Castrated Male Mice Research Articles

Abstract 1709: Activation of androgen signaling axis by NF-kappaB2/p52

Abstract INTRODUCTION: Castration-resistant progression of prostate cancer occurs in >80% of patients after failure of hormonal therapy but the underlying mechanisms are incompletely understood. Emerging evidence implies that ligand-independent activation of the androgen receptor (AR) is of critical importance during this process. Our previous studies show that the levels of NF-κB2/p52 are elevated in prostate cancer cells and that active NF-κB2/p52 promotes prostate cancer cell growth in vitro and in vivo. In this study, we examined the role of NF-κB2/p52 in the aberrant activation of the AR during castration resistant progression, in contrast to the interaction between the AR and RelA/p65. METHODS: The effects of NF-κB2/p52 on cell survival, growth and androgen responsiveness were examined in prostate cancer cells. Modulation of LNCaP tumor growth by p52 was examined in intact and castrated male nude mice. Effect of p52 or p65 on AR activation was examined using luciferase assays, EMSA, ChIP assays and ELISA. RESULTS: Expression of NF-κB2/p52 enhanced androgen-sensitive LNCaP human CaP cell growth and clonogenic ability in androgen-deprived conditions in vitro. LNCaP cells expressing p52 exhibited protection from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. Adenoviral mediated p52 expression in LNCaP cells induced tumor growth in castrated male nude mice. NF-κB/p52 induced the activation of the AR resulting in increased transactivation of AR-responsive genes such as PSA and NKX3.1 in a ligand-independent manner. NF-κB2/p52 enhanced nuclear translocation and transactivation of AR by interacting with its N-terminal domain and enhanced the recruitment of co-activators like p300 to the promoters of AR-dependent genes. These results were confirmed in 3 different prostate cancer cell lines: LAPC-4 (wild type AR), LNCaP (mutant AR) and C4-2 (androgen-insensitive). Transfection of p52 into LAPC-4 and LNCaP cells (which express low levels of p52) showed increased activation of the endogenous AR. Downregulation of endogenous p52 in C4-2 cells resulted in abrogation of AR constitutive activation. In contrast, we observed that p65 exhibited transcriptional repression of the AR, and did not induce expression of AR-responsive genes, PSA and NKX3.1. CONCLUSIONS: These findings demonstrate that overexpression of NF-κB2/p52 induces castration-resistant growth and aberrant activation of the AR in LNCaP cells. NF-κB2/p52 may promote castration resistant progression by activating the AR under conditions of androgen deprivation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1709.

Abstract 2105: miR-125b promotes the tumor growth of prostate cancer cells in intact and castrated male nude mice

Abstract Non-coding microRNAs (miRNAs) negatively regulate protein-coding genes, and aberrant expression of certain miRNAs has been reported to play a key role in the pathogenesis of human cancer including prostate cancer. Our previous studies have shown that miR-125b was highly expressed in a majority of clinical prostate cancer tissues we examined and ectopic expression of miR-125b stimulated the in vitro growth of LNCaP cells in the absence of androgens. In this study, we performed in vivo experiments to further determine whether this miRNA promotes prostatic xenograft growth. To this end, a miR-125b lentiviral vector (lenti-miR-125b) that expresses a ∼500-base pre-miR-125b was used to infect androgen-dependent PC-346C prostate cancer cells that have a limited ability to form tumors in intact nude mice and fail to grow in castrated nude mice. Quantitative RT-PCR exhibited a 39-fold increase in miR-125b level in lenti-miR-125b infected cells compared to that in lenti-vector control cells. The effect of miR-125b on xenograft tumor growth was then evaluated in intact and castrated male nude mice. After inoculation with lenti-miR-125b cells or control cells, tumors formed in all intact mice. However, the palpable lenti-miR-125b tumors appeared in approximately 7 days, while the vector control tumors took 15-20 days. The volume of xenograft tumor was markedly enlarged in all lenti-miR-125b mice compared to that in control mice. Surgery castration was performed when the tumor volume reached ∼0.5 cm3. Castratation resulted in temporary growth regression of miR-125b tumors followed by rapid growth of these tumors. To understand the molecular mechanism by which miR-125b promotes tumor growth in intact and castrated nude mice, xenograft tumors were examined by Western blot analyses of the expression of p53, Puma and Bak1 that have been validated to be the direct targets of miR-125b. Marked reduction of these three pro-apoptotic molecules was observed in miR-125b tumors compared to that in control tumors. Our data indicate that miR-125b acts as an oncogenic molecule, and overexpression of miR-125b stimulates the in vivo growth of prostate cancer cells as well as promotes the progression of prostate cancer cells to an androgen-independent status. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2105.

Abstract 1583: Plumbagin, a medicinal plant-derived naphthoquinone with both chemopreventive and therapeutic properties, selectively inhibits expression of Protein Kinase Cε, a predictive biomarker of prostate cancer progression

Abstract PKC is a major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). PKC represents a large family of phosphatidylserine-dependent serine/threonine kinases. Protein kinase Cε (PKCε) is a member of the novel PKC sub-family. PKCε is a transforming oncogene and a predictive biomarker of various human cancers, including prostate cancer (PCa). We have shown that PKCε expression level correlates with human PCa aggressiveness. PKCε overexpression is sufficient to transform androgen-dependent LNCaP cells into an androgen-independent variant, which rapidly initiates growth in vivo in both intact and castrated male nude mice. PKCε silencing using specific siRNA in DU145 cells inhibits Stat3-regulated gene expression (COX-2) and cell invasion. Also, PKCε deletion (even one allele) in TRAMP mice prevents development of PCa. Taken together, the evidence indicates an association of PKCε to PCa development and aggressiveness. Thus, PKCε may be a potential molecular target for prevention and treatment of PCa. To screen for PKCε inhibitors, we evaluated by Western Blot analyses 19 plant-derived chemopreventive agents and found plumbagin (PL) to be a selective inhibitor of PKCε expression. In this experiment, human prostate DU145 cells at 70% to 80% confluency were serum starved for 24 hr. Cells were treated with various natural agents: curcurmin (50μM), capsaicin (50μM), silibinin (200μM), epigallocatechin gallate (25μM), resveratrol (50μM), diallyl sulfide (50μM), lycopene (10μM), apigenin (10μM), docosahexacnoic acid (10μM), caffeic acid (25μM), genistein (10μM), tocopherol (10μM), β-carotene (10%), guggul (50μM), PL (50μM), ursodeoxycholic acid (50μM) in DMSO (final concentration, 0.1%) for 6 hr. Whole-cell lysates were prepared and used for Western blot analysis of PKC isoforms (α, βI, βII, δ, ε, γ, η, μ. ζ). Proteins, normalized to β-actin, were quantitated and only PL inhibited PKCε expression. In a more detailed dose-response study (5-20μM) with PL determined 10μM as IC50 for PKCε. Treatment of PCa cells (DU145 and CW22Rv1) with PL inhibits activation of transcription factors (AP-1, Stat3 and NF-kB), cell invasion and PCa cell xenografts in nude mice (J Biol Chem 2006, 281:17023; Cancer Res. 2008, 68:9024). PL (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone, was isolated from the roots of the Plumbago zeylanica L. (also known as Chitrak;). The roots of Plumbago zeylanica have been used in Indian medicine for more than 2,500 years for treatments of various ailments. PL is also present in black walnut and other various medicinal plants. We conclude that PL may be explored as a naturally occurring agent in the prevention and treatment of prostate cancer (Support: NIH grant CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1583.

1286 ABERRANT ACTIVATION OF THE ANDROGEN RECEPTOR BY NF-KAPPAB2/P52

You have accessJournal of UrologyProstate Cancer: Basic Research VII1 Apr 20101286 ABERRANT ACTIVATION OF THE ANDROGEN RECEPTOR BY NF-KAPPAB2/P52 Nagalakshmi Nadiminty, Wei Lou, and Allen Gao Nagalakshmi NadimintyNagalakshmi Nadiminty More articles by this author , Wei LouWei Lou More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.870AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Castration-resistant progression of prostate cancer occurs in >80% of patients after failure of hormonal therapy but the underlying mechanisms are incompletely understood. Emerging evidence implies that ligand-independent activation of the androgen receptor (AR) is of critical importance during this process. Our previous studies show that the levels of NF-κB2/p52 are elevated in prostate cancer cells and that active NF-κB2/p52 promotes prostate cancer cell growth in vitro and in vivo. In this study, we examined the role of NF-κB2/p52 in the aberrant activation of the AR during castration resistant progression, in contrast to the interaction between the AR and RelA/p65. METHODS The effects of NF-κB2/p52 on cell survival, growth and androgen responsiveness were examined in prostate cancer cells. Modulation of LNCaP tumor growth by p52 was examined in intact and castrated male nude mice. Effect of p52 or p65 on AR activation was examined using luciferase assays, EMSA, ChIP assays and ELISA. RESULTS Expression of NF-kB2/p52 enhanced androgen-sensitive LNCaP human CaP cell growth and clonogenic ability in androgen-deprived conditions in vitro. LNCaP cells expressing p52 exhibited protection from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. Adenoviral mediated p52 expression in LNCaP cells induced tumor growth in castrated male nude mice. NF-kB/p52 induced the activation of the AR resulting in increased transactivation of AR-responsive genes such as PSA and NKX3.1 in a ligand-independent manner. NF-kB2/p52 enhanced nuclear translocation and transactivation of AR by interacting with its N-terminal domain and enhanced the recruitment of co-activators like p300 to the promoters of AR-dependent genes. These results were confirmed in 3 different prostate cancer cell lines: LAPC-4 (wild type AR), LNCaP (mutant AR) and C4-2 (androgen-insensitive). Transfection of p52 into LAPC-4 and LNCaP cells (which express low levels of p52) showed increased activation of the endogenous AR. Downregulation of endogenous p52 in C4-2 cells resulted in abrogation of AR constitutive activation. In contrast, we observed that p65 exhibited transcriptional repression of the AR, and did not induce expression of AR-responsive genes, PSA and NKX3.1. CONCLUSIONS These findings demonstrate that overexpression of NF-kB2/p52 induces castration-resistant growth and aberrant activation of the AR in LNCaP cells. NF-kB2/p52 may promote castration resistant progression by activating the AR under conditions of androgen deprivation. Sacramento, CA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e498 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nagalakshmi Nadiminty More articles by this author Wei Lou More articles by this author Allen Gao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Generation of a Novel Transgenic Mouse Model for Bioluminescent Monitoring of Survivin Gene Activity in Vivo at Various Pathophysiological Processes : Survivin Expression Overlaps with Stem Cell Markers

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.

Prolonged treatment with bicalutamide induces androgen receptor overexpression and androgen hypersensitivity

Various hormone refractory prostate cancer cell models have been established with androgen depletion and have helped to clarify the mechanism for the transition into androgen-depletion independent status. However, the mechanism of bicalutamide resistance remains unclear because few cell models have been generated. We generated a bicalutamide-resistant subline, LNCaP-BC2, from LNCaP after prolonged treatment with bicalutamide. Androgen and/or bicalutamide responsiveness for proliferation and prostate-specific antigen (PSA) secretion were examined in vitro and in vivo. Testosterone and dihydrotestosterone (DHT) levels in xenografted tumors were analyzed by liquid chromatography-tandem mass spectrometry. Androgen receptor (AR) gene mutation and amplification and AR and pAR(210) expression were determined. LNCaP-BC2 did not grow in an androgen-depleted medium and proliferation was stimulated in a tenfold lower concentration of androgen than that of LNCaP. LNCaP-BC2 grew in castrated male mice, and the DHT level in grafted LNCaP-BC2 tumors was 7.7-fold lower than in LNCaP tumors. Bicalutamide stimulated LNCaP-BC2 proliferation and PSA secretion in vitro and the antitumor activity of bicalutamide against LNCaP-BC2 was weaker than that of LNCaP in vivo. Additional AR mutation and AR gene amplification were not detected in LNCaP-BC2, but AR and pAR(210) expression and PSA secretion in LNCaP-BC2 were higher than in LNCaP. Bicalutamide-resistant LNCaP-BC2 exhibited AR overexpression and hypersensitivity to low levels of androgen. Our data suggests that AR overexpression is a significant mechanism of bicalutamide resistance similar to resistance from chronic androgen depletion. In addition, pAR(210) overexpression could be a potential mechanism for hypersensitivity to low androgen in LNCaP-BC2.

Androgen regulated genes in human prostate xenografts in mice: relation to BPH and prostate cancer.

Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues.

318 EFFECT OF 6-N-PROPYL-2-THIOURACIL-INDUCED HYPOTHYROIDISM IN THE HOST MOUSE ON THE DEVELOPMENT OF BOVINE TESTIS XENOGRAFTS

In rodents, thyroid hormones inhibit Sertoli cell proliferation, promote Sertoli cell differentiation, and accelerate lumen formation in the seminiferous tubules. Conversely, transient hypothyroidism prolongs Sertoli cell proliferation, leading to increased Sertoli cell number and testicular size. In order to evaluate whether 6-N-propyl-2-thiouracil (PTU)-induced hypothyroidism in the host mouse would affect seminiferous tubule development and germ cell differentiation, and subsequently increase spermatogenesis in bovine testis xenografts, fragments (∼1 mm3) of testes from 1-wk-old Holstein calves (n = 6) were transplanted ectopically to castrated immunodeficient male mice (n = 6/donor). Mice (n = 3/donor) were treated with 0.1% (w/v) PTU in drinking water for 4 weeks or left as control. At 5 and 7 months after grafting, grafts were analyzed by morphometry and immunohistochemistry for expression of protein gene product 9.5 (PGP 9.5) as a germ cell marker, and Mullerian-inhibiting substance (MIS) and androgen receptor (AR) to assess Sertoli cell maturation. For each variable, averages of each group were compared at each collection point by t-test PTU treatment to the drinking water for 1 month suppressed thyroid hormone levels (T4) in host mice without negative systemic effects (0.3 ± 0.2 v. 4 ± 0.3 μg dL-1 at 4 weeks in treated v. control mice, respectively, P < 0.05). Spermatogenesis in recovered grafts was arrested at meiosis regardless of treatment and collection time. Graft weight was lower in treated mice than in controls (21 ± 4 v. 42 ± 5 and 24 ± 9 v. 51 ± 5 mg, at 5 and 7 months, respectively, P < 0.05). Volume density of the tubular and intertubular compartments, and seminiferous epithelium, was not affected by treatment (P > 0.05); however, treatment reduced lumen density compared to controls (9 ± 2 v. 19 ± 3 and 12 ± 1 v. 24 ± 4%) and tubular diameter (121 ± 3 v. 140 ± 7 and 144 ± 2v. 170 ± 2 (im, at 5 and 7 months, respectively (P < 0.05). Tubule length per milligram was not different at 5 months between control and treated groups (P > 0.05) but was increased at 7 months in the treated grafts (50 ± 1 v. 30 ± 1 cm, P < 0.05). Number of Sertoli cells per milligram was not affected by treatment (P > 0.05). However, Sertoli cell volume was increased in controls (440 ± 19 v. 341 ± 14 and 504 ± 6 v. 388 ± 18 μm3, at 5 and 7 months, respectively, P < 0.05). The number of germ cells per 100 Sertoli cells was not different between groups at any collection time (P > 0.05). Sertoli cells showed variable MIS expression and lack of or weak AR expression regardless of treatment and collection time, indicating an immature phenotype. In conclusion, suppression of thyroid hormone levels in host mice affects seminiferous tubule development in bovine testis xenografts, demonstrating that endocrine manipulation of the mouse host will affect xenografts in a predictable manner. However, treatment did not affect number and differentiation of germ cells. Rather, incomplete Sertoli cell maturation appears to lead to incomplete germ cell differentiation in bovine testis xenografts. Supported by USDA (2007-35203-18213).

Sexual dimorphism in the white matter of rodents

Sexual dimorphism of astrocytes and neurons is well documented in many brain and spinal cord structures. Sexual dimorphism of oligodendrocytes (Olgs) and myelin has received less attention. We recently showed that density of Olgs in corpus callosum, fornix, and spinal cord of wild-type male rodents is more densely packed than in females; myelin proteins and myelin gene expression are likewise greater in males than in female rodents. However, glial cell proliferation and cell death were two times greater in female corpus callosum. Endogenous sex hormones, specifically lack of androgens, produce an Olg female phenotype in castrated male mouse. In vitro studies using Olgs culture also showed differences between males and females Olg survival and signaling pathways in response to sexual hormones. Sexual dimorphism of white matter tracts and glia in rodents indicates the necessity for controlling gender in the experimental studies of neurodegenerative disorders. Most importantly, our studies suggest that hormones may contribute to sexual dimorphism observed in certain human diseases including multiple sclerosis.

Regulation of Human CYP2C18 and CYP2C19 in Transgenic Mice: Influence of Castration, Testosterone, and Growth Hormone

The hormonal regulation of human CYP2C18 and CYP2C19, which are expressed in a male-specific manner in liver and kidney in a mouse transgenic model, was examined. The influence of prepubertal castration in male mice and testosterone treatment of female mice was investigated, as was the effect of continuous administration of growth hormone (GH) to transgenic males. Prepubertal castration of transgenic male mice suppressed the expression of CYP2C18 and CYP2C19 in liver and kidney to female levels, whereas expression was increased for the endogenous female-specific mouse hepatic genes Cyp2c37, Cyp2c38, Cyp2c39, and Cyp2c40. Testosterone treatment of female mice increased CYP2C18 and CYP2C19 expression in kidney, and to a lesser extent in liver, but was without effect in brain or small intestine, where gene expression was not gender-dependent. Continuous GH treatment of transgenic males for 7 days suppressed hepatic expression of CYP2C19 (>90% decrease) and CYP2C18 ( approximately 50% decrease) but had minimal effect on the expression of these genes in kidney, brain, or small intestine. Under these conditions, continuous GH induced all four female-specific mouse liver Cyp2c genes in males to normal female levels. These studies indicate that the human CYP2C18 and CYP2C19 genes contain regulatory elements that respond to the endogenous mouse hormonal profiles, with androgen being the primary regulator of male-specific expression in kidney, whereas the androgen-dependent pituitary GH secretory pattern is the primary regulator of male-specific expression in liver in a manner that is similar to the regulation of the endogenous gender-specific hepatic genes.

Effects of DDE on development and progression of autoimmunity (137.36)

Abstract DDE, a metabolic breakdown product of the pesticide DDT, persists in the environment and acts as an androgen receptor antagonist. Androgen deprivation by castration of male mice causes changes in immune function and the lupus-like disease of NZB/W F1 hybrid mice is treated by androgens. Therefore, we hypothesized that the anti-androgenic effects of DDE may alter immune function, with consequences for autoimmunity. We treated both normal C57Bl/6 female mice and lupus-prone NZB/W F1 female mice with oil control or DDE, at 200 mg/kg for 3 days at the study's beginning. NZB/W mice were studied over 6 months and C57Bl/6 mice for 18 months. DDE did not alter organ or body weights. Proteinurea was increased in DDE-treated mice compared to age-matched oil-treated controls, as early as one month after treatment with DDE. By histopathology we observed glomerulonephritis sooner in kidneys of NZB/W mice treated with DDE than control. Kidneys of DDE-treated C57Bl/6 mice showed increased glomerular mesangium compared to kidneys of age-matched controls. By immunohistochemistry we saw increased glomerular expression of monocyte chemoattractant protein-1 and increased tubulointerstitial expression of osteopontin in response to DDE; upregulation of both chemokines occurs in murine lupus nephritis. We conclude that DDE impacts development and progression of autoimmune disease and continue to explore the mechanism of action.

Regulatory Role of Testosterone in Organic Cation Transport: in Vivo and in Vitro Studies

The renal proximal tubule (RPT) plays a crucial role in organic cation (OC) secretion and has a major impact on pharmacokinetics of OC drugs. Secretory transport is vectorial. Thus, it involves transporters located at both basolateral and apical membranes. Although sex hormones have been shown to regulate OC transport, there is little data on the effect of testosterone on OC secretion in a whole animal. Therefore, we determined the clearance of tetraethylammonium (TEA), a model OC substrate, in intact and castrated male mice. Castration significantly decreased renal TEA secretion by 30%, and testosterone supplementation returned TEA secretion to control levels in castrated mice. The mechanism of this effect was further examined in isolated mouse renal proximal tubules (mRPT). TEA uptake in isolated mRPT from castrated mice was reduced by 36%. This effect was reversed in tubules from castrated mice supplemented with testosterone. Kinetic analysis of [(3)H]-TEA uptake in isolated mRPT showed a decreased V(max) with no change in K(m), implying that the decrease in transport rate was caused by lowering in the number of transporters in castrated mice rather than a change in transporter affinity. Quantitative real time polymerase chain reaction (real time PCR) revealed that organic cation transporter (OCT)2 is the major TEA transporter in male mice. Moreover, OCT2 mRNA level was significantly reduced after castration. Castrated mice also showed a modest increase in organic cation/carnitine transporter 1 (OCTN1) mRNA level, indicating that testosterone may also regulate apical OCTN1 expression. These data suggest that testosterone regulates transepithelial transport of OC through modulation of OCT2 expression in male mice.

NF‐κB2/p52 enhances androgen‐independent growth of human LNCaP cells via protection from apoptotic cell death and cell cycle arrest induced by androgen‐deprivation

Androgen-deprivation therapy only causes a temporary regression of prostate cancer, as all tumors will eventually progress to refractory to hormonal therapy after 1-3 years of treatment. The underlying mechanisms of prostate cancer androgen refractory progression are incompletely understood. In this study, we employed in vitro as well as in vivo models to examine the role of NF-kappaB2/p52 in prostate cancer growth and androgen independent progression. The effects of NF-kappaB2/p52 on cell growth, androgen responsiveness, cell cycle and apoptosis were examined in androgen sensitive LNCaP cells. The effect of NF-kappaB2/p52 on tumor growth was examined in intact and castrated male mice. Overexpression of NF-kappaB2/p52 enhances androgen-sensitive LNCaP human prostate cancer cell growth and clonogenic ability in androgen-deprived condition in vitro. NF-kappaB2/p52 induced androgen-independent growth is through protecting LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. In addition, NF-kappaB2/p52 stimulates Cyclin D1 expression and knock down of Cyclin D1 expression by siRNA abolished NF-kappaB2/p52-induced cell growth in vitro. Adenoviral mediated NF-kappaB2/p52 expression in LNCaP cells enhances tumor growth in intact male nude mice and induces tumor growth in castrated male nude mice, suggesting that overexpression of NF-kappaB2/p52 induces androgen-independent growth of androgen-sensitive LNCaP cells. Overexpression of NF-kappaB2/p52 protects androgen sensitive LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. NF-kappaB2/p52 activation induces androgen-independent growth in vitro and in vivo.

Choline Autoradiography of Human Prostate Cancer Xenograft: Effect of Castration

The purpose of this study was to investigate the effects of castration and tracer uptake time interval on the level of radiolabeled choline accumulation in murine-implanted human prostate tumor xenografts using quantitative autoradiography. We implanted androgen-dependent (CWR22) and androgen-independent (PC3) human prostate cancer cells in castrated (n = 9) and noncastrated (n = 9) athymic male mice and allowed tumors to grow to 1 cm3. The mice were euthanized at 5, 10, and 20 minutes after injection of 5 microCi [14C]-choline. Mice were prepared for quantitative autoradiography with density light units of viable tumor sections converted to units of radioactivity (nCi/mm2) using calibration. Two-group comparisons were performed using a two-tailed Student t-test with unequal variance and with a significance probability level of less than .05. Two-group comparisons between the means of the tracer uptake level for each tumor type at each of three time points for each of two host types showed that (1) the level of tracer localization in the two tumor types was affected little in relation to the host type and (2) PC3 tumor uptake level tended to increase slowly with time only in the noncastrated host, whereas this was not observed in the castrated host or with CWR22 tumor in either host type. The uptake time interval and castration do not appear to significantly affect the level of radiolabeled choline uptake by the human prostate cancer xenograft.

Androgen Increases AT1a Receptor Expression in Abdominal Aortas to Promote Angiotensin II–Induced AAAs in Apolipoprotein E–Deficient Mice

Castration of male apolipoprotein E-deficient (apoE-/-) mice reduces angiotensin II (Ang II)-induced abdominal aorta aneurysms (AAAs) to that of female mice. The purpose of this study was to determine whether this reduction is attributable to androgen-mediated regulation of aortic Ang II type 1A receptors (AT1aR). AT1aR mRNA abundance in the AAA-prone region of abdominal aortas was 8-fold greater compared to thoracic aortas of male but not female mice. AT1aR mRNA abundance decreased after castration in abdominal but not thoracic aortas of male mice. Dihydrotestosterone (DHT, 0.16 mg/d) administration to castrated male mice restored AT1aR mRNA abundance in abdominal aortas but had no effect in thoracic aortas. DHT also increased AT1aR mRNA abundance in abdominal aortas from female mice. Castrated male or female apoE-/- mice were administered DHT during infusion of saline or Ang II (1000 ng/kg/min for 28 days). DHT administration did not alter serum cholesterol concentrations, lipoprotein distributions, or atherosclerotic lesion areas in either male or female mice. However, administration of DHT increased AAA incidence in male (27% placebo versus 75% DHT) and female mice (28% placebo versus 64% DHT). Androgen promotes AT1aR mRNA abundance in abdominal aortas associated with increased Ang II-induced AAAs.

Interleukin‐4 stimulates androgen‐independent growth in LNCaP human prostate cancer cells

Clinical data showed that the levels of interleukin-4 (IL-4) are significantly elevated in serum of patients with ablation resistant prostate cancer. Previous studies demonstrated that IL-4 enhances androgen receptor (AR) activation mediated by NF-kappaB in the absence or in the very low levels of androgen in prostate cancer cells. In this study, the role of IL-4 in promoting the growth of androgen-independent prostate cancer cells was examined. LNCaP cells were transfected with a full-length IL-4 cDNA and stable clones expressing IL-4 were selected. The growth of LNCaP cells expressing IL-4 was analyzed in vitro and in vivo both in the presence and absence of androgen. Overexpression of IL-4 enhances the growth of androgen-sensitive LNCaP cells in culture media containing charcoal-stripped FBS condition (CS-FBC), and increases the sensitivity of LNCaP cells in response to androgen stimulation. The DHT-mediated cell growth could not be blocked by bicalutamide in IL-4 overexpressing LNCaP cells, but can be neutralized by bicalutamide in parental LNCaP and neo control cells. Furthermore, overexpression of IL-4 stimulates tumor growth of androgen-sensitive LNCaP cells both in intact and castrated male mice. Overexpression of IL-4 increases the sensitivity of androgen-sensitive LNCaP prostate cancer cells in response to androgen stimulation and enhances the growth of LNCaP cells both in the presence and absence of androgen in vitro and in vivo. These studies suggest that IL-4 plays an important role in promoting androgen-independent prostate cancer growth.

Development of an androgen‐deprivation induced and androgen suppressed human prostate cancer cell line

Emergence of recurrent cells during androgen-deprivation therapy and intermittent androgen suppression therapy suggest that a subset of prostate cancer cells survive and proliferate in the androgen deprivation condition. Some of the recurrent cells that emerge during the androgen-deprivation therapy and intermittent androgen suppression therapy could be suppressed by replacement of androgen. In an attempt to recapitulate the clinical phenomenon, we developed an androgen-deprivation induced and androgen suppressed human prostate cancer cell line. LNCS, an androgen-deprivation induced and androgen suppressed human prostate cancer cell line, was generated from an androgen-sensitive LNCaP cells cultured in PRMI-1640 media containing charcoal-stripped FBS for a prolong period for more than a year. The responsiveness to androgen in vitro and in vivo was examined. The characteristics of this subline including activation of signaling pathways were investigated. LNCS, an androgen-deprivation induced and androgen suppressed human prostate cancer cell line, was developed. LNCS cells express considerably lower levels of androgen receptor than LNCaP cells and grow vigorously in androgen deprived condition in vitro, and develop tumors in castrated male mice in vivo. Addition of androgen inhibits cell growth in vitro and tumor growth in vivo. LNCS cells are more resistant to etoposide, a typical apoptotic inducing agent. Although AR signaling is less active in LNCS cells, Stat3 is constitutively activated. Down regulation of Stat3 activation inhibits LNCS cell growth in vitro. We have developed an androgen-deprivation induced and androgen suppressed human prostate cancer cell line. This cell line is a valuable tool to study molecular mechanisms of the progression of androgen refractory prostate cancer and intermittent androgen suppression therapy.

TESTIS TISSUE ALLOGRAFTING REVEALS A PHENOTYPE OF MEIOTIC ARREST IN NEONATALLY LETHAL LSH KNOCK OUT MICE

Lymphoid specific helicase (Lsh) is a member of the SNF2-helicase family of chromatin remodeling proteins. Lsh is ubiquitously expressed and essential in embryonic development. We demonstrated recently that Lsh plays a critical role in female meiosis. Because Lsh null mice die at birth, a potential phenotype in male meiosis cannot be studied in these animals. Grafting of tissue from mutant mice to immuno-tolerant wild type recipients allows the study of postnatal tissue differentiation even in animals with a neonatal lethal phenotype. Therefore, we employed allografting of testis tissue from Lsh -/- mice to immuno-deficient wild type host mice to enable the study of postnatal germ cell development and differentiation. Testes were harvested at day 18.5 of gestation from Lsh -/-, +/- and +/+ mice of 24 litters and grafted ectopically under the back skin of 30 immuno-deficient, castrated male recipient mice (3-6 grafts/mouse). Each mouse received at least one graft from each genotype. To compare proliferative activity of germ cells between Lsh +/+ and -/- testis tissue, 3 recipient mice were treated with BrdU for 1 h before grafts were harvested at 1 wk post grafting. Double immunocytochemistry for BrdU incorporation and a germ cell marker (UCHL1) revealed significantly fewer dividing germ cells in tissue grafts from Lsh -/- mice than in grafts from Lsh +/+ animals (22.4 + 3.8 % vs 39.2 + 11.8 %, respectively, P<0.05). Grafted tissue from the remaining mice was harvested at 4, 6 and 8 weeks after grafting (n= 6, 10 and 11 mice, respectively) and analyzed histologically for spermatogenic differentiation. While spermatogenesis proceeded through meiosis with the appearance of round and elongated spermatids in the seminiferous tubules in grafts from Lsh heterozygote and wild type donor testes, pachytene spermatocytes were the most advanced germ cell type present in grafts from Lsh -/- mice at all analysis time points. These results indicate that proliferation of spermatogonia is reduced and that germ cell differentiation is arrested in the absence of Lsh, indicating an essential role for Lsh also during male meiosis. This study also highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity. Meiotic germ cells harvested from testis tissue allografts can now be studied to define the mechanism underlying the observed arrest of spermatogenesis and to further characterize the role of Lsh in meiotic chromatin remodeling. Supported by RR17359 (NCRR/NIH) and by NCI contract # N01-C0-12400. (platform)

Raloxifen prevents bone loss in castrated male mice

Raloxifen is a selective estrogen receptor modulator which prevents bone loss in ovariectomized female mice in a fashion similar to estrogens. Since testosterone-deficient male mice also lose bone mass, we were interested in testing the effects of raloxifen on bones in intact and castrated male mice. Bone density was significantly reduced in castrated animals (1.36+/-0.04 g/ml) as compared to intact animals (1.42+/-0.03 g/ml) (p<0.01). When castrated mice with extraordinarily low concentrations of testosterone and with reduced weight of seminal vesicles were treated with raloxifen, the changes in bone density and bone minerals resulting from castration (1.36+/-0.04 g/ml) were entirely prevented (1.40+/-0.01 g/ml). Cortical bone was lost in orchidectomized mice, and this decrease in cortical thickness of the femur was prevented by raloxifen administration. Raloxifen in a dose used in humans for treatment of osteoporosis decreased the weight of seminal vesicles, an organ which is highly sensitive to the androgenic effect, decreased the concentration of testosterone (12.5+/-2.8 micromol/l) (p<0.01) but not to the same level as in the case of castrated animals (0.6+/-0.3 micromol/l), and did not have any effect on bone density or mineral content in intact mice. The results of the present study may thus be interpreted as supporting the hypothesis that raloxifen is an effective agent against the deleterious effects of castration-induced osteopenia in male mice and also support the hypothesis that estrogens may have physiological skeletal effects in male mice.

Male sex hormones promote vagally mediated reflex airway responsiveness to cholinergic stimulation

A sex disparity in airway responsiveness to cholinergic stimulation has been observed in laboratory mice in that males are considerably more responsive than females, but the basis for this difference is unclear. In this report, we demonstrate that male sex hormones promote murine airway responsiveness to cholinergic stimulation via vagus nerve-mediated reflex mechanisms. In tissue bath preparations, no sex-based differences were observed in the contractile responses of isolated tracheal and bronchial ring segments to carbachol, indicating that the mechanism(s) responsible for the in vivo sex difference is (are) absent ex vivo. Bilateral cervical vagotomy was found to abolish in vivo airway responsiveness to methacholine in male mice, whereas it did not alter the responses of females, suggesting a regulatory role for male sex hormones in promoting reflex airway constriction. To test this possibility, we next studied mice with altered circulating male sex hormone levels. Castrated male mice displayed airway responsiveness equivalent to that observed in intact females, whereas administration of exogenous testosterone to castrated males restored responsiveness, albeit not to the level observed in intact males. Administration of exogenous testosterone to intact female mice similarly enhanced responsiveness. Importantly, the promotive effects of exogenous testosterone in castrated male and intact female mice were absent when bilateral vagotomy was performed. Together, these data indicate that male sex hormones promote cholinergic airway responsiveness via a vagally mediated reflex mechanism that may be important in the regulation of airway tone in the normal and diseased lung.