Although Apc is well characterized as a tumor-suppressor gene in the intestine, the precise mechanism of this suppression remains to be defined. Using a novel inducible Ahcre transgenic line in conjunction with a loxPflanked Apc allele we, show that loss of Apc acutely activates Wnt signaling through the nuclear accumulation of -catenin. Coincidentally, it perturbs differentiation, migration, proliferation, and apoptosis, such that Apc-deficient cells maintain a "crypt progenitor-like" phenotype. Critically, for the first time we confirm a series of Wnt target molecules in an in vivo setting and also identify a series of new candidate targets within the same setting.Supplemental material is available at http://www.genesdev.org.
Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.Analysis of histone-modifying enzymes such as the histone acetyltransferases (HATs) 1 and deacetylases has resulted in significant advances in our understanding of transcriptional regulation (1-4). These studies have resulted in a model of transcription in which transcriptionally competent genes are transcribed or repressed dependent upon their ability to recruit either HATs or histone deacetylases to the promoter (4). In these models, recruited histone acetyltransferases associate with transcription factor complexes (5-8), resulting in the acetylation of nucleosomal histones, relaxation of nucleosomal integrity, and hence transcription. Conversely, transcriptional repression occurs when histone deacetylases (and cofactors) are recruited to DNA-bound transcription factors, resulting in the removal of acetyl groups from NH 2 -terminal lysines causing a "tightening" of nucleosomal integrity and a suppression of transcription (9 -13).The isolation and synthesis of new and potent inhibitors of histone deacetylase enzymes (HDACs) has allowed us to identify some of the biological outcomes resulting from manipulation of histone deacetylase activity. For example, it is now established that treatment of cells in vitro and in vivo with HDAC inhibitors can result in specific functional outcomes such as cell cycle arrest (14 -16), apoptosis/cell death (17-19), or differentiation (19 -21). These outcomes to a large extent are cell type-specific and have raised the potential that the HDAC inhibitors may represent a new and important class of anticancer therapeutic agents (4).HDAC inhibitors comprise a diverse range of unrelated compounds that...
The Neurokinin 1 receptor is a member of the tachykinin receptor sub‐family. It is distributed throughout the CNS, although to a lesser extent than its natural agonist, Substance P.There is much evidence to implicate the NK1 receptor within the physiology of acute pain, such as the anatomical localisation of Substance P and NK1 receptors within lamina I of the spinal cord, as well as correlative increases in Substance P production and NK1 receptor internalisation in response to increasing noxious stimuli. Reproduction of in vivo efficacy in the clinic has, however, been unsuccessful.Selective NR2 subunit mediated NMDA antagonism has also shown in vivo efficacy, expression of this subunit within the superficial lamina of the spinal cord adding anatomical evidence to support this mechanism. However, co‐expression of the NK1 receptor and the NMDA receptor has not been reported.We have developed a methodology to allow the isolation of high quality RNA, via Laser Capture Microdissection, following the immunolabelling of specific proteins of interest.Using this technology, we validated methodology via PCR of dissected samples, as well as looking at differential expression of the NR2A and NR2B receptor subunits on NK1 +ve and NK1 –ve samples taken from the rat striatum.
The aim of the study was to explore novel tissue based techniques to generate gene expression profiles of both afferent (sensory) and efferent (motor) neurons that specifically innervate the rat bladder and urethra.Retrograde neuronal tracing studies in combination with laser capture microdissection (LCM) and microarray analysis were used to generate gene expression profiles of both afferent and efferent neurons that specifically innervate the rat bladder and urethra. A complete rat study (n=12) was performed in which fluorescently labelled cholera toxin B was injected into the bladder and urethra. Traced neurons were isolated by LCM, RNA was extracted, amplified and analysed by microarray.Extensive analysis of all the data derived from this study give very sensitive and powerful gene expression data from only 50–100 microdissected neurons. Expression of sensory neuron specific markers could be confirmed in dorsal root ganglion neurons whereas expression of genes involved in motor function of the lower urinary tract could be confirmed in efferent neurons e.g. Onuf's Nucleus.The resulting gene expression profile will increase our understanding of signalling pathways involved in the control of the lower urinary tract. Data derived from this study will not only be relevant for safety and efficacy but will also enable us to identify targets for the treatment of urinary frequency and urge incontinence (UFUI).
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