The epidermal growth factor receptor (EGFR) and one of its ligands, transforming growth factor alpha (TGF-alpha), are thought to function as a potential autocrine loop in non-small cell lung cancer (NSCLC). However, the expression pattern of EGFR and the TGF-alpha-related ligands have not been fully characterized in primary NSCLC and adjacent benign lung tissue. For this reason, we comprehensively examined the coexpression and differential expression of EGFR and its ligands, TGF-alpha, epidermal growth factor (EGF), and amphiregulin (AR), by Northern analysis, in paired samples of primary tumors and uninvolved lung. For those RNA species overexpressed in malignant lung, single cell expression patterns were studied by immunohistochemistry. Specimens were obtained from 57 consecutive patients who underwent resection of carefully staged resectable NSCLC and were followed prospectively. Most (112 of 114) tissue samples yielded high-quality RNA. EGFR was expressed in 82 of 88 (93%) tissue samples, while TGF-alpha was expressed in 62 of 72 (86%) samples, and AR was expressed in 64 of 70 (92%) samples. EGF was unexpressed in total cellular RNA in both tumor and uninvolved lung. In a comparison of RNA expression patterns in tumors and uninvolved lung, overexpression of EGFR was found in 45% (22 of 44) of tumors, while overexpression of TGF-alpha was seen in 61% (22 of 36) of tumors, and decreased expression of AR was seen in 63% (22 of 35) of tumors. Cell type and stage did not influence differential expression, indicating that this is a frequent event in primary NSCLC. Simultaneous overexpression of EGFR and TGF-alpha was seen in only 38% of tumors. Simultaneous overexpression of EGFR and decreased expression of AR were seen in only 21% of tumors. Thus far, the differential expression of EGFR, TGF-alpha, and AR does not correlate with either disease-free or overall survival. These findings indicate that histologically dissimilar tumors can express similar components of autocrine or paracrine growth factor loops. Differential expression of EGFR and its ligands in tumor specimens compared to uninvolved lung is a common event in NSCLC and may participate in tumor growth without necessarily influencing tumor progression or histology.

BACKGROUND

Transgenic cardiac beta(2)-adrenergic receptor (AR) overexpression has resulted in enhanced signaling and cardiac function in mice, whereas relatively low levels of transgenically expressed G(alphas) or beta(1)AR have resulted in phenotypes of ventricular failure. Potential relationships between the levels of betaAR overexpression and biochemical, molecular, and physiological consequences have not been reported.

RESULTS

We generated transgenic mice expressing beta(2)AR at 3690, 7120, 9670, and 23 300 fmol/mg in the heart, representing 60, 100, 150, and 350 times background betaAR expression. All lines showed enhanced basal adenylyl cyclase activation but a decrease in forskolin- and NaF-stimulated adenylyl cyclase activities. Mice of the highest-expressing line developed a rapidly progressive fibrotic dilated cardiomyopathy and died of heart failure at 25+/-1 weeks of age. The 60-fold line exhibited enhanced basal cardiac function without increased mortality when followed for 1 year, whereas 100-fold overexpressors developed a fibrotic cardiomyopathy and heart failure, with death occurring at 41+/-1 weeks of age. Adenylyl cyclase activation did not correlate with early or delayed decompensation. Propranolol administration reduced baseline +dP/dt(max) to nontransgenic levels in all beta(2)AR transgenics except the 350-fold overexpressors, indicating that spontaneous activation of beta(2)AR was present at this level of expression.

CONCLUSIONS

These data demonstrate that the heart tolerates enhanced contractile function via 60-fold beta(2)AR overexpression without detriment for a period of>>/=1 year and that higher levels of expression result in either aggressive or delayed cardiomyopathy. The consequences for enhanced betaAR function in the heart appear to be highly dependent on which signaling elements are increased and to what extent.

Studies of ETS-mediated prostate oncogenesis have been hampered by a lack of suitable experimental systems. Here we describe a new conditional mouse model that shows robust, homogenous ERG expression throughout the prostate. When combined with homozygous Pten loss, the mice developed accelerated, highly penetrant invasive prostate cancer. In mouse prostate tissue, ERG markedly increased androgen receptor (AR) binding. Robust ERG-mediated transcriptional changes, observed only in the setting of Pten loss, included the restoration of AR transcriptional output and upregulation of genes involved in cell death, migration, inflammation and angiogenesis. Similarly, ETS variant 1 (ETV1) positively regulated the AR cistrome and transcriptional output in ETV1-translocated, PTEN-deficient human prostate cancer cells. In two large clinical cohorts, expression of ERG and ETV1 correlated with higher AR transcriptional output in PTEN-deficient prostate cancer specimens. We propose that ETS factors cause prostate-specific transformation by altering the AR cistrome, priming the prostate epithelium to respond to aberrant upstream signals such as PTEN loss.

It is unclear whether a single clone metastasizes and remains dominant over the course of lethal prostate cancer. We describe the clonal architectural heterogeneity at different stages of disease progression by sequencing serial plasma and tumor samples from 16 ERG-positive patients. By characterizing the clonality of commonly occurring deletions at 21q22, 8p21, and 10q23, we identified multiple independent clones in metastatic disease that are differentially represented in tissue and circulation. To exemplify the clinical utility of our studies, we then showed a temporal association between clinical progression and emergence of androgen receptor (AR) mutations activated by glucocorticoids in about 20% of patients progressing on abiraterone and prednisolone or dexamethasone. Resistant clones showed a complex dynamic with temporal and spatial heterogeneity, suggesting distinct mechanisms of resistance at different sites that emerged and regressed depending on treatment selection pressure. This introduces a management paradigm requiring sequential monitoring of advanced prostate cancer patients with plasma and tumor biopsies to ensure early discontinuation of agents when they become potential disease drivers.

BACKGROUND

The traditional belief that prostate cancer (PCa) growth is dependent on serum testosterone (T) level has been challenged by recent negative studies in noncastrated men.

OBJECTIVE

To provide an improved framework for understanding the relationship of PCa to serum T level that is consistent with current evidence and is based on established biochemical principles of androgen action within the prostate.

METHODS

A literature search was performed of publications dating from 1941 to 2008 that addressed experimental and clinical effects of androgens on prostate growth. Review of studies investigating the prostatic effects of manipulation of androgen concentrations in human and animal studies, and in PCa cell lines.

RESULTS

Prostate growth is exquisitely sensitive to variations in androgen concentrations at very low concentrations, but becomes insensitive to changes in androgen concentrations at higher levels. This pattern is consistent with the observation that androgens exert their prostatic effects primarily via binding to the androgen receptor (AR), and that maximal androgen-AR binding is achieved at serum T concentrations well below the physiologic range. A Saturation Model is proposed that accounts for the seemingly contradictory results in human PCa studies. Changes in serum T concentrations below the point of maximal androgen-AR binding will elicit substantial changes in PCa growth, as seen with castration, or with T administration to previously castrated men. In contrast, once maximal androgen-AR binding is reached the presence of additional androgen produces little further effect.

CONCLUSIONS

The evidence clearly indicates that there is a limit to the ability of androgens to stimulate PCa growth. A Saturation Model based on androgen-AR binding provides a satisfactory conceptual framework to account for the dramatic effects seen with castration as well as the minor impact of T administration in noncastrated men.

BACKGROUND

Wnt-1 and beta-catenin expression levels play an important role in several malignancies. The authors determined the level of production of Wnt-1 and beta-catenin in normal and malignant human prostate carcinoma cell lines. Surgical pathology specimens from primary prostatic adenocarcinoma, lymph node metastases, and skeletal metastases were used to establish a correlation between the level of Wnt-1/beta-catenin expression, Gleason score, serum prostate-specific antigen (PSA) status, and androgen receptor (AR) status.

METHODS

Immunohistochemical analysis was used to investigate the expression of Wnt-1 and beta-catenin in human prostate carcinoma cell lines and in paraffin embedded sections of archival samples from 67 patients with prostate carcinoma. Comparison was made with the expression of tumoral AR and lymph node and skeletal metastases. These results were used to establish a correlation with the clinicopathologic status of patients with prostate carcinoma.

RESULTS

Levels of both Wnt-1 and beta-catenin were low in normal prostate cells and were expressed highly in human prostate carcinoma cell lines. Wnt-1 and cytoplasmic/nuclear beta-catenin expression was observed in 52% and 34%, respectively, of primary prostate carcinoma specimens. High levels of expression of Wnt-1 and beta-catenin were seen in 77% of lymph node metastases and in 85% of skeletal metastases. These increased levels of expression were related directly to the Gleason score and to serum PSA levels in these patients. Maximum levels of Wnt-1 and beta-catenin production were observed in skeletal metastases, whereas normal prostatic tissue failed to exhibit any detectable nuclear staining for beta-catenin.

CONCLUSIONS

High levels of Wnt-1 and beta-catenin expression were associated with advanced, metastatic, hormone-refractory prostate carcinoma, in which they can serve as markers of disease progression.

Yeast chromosome ends are similar in structure and function to chromosome ends in most, if not all, eukaryotic organisms. There is a G-rich terminal repeat at the ends which is maintained by telomerase. In addition to the classical functions of protecting the end from degradation and end-to-end fusions, and completing replication, yeast telomeres have several interesting properties including: non-nucleosomal chromatin structure; transcriptional position effect variegation for genes with adjacent telomeres; nuclear peripheral localization; apparent physical clustering; non-random recombinational interactions. A number of genes have been identified that are involved in modifying one or more of these properties. These include genes involved in general DNA metabolism, chromatin structure and telomere maintenance. Adjacent to the terminal repeat is a mosaic of middle repetitive elements that exhibit a great deal of polymorphism both between individual strains and among different chromosome ends. Much of the sequence redundancy in the yeast genome is found in the sub-telomeric regions (within the last 25 kb of each end). The sub-telomeric regions are generally low in gene density, low in transcription, low in recombination, and they are late replicating. The only element which appears to be shared by all chromosome ends is part of the previously defined X element containing an ARS consensus. Most of the 'core' X elements also contain an Abf1p binding site and a URS1-like element, which may have consequences for the chromatin structure, nuclear architecture and transcription of native telomeres. Possible functions of sub-telomeric repeats include: fillers for increasing chromosome size to some minimum threshold level necessary for chromosome stability; barrier against transcriptional silencing; a suitable region for adaptive amplification of genes; secondary mechanism of telomere maintenance via recombination when telomerase activity is absent.

Androgens play a major role in promoting the development and progression of prostate cancer. As a result, androgen ablation or blockade of androgen action through the androgen receptor (AR) has been the cornerstone of treatment of advanced prostate cancer. Different strategies involving this hormonal therapy produce a significant clinical response in most of the patients, but most responders eventually lose dependency, resulting in mortality. Thus, whether hormonal therapy contributes to the improvement of overall survival rates, especially in patients with advanced prostate cancer, remains controversial. However, patients with advanced disease clearly have a benefit from androgen deprivation-based treatment for palliating their symptoms and for improving the quality of their lives. In order to improve overall survival, novel treatment strategies that prolong the androgen-dependent state and that are useful for androgen-independent disease based on specific molecular mechanisms need to be identified.

BACKGROUND

The diversity and complexity of the human androgen receptor (AR) splicing variants are well appreciated but not fully understood. The goal of this study is to generate a comprehensive expression signature of AR variants in castration-resistant prostate cancer (CRPC), and to address the relative importance of the individual variants in conferring the castration-resistant phenotype.

METHODS

A modified RNA amplification method, termed selective linear amplification of sense RNA, was developed to amplify all AR transcripts containing AR exon 3 in CRPC specimens, which were profiled using tiling expression microarrays. Coding sequences for the AR variants were cloned into expression vectors and assessed for their transcriptional activities. Quantitative RT-PCR was used to determine their in vivo expression patterns in an expanded set of clinical specimens.

RESULTS

In addition to expression peaks in AR intron 3, a novel AR exon, termed exon 9, was discovered. Exon 9 was spliced into multiple novel AR variants. Different AR splicing variants were functionally distinctive, with some demonstrating constitutive activity while others were conditionally active. Conditionally active AR-Vs may activate AR signaling depending on the cellular context. Importantly, AR variant functions did not appear to depend on the full-length AR.

CONCLUSIONS

This study provided the first unbiased snapshot of the AR variant signature consisting of multiple AR variants with distinctive functional properties, directly in CRPC specimens. Study findings suggest that the aggregate function of multiple AR variants may confer a castration-resistant phenotype independent of the full-length AR.

Mouse models with cell-specific deletion of the estrogen receptor (ER) α, the androgen receptor (AR) or the receptor activator of nuclear factor κB ligand (RANKL), as well as cascade-selective estrogenic compounds have provided novel insights into the function and signalling of ERα and AR. The studies reveal that the effects of estrogens on trabecular versus cortical bone mass are mediated by direct effects on osteoclasts and osteoblasts, respectively. The protection of cortical bone mass by estrogens is mediated via ERα, using a non-nucleus-initiated mechanism. By contrast, the AR of mature osteoblasts is indispensable for the maintenance of trabecular bone mass in male mammals, but not required for the anabolic effects of androgens on cortical bone. Most unexpectedly, and independently of estrogens, ERα in osteoblast progenitors stimulates Wnt signalling and periosteal bone accrual in response to mechanical strain. RANKL expression in B lymphocytes, but not T lymphocytes, contributes to the loss of trabecular bone caused by estrogen deficiency. In this Review, we summarize this evidence and discuss its implications for understanding the regulation of trabecular and cortical bone mass; the integration of hormonal and mechanical signals; the relative importance of estrogens versus androgens in the male skeleton; and, finally, the pathogenesis and treatment of osteoporosis.

Catecholamines stimulate cardiac contractility through beta(1)-adrenergic receptors (beta(1)-ARs), which in humans are polymorphic at amino acid residue 389 (Arg/Gly). We used cardiac-targeted transgenesis in a mouse model to delineate mechanisms accounting for the association of Arg389 with human heart failure phenotypes. Hearts from young Arg389 mice had enhanced receptor function and contractility compared with Gly389 hearts. Older Arg389 mice displayed a phenotypic switch, with decreased beta-agonist signaling to adenylyl cyclase and decreased cardiac contractility compared with Gly 389 hearts. Arg389 hearts had abnormal expression of fetal and hypertrophy genes and calcium-cycling proteins, decreased adenylyl cyclase and G alpha(s) expression, and fibrosis with heart failure This phenotype was recapitulated in homozygous, end-stage, failing human hearts. In addition, hemodynamic responses to beta-receptor blockade were greater in Arg389 mice, and homozygosity for Arg389 was associated with improvement in ventricular function during carvedilol treatment in heart failure patients. Thus the human Arg389 variant predisposes to heart failure by instigating hyperactive signaling programs leading to depressed receptor coupling and ventricular dysfunction, and influences the therapeutic response to beta-receptor blockade.

Chromosomal rearrangements involving erythroblast transformation specific (ETS) family transcription factors were recently defined as the most common genetic alterations in human prostate cancer. Despite their prevalence, it is unclear what quantitative role they play in either initiation or progression of the disease. Using a lentiviral transduction and dissociated cell prostate regeneration approach, we find that acutely increased expression of ETS proteins in adult murine prostate epithelial cells is sufficient to induce the formation of epithelial hyperplasia and focal prostatic intraepithelial neoplasia (PIN) lesions, but not progression to carcinoma. However, combined expression of ERG with additional genetic alternations associated with human prostate cancer can lead to aggressive disease. Although ERG overexpression does not cooperate with loss of the tumor suppressor p53, it does collaborate with alterations in PI3K signaling, such as Pten knockdown or AKT up-regulation, to produce a well-differentiated adenocarcinoma. Most striking is our finding that overexpression of androgen receptor (AR) does not give rise to any hyperplastic lesions, but when combined with high levels of ERG, it promotes the development of a more poorly differentiated, invasive adenocarcinoma. These findings suggest that in human prostate cancer, the most potent function of ETS gene fusions may be to synergize with alternative genetic events and provide different pathways for carcinoma production and invasive behavior. Our results provide direct evidence for selective cooperating events in ERG-induced prostate tumorigenesis and offer a rational basis for combined therapeutic interventions against multiple oncogenic pathways in prostate cancer.

Discoveries over the past decade suggest that castration-resistant prostate cancer (CRPC) is sensitive, but not resistant to, further manipulation of the androgen-androgen receptor (AR) axis. Several new therapies that target this axis have demonstrated clinical activity. In this article, preclinical and clinical findings occurring in the field of AR-targeted therapies are reviewed. Reviews of scientific and clinical development are divided into those occurring prereceptor (androgen production and conversion) and at the level of the receptor (AR aberrations and therapies targeting AR directly). Intracrine androgen production and AR amplification, among others, are among the principal aberrancies driving CRPC growth. Phase III data with abiraterone acetate and phase II data with MDV-3100, along with other similar therapies, confirm for the clinician that the scientific findings related to persistent AR signaling in a castrate milieu can be harnessed to produce significant clinical benefit for patients with the disease. Studies aimed at optimizing the timing of their use and exploring the mechanisms of resistance to these therapies are under way. The clinical success of therapies that directly target androgen synthesis as well as the most common aberrancies of the AR confirm that prostate cancer retains dependence on AR signaling, even in the castrate state.

Clinical and neuropathologic data in 45 patients with Parkinson's disease (PD) were compared. Twenty-seven patients suffered from marked akinesia and rigidity (AR-type) and 18 patients from predominant resting tremor (T-type). Dementia, depression, and psychosis occurred in 26, 18, and 18 patients, respectively. Neuronal counts were performed in defined areas of the medial and lateral substantia nigra (SNM, SNL), locus ceruleus (LC), and dorsal raphe nucleus (DRN). The AR-type (compared with the T-type) showed higher neuronal loss of LC, SNL, SNM, and more severe gliosis, extraneuronal melanin deposits, and neuroaxonal dystrophy in substantia nigra. Demented PD patients showed more intense cortical Alzheimer lesions and higher neuronal depletion in the SNM, whereas PD subjects with moderate or marked dementia differed from mildly or not demented ones only in the higher degree of cortical Alzheimer lesions. More severe neuronal cell loss of DRN was observed in PD patients with depression. Occurrence of psychosis was not associated with any pathologic feature. Our findings indicate that some major clinical features of PD are related to distinct neuropathologic lesions.

Autosomal recessive juvenile parkinsonism (AR-JP, PARK2; OMIM 602544), one of the monogenic forms of Parkinson's disease (PD), was initially described in Japan. It is characterized by early onset (before age 40), marked response to levodopa treatment and levodopa-induced dyskinesias. The gene responsible for AR-JP was recently identified and designated parkin. We have analysed the 12 coding exons of the parkin gene in 35 mostly European families with early onset autosomal recessive parkinsonism. In one family, a homozygous deletion of exon 4 could be demonstrated. By direct sequencing of the exons in the index patients of the remaining 34 families, eight previously undescribed point mutations (homozygous or heterozygous) were detected in eight families that included 20 patients. The mutations segregated with the disease in the families and were not detected on 110-166 control chromosomes. Four mutations caused truncation of the parkin protein. Three were frameshifts (202-203delAG, 255delA and 321-322insGT) and one a nonsense mutation (Trp453Stop). The other four were missense mutations (Lys161Asn, Arg256Cys, Arg275Trp and Thr415Asn) that probably affect amino acids that are important for the function of the parkin protein, since they result in the same phenotype as truncating mutations or homozygous exon deletions. Mean age at onset was 38 +/- 12 years, but onset up to age 58 was observed. Mutations in the parkin gene are therefore not invariably associated with early onset parkinsonism. In many patients, the phenotype is indistinguishable from that of idiopathic PD. This study has shown that a wide variety of different mutations in the parkin gene are a common cause of autosomal recessive parkinsonism in Europe and that different types of point mutations seem to be more frequently responsible for the disease phenotype than are deletions.

Sequences between a pair of divergently transcribed histone genes in Saccharomyces cerevisiae are able to confer periodic transcription during the cell cycle. This conclusion contrasts to our previous hypothesis that an ars (autonomously replicating sequence) 3' to this locus is a transcription timer for yeast histone genes. The promoter sequences required for periodic expression have been localized by deletion analysis, and isolated elements have been analyzed by insertion into a heterologous promoter. Two cell-cycle-specific promoter functions have been identified. One function activates transcription in a cell-cycle-dependent manner. The other periodically represses transcription. Negative regulation may be the predominant form of cell-cycle control, because removal of the repressing function results in constitutive expression of the histone genes.

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARSARSARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.

Mutations in DJ-1 gene have been linked to autosomal recessive early onset parkinsonism (AR-EOP). Although the mechanism of neuronal cell death due to DJ-1 mutation has not been fully elucidated, loss of DJ-1 function was considered to cause the phenotype. Here, we demonstrated that the down regulation of endogenous DJ-1 of the neuronal cell line by siRNA enhanced the cell death which was induced by oxidative stress, ER stress, and proteasome inhibition, but not by pro-apoptotic stimulus. The cell death with hydrogen peroxide was dramatically rescued by over-expression of wild-type DJ-1, but not by that of L166P mutant DJ-1. Furthermore, DJ-1 rescued the cell death caused by over-expression of Pael receptor, which was a substrate of Parkin, another gene product for autosomal recessive juvenile parkinsonism. These results suggest that loss of protective activity of DJ-1 from neuro-toxicity induced by these stresses contributes to neuronal cell death in AR-EOP with mutant DJ-1.

BACKGROUND

Human prostate cancers are initially androgen dependent but ultimately become androgen independent. Overexpression of the Her-2-neu receptor tyrosine kinase has been associated with the progression to androgen independence in prostate cancer cells. We examined the expression of Her-2-neu in normal and cancerous prostate tissues to assess its role in the progression to androgen independence.

METHODS

Prostate cancer tissue sections were obtained from 67 patients treated by surgery alone (UNT tumors), 34 patients treated with total androgen ablation therapy before surgery (TAA tumors), and 18 patients in whom total androgen ablation therapy failed and who developed bone metastases (androgen-independent [AI] disease). The sections were immunostained for Her-2-neu, androgen receptor (AR), prostate-specific antigen (PSA), and Ki-67 (a marker of cell proliferation) protein expression. Messenger RNA (mRNA) levels and gene amplification of Her-2-neu were examined by RNA in situ hybridization and fluorescent in situ hybridization(FISH), respectively, in a subset of 27 tumors (nine UNT, 11 TAA, and seven AI). All statistical tests were two-sided.

RESULTS

Her-2-neu protein expression was statistically significantly higher in TAA tumors than in UNT tumors with the use of two different scoring methods (P =.008 and P =.002). The proportion of Her-2-neu-positive tumors increased from the UNT group (17 of 67) to the TAA group (20 of 34) to the AI group (14 of 18) (P<.001). When compared with UNT tumors, tumor cell proliferation was higher in AI tumors (P =.014) and lower in TAA tumors (P<.001). All tumors expressed AR and PSA proteins. Although Her-2-neu mRNA expression was high in TAA and AI tumors, no Her-2-neu gene amplification was detected by FISH in any of the tumor types.

CONCLUSIONS

Her-2-neu expression appears to increase with progression to androgen independence. Thus, therapeutic targeting of this tyrosine kinase in prostate cancer may be warranted.

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.

A(2A) adenosine receptors (A(2A)AR) inhibit inflammation, although the mechanisms through which adenosine exerts its effects remain unclear. Although the transfer of regulatory Th cells blocks colitis induced by pathogenic CD45RB(high) Th cells, we show that CD45RB(low) or CD25+ Th cells from A(2A)AR-deficient mice do not prevent disease. Moreover, CD45RB(high) Th cells from A(2A)AR-deficient mice were not suppressed by control CD45RB(low) Th cells. A(2A)AR agonists suppressed the production of proinflammatory cytokines by CD45RB(high) and CD45RB(low) T cells in association with a loss of mRNA stability. In contrast, anti-inflammatory cytokines, including IL-10 and TGF-beta, were minimally affected. Oral administration of the A(2A)AR agonist ATL313 attenuated disease in mice receiving CD45RB(high) Th cells. These data suggest that A(2A)AR play a novel role in the control of T cell-mediated colitis by suppressing the expression of proinflammatory cytokines while sparing anti-inflammatory activity mediated by IL-10 and TGF-beta.

Fully understanding the mechanisms of signaling proteins such as G protein-coupled receptors (GPCRs) will require the characterization of their conformational states and the pathways connecting those states. The recent crystal structures of the beta(2)- and beta(1)-adrenergic receptors in a nominally inactive state constituted a major advance toward this goal, but also raised new questions. Although earlier biochemical observations had suggested that these receptors possessed a set of contacts between helices 3 and 6, known as the ionic lock, which was believed to form a molecular switch for receptor activation, the crystal structures lacked these contacts. The unexpectedly broken ionic lock has raised questions about the true conformation(s) of the inactive state and the role of the ionic lock in receptor activation and signaling. To address these questions, we performed microsecond-timescale molecular dynamics simulations of the beta(2)-adrenergic receptor (beta(2)AR) in multiple wild-type and mutant forms. In wild-type simulations, the ionic lock formed reproducibly, bringing the intracellular ends of helices 3 and 6 together to adopt a conformation similar to that found in inactive rhodopsin. Our results suggest that inactive beta(2)AR exists in equilibrium between conformations with the lock formed and the lock broken, whether or not the cocrystallized ligand is present. These findings, along with the formation of several secondary structural elements in the beta(2)AR loops during our simulations, may provide a more comprehensive picture of the inactive state of the beta-adrenergic receptors, reconciling the crystal structures with biochemical studies.

Most prostate cancers (PCs) become resistant to combined androgen blockade therapy with surgical or medical castration and antiandrogens after several years. Some of these refractory PCs regress after discontinuation of antiandrogen administration [antiandrogen withdrawal syndrome (AWS)]. Although the molecular mechanisms of the AWS are not fully understood because of the lack of suitable experimental models, one hypothesis of the mechanism is mutation of androgen receptor (AR). However, bicalutamide, which has become the most prevalent pure antiandrogen, does not work as an agonist for any mutant AR detected thus far in PC. To elucidate the mechanisms of the AWS, we established and characterized novel LNCaP cell sublines, LNCaP-cxDs, which were generated in vitro by culturing androgen-dependent LNCaP-FGC human PC cells in androgen-depleted medium with bicalutamide to mimic the combined androgen blockade therapy. LNCaP-FGC cells did not grow at first, but they started to grow after 6-13 weeks of culture. Bicalutamide stimulated LNCaP-cxD cell growth and increased prostate-specific antigen secretion from LNCaP-cxD cells both in vitro and in vivo. Sequencing of AR transcripts revealed that the AR in LNCaP-cxD cells harbors a novel mutation in codon 741, TGG (tryptophan) to TGT (cysteine; W741C), or in codon 741, TGG to TTG (leucine; W741L), in the ligand-binding domain. Transactivation assays showed that bicalutamide worked as an agonist for both W741C and W741L mutant ARs. Importantly, another antiandrogen, hydroxyflutamide, worked as an antagonist for these mutant ARs. In summary, we demonstrate for the first time that within only 6-13 weeks of in vitro exposure to bicalutamide, LNCaP-FGC cells, whose growth had initially been suppressed, came to use bicalutamide as an AR agonist via W741 AR mutation to survive. Our data strongly support the hypothesis that AR mutation is one possible mechanism of the AWS and suggest that flutamide might be effective as a second-line therapy for refractory PC previously treated with bicalutamide.

Although formerly regarded as a nuisance disease, allergic rhinitis (AR) has a considerable effect on quality of life and can have significant consequences if left untreated. The total burden of this disease lies not only in impaired physical and social functioning but also in a financial burden made greater when considering evidence that AR is a possible causal factor in comorbid diseases such as asthma or sinusitis. Compared with matched controls, patients with AR have an approximate twofold increase in medication costs and 1.8-fold the number of visits to health practitioners. Hidden direct costs include the treatment of comorbid asthma, chronic sinusitis, otitis media, upper respiratory infection, and nasal polyposis. Nasal congestion, the most prominent symptom in AR, is associated with sleep-disordered breathing, a condition that can have a profound effect on mental health, including increased psychiatric disorders, depression, anxiety, and alcohol abuse. Furthermore, sleep-disordered breathing in childhood and adolescence is associated with increased disorders of learning performance, behavior, and attention. In the United States, AR results in 3.5 million lost workdays and 2 million lost schooldays annually. Patients struggle to alleviate their misery, frequently self-adjusting their treatment regimen of over-the-counter and prescription medications because of lack of efficacy, deterioration of efficacy, lack of 24-hour relief, and bothersome side effects. Ironically, health care providers overestimate patient satisfaction with therapy. Therefore, improvement in patient-practitioner communication may enhance patient adherence with prescribed regimens.