Whereas several apoptosis-related proteins have been linked to the antiapoptotic effects of Akt serine-threonine kinase, the search continues to explain the Akt signaling role in promoting cell survival via antiapoptotic effects. Here, we demonstrate that Akt phosphorylates the androgen receptor (AR) at Ser-210 and Ser-790. A mutation at AR Ser-210 results in the reversal of Akt-mediated suppression of AR transactivation. Activation of the phosphatidylinositol-3-OH kinase/Akt pathway results in the suppression of AR target genes, such as p21, and the decrease of androgen/AR-mediated apoptosis, which may involve the inhibition of interaction between AR and AR coregulators. Together, these findings provide a molecular basis for cross-talk between two signaling pathways at the level of Akt and AR-AR coregulators that may help us to better understand the roles of Akt in the androgen/AR-mediated apoptosis.

SoyBase, the USDA-ARS soybean genetic database, is a comprehensive repository for professionally curated genetics, genomics and related data resources for soybean. SoyBase contains the most current genetic, physical and genomic sequence maps integrated with qualitative and quantitative traits. The quantitative trait loci (QTL) represent more than 18 years of QTL mapping of more than 90 unique traits. SoyBase also contains the well-annotated 'Williams 82' genomic sequence and associated data mining tools. The genetic and sequence views of the soybean chromosomes and the extensive data on traits and phenotypes are extensively interlinked. This allows entry to the database using almost any kind of available information, such as genetic map symbols, soybean gene names or phenotypic traits. SoyBase is the repository for controlled vocabularies for soybean growth, development and trait terms, which are also linked to the more general plant ontologies. SoyBase can be accessed at http://soybase.org.

The 'silent' yeast mating-type loci (HML and HMR) are repressed by sequences (HMLE and HMRE) located over 1 kb from their promoters which have properties opposite those of enhancers, and are called 'silencers'. Both silencers contain autonomously replicating sequences (<em>ARS</em>). Silencer activity requires four trans-acting genes called SIR (silent information regulator). We have identified two DNA binding factors , SBF-B and SBF-E, which bind to known regulatory elements at HMRE. SBF-B binds to a region involved in both the silencer and <em>ARS</em> functions of HMRE, but doesn not bind to HMLE. This factor also binds to the unlinked <em>ARS</em>1 element. SBF-E recognizes a sequence found at both silencers. These results suggest that the two silencers may be composed of different combinations of regulatory elements at least one of which is common to both. Neither factor appears to be a SIR gene product. Hence the SIR proteins may not directly interact with the silencer control sites.

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold-induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Galphai2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein-protein and lipid-lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP(3) production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Galphai2. Because both Galphai2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Galphai2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

OBJECTIVE

To better direct targeted therapies to the patients with tumors that express the target, there is an urgent need for blood-based assays that provide expression information on a consistent basis in real time with minimal patient discomfort. We aimed to use immunomagnetic-capture technology to isolate and analyze circulating tumor cells (CTC) from small volumes of peripheral blood of patients with advanced prostate cancer.

METHODS

Blood was collected from 63 patients with metastatic prostate cancer. CTCs were isolated by the Cell Search system, which uses antibodies to epithelial cell adhesion marker and immunomagnetic capture. CTCs were defined as nucleated cells positive for cytokeratins and negative for CD45. Captured cells were analyzed by immunofluorescence, Papanicolau staining, and fluorescence in situ hybridization.

RESULTS

Most patients (65%) had 5 or more CTCs per 7.5 mL blood sample. Cell counts were consistent between laboratories (c = 0.99) and did not change significantly over 72 or 96 h of storage before processing (c = 0.99). Their identity as prostate cancer cells was confirmed by conventional cytologic analysis. Molecular profiling, including analysis of epidermal growth factor receptor (EGFR) expression, chromosome ploidy, and androgen receptor (AR) gene amplification, was possible for all prostate cancer patients with>>or=5 CTCs.

CONCLUSIONS

The analysis of cancer-related alterations at the DNA and protein level from CTCs is feasible in a hospital-based clinical laboratory. The alterations observed in EGFR and AR suggest that the methodology may have a role in clinical decision making.

Dyskeratosis congenita (DC) is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. It exhibits marked clinical and genetic heterogeneity. DKC1 encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein (snoRNP) particles is mutated in X-linked recessive DC. Telomerase RNA component (TERC), the RNA component and TERT the enzymatic component of telomerase, are mutated in autosomal dominant DC, suggesting that DC is primarily a disease of defective telomere maintenance. The gene(s) involved in autosomal recessive DC remains elusive. This paper describes studies aimed at defining the genetic basis of AR-DC. Homozygosity mapping in 16 consanguineous families with 25 affected individuals demonstrates that there is no single genetic locus for AR-DC. However, we show that NOP10, a component of H/ACA snoRNP complexes including telomerase is mutated in a large consanguineous family with classical DC. Affected homozygous individuals have significant telomere shortening and reduced TERC levels. While a reduction of TERC levels is not a universal feature of DC, it can be brought about through a reduction of NOP10 transcripts, as demonstrated by siRNA interference studies. A similar reduction in TERC levels is also seen when the mutant NOP10 is expressed in HeLa cells. These findings identify the genetic basis of one subtype of AR-DC being due to the first documented mutations in NOP10. This further strengthens the model that defective telomere maintenance is the primary pathology in DC and substantiates the evidence in humans for the involvement of NOP10 in the telomerase complex and telomere maintenance.

Targeting androgens/androgen receptor (AR) functions via androgen deprivation therapy (ADT) remains the standard treatment for prostate cancer. However, most tumors eventually recur despite ADT. Here we demonstrate that the prostate AR may function as both a suppressor and a proliferator to suppress or promote prostate cancer metastasis. Results from orthotopically recombining stromal WPMY1 cells with epithelial PC3 prostate cancer cells in mice demonstrated that restoring AR in epithelial PC3 cells or knockdown of AR in stromal WPMY1 cells suppressed prostate cancer metastasis. Knockdown of the AR in epithelial CWR22rv1 prostate cancer cells also resulted in increased cell invasion in vitro and in vivo. Restoring AR in PC3 cells (PC3-ARAR have increased apoptosis in epithelial luminal cells and increased proliferation in epithelial basal cells. The consequences of these two contrasting results led to the expansion of CK5/CK8-positive intermediate cells, and mice developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that androgens/AR might directly or indirectly modulate metastasis-related genes and suppression of TGFbeta1 signals results in the partial inhibition of AR-mediated metastasis. Collectively, our understanding of these opposing roles of prostatic AR may revolutionize the way we combat prostate cancer, and allow the development of new and better therapies by targeting only the proliferative role of AR.

BACKGROUND

Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa).

RESULTS

Using 2 antibodies against different regions of AR protein (N- or C-terminus), we successfully showed the existence of AR variant in the LuCaP 86.2 xenograft. To evaluate the prevalence of AR variants in human PCa tissue, we used this method on tissue microarrays including 50 primary PCa and 162 metastatic CRPC tissues. RT-PCR was used to confirm AR variants. We observed a significant decrease in nuclear C-terminal AR staining in CRPC but no difference between N- and C-terminal AR nuclear staining in primary PCa. The expression of the AR regulated proteins PSA and PSMA were marginally affected by the decrease in C-terminal staining in CRPC samples. These data suggest that there is an increase in the prevalence of AR variants in CRPC based on our ability to differentiate nuclear AR expression using N- and C-terminal AR antibodies. These findings were validated using RT-PCR. Importantly, the loss of C-terminal immunoreactivity and the identification of AR variants were different depending on the site of metastasis in the same patient.

CONCLUSIONS

We successfully developed a novel immunohistochemical approach which was used to ascertain the prevalence of AR variants in a large number of primary PCa and metastatic CRPC. Our results showed a snapshot of overall high frequency of C-terminal truncated AR splice variants and site specific AR loss in CRPC, which could have utility in stratifying patients for AR targeted therapeutics.

Inhaled beta-adrenergic agonists are the most commonly used medications for the treatment of asthma although there is evidence that regular use may produce adverse effects in some patients. Polymorphisms of the beta(2)-adrenergic receptor (beta(2)-AR) can affect regulation of the receptor. Smaller studies examining the effects of such polymorphisms on the response to beta-agonist therapy have produced inconsistent results. We examined whether polymorphisms at codon 16 (beta(2)-AR-16) and codon 27 (beta(2)-AR-27) of the beta(2)-AR might affect the response to regular versus as-needed use of albuterol by genotyping the 190 asthmatics who had participated in a trial examining the effects of regular versus as needed albuterol use. During the 16-wk treatment period there was a small decline in morning peak expiratory flow in patients homozygous for arginine at B(2)-AR-16 (Arg/Arg) who used albuterol regularly. This effect was magnified during a 4-wk run out period, during which all patients returned to using as-needed albuterol, so that by the end of the study Arg Arg patients who had regularly used albuterol had a morning peak expiratory flow 30. 5 +/- 12.1 L/min lower (p = 0.012) than Arg/Arg patients who had used albuterol on an as needed basis. There was no decline in peak flow with regular use of albuterol in patients who were homozygous for glycine at beta(2)-AR-16. Evening peak expiratory flow also declined in the Arg/Arg patients who used albuterol regularly but not in those who used albuterol on an as-needed basis. No significant differences in outcomes between regular and as-needed treatment were associated with polymorphisms at position 27 of the beta(2)-AR. No other differences in asthma outcomes that we investigated occurred in relation to these beta(2)-AR polymorphisms. Polymorphisms of the beta(2)-AR may influence airway responses to regular inhaled beta-agonist treatment.

Immunohistochemistry is an integral technique in many veterinary laboratories for diagnostic and research purposes. In the last decade, the ability to detect antigens (Ags) in tissue sections has improved dramatically, mainly by countering the deleterious effects of formaldehyde with antigen retrieval (AR) and increasing sensitivity of the detection systems. In this review, I address these topics and provide an overview of technical aspects of immunohistochemistry, including those related to antibodies (Abs) and Ags, fixation, AR, detection methods, background, and troubleshooting. Microarray technology and the use of rabbit monoclonal Abs in immunohistochemistry are also discussed.

The interaction between nanoparticles (NPs) and cells has been studied extensively, but the effect of particle shape on cell behavior has received little attention. Herein three different shaped monodisperse mesoporous silica nanoparticles (MSNs) of similar particle diameter, chemical composition and surface charge but with different aspect ratios (ARs, 1, 2, 4) were specially designed. Then the effects of particle shape of these three different shaped particles on cellular uptake and behavior were studied. The results indicated that these different shaped particles were readily internalized in A375 human melanoma (A375) cells by nonspecific cellular uptake. Particles with larger ARs were taken up in larger amounts and had faster internalization rates. Likewise, it was also found that particles with larger ARs had a greater impact on different aspects of cellular function including cell proliferation, apoptosis, cytoskeleton formation, adhesion and migration. These results show that nanoparticles should no longer be viewed as simple carriers for biomedical applications, but can also play an active role in mediating biological effects. Therefore, our findings may provide useful information for the development of new strategies for the design of efficient drug delivery nanocarriers and therapeutic systems and provide insights into nanotoxicity.

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.

Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether beta-adrenergic receptor (beta-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca](i)) and SR Ca load ([Ca](SRT)) were assessed using fluo-4. beta-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca](i) decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2-related inhibitory peptide II or beta-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the beta-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca](i) decline rate. The results suggest that beta-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca](i).

Oncogenic BRAF mutations are found in several tumor types, including melanomas and colorectal cancers. Tumors with BRAF mutations have increased mitogen-activated protein kinase pathway activity and heightened sensitivity to BRAF and MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase) inhibitors. To identify potential mechanisms of acquired drug resistance, we generated clones resistant to the allosteric MEK inhibitor AZD6244 from two BRAF V600E mutant colorectal cancer cell lines that are highly sensitive to MEK or BRAF inhibition. These AZD6244-resistant (AR) clones, which exhibited cross-resistance to BRAF inhibitors, acquired resistance through amplification of the BRAF gene. A small percentage of treatment-naïve parental cells showed preexisting BRAF amplification. We observed similar amplification in a subset of cells in a BRAF-mutant colorectal cancer. In cell lines, BRAF amplification increased the abundance of phosphorylated MEK and impaired the ability of AZD6244 to inhibit ERK (extracellular signal-regulated kinase) phosphorylation. The ability of AZD6244 to inhibit ERK phosphorylation in AR cells was restored by treatment with a BRAF inhibitor at low concentrations that reduced the abundance of phosphorylated MEK to amounts observed in parental cells. Combined MEK and BRAF inhibition fully overcame resistance to MEK or BRAF inhibitors alone and was also more effective in parental cells compared to treatment with either inhibitor alone. These findings implicate BRAF amplification as a mechanism of resistance to both MEK and BRAF inhibitors and suggest combined MEK and BRAF inhibition as a clinical strategy to overcome, or possibly prevent, this mechanism of resistance.

OBJECTIVE

We report the isolation and characterization of a novel prostate cancer cell line derived from a vertebral metastatic lesion, Vertebral-Cancer of the Prostate (VCaP).

METHODS

Prostate cancer tissue was harvested at autopsy from a metastatic lesion to a lumbar vertebral body of a patient with hormone refractory prostate cancer. This tissue was aseptically xenografted into SCID mice and later harvested and plated on tissue culture dishes. For characterization, soft agar clonegenic assay, in vivo xenograft growth, in vitro doubling time, karyotype analysis, immunocytochemistry for cytokeratin-18 expression immunochemistry for PSA (prostate specific antigen), RT PCR for PAP (prostatic acid phosphatase) and northern blot and western blot analysis to determine expression of Rb and p53, were performed. Androgen receptor expression was measured by transient transfection with a luciferase reporter construct.

RESULTS

VCaP cells are immortal in vitro and can be passaged serially in vivo. They express large quantities of prostate specific antigen (PSA). This cell line also expresses prostatic acid phosphatase (PAP), cytokeratin-18 and the androgen receptor, and is androgen sensitive in vitro and in vivo.

CONCLUSIONS

This cell line was derived from a metastatic tumor to the vertebrae of a prostate cancer patient. It exhibits many of the characteristics of clinical prostate carcinoma, including expression of PSA, PAP, and AR. We believe that VCaP will be a useful addition to the existing models of prostate cancer, and enable more advanced study of the mechanisms of prostate cancer progression and metastasis.

Despite earlier detection and recent advances in surgery and radiation, prostate cancer is second only to lung cancer in male cancer deaths in the United States. Hormone therapy in the form of medical or surgical castration remains the mainstay of systemic treatment in prostate cancer. Over the last 15 years with the clinical use of prostate specific antigen (PSA), there has been a shift to using hormone therapy earlier in the disease course and for longer duration. Despite initial favorable response to hormone therapy, over a period of time these tumors will develop androgen-independence that results in death. The androgen receptor (AR) is central to the initiation and growth of prostate cancer and to its response to hormone therapy. Analyses have shown that AR continues to be expressed in androgen-independent tumors and AR signaling remains intact as demonstrated by the expression of the AR regulated gene, PSA. Androgen-independent prostate cancers have demonstrated a variety of AR alterations that are either not found in hormone naïve tumors or found at lower frequency. These changes include AR amplification, AR point mutation, and changes in expression of AR co-regulatory proteins. These AR changes result in a "super AR" that can respond to lower concentrations of androgens or to a wider variety of agonistic ligands. There is also mounting evidence that AR can be activated in a ligand independent fashion by compounds such as growth factors or cytokines working independently or in combination. These growth factors working through receptor tyrosine kinase pathways may promote AR activation and growth in low androgen environments. The clinical significance of these AR alterations in the development and progression of androgen-independent prostate cancer remains to be determined. Understanding the changes in AR signaling in the evolution of androgen-independent prostate cancer will be key to the development of more effective hormone therapy.

We investigated the effects of 6 mo of near-physiological testosterone administration to older men on skeletal muscle function and muscle protein metabolism. Twelve older men >> or =60 yr) with serum total testosterone concentrations <17 nmol/l (480 ng/dl) were randomly assigned in double-blind manner to receive either placebo (n = 5) or testosterone enanthate (TE; n = 7) injections. Weekly intramuscular injections were given for the 1st mo to establish increased blood testosterone concentrations at 1 mo and then changed to biweekly injections until the 6-mo time point. TE doses were adjusted to maintain nadir serum testosterone concentrations between 17 and 28 nmol/l. Lean body mass (LBM), muscle volume, prostate size, and urinary flow were measured at baseline and at 6 mo. Protein expression of androgen receptor (AR) and insulin-like growth factor I, along with muscle strength and muscle protein metabolism, were measured at baseline and at 1 and 6 mo of treatment. Hematological parameters were followed monthly throughout the study. Older men receiving testosterone increased total and leg LBM, muscle volume, and leg and arm muscle strength after 6 mo. LBM accretion resulted from an increase in muscle protein net balance, due to a decrease in muscle protein breakdown. TE treatment increased expression of AR protein at 1 mo, but expression returned to pre-TE treatment levels by 6 mo. IGF-I protein expression increased at 1 mo and remained increased throughout TE administration. We conclude that physiological and near-physiological increases of testosterone in older men will increase muscle protein anabolism and muscle strength.

Communication between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signalling systems involves cell surface proteolysis of EGF-like precursors. The underlying mechanisms of EGFR signal transactivation pathways, however, are largely unknown. We demonstrate that in squamous cell carcinoma cells, stimulation with the GPCR agonists LPA or carbachol specifically results in metalloprotease cleavage and release of amphiregulin (AR). Moreover, AR gene silencing by siRNA or inhibition of AR biological activity by neutralizing antibodies and heparin prevents GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signalling events, cell proliferation, migration and activation of the survival mediator Akt/PKB. Therefore, despite some functional redundancy among EGF family ligands, the present study reveals a distinct and essential role for AR in GPCR-triggered cellular responses. Furthermore, we present evidence that blockade of the metalloprotease-disintegrin tumour necrosis factor-alpha-converting enzyme (TACE) by the tissue inhibitor of metalloprotease-3, a dominant-negative TACE mutant or RNA interference suppresses GPCR-stimulated AR release, EGFR activation and downstream events. Thus, TACE can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network.

The second-generation antiandrogen enzalutamide was recently approved for patients with castration-resistant prostate cancer. Despite its success, the duration of response is often limited. For previous antiandrogens, one mechanism of resistance is mutation of the androgen receptor (AR). To prospectively identify AR mutations that might confer resistance to enzalutamide, we performed a reporter-based mutagenesis screen and identified a novel mutation, F876L, which converted enzalutamide into an AR agonist. Ectopic expression of AR F876L rescued the growth inhibition of enzalutamide treatment. Molecular dynamics simulations performed on antiandrogen-AR complexes suggested a mechanism by which the F876L substitution alleviates antagonism through repositioning of the coactivator recruiting helix 12. This model then provided the rationale for a focused chemical screen which, based on existing antiandrogen scaffolds, identified three novel compounds that effectively antagonized AR F876L (and AR WT) to suppress the growth of prostate cancer cells resistant to enzalutamide. DOI:http://dx.doi.org/10.7554/eLife.00499.001.

UNASSIGNED

A critical decision in the management of metastatic castration-resistant prostate cancer (mCRPC) is when to administer an androgen receptor signaling (ARS) inhibitor or a taxane.

UNASSIGNED

To determine if pretherapy nuclear androgen-receptor splice variant 7 (AR-V7) protein expression and localization on circulating tumor cells (CTCs) is a treatment-specific marker for response and outcomes between ARS inhibitors and taxanes.

UNASSIGNED

For this cross-sectional cohort study at Memorial Sloan Kettering Cancer Center, 265 men with progressive mCRPC undergoing a change in treatment were considered; 86 were excluded because they were not initiating ARS or taxane therapy; and 18 were excluded for processing time constraints, leaving 161 patients for analysis. Between December 2012 and March 2015, blood was collected and processed from patients with progressive mCRPC immediately prior to new line of systemic therapy. Patients were followed up to 3 years.

UNASSIGNED

Prostate-specific antigen (PSA) response, time receiving therapy, radiographic progression-free survival (rPFS), and overall survival (OS).

UNASSIGNED

Overall, of 193 prospectively collected blood samples from 161 men with mCRPC, 191 were evaluable (128 pre-ARS inhibitor and 63 pretaxane). AR-V7-positive CTCs were found in 34 samples (18%), including 3% of first-line, 18% of second-line, and 31% of third- or greater line samples. Patients whose samples had AR-V7-positive CTCs before ARS inhibition had resistant posttherapy PSA changes (PTPC), shorter rPFS, shorter time on therapy, and shorter OS than those without AR-V7-positive CTCs. Overall, resistant PTPC were seen in 65 of 112 samples (58%) without detectable AR-V7-positive CTCs prior to ARS inhibition. There were statistically significant differences in OS but not in PTPC, time on therapy, or rPFS for patients with or without pretherapy AR-V7-positive CTCs treated with a taxane. A multivariable model adjusting for baseline factors associated with survival showed superior OS with taxanes relative to ARS inhibitors when AR-V7-positive CTCs were detected pretherapy (hazard ratio, 0.24; 95% CI, 0.10-0.57; P = .035).

UNASSIGNED

The results validate CTC nuclear expression of AR-V7 protein in men with mCRPC as a treatment-specific biomarker that is associated with superior survival on taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further aid clinical utility.

Aldose reductase (AR) is widely expressed aldehyde-metabolizing enzyme. The reduction of glucose by the AR-catalyzed polyol pathway has been linked to the development of secondary diabetic complications. Although treatment with AR inhibitors has been shown to prevent tissue injury in animal models of diabetes, the clinical efficacy of these drugs remains to be established. Recent studies suggest that glucose may be an incidental substrate of AR, which appears to be more adept in catalyzing the reduction of a wide range of aldehydes generated from lipid peroxidation. Moreover, inhibition of the enzyme has been shown to increase inflammation-induced vascular oxidative stress and prevent myocardial protection associated with the late phase of ischemic preconditioning. On the basis of these studies, several investigators have ascribed an important antioxidant role to the enzyme. Additionally, ongoing work indicates that AR is a critical component of intracellular signaling, and inhibition of the enzyme prevents high glucose-, cytokine-, or growth factor-induced activation of protein kinase C and nuclear factor-kappa-binding protein. Thus, treatment with AR inhibitors prevents vascular smooth muscle cell growth and endothelial cell apoptosis in culture and inflammation and restenosis in vivo. Additional studies indicate that the antioxidant and signaling roles of AR are interlinked and that AR regulates protein kinase C and nuclear factor-kappaB via redox-sensitive mechanisms. These data underscore the need for reevaluating anti-AR interventions for the treatment of diabetic complications. Potentially, the development of newer drugs that selectively inhibit AR-mediated glucose metabolism and signaling, without affecting aldehyde detoxification, may be useful in preventing inflammation associated with the development of diabetic complications, particularly micro- and macrovascular diseases.

The Charcot-Marie-Tooth (CMT) disorders comprise a group of clinically and genetically heterogeneous hereditary motor and sensory neuropathies, which are mainly characterized by muscle weakness and wasting, foot deformities, and electrophysiological, as well as histological, changes. A subtype, CMT2, is defined by a slight or absent reduction of nerve-conduction velocities together with the loss of large myelinated fibers and axonal degeneration. CMT2 phenotypes are also characterized by a large genetic heterogeneity, although only two genes---NF-L and KIF1Bbeta---have been identified to date. Homozygosity mapping in inbred Algerian families with autosomal recessive CMT2 (AR-CMT2) provided evidence of linkage to chromosome 1q21.2-q21.3 in two families (Zmax=4.14). All patients shared a common homozygous ancestral haplotype that was suggestive of a founder mutation as the cause of the phenotype. A unique homozygous mutation in LMNA (which encodes lamin A/C, a component of the nuclear envelope) was identified in all affected members and in additional patients with CMT2 from a third, unrelated family. Ultrastructural exploration of sciatic nerves of LMNA null (i.e., -/-) mice was performed and revealed a strong reduction of axon density, axonal enlargement, and the presence of nonmyelinated axons, all of which were highly similar to the phenotypes of human peripheral axonopathies. The finding of site-specific amino acid substitutions in limb-girdle muscular dystrophy type 1B, autosomal dominant Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy type 1A, autosomal dominant partial lipodystrophy, and, now, AR-CMT2 suggests the existence of distinct functional domains in lamin A/C that are essential for the maintenance and integrity of different cell lineages. To our knowledge, this report constitutes the first evidence of the recessive inheritance of a mutation that causes CMT2; additionally, we suggest that mutations in LMNA may also be the cause of the genetically overlapping disorder CMT2B1.

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.