Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine with many components of ubiquitin-proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered degradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP) chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whether increasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with mice overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor function of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the large complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the enhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for the development of an HSP70-related therapy for SBMA and other polyQ diseases.

TRP1ARSARSARSARS sequences, and are stable at low and high levels of digestion. The TRP1 gene region is covered by four nucleosomes, but they are neither randomly arranged nor precisely positioned. They are not stable and rearrange or disintegrate during digestion. The nucleosomal regions are separated by two segments of DNA (A, B), each about 180 base-pairs long, which are very sensitive to DNAase I and micrococcal nuclease and therefore presumably not packed in nucleosomes. Region B is found 5' to the TRP1 gene and might be related to transcription, whereas region A is centered around the termination codon of the TRP1 gene and the putative origin of replication.

Alterations in DNA repair promote tumor development, but the impact on tumor progression is poorly understood. Here, discovery of a biochemical circuit linking hormone signaling to DNA repair and therapeutic resistance is reported. Findings show that androgen receptor (AR) activity is induced by DNA damage and promotes expression and activation of a gene expression program governing DNA repair. Subsequent investigation revealed that activated AR promotes resolution of double-strand breaks and resistance to DNA damage both in vitro and in vivo. Mechanistically, DNA-dependent protein kinase catalytic subunit (DNAPKcs) was identified as a key target of AR after damage, controlling AR-mediated DNA repair and cell survival after genotoxic insult. Finally, DNAPKcs was shown to potentiate AR function, consistent with a dual role in both DNA repair and transcriptional regulation. Combined, these studies identify the AR-DNAPKcs circuit as a major effector of DNA repair and therapeutic resistance and establish a new node for therapeutic intervention in advanced disease.

CONCLUSIONS

The present study identifies for the fi rst time a positive feedback circuit linking hormone action to the DNA damage response and shows the significant impact of this process on tumor progression and therapeutic response. These provocative findings provide the foundation for development of novel nodes of therapeutic intervention for advanced disease.

DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important antiinflammatory molecule in tissue inflammation, and adenosine 2A receptor (A₂AR) agonists protect kidneys from IRI through their actions on leukocytes. In this study, we showed that mice with A₂AR-deficient DCs are more susceptible to kidney IRI and are not protected from injury by A₂AR agonists. In addition, administration of DCs treated ex vivo with an A₂AR agonist protected the kidneys of WT mice from IRI by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. A₂AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A₂AR-induced tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to attenuate organ IRI.

Sex differences in neural development are established via a number of cellular processes (i.e., migration, death and survival). One critical factor identified is the neonatal rise in testosterone (T) which activates gene transcription via androgen (AR) and, after aromatization to estradiol, estrogen receptors (ERalpha and beta). Recent evidence shows that AR and ERs interact with histone modifying enzymes. Post-translational modifications of histones, including acetylation and methylation, are involved in transcriptional regulation during normal development. Therefore, we hypothesized that acetylation and/or methylation of histone H3 may underlie sexual differentiation, at least in some regions of the brain. We measured levels of acetylated (H3K9/14Ac) and trimethylated (H3K9Me3) H3 in whole neonatal mouse brains and in three regions: preoptic area + hypothalamus, amygdala and cortex + hippocampus (CTX/HIP). Sex differences in H3K9/14Ac and H3K9Me3 (males>> females) were noted in the CTX/HIP on embryonic day 18, the day of birth, and six days later. To determine if T mediates these changes in H3 modifications, pregnant dams received vehicle or T for the final four days of gestation; pup brains were collected at birth. Methylation of H3 was sexually dimorphic despite hormone treatment. In contrast, H3 acetylation in the CTX/HIP of females from T-treated dams rose to levels equivalent to males. Thus, H3 modifications are sexually dimorphic in the developing mouse CTX/HIP and acetylation, but not methylation, is masculinized in females by T in utero. This is the first demonstration that histone modification is associated with neural sexual differentiation.

A cure for prostate cancer (CaP) will be possible only after a complete understanding of the mechanisms causing this disease to progress from androgen dependence to androgen independence. To carry on a careful characterization of the phenotypes of CaP cell lines before and after acquisition of androgen independence, we used two human CaP LNCaP sublines: LNCaP(nan), which is androgen dependent (AD), and LNCaP-HP, which is androgen independent (AI). In AD LNCaP(nan) cells, dihydrotestosterone (DHT) stimulated in an androgen receptor (AR)-dependent way a phosphorylation signaling pathway involving steroid receptor coactivator (Src)-mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2-ERK-1/2-cAMP-response element binding-protein (CREB). Activation of this pathway was associated with increased [(3)H]thymidine incorporation and resistance to apoptosis. Use of dominant-negative forms of MEK-1/2 and CREB demonstrated in LNCaP(nan) cells that DHT induced [(3)H]thymidiine incorporation through a thus far unidentified molecule activated downstream of MEK-1/2, and antiapoptosis through phosphorylation of the transcription factor CREB. In contrast, in AI LNCaP-HP cells, the Src-MEK-1/2-ERK-1/2-CREB pathway was constitutively active. Because it was not further stimulated by addition of DHT, no increase of [(3)H]thymidine incorporation or apoptosis resistance was demonstrated in LNCaP-HP cells. Additional experiments showed that Src and the scaffold protein MNAR coimmunoprecipitated with AR, indicating a role for Src as an apical molecule in the Src-MEK-1/2-ERK-1/2-CREB pathway. Interestingly, differences between the two cell lines were that in LNCaP-HP cells presence of an AI phenotype and lack of response to DHT were associated with constitutive activation of the protein kinase Src and interaction among Src, AR, and MNAR. In contrast, in LNCaP(nan) cells, presence of an AD phenotype and ability to respond to DHT were associated with DHT-dependent activation of Src kinase activity and interaction among Src, AR, and MNAR. Intriguingly, in LNCaP(nan) cells, we found that transcription through the prototypical CREB-responsive promoter c-fos could be induced in a DHT-dependent way, and this action was inhibited by the AR antagonist Casodex and MEK-1 inhibitor PD98059. In contrast, transcription through the PSA P/E promoter, a prototypical AR-dependent promoter directly activated by agonist, was obliterated only by Casodex. Additional experiments with genital skin fibroblasts derived from patients with a variety of AR abnormalities indicated that nongenotropic AR signaling does not depend on an intact DNA-binding domain or on the ability of AR to translocate to the nucleus. The results suggest the following: (1) Constitutive activation of the Src-MEK-1/2-ERK-1/2-CREB pathway is associated with the AI phenotype observed in LNCaP-HP cells. (2) Activation of the Src-MEK-1/2-ERK-1/2-CREB pathway is DHT dependent in AD LNCaP(nan) cells. (3) DHT activation of this pathway is associated with induction of [(3)H]thymidine incorporation by a molecule activated downstream of MEK-1/2 and of antiapoptosis through activation of the transcription factor CREB in AD LNCaP(nan) cells. (4) AR regulates transcription either directly upon ligand binding and nuclear translocation or indirectly through kinase pathways leading to activation of downstream transcription factors. (5) Nuclear translocation and ability of the DNA-binding domain of AR to interact with DNA are not prerequisites for nongenotropic AR activity.

Although aminoacyl-tRNA synthetases (ARSs) are housekeeping enzymes essential for protein synthesis, they can play non-catalytic roles in diverse biological processes. Some ARSs are capable of forming complexes with each other and additional proteins. This characteristic is most pronounced in mammals, which produce a macromolecular complex comprising nine different ARSs and three additional factors: p43, p38 and p18. We have been aware of the existence of this complex for a long time, but its structure and function have not been well understood. The only apparent distinction between the complex-forming ARSs and those that do not form complexes is their ability to interact with the three non-enzymatic factors. These factors are required not only for the catalytic activity and stability of the associated ARSs, such as isoleucyl-, methionyl-, and arginyl-tRNA synthetase, but also for diverse signal transduction pathways. They may thus have joined the ARS community to coordinate protein synthesis with other biological processes.

Mal de Meleda is an autosomal recessive inflammatory and keratotic palmoplantar skin disorder due to mutations in the ARS B gene, encoding for SLURP-1 (secreted mammalian Ly-6/uPAR-related protein 1). SLURP-1 belongs to the Ly-6/uPAR superfamily of receptor and secreted proteins, which participate in signal transduction, immune cell activation or cellular adhesion. The high degree of structural similarity between SLURP-1 and the three fingers motif of snake neurotoxins and Lynx1 suggests that this protein interacts with the neuronal acetylcholine receptors. We found that SLURP-1 potentiates the human alpha 7 nicotinic acetylcholine receptors that are present in keratinocytes. These results identify SLURP-1 as a secreted epidermal neuromodulator which is likely to be essential for both epidermal homeostasis and inhibition of TNF-alpha release by macrophages during wound healing. This explains both the hyperproliferative as well as the inflammatory clinical phenotype of Mal de Meleda.

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease caused by the expansion of a trinucleotide CAG repeat encoding the polyglutamine tract in the first exon of the androgen receptor gene (AR). The pathogenic, polyglutamine-expanded AR protein accumulates in the cell nucleus in a ligand-dependent manner and inhibits transcription by interfering with transcriptional factors and coactivators. Heat-shock proteins (HSPs) are stress-induced chaperones that facilitate the refolding and, thus, the degradation of abnormal proteins. Geranylgeranylacetone (GGA), a nontoxic antiulcer drug, has been shown to potently induce HSP expression in various tissues, including the central nervous system. In a cell model of SBMA, GGA increased the levels of Hsp70, Hsp90, and Hsp105 and inhibited cell death and the accumulation of pathogenic AR. Oral administration of GGA also up-regulated the expression of HSPs in the central nervous system of SBMA-transgenic mice and suppressed nuclear accumulation of the pathogenic AR protein, resulting in amelioration of polyglutamine-dependent neuromuscular phenotypes. These observations suggest that, although a high dose appears to be needed for clinical effects, oral GGA administration is a safe and promising therapeutic candidate for polyglutamine-mediated neurodegenerative diseases, including SBMA.

Deletion or mutation of the androgen receptor (AR) renders prostate tumors refractory to apoptosis by androgen ablation, the mainstay of prostate cancer therapy. To identify novel therapeutics that can induce apoptosis regardless of the AR status of prostate cancer cells, we screened dietary herbal compounds using a reporter assay for the prostate apoptosis response-4 (Par-4) gene, which induces p53- and PTEN-independent and cancer-selective apoptosis. One of the compounds, withaferin A (WA), a major constituent of the dietary compound Withania somnifera, induced Par-4-dependent apoptosis in androgen-refractory prostate cancer cells and regression of PC-3 xenografts in nude mice. Interestingly, restoration of wild-type AR in PC-3 (AR negative) cells abrogated both Par-4 induction and apoptosis by WA. Individually, WA and anti-androgens induced neither Par-4 nor apoptosis in androgen-responsive prostate cancer cells, yet in combination, WA and anti-androgen synergistically induced Par-4 and apoptosis in androgen-responsive prostate cancer cells. Thus, when judiciously combined with anti-androgens, WA inhibits survival of both androgen-responsive and androgen-refractory prostate cancer cells by a Par-4-dependent mechanism. As Par-4 up-regulation induces apoptosis in most tumor cells, our findings can be extended to high-throughput screens to identify synergistic combinations for both therapy-sensitive and therapy-resistant cancers.

PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.

Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y' telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques. Replication origins (ARSs) at internal Y' elements were found to fire in early-mid-S phase, while ARSs at the terminal Y' elements were confirmed to fire late. An unfired Y' ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase. The pause at telomeric sequence TG(1-3) repeats was stronger at the terminal tract than at the internal TG(1-3) sequences located between tandem Y' elements. We show that the telomeric replication fork pause associated with the terminal TG(1-3) tracts begins approximately 100 bp upstream of the telomeric repeat tract sequence. Telomeric pause strength was dependent upon telomere length per se and did not require the presence of a variety of factors implicated in telomere metabolism and/or known to cause telomere shortening. The telomeric replication fork pause was specific to yeast telomeric sequence and was independent of the Sir and Rif proteins, major known components of yeast telomeric heterochromatin.

We hypothesize the existence of an inherited predisposition for a spectrum of behaviors and traits characterized by behavioral disinhibition. This externalizing spectrum includes childhood disruptive disorders, antisocial behavior, substance use disorders, personality traits related to behavioral undercontrol, and the precocious expression of problem behavior. We further hypothesize that a genetically influenced central nervous system diathesis underlies this spectrum and is reflected in reduced P300 amplitude in a visual oddball event-related potential task. A review of evidence bearing on the model is derived from findings from the Minnesota Twin Family Study, a population-based, longitudinal investigation of twin youth. These findings indicate that the collection of attributes related to behavioral disinhibition is familial, heritable, and interrelated. Evidence supporting P3 amplitude reduction (P3-AR) as an index of genetic vulnerability for this externalizing spectrum includes its association with (a) familial risk for substance use and antisocial personality disorders, (b) diagnoses of childhood disruptive disorders and substance use disorders, (c) early onset of undersocialized behavior, and (d) quantitative phenotypes related to externalizing problems. In addition, the development of substance use disorders over a 3-year period is associated with P3-AR measured prior to their expression. These findings suggest that P3-AR indexes one aspect of the genetic diathesis for a spectrum of externalizing problem behavior.

The NF-kappaB family of transcription factors has been shown to be constitutively activated in various human malignancies, including leukemias, lymphomas, and a number of solid tumors. NF-kappaB is hypothesized to contribute to development and/or progression of malignancy by regulating the expression of genes involved in cell growth and proliferation, anti-apoptosis, angiogenesis, and metastasis. Prostate cancer cells have been reported to have constitutive NF-kappaB activity due to increased activity of the IkappaB kinase complex. Furthermore, an inverse correlation between androgen receptor (AR) status and NF-kappaB activity was observed in prostate cancer cell lines. NF-kappaB may promote cell growth and proliferation in prostate cancer cells by regulating expression of genes such as c-myc, cyclin D1, and IL-6. NF-kappaB may also inhibit apoptosis in prostate cancer cells through activation of expression of anti-apoptotic genes, such as Bcl-2, although pro-apoptotic activity of NF-kappaB has also been reported. NF-kappaB-mediated expression of genes involved in angiogenesis (IL-8, VEGF), and invasion and metastasis (MMP9, uPA, uPA receptor) may further contribute to the progression of prostate cancer. Constitutive NF-kappaB activity has also been demonstrated in primary prostate cancer tissue samples and suggested to have prognostic importance for a subset of primary tumors. The limited number of samples analyzed in those studies and the relative lack of NF-kappaB target genes identified in RNA expression microarray analyses of prostate cancer cells suggest that further studies will be required in order to determine if NF-kappaB actually plays a role in human prostate cancer development, and/or progression, and to characterize its potential as a therapeutic target.

A 47-kilodalton neutrophil cytosol factor (NCF-47k), required for activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase superoxide (O2-.) production, is absent in most patients with autosomal recessive chronic granulomatous disease (AR-CGD). NCF-47k cDNAs were cloned from an expression library. The largest clone predicted a 41.9-kD protein that contained an arginine and serine-rich COOH-terminal domain with potential protein kinase C phosphorylation sites. A 33-amino acid segment of NCF-47k shared 49% identity with ras p21 guanosine triphosphatase activating protein. Recombinant NCF-47k restored O2-. -producing activity to AR-CGD neutrophil cytosol in a cell-free assay. Production of active recombinant NCF-47k will enable functional regions of this molecule to be mapped.

BACKGROUND

Prostate stem/progenitor cells function in glandular development and maintenance. They may be targets for tumor initiation, so characterization of these cells may have therapeutic implications. Cells from dissociated tissues that form spheres in vitro often represent stem/progenitor cells. A subset of human prostate cells that form prostaspheres were evaluated for self-renewal and tissue regeneration capability in the present study.

METHODS

Prostaspheres were generated from 59 prostatectomy specimens. Lineage marker expression and TMPRSS-ERG status was determined via immunohistochemistry and fluorescence in situ hybridization (FISH). Subpopulations of prostate epithelial cells were isolated by cell sorting and interrogated for sphere-forming activity. Tissue regeneration potential was assessed by combining sphere-forming cells with rat urogenital sinus mesenchyme (rUGSM) subcutaneously in immunocompromised mice.

RESULTS

Prostate tissue specimens were heterogeneous, containing both benign and malignant (Gleason 3-5) glands. TMPRSS-ERG fusion was found in approximately 70% of cancers examined. Prostaspheres developed from single cells at a variable rate (0.5-4%) and could be serially passaged. A basal phenotype (CD44+CD49f+CK5+p63+CK8-AR-PSA-) was observed among sphere-forming cells. Subpopulations of prostate cells expressing tumor-associated calcium signal transducer 2 (Trop2), CD44, and CD49f preferentially formed spheres. In vivo implantation of sphere-forming cells and rUGSM regenerated tubular structures containing discreet basal and luminal layers. The TMPRSS-ERG fusion was absent in prostaspheres derived from fusion-positive tumor tissue, suggesting a survival/growth advantage of benign prostate epithelial cells.

CONCLUSIONS

Human prostate sphere-forming cells self-renew, have tissue regeneration capability, and represent a subpopulation of basal cells.

An age-dependent association between variation at the FTO locus and BMI in children has been suggested. We meta-analyzed associations between the FTO locus (rs9939609) and BMI in samples, aged from early infancy to 13 years, from 8 cohorts of European ancestry. We found a positive association between additional minor (A) alleles and BMI from 5.5 years onwards, but an inverse association below age 2.5 years. Modelling median BMI curves for each genotype using the LMS method, we found that carriers of minor alleles showed lower BMI in infancy, earlier adiposity rebound (AR), and higher BMI later in childhood. Differences by allele were consistent with two independent processes: earlier AR equivalent to accelerating developmental age by 2.37% (95% CI 1.87, 2.87, p = 10(-20)) per A allele and a positive age by genotype interaction such that BMI increased faster with age (p = 10(-23)). We also fitted a linear mixed effects model to relate genotype to the BMI curve inflection points adiposity peak (AP) in infancy and AR. Carriage of two minor alleles at rs9939609 was associated with lower BMI at AP (-0.40% (95% CI: -0.74, -0.06), p = 0.02), higher BMI at AR (0.93% (95% CI: 0.22, 1.64), p = 0.01), and earlier AR (-4.72% (-5.81, -3.63), p = 10(-17)), supporting cross-sectional results. Overall, we confirm the expected association between variation at rs9939609 and BMI in childhood, but only after an inverse association between the same variant and BMI in infancy. Patterns are consistent with a shift on the developmental scale, which is reflected in association with the timing of AR rather than just a global increase in BMI. Results provide important information about longitudinal gene effects and about the role of FTO in adiposity. The associated shifts in developmental timing have clinical importance with respect to known relationships between AR and both later-life BMI and metabolic disease risk.

Modification by acetylation occurs at epsilon-amino lysine residues of histones and transcription factors. Unlike phosphorylation, a direct link between transcription factor acetylation and cellular growth or apoptosis has not been established. We show that the nuclear androgen receptor (AR), a DNA-binding transcriptional regulator, is acetylated in vivo. The acetylation of the AR is induced by ligand dihydrotestosterone and by histone deacetylase (HDAC) inhibitors in living cells. Direct AR acetylation augmented p300 binding in vitro. Constructs mimicking neutral polar substitution acetylation (AR(K630Q), AR(K630T)) enhanced p300 binding and reduced N-CoR/HDAC/Smad3 corepressor binding, whereas charged residue substitution (AR(K630R)) reduced p300 binding and enhanced corepressor binding. The AR acetylation mimics promoted cell survival and growth of prostate cancer cells in soft agar and in nude mice and augmented transcription of a subset of growth control target gene promoters. Thus, transcription factor acetylation regulates coactivator/corepressor complex binding, altering expression of specific growth control genes to promote aberrant cellular growth in vivo.

Interleukin-6 (IL-6) is involved in regulation of immune reaction and cell growth and differentiation. It causes multifunctional responses ranging from inhibition of proliferation to promotion of cell survival. IL-6 effects may depend on experimental conditions such as passage numbers and serum composition. IL-6 signals in target tissues through the receptor that is composed of the ligand-binding and signal-transducing subunits. IL-6 is expressed in benign and malignant prostate tissue and the levels of the cytokine and its receptor increase during prostate carcinogenesis. IL-6 is considered a positive growth factor for most prostate cells. The only exemption seems to be the LNCaP cell line, in which IL-6 causes growth arrest and induces differentiation function. In contrast, IL-6 acts as an autocrine growth factor in the subline LNCaP-IL-6+ established after chronic treatment with IL-6. IL-6 is a candidate for targeted therapy in prostate cancer because of its association with morbidity. Activation of signaling pathways of Janus kinase/signal transducers and activators of transcription factors, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase has been reported in various prostate cancer cell lines. IL-6 and the related cytokine oncostatin M induce activation of the androgen receptor (AR) in the absence of androgen. IL-6 is also involved in regulation of vascular endothelial growth factor expression as well as neuroendocrine differentiation in prostate. Anti-IL-6 antibodies showed an inhibitory effect on the PC-3 xenograft. However, the development of this therapy in prostate cancer is in early stages.

OBJECTIVE

To assess the feasibility of characterizing gene copy number alteration by fluorescence in situ hybridization (FISH) of circulating tumor cells (CTC) isolated using the CellSearch system in patients with progressive castration-resistant metastatic prostate cancer.

METHODS

We used probe combinations that included the androgen receptor (AR) and MYC genes for FISH analysis of CTC samples collected from 77 men with castration-resistant metastatic prostate cancer.

RESULTS

High-level chromosomal amplification of AR was detected in 38% and relative gain of MYC in 56% of samples analyzed. No such abnormalities were detected in samples with CTC counts of <10, reflecting ascertainment difficulty in these lower count samples.

CONCLUSIONS

The CTC isolated from our patient cohort present a very similar molecular cytogenetic profile to that reported for late-stage tumors and show that FISH analysis of CTC can be a valuable, noninvasive surrogate for routine tumor profiling. That as many as 50% of these patients have substantial amplification of the AR locus indicates that androgen signaling continues to play an important role in late-stage prostate cancer.

Twenty-four pesticides were tested for interactions with the estrogen receptor (ER) and the androgen receptor (AR) in transactivation assays. Estrogen-like effects on MCF-7 cell proliferation and effects on CYP19 aromatase activity in human placental microsomes were also investigated. Pesticides (endosulfan, methiocarb, methomyl, pirimicarb, propamocarb, deltamethrin, fenpropathrin, dimethoate, chlorpyriphos, dichlorvos, tolchlofos-methyl, vinclozolin, iprodion, fenarimol, prochloraz, fosetyl-aluminum, chlorothalonil, daminozid, paclobutrazol, chlormequat chlorid, and ethephon) were selected according to their frequent use in Danish greenhouses. In addition, the metabolite mercaptodimethur sulfoxide, the herbicide tribenuron-methyl, and the organochlorine dieldrin, were included. Several of the pesticides, dieldrin, endosulfan, methiocarb, and fenarimol, acted both as estrogen agonists and androgen antagonists. Prochloraz reacted as both an estrogen and an androgen antagonist. Furthermore, fenarimol and prochloraz were potent aromatase inhibitors while endosulfan was a weak inhibitor. Hence, these three pesticides possess at least three different ways to potentially disturb sex hormone actions. In addition, chlorpyrifos, deltamethrin, tolclofos-methyl, and tribenuron-methyl induced weak responses in one or both estrogenicity assays. Upon cotreatment with 17beta-estradiol, the response was potentiated by endosulfan in the proliferation assay and by pirimicarb, propamocarb, and daminozid in the ER transactivation assay. Vinclozolin reacted as a potent AR antagonist and dichlorvos as a very weak one. Methomyl, pirimicarb, propamocarb, and iprodion weakly stimulated aromatase activity. Although the potencies of the pesticides to react as hormone agonists or antagonists are low compared to the natural ligands, the integrated response in the organism might be amplified by the ability of the pesticides to act via several mechanism and the frequent simultaneous exposure to several pesticides.

In the United States, prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in males and the second leading cause of cancer-related death for men. The prostate is an androgen-dependent organ and PCa is an androgen-dependent disease. Androgen action is mediated by the androgen receptor (AR), a hormone activated transcription factor. The primary treatment for metastatic PCa is androgen deprivation therapy (ADT). For the most part, tumors respond to ADT, but most become resistant to therapy within two years. There is persuasive evidence that castration resistant (also termed castration recurrent) PCa (CRPC) remains AR dependent. Recent studies have shown that there are numerous factors that contribute to AR reactivation despite castrate serum levels of androgens. These include changes in AR expression and structure through gene amplification, mutation, and alternative splicing. Changes in steroid metabolism, cell signaling, and coregulator proteins are also important contributors to AR reactivation in CRPC. Most AR targeted therapies have been directed at the hormone binding domain. The finding that constitutively active AR splice variants that lack the hormone binding domain are frequently expressed in CRPC highlights the need to develop therapies that target other portions of AR. In this review, the role of AR in normal prostate, in PCa, and particularly the mechanisms for its reactivation subsequent to ADT are summarized. In addition, recent clinical trials and novel approaches to target AR are discussed.

We examined the controversial question concerning DNA sequences required for replication in Xenopus eggs. First we used yeast to isolate ARS elements from the Xenopus genome. They show a striking sequence homology with the yeast ARS consensus sequence. The cloning vector and the ARS-containing plasmids replicate equally after injection into Xenopus eggs. Second, we compared a wide range of DNA templates from procaryotes and eucaryotes. All DNA molecules tested replicate as monomeric molecules, and the efficiency is proportional to their size for templates between 4 and 12 kb. Third, we re-examined two reports of replication origins from the Xenopus genome. In both cases, the vector and the recombinant molecules replicate equally under all conditions tested. The apparent lack of sequence specificity for replication in Xenopus eggs does not prevent the injected molecule from being under cellular temporal control of replication. These results are compared with those from yeast.