnostic or prognostic value. By contrast currently, no oncogene has been correlated conclusively with the initiation or progression of prostate cancer. The ras oncogene has been investigated the most thoroughly for its involvement in prostate cancer, but ras does not appear to play a significant role in the development of this malignancy. Several years ago, limited studies hinted at the possibility of overexpression of the myc oncogene and aberrant expression of the sis oncogene in prostate cancer, but additional studies to clarify the involvement of these oncogenes have not been done. Oncogenic activity of growth factors or growth factor receptors in prostate cancer has been suggested but not amply demonstrated. Current dogma indicates that oncogenes exist in prostate cancer, but these will be identified only by more intensive investigation.

    View details for Web of Science ID A1993KM67400038

    View details for PubMedID 8428339

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