The androgen receptor is unusual among nuclear receptors in that most, if not all, of its activity is mediated via the constitutive activation function in the N terminus. Here we demonstrate that p160 coactivators such as SRC1 (steroid receptor coactivator 1) interact directly with the N terminus in a ligand-independent manner via a conserved glutamine-rich region between residues 1053 and 1123. Although SRC1 is capable of interacting with the ligand-binding domain by means of LXXLL motifs, this interaction is not essential since an SRC1 mutant with no functional LXXLL motifs retains its ability to potentiate androgen receptor activity. In contrast, mutants lacking the glutamine-rich region are inactive, indicating that this region is both necessary and sufficient for recruitment of SRC1 to the androgen receptor. This recruitment is in direct contrast to the recruitment of SRC1 to the estrogen receptor, which requires interaction with the ligand-binding domain.The androgen receptor (AR), a member of the nuclear receptor superfamily (6,45), is a ligand-activated transcription factor with the major ligands testosterone and dihydrotestosterone. It has the overall domain structure common to nuclear receptors, comprising an N-terminal activation domain (activation function 1 [AF1]), a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). A second, ligand-dependent activation function (AF2) in several nuclear receptors, including other steroid hormone receptors, has been well characterized (5,15,19,66), but until recently there was no evidence to support such a function for the AR LBD. Deletion of the LBD results in a molecule with constitutive activity which in many reporter activation assays is equivalent to the maximum activity of the full-length receptor in the presence of ligand, implying that AF1 contributes all the activity of the receptor (35,56,61,82). This finding is in contrast to what occurs with the closely related estrogen receptor (ER), in which AF2 is the major activation domain and AF1 has little independent activity (67). The situation in the AR is still more complex, in that two discrete, overlapping activation domains exist in the N-terminal domain and their usage is context dependent. While almost the entire N terminus (residues 1 to 494) is required for full activity of the full-length receptor, a core that contributes 50% of activity is located between residues 101 and 360, and this region has been termed TAU1. However, in the absence of the LBD a different region, termed TAU5 (residues 370 to 494), mediates activation (34).Upon binding of ligand, steroid hormone receptors adopt an active conformation that facilitates the dissociation of heat shock proteins, dimerization, and binding to response elements in the promoters of responsive genes. These receptors have been shown to interact with some components of the basal transcriptional machinery (3, 9, 10, 32, 46, 59) and also to promote transcription of the gene by interacting with coactivator proteins (23, 68). Coactiva...

Concurrent with the explosion in the number of publications reporting biomarker discovery by profiling technologies, such as proteomics and pattern recognition, has been the increase in evidence highlighting the susceptibility of these approaches to analytical and experimental bias. The work presented here addresses these timely issues by delivering a detailed characterization of the effect of common sources of bias in clinical studies on serum and plasma profiles generated by a key technology in metabonomics, NMR spectroscopy. Specifically, differences in composition when blood samples were collected onto and in the absence of ice, over a series of serum-clot contact times, the stability of NMR-prepared samples over time and the effect on the metabolic profile of freeze-thawing were examined. While differences between individuals were far greater than variation from any other experimental factor, each of the conditions examined did cause slight alterations to the NMR profile that could produce a systematic bias. Variation due to clotting time caused changes in energy metabolites, which were delayed by ice with no other spectral effects. Room-temperature stability and hence NMR spectral repeatability were high (<1% intrasample variation). Higher molecular weight species such as lipoproteins were more susceptible to the variations present in the examined factors. These observations have implications for profiling study design, and hence, our results form a new and valuable resource for those attempting clinical metabolic profiling, for regulatory agencies involved in the licensing of clinical tests and in the generation of international reporting standards for metabonomics.

Background:Surgery is considered to be the first line treatment for solid tumours. Recently, retrospective studies reported that general anaesthesia was associated with worse long-term cancer-free survival when compared with regional anaesthesia. This has important clinical implications; however, the mechanisms underlying those observations remain unclear. We aim to investigate the effect of anaesthetics isoflurane and propofol on prostate cancer malignancy.Methods:Prostate cancer (PC3) cell line was exposed to commonly used anaesthetic isoflurane and propofol. Malignant potential was assessed through evaluation of expression level of hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, cell proliferation and migration as well as development of chemoresistance.Results:We demonstrated that isoflurane, at a clinically relevant concentration induced upregulation of HIF-1α and its downstream effectors in PC3 cell line. Consequently, cancer cell characteristics associated with malignancy were enhanced, with an increase of proliferation and migration, as well as development of chemoresistance. Inhibition of HIF-1α neosynthesis through upper pathway blocking by a PI-3K-Akt inhibitor or HIF-1α siRNA abolished isoflurane-induced effects. In contrast, the intravenous anaesthetic propofol inhibited HIF-1α activation induced by hypoxia or CoCl2. Propofol also prevented isoflurane-induced HIF-1α activation, and partially reduced cancer cell malignant activities.Conclusions:Our findings suggest that modulation of HIF-1α activity by anaesthetics may affect cancer recurrence following surgery. If our data were to be extrapolated to the clinical setting, isoflurane but not propofol should be avoided for use in cancer surgery. Further work involving in vivo models and clinical trials is urgently needed to determine the optimal anaesthetic regimen for cancer patients.

Hey1 is a member of the basic helix-loop-helix-Orange family of transcriptional repressors that mediate Notch signaling. Here we show that transcription from androgen-dependent target genes is inhibited by Hey1 and that expression of a constitutively active form of Notch is capable of repressing transactivation by the endogenous androgen receptor (AR). Our results indicate that Hey1 functions as a corepressor for AF1 in the AR, providing a mechanism for cross talk between Notch and androgen-signaling pathways. Hey1 colocalizes with AR in the epithelia of patients with benign prostatic hyperplasia, where it is found in both the cytoplasm and the nucleus. In marked contrast, we demonstrate that Hey1 is excluded from the nucleus in most human prostate cancers, raising the possibility that an abnormal Hey1 subcellular distribution may have a role in the aberrant hormonal responses observed in prostate cancer.

Since they were first described in the 1990s, circulating microRNAs (miRNAs) have provided an active and rapidly evolving area of current research that has the potential to transform cancer diagnostics and therapeutics. In particular, miRNAs could provide potential new biomarkers for prostate cancer, the most common cause of cancer in UK men. Current diagnostic tests for prostate cancer have low specificity and poor sensitivity. Further, although many prostate cancers are so slow growing as not to pose a major risk to health, there is currently no test to distinguish between these and cancers that will become aggressive and life threatening. Circulating miRNAs are highly stable and are both detectable and quantifiable in a range of accessible bio fluids, thus have the potential to be useful diagnostic, prognostic and predictive biomarkers. This review aims to summarise the current understanding of circulating miRNAs in prostate cancer patients and their potential role as biomarkers.

Prostate tumour growth is almost always dependent upon the androgen receptor pathway and hence therapies aimed at blocking this signalling axis are useful tools in the management of this disease. Unfortunately such therapies invariably fail; and the tumour progresses to an “androgen-independent” stage. In such cases androgen receptor expression is almost always maintained and much evidence exists to suggest that it may still be driving growth. One mechanism by which the receptor is thought to remain active is mutation. This review summarises the present data on androgen receptor mutations in prostate cancer, and how such substitutions offer a growth advantage by affecting cofactor interactions or by reducing ligand specificity. Such alterations appear to have a subsequent effect upon gene expression suggesting that tumours may “behave” differently dependent upon the ligand promoting growth and if a mutation is present.

The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300. SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300. In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two ␣-helices. We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID). We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300. In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region. In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligandinduced activity. Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays.The nuclear receptors (NRs) are ligand-regulated transcription factors that mediate the effects of steroids, retinoids, and other lipophilic hormones on gene expression (32). In common with other transcriptional activators, NRs stimulate transcription by promoting the local modification of chromatin structure and recruitment of a preinitiation complex (59). This is achieved via two transcriptional activation functions (AF1 and AF2) which provide molecular surfaces for the recruitment of transcriptional coactivator proteins (17,28,36,60).The AF2 surfaces of the ligand binding domains (LBDs) of NRs appear to be the principal sites for coactivator recruitment. Far-Western experiments detected two major classes of proteins in nuclear extracts (with apparent molecular masses of 160 and 140 kDa) which bind to the LBD of the estrogen receptor (ER) in the presence of ligand (5, 14). At least three distinct p160 proteins have been identified, including steroid receptor coactivator 1 (SRC1) (39), transcription intermediary factor 2 (TIF2) (54) and its murine homologue GRIP1 (18), and p300-CBP cointegrator-associated protein (pCIP) (50), which is the mouse homologue of the human protein AIB1 (1), also known as ACTR (8), RAC3 (29), or TRAM1 (49). These proteins appear to be bona fide coactivators, as they enhance the activity of NRs in both in vitro and in vivo experimental systems. The p140 class appears to consist chiefly of the nuclear protein RIP140 (6). The function of RIP140 is unknown, although it has been shown to down-regulate NR-mediated transcription in transient-reporter assays, possibly via competition with p160s for the LBD (15,27,35,51). Other AF2 binding proteins ...

Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic β-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.