Diabetes Erectile Dysfunction And Apoptosis Of Cavernous Tissues

Rajvir Dahiya, Professor And Director
Northern California Institute Res & Educ

Grant 5R01DK075524-03 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG1

Abstract: The main goal of this proposal is to investigate whether oxidative stress and apoptosis of cavernous tissues are major pathways in erectile dysfunction. We will also investigate whether inhibition of oxidative stress and apoptosis can restore erectile function. The rationale for this project is that erectile dysfunction affects about 30 million men in the United States and more than 200 million men worldwide. Based on our preliminary data and prior publications, we hypothesize that apoptosis of cavernous tissues are down stream events in erectile dysfunction. We further hypothesize that anti-oxidative agent or Bcl2 gene therapy can restore erectile function in animal model. We will test these hypotheses through the following experiments Specific Aim# 1. To test the hypothesis that diabetes-induced erectile dysfunction is due to apoptosis of cavernous tissues. Under this specific aim, we will analyze erectile function in diabetic rats and then analyze apoptotic index, anti-apoptotic genes, pro-apoptotic genes, cytochrome c, superoxide dismutase and smooth muscle differentiation markers. We will also investigate whether an anti-oxidative agent (TEMPOL) can restore erectile function in diabetic rats. We will use the following techniques RT-PCR, real-time RT-PCR, in situ hybridization, immunohistochemistry, electro-stimulation of cavernous nerve for pressure measurements, gene delivery, histology, light and electron microscopy. Specific Aim # 2. To test the hypothesis that insulin treatment can restore erectile function through restoration of anti-apoptotic factors in cavernous tissues of diabetic rats. Under this specific aim, we will treat diabetic rats with insulin and then investigate whether erectile function is restored through restoration of anti-apoptotic pathways described under specific aim # 1. Specific Aim # 3. To test the hypothesis that gene therapy with the Bcl2 anti-apoptotic gene can restore functional, cellular and molecular mechanisms of erectile dysfunction in diabetic rats. Under this specific aim, the Bcl2 gene will be transfected into the cavernous tissue of diabetic rats using an adeno-associated virus vector. We will do the following experiments (a) Functional assessment of the effects of Bcl2 gene therapy on penile erection in diabetic rats, (b) Analysis of the effects of Bcl2 gene therapy on the oxidative stress pathways and the apoptotic pathways described under specific aim 1. (c) Analysis of the effects of Bcl2 gene therapy on the expression of smooth muscle markers in diabetic rat penis. This proposal is novel because oxidative stress and apoptotic pathways in diabetes-induced erectile dysfunction have never been investigated. Accomplishment of these experiments will provide novel molecular mechanisms of erectile dysfunction and can help guide us towards the development of new therapies for the treatment of erectile dysfunction

Keywords: apoptosis, diabetes mellitus, diabetes mellitus therapy, gene therapy, impotence, medical complication, nonhuman therapy evaluation, oxidative stress antioxidant, biomarker, cell differentiation, cytochrome c, disease /disorder model, gene expression, insulin, penis erection, smooth muscle, superoxide dismutase electron microscopy, histology, immunocytochemistry, in situ hybridization, laboratory rat, light microscopy, polymerase chain reaction

Project start date: 2006-09-15

Project end date: 2011-06-30

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Diabetes Erectile Dysfunction And Apoptosis Of Cavernous Tissues

Rajvir Dahiya, Professor And Director
Northern California Institute Res And Educ San Francisco, Ca 941211545

Grant 5R01DK075524-02 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG1

Abstract: The main goal of this proposal is to investigate whether oxidative stress and apoptosis of cavernous tissues are major pathways in erectile dysfunction. We will also investigate whether inhibition of oxidative stress and apoptosis can restore erectile function. The rationale for this project is that erectile dysfunction affects about 30 million men in the United States and more than 200 million men worldwide. Based on our preliminary data and prior publications, we hypothesize that apoptosis of cavernous tissues are down stream events in erectile dysfunction. We further hypothesize that anti-oxidative agent or Bcl2 gene therapy can restore erectile function in animal model. We will test these hypotheses through the following experiments Specific Aim# 1. To test the hypothesis that diabetes-induced erectile dysfunction is due to apoptosis of cavernous tissues. Under this specific aim, we will analyze erectile function in diabetic rats and then analyze apoptotic index, anti-apoptotic genes, pro-apoptotic genes, cytochrome c, superoxide dismutase and smooth muscle differentiation markers. We will also investigate whether an anti-oxidative agent (TEMPOL) can restore erectile function in diabetic rats. We will use the following techniques RT-PCR, real-time RT-PCR, in situ hybridization, immunohistochemistry, electro-stimulation of cavernous nerve for pressure measurements, gene delivery, histology, light and electron microscopy. Specific Aim # 2. To test the hypothesis that insulin treatment can restore erectile function through restoration of anti-apoptotic factors in cavernous tissues of diabetic rats. Under this specific aim, we will treat diabetic rats with insulin and then investigate whether erectile function is restored through restoration of anti-apoptotic pathways described under specific aim # 1. Specific Aim # 3. To test the hypothesis that gene therapy with the Bcl2 anti-apoptotic gene can restore functional, cellular and molecular mechanisms of erectile dysfunction in diabetic rats. Under this specific aim, the Bcl2 gene will be transfected into the cavernous tissue of diabetic rats using an adeno-associated virus vector. We will do the following experiments (a) Functional assessment of the effects of Bcl2 gene therapy on penile erection in diabetic rats, (b) Analysis of the effects of Bcl2 gene therapy on the oxidative stress pathways and the apoptotic pathways described under specific aim 1. (c) Analysis of the effects of Bcl2 gene therapy on the expression of smooth muscle markers in diabetic rat penis. This proposal is novel because oxidative stress and apoptotic pathways in diabetes-induced erectile dysfunction have never been investigated. Accomplishment of these experiments will provide novel molecular mechanisms of erectile dysfunction and can help guide us towards the development of new therapies for the treatment of erectile dysfunction.

Keywords: apoptosis, diabetes mellitus, diabetes mellitus therapy, gene therapy, impotence, medical complication, nonhuman therapy evaluation, oxidative stress, antioxidant, biomarker, cell differentiation, cytochrome c, disease /disorder model, gene expression, insulin, penis erection, smooth muscle, superoxide dismutase, electron microscopy, histology, immunocytochemistry, in situ hybridization, laboratory rat, light microscopy, polymerase chain reaction

Project start date: 2006-09-15

Project end date: 2011-06-30

5R01DK075524-02 (2007): $313711

Wnt Antagonist Genes In Kidney Tumor Progression And Metastasis

Rajvir Dahiya, Professor And Director
Northern California Institute Res & Educ
san Francisco, Ca 941211545

Grant 1R01CA130860-01 from National Cancer Institute, IRG: TPM

Abstract: Wnt antagonist genes in kidney tumor progression and metastasis. Main goal of this project is to investigate the role of Wnt antagonist genes in the progression and metastasis of renal cancer. The rationale is that Wnt antagonist genes are inactivated through CpG methylation pathways with the result that Wnt/ b-catenin pathways are activated and induce malignant transformation of various organs. However, such studies are lacking in kidney cancer. Three specific hypotheses will be tested to determine if 1) Inactivation of Wnt antagonist genes is involved in the progression and metastasis of kidney cancer. 2) The mechanisms of inactivation of the Wnt antagonist genes are through epigenetic pathways such as DNA methylation, histone modification and chromatin remodeling. 3) Transfection of Wnt antagonist genes suppresses the in vitro and in vivo growth and metastasis of kidney cancer. To test these hypotheses, we will pursue the following specific aims. Specific Aim # 1 To investigate whether inactivation of Wnt antagonist genes is involved in the progression and metastasis of human renal cell carcinoma. Based on the preliminary data, we have screened several Wnt antagonist genes and have identified six genes that are silenced in kidney cancer. These genes are secreted frizzled-related protein-1 (sFRP-1), sFRP-2, sFRP-4, sFRP-5, Wnt inhibitory factor-1 (Wif-1) and DICKKOPF-3 (DKK-3). Under this specific aim, we will determine the levels of mRNA and protein expression of Wnt antagonist genes in normal and different stages and grades of kidney cancer. The mRNA expression will be analyzed by real-time RT- PCR and protein expression by immunohistochemistry and Western blotting. Specific Aim # 2 To investigate the mechanisms of inactivation of Wnt antagonist genes in kidney cancer. Under this aim, we will analyze hypermethylation of CpG Islands in promoter regions of Wnt antagonist genes using sodium bisulfite methylation techniques and confirm by direct DNA sequencing. We will also investigate whether the mechanisms of inactivation of Wnt antagonist genes are due to DNA methyltransferase (DNMT-1, DNMT3a, DNMT3b), demethylase (MBD2) genes, histone acetylation and chromatin remodeling through analysis of these parameters in kidney cancer tissues. Specific Aim # 3 To investigate the functional role of Wnt antagonist genes in kidney cancer. Under this specific aim, we will transfect full-length Wnt antagonist gene cDNA in dominant-negative kidney cancer cells and establish the stable transfectant cells. We will analyze in vitro and in vivo growth of transfected kidney cancer cells. Also analyze in vitro invasiveness (extra-cellular matrix binding assay, invasion assay and soft agar colony forming efficiency) of transfected and parental cells. Successful completion of these experiments will demonstrate the functional role of Wnt antagonist genes in the suppression of kidney cancer growth, progression and metastasis and also the mechanism of inactivation of these genes in kidney cancer. In the future, these results may provide better strategies for the management of kidney cancer progression and metastasis. Renal cell carcinoma (RCC) is the third most common malignancy of the genitourinary system. Based on the published literature, it is clear that Wnt antagonist genes are associated with various cancers. However, such studies are lacking in kidney cancer. We have proposed to investigate the functional role of Wnt antagonist genes in the progression and metastasis of renal cell carcinoma using both in vitro and in vivo models. Successful completion of proposed experiments may provide us with the better strategies for the management of kidney cancer

Project start date: 2008-08-27

Project end date: 2013-05-31

Urological Research Training Program

Rajvir Dahiya, Professor And Director
Urologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962

Grant 5T32DK007790-07 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: DDK

Abstract: This proposal seeks continuation of our unique program in academic urology, which provides intensive training in urological research to postdoctoral fellows. The main goal of this program is to train the future leaders in the field of genitourinary (GU) diseases. UCSF has 29 faculty members (15 MD and 14 Ph.D.) with strong interests in GU research. All of these investigators have research funding from NIH / DOD / VA / other funding agencies and have an outstanding track record of training urologists and Ph.D. fellows. The training program is focused to train postdoctoral fellows in a specific research project involving modern laboratory techniques including molecular biology, basic biomedical sciences, clinical sciences, genetics and epigenetics technology, epidemiology, recombinant DNA technology, gene knock out technology, Si RNA technology, pathology, cell biology, biostatistics and magnetic resonance spectroscopic imaging in both benign and malignant GU diseases. This is a comprehensive multi-disciplinary program in which both biomedical and translational research training will be provided. Under this program, trainees from under-represented racial / ethnic groups will be accommodated. Based on the number of faculty members, we propose to train four T32 positions per year over five years. The first year of training will comprise of course work in basic sciences, research ethics, basic research techniques, immunology, biochemistry, epidemiology, pathology, and clinical research methods. In the remainder of the first year and in the second and third years, the trainees will work under the mentorship of faculty members in one of the research areas described in the proposal. All trainees will have the opportunity to take these courses offered by various Departments of the University of California San Francisco. All training will take place in the laboratories of senior scientists using a well-proven system of training fellows. Our focus is to help these bright young postdoctoral fellows bridge the transition from supervised research training to independent investigators in the field of GU research. The progress of the trainees will be evaluated through (a) close individual supervision in the laboratory and weekly one-on-one meeting with mentors plus monthly hour-long meetings with supervising subcommittees consisting of 3-4 mentors, (b) participation in weekly research conferences and journal clubs, (c) participation in program-wide conferences and courses. This intense supervision is key to the transition of the fellows to become independent scientists. The individual fellows will choose a principal mentor, and the executive training committee will choose 2-3 co-mentors to supplement the talents of the principal mentor. We have developed a strong recruitment plan to enroll women and minorities. Upon completion of this program, the trainees will be prepared to pursue an independent research career in academic urology. This will ensure an adequate pool of outstanding future scientists in urology research

Project start date: 2000-09-15

Project end date: 2012-08-31

2T32DK007790-06A2 (2007): $93414

Diabetes Erectile Dysfunction And Apoptosis Of Cavernous Tissues

Rajvir Dahiya, Professor And Director
Northern California Institute Res And Educ San Francisco, Ca 941211545

Grant 1R01DK075524-01 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG1

Abstract: The main goal of this proposal is to investigate whether oxidative stress and apoptosis of cavernous tissues are major pathways in erectile dysfunction. We will also investigate whether inhibition of oxidative stress and apoptosis can restore erectile function. The rationale for this project is that erectile dysfunction affects about 30 million men in the United States and more than 200 million men worldwide. Based on our preliminary data and prior publications, we hypothesize that apoptosis of cavernous tissues are down stream events in erectile dysfunction. We further hypothesize that anti-oxidative agent or Bcl2 gene therapy can restore erectile function in animal model. We will test these hypotheses through the following experiments Specific Aim# 1. To test the hypothesis that diabetes-induced erectile dysfunction is due to apoptosis of cavernous tissues. Under this specific aim, we will analyze erectile function in diabetic rats and then analyze apoptotic index, anti-apoptotic genes, pro-apoptotic genes, cytochrome c, superoxide dismutase and smooth muscle differentiation markers. We will also investigate whether an anti-oxidative agent (TEMPOL) can restore erectile function in diabetic rats. We will use the following techniques RT-PCR, real-time RT-PCR, in situ hybridization, immunohistochemistry, electro-stimulation of cavernous nerve for pressure measurements, gene delivery, histology, light and electron microscopy. Specific Aim # 2. To test the hypothesis that insulin treatment can restore erectile function through restoration of anti-apoptotic factors in cavernous tissues of diabetic rats. Under this specific aim, we will treat diabetic rats with insulin and then investigate whether erectile function is restored through restoration of anti-apoptotic pathways described under specific aim # 1. Specific Aim # 3. To test the hypothesis that gene therapy with the Bcl2 anti-apoptotic gene can restore functional, cellular and molecular mechanisms of erectile dysfunction in diabetic rats. Under this specific aim, the Bcl2 gene will be transfected into the cavernous tissue of diabetic rats using an adeno-associated virus vector. We will do the following experiments (a) Functional assessment of the effects of Bcl2 gene therapy on penile erection in diabetic rats, (b) Analysis of the effects of Bcl2 gene therapy on the oxidative stress pathways and the apoptotic pathways described under specific aim 1. (c) Analysis of the effects of Bcl2 gene therapy on the expression of smooth muscle markers in diabetic rat penis. This proposal is novel because oxidative stress and apoptotic pathways in diabetes-induced erectile dysfunction have never been investigated. Accomplishment of these experiments will provide novel molecular mechanisms of erectile dysfunction and can help guide us towards the development of new therapies for the treatment of erectile dysfunction.

Keywords: apoptosis, diabetes mellitus, diabetes mellitus therapy, gene therapy, impotence, medical complication, nonhuman therapy evaluation, oxidative stress, antioxidant, biomarker, cell differentiation, cytochrome c, disease /disorder model, gene expression, insulin, penis erection, smooth muscle, superoxide dismutase, electron microscopy, histology, immunocytochemistry, in situ hybridization, laboratory rat, light microscopy, polymerase chain reaction

Project start date: 2006-09-15

Project end date: 2011-08-31

1R01DK075524-01 (2006): $322721

Genistein And Prostate Cancer

Rajvir Dahiya, Professor And Director
Northern California Institute Res & Educ

Grant 5R01CA111470-03 from National Cancer Institute, IRG: CDP

Abstract: The main goal of this proposal is to investigate whether genistein inhibits prostate cancer growth through epigenetic pathways. The rationale for this project is that Western men have a 5-6 fold higher incidence of prostate cancer than Asians. One reason for this discrepancy may be the high soy diet (genistein) consumed by Asians. We hypothesize that genistein inhibits prostate cancer growth through epigenetic pathways. Specific Aim # 1. To investigate whether genistein can suppress prostate cancer growth through cell cycle arrest, cyclin genes and apoptosis. Based on the published literature and preliminary data, we lypothesize that genistein will inhibit prostate cancer cell growth in vitro. We will investigate whether genistein alone or in combination with demethylating agent (5-´-aza-2-deoxycytidine (5-AZA-C) and histone deacetylase inhibitor (trichostatin (TSA) can inhibit prostate cancer growth in vitro. A panel of human prostate cell lines will be studies for cell proliferation, cell cycle, cyclin genes and apoptosis. Specific Aim # 2. To investigate the molecular mechanisms of genistein action through epigenetic pathways. Based on our preliminary data, we hypothesize that genistein inhibits prostate cancer growth through modulation of epigenetic events, such as DNA methylation and histone acetylation. To test this hypothesis, we will analyze 1) Global methylation and enzymatic activity of DNA methyltransferase (DNMT) using radiolabeled S- adenosylmethionine (SAM) assays. 2) Promoter methylation of candidate genes using bisulfite modification of DNA based techniques. 3) Alterations in gene-specific histone acetylation using ChIP assay, and enzymatic activities of histone acetyl transferases (HATs). 4) Histone deacetylase (HDACs) analysis using biochemical assays. 5). Alterations in mRNA expression of genes that encode enzymes participating in DNA methylation and histone acetylation including DNMTs, demethylases, HATs, HDACs using RT-PCR and northern blot assays. 6) To analyze transcription factors which mediate the effects of genistein on prostate cancer. Specific Aim # 3. To investigate whether genistein has anti-tumor effects through reversal of epigenetic pathways in prostate cancer using an in vivo system. Based on the published literature, genistein or demethylating agents have anti-proliferative effects on various cancers. We hypothesize that genistein will inhibit prostate cancer growth through reversal of epigenetic pathways. Studies will be conducted in athymic nude mice implanted with androgen-responsive LNCaP and androgen nonresponsive DuPro prostate cancer cells. Mice will be fed a diet supplemented with genistein, or a combination of genistein with 5-AZA-C or TSA. The following experiments will be conducted 1) Determine the in vivo anti-tumor effects of genistein and in combination with 5-AZA-C or TSA. 2) To evaluate expression of steroid receptor genes and tumor suppressor genes at the mRNA and protein levels using Northern blot, and Western blotting analysi respectively. 3) To analyze whether genistein treatment can modulate global and candidate gene methylation in tumor tissue using a radiolabeled SAM assay and bisulfite modified genomic sequencing techniques respectively. 4) To analyze, in tumor tissue, gene-specific histone acetylation using ChIP assay and the enzymatic activities of HATs and HDACs using biochemical assays. Accomplishment of these experiments will demonstrate whether epigenetic events are modulated by genistein and provide rationale for a clinical trial of genistein alone or in combination with other epigenetic modulators for the treatment or prevention of prostate cancer

Keywords: genistein, prostate neoplasm 5 methylcytosine, Asian, DNA, DNA methylation, N glycosidase, S adenosylmethionine, acetylation, acyltransferase, adhesion, amidohydrolase, androgen, androgen receptor, apoptosis, athymic mouse, base, binding site, bioinformatics, cadherin, cancer prevention, cell, cell cycle, cell cycle protein, cell line, cell proliferation, chromatin immunoprecipitation, clinical trial, cyclin, death, deoxycytidine, diet, enzyme, enzyme inhibitor, epigenetics, estrogen, estrogen receptor, gene, gene expression, gene induction /repression, glutathione, histone, hormone, human, implant, lead, male, methylation, methyltransferase, neoplasm /cancer, neoplastic cell, neoplastic growth, northern blotting, nutrition, p53 gene /protein, phytoestrogen, plant, play, polymerase chain reaction, prevention, prostate, protein, protein kinase, radiotracer, receptor, repression, retinoblastoma, role, steroid, sulfite, therapy, tissue, transcription factor, transferase, tumor suppressor gene, university, western blotting

Project start date: 2006-08-09

Project end date: 2011-07-31

5R01CA111470-02 (2007): $271628

1R01CA111470-01A2 (2006): $279119

AGING AND PROSTATE GROWTH

Rajvir Dahiya, Professor And Director
Northern California Institute Res & Educ
san Francisco, Ca 941211545

Grant 5R01AG016870-04 from National Institute On Aging, IRG: ZRG4

Abstract: The main goal of this proposal is to investigate the molecular mechanisms of prostate growth in aging. The rationale for this proposal is that the mechanisms of neoformation of ductal-acinar tissue during the pathogenesis of benign prostatic hyperplasia (BPH) is similar (if not identical) to the process of ductal-acinar growth and development occurring during the organogenesis of the prostate in fetal and pubertal periods. This proposal is based on the hypothesis that the age- dependent endocrine control of prostatic cell growth is regulated by mesenchyme-epithelial cell interactions mediated by the local production and action of growth factors (KGF, TGFa, and TGFb). Based upon preliminary experiments, TGFa and KGF may up-regulate and TGFb down- regulate the prostatic epithelium growth. This hypothesis will be tested through pursuit of the following specific aims. 1. Analysis of paracrine mechanisms in the regulation of rat fetal prostatic growth and differentiation. Under this specific aim, rat fetal prostates (different ages) will be studied to determine growth kinetics, ductal morphogenesis, expression of differentiation markers and expression of KGF, TGFa and TGFb and their receptors. Specific Aim number 2. Analysis of the growth promoting and morphogenetic effects of rat urogenital sinus mesenchyme (rat UGM) on growth-quiescent adult rat prostatic epithelium. Under this specific aim we will test the hypothesis that rat UGM can induce the growth and differentiation of growth-quiescent adult rat prostatic epithelium. To test this hypothesis, rat UGM will be associated with rat prostatic epithelium from adult rats (age 3, 6, 12, 18 and 24 months) and the resultant tissue recombinants will be grown in kidney capsules of male athymic nude mice and will be characterized for growth, differentiation and expression of KGF, TGFa and TGFb and their receptors. Specific Aim number 3. Analysis of the inter- relationships between growth factor families during normal and impaired prostatic epithelial growth. Under this specific aim, we hypothesize that there is a complex interaction between androgen action and growth factors. Indeed, for some systems it has been shown that the actions of one growth factor may be mediated by other downstream signaling growth factors. To dissect this complex interplay between growth factors, various transgenic knockout mice will be utilized. The goal of this experiment is to elucidate the linkage between different growth factor pathways employing chimeric rat-mouse prostatic tissue recombinants

Keywords: aging, cell cell interaction, cell growth regulation, epithelium, histogenesis, mesenchyme, prostate acinar cell, benign prostate hyperplasia, biological signal transduction, cell differentiation, growth factor receptor, hormone regulation /control mechanism, paracrine, prostate preneoplastic state, receptor expression, transforming growth factor athymic mouse, embryo /fetus cell culture, gene targeting, laboratory rat, transgenic animal

Project start date: 1998-09-01

Project end date: 2003-11-30

5R01AG016870-04 (2002): $265161

5R01AG016870-03 (2001): $258117

5R01AG016870-02 (2000): $251278

1R01AG016870-01 (1998): $244637

SMOOTH MUSCLE-EPITHELIAL INTERACTIONS IN PROSTATE CANCER

Rajvir Dahiya, Professor And Director
Urologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962

Grant 1R01CA064872-01 from National Cancer Institute, IRG: SRC

Abstract: The main goal of this proposal is to investigate the cellular and molecular mechanisms of smooth muscle-epithelial interactions in normal and malignant prostatic epithelial growth and differentiation. The rationale of the overall approach of this proposal is that the initial differentiation of smooth muscle in the developing prostate is induced and maintained through interactions with epithelium. During prostatic carcinogenesis the ability of epithelium to induce and maintain smooth muscle differentiation will diminish as signaling from epithelium to smooth muscle becomes abnormal. Hypothesis This proposal is based upon the hypothesis that normal and malignant prostatic epithelium cell growth and differentiation as regulated by smooth muscle-epithelial cell interactions medicated by paracrine growth or differentiation factors. This hypothesis will be examined through pursuit of the following specific aims. (Specific aim #1) Analysis of expression of prostatic smooth muscle markers during normal prostatic development and prostatic carcinogenesis. Overall strategy for specific aim #1 will include (a) to examine the sequential expression of smooth muscle differentiation markers during normal prostatic development in rat and human by immunohistochemistry and western blot. (b) to examine expression of smooth muscle differentiation markers in malignant human prostate and during prostatic carcinogenesis in rats. (Specific aim #2) Analysis of induction and maintenance of prostatic smooth muscle differentiation by normal and malignant prostatic epithelial cells. The overall strategy will include (a) Analysis of cellular mechanisms of induction and maintenance of smooth muscle differentiation. (b) Analysis of hormonal mechanisms of induction of smooth muscle differentiation. (c) Analysis of molecular mechanisms of induction of smooth muscle differentiation. (Specific aim #3) Analysis of prostatic smooth muscle and Dunning tumor stroma as regulators of normal and malignant prostatic epithelial growth and differentiation. (Specific aim #4) Development and utilization of new methods of prostate carcinogenesis. The long-term objective of this research project is to develop the best strategy for regulation of human prostatic cancer growth by understanding the cellular and molecular mechanisms of smooth muscle-epithelial interactions

Keywords: cell cycle, cellular oncology, epithelium, molecular oncology, prostate neoplasm, smooth muscle autocrine, biological signal transduction, carcinogenesis, cell differentiation, cell growth regulation, endocrine gland /system, hormone regulation /control mechanism Rodentia, athymic mouse, human embryo /fetus tissue, human subject, immunocytochemistry, northern blotting, western blotting

Project start date: 1994-09-30

Project end date: 1997-09-29

1R01CA064872-01 (1994): $185386

DNA Methylation In Aging, Race And Prostate Cancer

Rajvir Dahiya, Professor And Director
Northern California Institute Res And Educ San Francisco, Ca 941211545

Grant 5R01AG021418-05 from National Institute On Aging, IRG: ZAG1

Abstract: The main goal of this proposal is to identify and characterize the epigenetic alterations that are involved in aging and race-related prostate cancer. Specific hypotheses 1) The DNA methylation status of most frequently deleted genes [ERs, E-cadherin, CD44 and GSTPI (glutathione S-transferase pi)] will be different in aging and race-related prostate cancer. 2) Identification of novel DNA methylated genes will provide us the potential biomarkers of aging and race-related prostate cancer. 3) DNA methyltransferase, DNA demethylase and histone deacetylase activities will correspond to DNA methylation status in aging and race-related prostate cancer. Specific Aim number 1. To test the hypothesis that inactivation of ER genes by DNA methylation can be used as biomarkers for aging and race-related prostate cancer. We hypothesize that ERalpha and ERbeta are differentially expressed in different aging and race related prostate cancer. To test this hypothesis we will investigate the mRNA and protein expression of ER types (alpha and beta) in different stages and grades of prostate cancer. Protein expression of ERs will be analyzed by immunohistochemistry (for localization) and western blotting (for quantitation). RT-PCR (for screening) and northern blotting (for quantitation) will analyze Gene expression. DNA methylation will be analyzed by sodium bisulfite methylation techniques and confirm by direct DNA sequencing. Specific Aim number 2. To test the hypothesis that inactivation of E-cadherin, CD44 and GSTPI genes by DNA methylation can be used as biomarkers for aging and race-related prostate cancer. Based on our preliminary data and prior publications, E-cadherin, CD44 and GSTPI genes are most frequently inactivated by DNA methylation in prostate cancer. We hypothesize that the expression or inactivation of these genes will be different in aging and race- related prostate cancer. To test this hypothesis, we will first determine the protein expression of these genes in prostate cancer using immunohistochemistry (for localization), and western blotting (for quantitation). RNA expression will be analyzed by RT-PCR (for screening) and northern blotting (for quantitation). CpG methylation will be analyzed by sodium bisulfite methylation techniques and confirm by direct DNA sequencing. Specific Aim number 3. To test the hypothesis that identification of new DNA methylated genes by restriction landmark genomic scanning (RLGS) can be used as novel biomarkers for aging and race-related prostate cancer. We hypothesize that identification of novel genes that are inactivated by DNA methylation will be important in understanding the mechanisms of aging and race-related prostate cancer. To test this hypothesis, we will analyze new genes that are methylated in aging and race-related prostate cancer. We will use Restriction landmark genomic scanning for methylation (RLGS) to survey genome wide methylation alterations in aging and race-related prostate cancer and subsequently verify potentially hypermethylated gene loci using sodium bisulfite sequencing technique. Specific Aim number 4. To investigate the mechanisms of DNA methylation in aging and race-related prostate cancer. We hypothesize that DNA methyltransferase, DNA demethylase, histone deacetylase are responsible for regulation of DNA hypermethylation. To test this hypothesis, we will analyze DNA methyltransferase, DNA demethylase and histone deacetylase enzyme activity, mRNA and protein expression in different age and race related prostate cancer. Accomplishment of these experiments will demonstrate whether DNA methylated genes are involved in aging and race-related prostate cancer. This information can be used for better management of prostate cancer using DNA methylated genes as diagnostic or prognostic biomarkers.

Keywords: DNA methylation, aging, neoplasm /cancer genetics, prostate neoplasm, racial /ethnic difference, CD44 molecule, CpG island, amidohydrolase, biomarker, cadherin, enzyme activity, estrogen receptor, gene expression, glutathione transferase, methyltransferase, molecular oncology, tumor suppressor gene, African American, Japanese, caucasian American, clinical research, human middle age (35-64), human old age (65+), human tissue, male, nucleic acid sequence, patient oriented research, restriction fragment length polymorphism

Project start date: 2002-07-15

Project end date: 2007-06-30

5R01AG021418-05 (2006): $322245

5R01AG021418-04 (2005): $330000

5R01AG021418-03 (2004): $303000

5R01AG021418-02 (2003): $303000

1R01AG021418-01 (2002): $303000

MECHANISMS OF PROSTATE DEVELOPMENT

Rajvir Dahiya, Professor And Director
Urologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962

Grant 5R01DK047517-02 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: DDK

Abstract: The MAIN GOAL of this project is to understand the mechanisms regulating cellular growth of human fetal and adult prostate with the emphasis on the role of mesenchyme-epithelial interactions and growth factors (KGF, TGFalpha, and TGFbeta). The RATIONALE for this proposal is that the developing prostate is an appropriate paradigm for mechanistic studies of growth regulation by paracrine mechanisms in normal and hyperplastic prostates. This project is based on the HYPOTHESIS that the endocrine and developmental control of prostatic cell growth may be regulated by cell-cell interactions mediated by the local production and action of peptide growth factors. In order to test this hypothesis that paracrine mechanisms may be involved in growth and differentiation of developing and adult prostate, we will pursue the following SPECIFIC AIMS 1) To elucidate the paracrine mechanisms in the regulation of human prostatic growth and differentiation. This will be achieved by grafting human fetal prostates into male athymic nude mice. The resultant growing human fetal prostatic tissue will be examined for a) growth kinetics (doubling time and 3H-thymidine labeling index), ductal elongation and branching morphogenesis, and expression of differentiation markers (PSA, PSAP) by immunohistochemistry and western blot; b) expression of KGF, TGFalpha, and TGFbeta and their receptors by RT-PCR, nuclease protection assay, northern blot, and immunohistochemistry; c) the effects of exogenous KGF, TGFalpha, and TGFbeta and their neutralizing antibodies on organ cultures of growing human fetal vs. adult human prostate (BPH) tissue; d) regulation of KGF, TGFalpha, TBFbeta levels by androgens; e) determination of temporal and spatial aspects of growth factors and their receptors in fetal and BPH tissue. 2) To determine the growth promoting and morphogenetic effects of human urogenital sinus mesenchyme (UGM) on growth-quiescent adult human prostatic epithelium. For this purpose we will isolate UGM and combine it with human prostatic epithelial cells. The resultant tissue recombinant will be grown in athymic nude mice and will be characterized for growth and development as described under Specific Aim #1. 3) To investigate the role of growth factors and cell- cell interactions in the prostate using a new BPH-derived epithelial cell line. This specific aim will be achieved by determining KGF, TGFalpha, and TGFbeta as regulators of prostatic epithelial growth and development. We will also examine the role of prostatic epithelium in the induction of smooth muscle differentiation. Accomplishment of these objectives will elucidate the mechanisms regulating prostatic growth and development by mesenchymal epithelial interaction mediated by the local production and action of growth factors. Through this multidisciplinary approach, we will definitely establish the role of these growth factors as paracrine/autocrine growth regulators in the human prostatic development. The LONG-TERM OBJECTIVES of the proposal are to develop the best strategy for regulation of prostatic growth

Keywords: cell cell interaction, cell differentiation, cell growth regulation, growth factor, prostate, transforming growth factor androgen, benign prostate hyperplasia, epithelium, growth factor receptor, hormone regulation /control mechanism, mesenchyme, paracrine, smooth muscle RNase protection assay, athymic mouse, human embryo /fetus tissue, immunocytochemistry, northern blotting, polymerase chain reaction, western blotting

Project start date: 1993-09-30

Project end date: 1997-08-31

5R01DK047517-02 (1994): $190312

1R01DK047517-01 (1993): $182438

Cytochrome-P-450 Genes In Pathobiology Of Endometrial Cancer

Rajvir Dahiya, Professor And Director
Urologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962

Grant 5R01CA108612-04 from National Cancer Institute, IRG: CE

Abstract: The main goal of this project is to investigate whether the cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1) genes are risk factors for pathobiology of endometrial cancer. The rationale is that CYP1A1 and CYP1B1 catalyze the hydroxylation of estrogen to highly carcinogenic 2-hydroxy catechol estrogen (2-OH-E2) and 4-hydroxy catechol estrogen (4-OH-E2) metabolites, respectively. However, such studies are lacking in endometrial cancer. Based on our preliminary data and prior publications, we hypothesize that CYP1A1 and CYP1B1 genes are risk factor for pathobiology of endometrial cancer. Specific Aim # 1. To investigate whether the CYP1A1 and CYP1B1 genes are hyper-activated in endometrial cancer. We hypothesize that the CYP1A1 and CYP1B1 genes are hyper-activated during malignant transformation of the endometrium. To test this hypothesis, we will (a) Analyze the level of CYP1A1 and CYP1B1 mRNA expression by real-time PCR (for quantification) in benign endometrium and in different stages and grades of endometrial cancer. In addition, in situ hybridization will be used to localize CYP1A1 and CYP1B1 mRNA expression in endometrial tissues, (b) Analysis of protein expression of CYP1A1 and CYP1B1 in benign endometrium and in different stages and grades of endometrial cancer using immunohistochemistry (for localization) and western blotting for quantification. (c) Analysis of the level of CYP1A1 and CYP1B1 enzymatic activity in endometrial tissues by biochemical techniques as well as determine the level of estrogen metabolites (2-OH-E2 and 4-OH-E2) in endometrial tissues by gas chromatography methods, (d) To analyze whether CYP1A1 and CYP1B1 genes, protein, enzymatic activity and/or the levels of 2-OH-E2 and 4-OH-E2 metabolites in endometrial cancer are predictors of recurrence and patient survival. Specific Aim # 2 To investigate whether single nucleotide polymorphisms of the CYP1 Al and CYP1B1 genes are risk factors for endometrial cancer. We hypothesize that the analysis of SNPs in the CYP1A1 and CYP1B1 genes can identify populations with a higher risk of endometrial cancer. To test this hypothesis, we will (a) Analyze SNPs of the CYP1A1 and CYP1B1 genes in blood and tissue DNA from benign and endometrial cancer patients using PCR-RFLP (restriction fragment length polymorphism) and direct genomic sequencing, (b) Analyze whether SNPs of CYP1A1 and CYP1B1 genes are associated with altered enzymatic activity and the altered production of estrogen metabolites in endometrial cancer, (c) Analyze whether SNPs of the CYP1A1 and CYP1B1 genes are associated with endometrial cancer recurrence and patient survival. Specific Aim # 3 To investigate whether polymorphic variants of the CYP1A1 and CYP1B1 genes can modulate estrogen hydroxylase activity. We hypothesize that SNPs in exon regions of CYP1A1 and CYP1B1 genes can modulate their enzymatic activity in endometrial cancer. To test this hypothesis we will express wild-type and polymorphic variant forms of CYP1A1 and CYP1B1 (which contain a NH2-terminal hexahistidine tag) using a ThioFusion expression vector (pThioHis). CYP1A1 and CYP1B1 mRNA isolated from normal endometrium will serve as the source of wild-type cDNAs, which will be inserted into the pThioHis expression vector. We will then use the wild-type pThioHis CYP1A1 and CYP1B1 expression plasmids as templates to generate polymorphic variants of CYP1A1 and CYP1B1 using site-directed mutagenesis and determine their estrogen hydroxylase activity. This project will provide us with novel mechanisms involving the pathobiology of endometrial cancer

Keywords: cancer risk, cytochrome P450, enzyme induction /repression, genetic susceptibility, neoplasm /cancer genetics, steroid hormone metabolism, uterus neoplasm enzyme activity, estrogen, gene expression, hormone related neoplasm /cancer, oxygenase, single nucleotide polymorphism female, human tissue, in situ hybridization, patient oriented research

Project start date: 2005-06-10

Project end date: 2010-04-30

5R01CA108612-03 (2007): $283708

5R01CA108612-02 (2006): $292181

1R01CA108612-01A1 (2005): $299213

CYP1A1 And CYP1B1 Genes In Race-related Prostate Cancer

Rajvir Dahiya, Professor And Director
Northern California Institute Res And Educ San Francisco, Ca 941211545

Grant 5R01CA101844-05 from National Cancer Institute, IRG: REN

Abstract: The main goal of this project is to investigate whether over-expression of cytochrome P450 1A1 (CYP1A1) and cytochrome P4501 B1 (CYP1B1) genes can be risk factors for race-related prostate cancer. Rationale is that African-Americans have twice the risk of Whites for presenting with advanced-stage prostate cancer. The mortality of African-American men 40-60 years of age due to prostate cancer is almost 2-3 folds greater than that of White men of the same age. The genetic basis for racial / ethnic differences in prostate cancer incidence is not known. To address this problem, we have targeted the CYP1A1 and CYP1 B1 genes because their products (2-OH-E2 and 4-OH-E2) are highly carcinogenic. This is a first step towards investigating the genetic basis for racial / ethnic differences in prostate cancer incidence. Based on our prior publications and preliminary data, we hypothesize that the CYP1A1 and CYP1 B1 genes are risk factors for race-related prostate cancer. Specific Aim # 1 To test the hypothesis that CYP1A1 and CYP1 B1 genes are hyper-activated during malignant transformation of race-related prostate cancer. Under this specific aim, we will analyze mRNA expression of the CYP1A1 and CYP1 B1 genes by real-time PCR in different stages and grades of race-related prostate cancer. In situ hybridization will be used to localize gene expression in prostate tissues. Enzyme activities of CYP1 A1 and CYP1 B1 will be analyzed in prostate tissues by radio-isotopic methods. Specific Aim # 2 To test the hypothesis that single nucleotide polymorphisms of CYP1A1 and CYP1 B1 genes are risk factors for prostate cancer. Under this specific aim, we will analyze single nucleotide polymorphisms of four polymorphic sites of the CYPIA1 gene and six polymorphic sites of the CYP1 B1 gene in different stages and grades of race-related prostate cancer. SNPs will be analyzed by PCR-RFLP (restriction fragment length polymorphism) and direct genomic sequencing. Specific Aim # 3 To test the hypothesis that transfection of polymorphic variants of CYP1A1 and CYP1 B1 in E.Coli can hyperactivate estrogen hydoxylase activities using site-directed mutagenesis assays. We will investigate whether polymorphic variants of CYP1 A1 and CYP1 B1 have higher estrogen hydroxylase activities as compared to wild-type. We will also investigate whether estrogen hydroxylase activities and estrogen metabolites (2-OHE2 and 4-OH-E2) are associated with prostate cancer recurrence.

Keywords: African American, cytochrome P450, genetic polymorphism, genetic susceptibility, neoplasm /cancer genetics, prostate neoplasm, racial /ethnic difference, enzyme activity, neoplasm /cancer relapse /recurrence, single nucleotide polymorphism, steroid hormone metabolism, clinical research, human genetic material tag, human tissue, in situ hybridization, male, site directed mutagenesis, transfection

Project start date: 2003-09-05

Project end date: 2008-06-30

5R01CA101844-05 (2007): $278480

5R01CA101844-04 (2006): $286798

5R01CA101844-03 (2005): $293700

5R01CA101844-02 (2004): $269670

1R01CA101844-01 (2003): $269670

ESTROGEN AND PROSTATIC GROWTH

Rajvir Dahiya, Professor And Director
Urologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962

Grant 5R01DK047517-08 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: REN

Abstract: adapted from s ) and his co-investigator, Dr. Gerry Cunha, have focused their studies on the cellular and molecular mechanisms of estrogen action on developing and adult prostate with an emphasis on the role of tissue age on cell-cell interactions, estrogen receptors, growth factor pathways. The hypothesis suggested states that estrogens and androgens and the developmental control of prostatic cell growth may be regulated by cell-cell interactions mediated by the local production and action of peptide growth factors. In the first aim, Dr. Dahiya proposes to look at the estrogenic effects on prostate of the neonate and adult mouse using both wild-type and estrogen receptor knock-out mice. Next he will categorize growth factor and receptors during estrogen induced prostatic squamous metaplasia. The role of mesenchymal-epithelial interactions in the estrogenic response of the prostate will be examined. Finally, will analyze cellular and molecular pathways of estrogen action in neonatal prostates

Keywords: cell cell interaction, cell differentiation, cell growth regulation, estrogen, growth factor, hormone regulation /control mechanism, prostate androgen, benign prostate hyperplasia, diethylstilbestrol, estrogen receptor, gene expression, growth factor receptor, mesenchyme, metaplasia, receptor expression athymic mouse, gene targeting, immunocytochemistry, mature animal, newborn animal, polymerase chain reaction, transgenic animal

Project start date: 1993-09-30

Project end date: 2002-06-30

5R01DK047517-08 (2001): $271123

5R01DK047517-07 (2000): $263226

2R01DK047517-05 (1998): $247995

PARACRINE MECHANISMS IN HUMAN BENIGN PROSTATIC GROWTH.

Rajvir Dahiya, Professor And Director
Urologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962

Grant 1R01DK045861-01A3 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: REN

Abstract: Adapted from ) This new R01 application, currently in its 4th version seeks to understand the cellular and molecular mechanisms of human benign prostatic hyperplasia. The central hypothesis is that the growth and differentiation of the prostate is regulated by mesenchyme-epithelial interactions mediated by the local production and action of peptide growth factors such as TGFa, TGFB and KGF. Two specific aims are planned to test this hypothesis. The first aim is to characterize a human cell line (BPH-1) that was generated by SV-40 large T antigen transformation of epithelial cells obtained from a BPH specimen. The characterization of these cells will include the testing for the expression of cytokeratins, androgen receptor, and prostate-specific secretory proteins. In addition, the effects of various growth factors on this cell line and the regulation of growth factor production by androgens will also be studied. The second aim is to investigate the role of mesenchyme-epithelial interaction in the regulation of the growth and development of the prostate. Tissue recombinants will be grown in athymic nude mice. Growth kinetics, tissue morphology and the expression of markers of prostatic differentiation as well as growth factor expression will be studied

Keywords: autocrine, benign prostate hyperplasia, paracrine androgen receptor, biomarker, cell cell interaction, cell differentiation, epithelium, hormone regulation /control mechanism, insulinlike growth factor, keratin, mesenchyme, receptor expression, secretory protein athymic mouse, human tissue, recombinant protein, tissue /cell culture

Project start date: 1994-09-30

Project end date: 1998-08-31

1R01DK045861-01A3 (1994): $169333

5R01DK045861-04 (1997): $193662

5R01DK045861-02 (1995): $177020

UROLOGICAL RESEARCH TRAINING PROGRAM

Rajvir Dahiya, Professor And Director
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962

Grant 5T32DK007790-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: DDK

Abstract: adapted ) The main goal of this application is to provide clinical and basic biomedical research training to urologists and Ph.D. scientists that are interested in genitourinary (GU) diseases. The ongoing need for training academic urologists and Ph.D. scientists derives from the current inadequate understanding of the GU diseases. The training of highly qualified urologists and Ph.D. scientists to pursue contemporary research relevant to diseases of the GU system is an investment that is essential to the nation s current and future health care needs. Our training program will develop scientists who can meet this medical challenge. We are fortunate to have 24 senior level investigators that are doing research on GU diseases. All these investigators have research funding from NIH and other funding agencies. These investigators have outstanding track records of training urologists and Ph.D. scientists for many years and most trainees have gone onto careers in academic medicine and many are currently funded by NIH and other granting agencies. This training program is a comprehensive multidisciplinary program in which both basic science and translational research will be conducted and both clinical and basic biomedical scientists will be benefited. Under this program trainees from underrepresented racial/ethnic group will be accommodated. Training opportunities in basic biomedical science, clinical science, epidemiology, biostatistics, recombinant DNA technology and cell biology will be provided with the goal of producing well-rounded interdisciplinary scientists. Specific mentoring by coordinated teams of clinician-investigators and basic scientists in specific GU research areas ensures comprehensive multidisciplinary research training. Under this program, 3 positions will be for Ph.D. post-doctoral fellows and two for M.D. fellows (these positions are based on the number of investigators and funded projects in this program). The first year of training will consist of course work in basic sciences and research ethics plus basic research laboratory techniques, or epidemiology and clinical research methods. In the remainder of first year and in second and third years, the trainees will work under the mentorship of faculty members in one of the research areas described in the proposal. Progress in the program will be evaluated through (a) close individual supervision in the laboratory, (b) attendance of weekly research conferences and journal clubs, (c) participation at program-wide conferences and courses. UCSF provides a rich scientific environment through series of seminars, visiting professors, and conferences that create stimulating exchanges among trainees and mentors.

Project start date: 2000-09-15

Project end date: 2007-08-31

5T32DK007790-05 (2004): $83826

5T32DK007790-04 (2003): $26523

5T32DK007790-03 (2002): $105955

5T32DK007790-02 (2001): $108228

1T32DK007790-01 (2000): $51051

DIABETES AND ERECTILE FUNCTION

Rajvir Dahiya, Professor And Director
Northern California Institute Res & Educ
san Francisco, Ca 941211545

Grant 5R01DK055040-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZDK1

Abstract: adapted ) The long term goal of this application is to investigate the cellular and molecular mechanisms of diabetic impotence. Based on prior publications and preliminary data, we hypothesize that diabetic impotence is associated with alterations in signaling pathways of noradrenergic non-cholinergic (NANC) neurotransmission adenosine 3´, 5´ cyclic monophosphate (cAMP), guanosine 3´, 5´ cyclic monophosphate (cGMP), nitric oxide synthase (NOS), adrenoreceptor alpha, and growth factors (TGFa, TGFb1, TGFb2, TGFb3, IGF, and NGF). We will induce diabetes in Fisher rats by streptozotocin (35 mg /kg) and pursue the following experiments to test our hypothesis In Specific Aim 1, we will test the hypothesis that streptozotocin-induced diabetes will alter NANC vasodilator system in rats. Under this aim we will analyze intracavernous pressure (functional assessment) of penile erection in streptozotocin-induced diabetic (2, 4, 6, 8 weeks) rats. Immunohistochemical examination (NADPH diaphorase and NOS antibodies staining) of NOS-containing neurons and nerve fibers of the following sites major pelvic ganglion and intra-penile nerves (dorsal, cavernous and spongiosal) and endothelium in early and late diabetic rats. In Specific Aim 2, we will test the hypothesis that the cellular and molecular mechanisms of impotence associated with streptozotocin-induced diabetes are due to altered gene and protein expression of NOS, transforming growth factor (TGF), nerve growth factor (NGF), insulin like growth factor (IGF), and adrenoreceptor in rats. Under this specific aim, we will analyze NOS activity by enzymatic assay, NOS gene expression by Norther blot, cAMP, cGMP by radioimmunoassay in diabetic rats. The protein and gene expression for adrenoreceptor, NOS, TGFa, TGFb, NGF, and IGF-I will be done by immunohistochemistry/ Western blotting and Norther blot / reverse transcription/polymerase chain reaction (RT-PCR), respectively. In Specific Aim 3, we will test the hypothesis that insulin treatment can alter diabetes induced cellular and molecular mechanisms of importance in rats. Under this we will first induced diabetes in rats (streptozotocin injection/35 mg/kg i.p., for 2, 4, 6, and 8 weeks, and then treat them with insulin 915 IU/Kg wt, subcutaneous injection). We will study (1) functional assessment of penile erection, (2) immunohistochemical parameters (NADPH diaphorase and NOS antibodies staining) of NOS containing neurons and nerve fibers, and (3) protein and gene expression for adrenoreceptor, NOS, TGFa, TGFb, NGF, and IGF-I will be done by immunohistochemistry/ Western blotting and Norther blot / RT-PCR, respectively

Keywords: biological signal transduction, diabetes mellitus, impotence, neural transmission, penis erection adrenergic receptor, cyclic AMP, cyclic GMP, diabetes mellitus therapy, gene expression, insulin, insulinlike growth factor, neuroregulation, neurotrophic factor, nitric oxide synthase, nonhuman therapy evaluation, transforming growth factor, vasodilation immunocytochemistry, laboratory rat, male, northern blotting, polymerase chain reaction, streptozotocin, western blotting

Project start date: 1998-09-30

Project end date: 2003-08-31

5R01DK055040-05 (2002): $239686

5R01DK055040-04 (2001): $232705

5R01DK055040-03 (2000): $225927

1R01DK055040-01 (1998): $212958

SMOOTH MUSCLE-EPITHELIAL INTERACTIONS IN PROSTATE CANCER

Rajvir Dahiya, Professor And Director
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962

Grant 5R01CA064872-03 from National Cancer Institute, IRG: SRC

Project start date: 1994-09-30

Project end date: 1997-09-29

5R01CA064872-03 (1996): $203807

MECHANISMS OF PROSTATE DEVELOPMENT

Rajvir Dahiya, Professor And Director
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962

Grant 5R01DK047517-04 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: DDK

Project start date: 1993-09-30

Project end date: 1997-08-31

5R01DK047517-04 (1996): $209403

PARACRINE MECHANISMS IN HUMAN BENIGN PROSTATIC GROWTH.

Rajvir Dahiya, Professor And Director
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962

Grant 5R01DK045861-03 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: REN

Project start date: 1994-09-30

Project end date: 1998-08-31

5R01DK045861-03 (1996): $186214