The E-cadherin repressor slug and progression of human extrahepatic hilar cholangiocarcinoma

<p>Abstract</p> <p>Objectives</p> <p>This study explored the expression and function of Slug in human extrahepatic hilar cholangiocarcinoma (EHC) to identify its role in tumor progression.</p> <p>Methods</p> <p>The expression of Snail and Slug mRNA in 52 human tissue samples of EHC was investigated. The mRNA of Snail and Slug were quantified using reverse transcriptase-PCR, and correlations with E-cadherin expression and clinicopathological factors were investigated. We then investigated transfection of Slug cDNA in endogenous E-cadherin-positive human EHC FRH0201 cells, selectively induced the loss of E-cadherin protein expression, and then small interfering RNA (siRNA) for inhibition of Slug expression in endogenous Slug-positive human EHC QBC939 cells, selectively induced the loss of Slug protein expression. A Boyden chamber transwell assay was used for invasion.</p> <p>Results</p> <p>Slug mRNA was overexpressed in 18 cases (34.6%) of EHC compared with adjacent noncancerous tissue. E-Cadherin protein expression determined in the same 52 cases by immunohistochemistry was significantly down-regulated in those cases with Slug mRNA overexpression (P = 0.0001). The tumor and nontumor ratio of Slug mRNA was correlated with nodal metastasis(p = 0.0102), distant metastasis (p = 0.0001)and Survival time(p = 0.0443). However, Snail mRNA correlated with neither E-cadherin expression nor tumor invasiveness. By inhibiting Slug expression by RNA interference, we found that reduced Slug levels upregulated E-cadherin and decreased invasion in QBC939 cell. When the QBC939 cells was infected with Slug cDNA,, significant E-cadherin was downregulated and increased invasion in QBC939 cell.</p> <p>Conclusions</p> <p>The results suggested that Slug expression plays an important role in both the regulation of E-cadherin expression and in the acquisition of invasive potential in human EHC. Slug is possibly a potential target for an antitumor therapy blocking the functions of invasion and metastasis in human EHCs.</p

Functional epigenomics approach to identify methylated candidate tumour suppressor genes in renal cell carcinoma

Promoter region hypermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many human cancers. Previously, to identify candidate epigenetically inactivated TSGs in renal cell carcinoma (RCC), we monitored changes in gene expression in four RCC cell lines after treatment with the demethylating agent 5-azacytidine. This enabled us to identify HAI-2/SPINT2 as a novel epigenetically inactivated candidate RCC TSG. To identify further candidate TSGs, we undertook bioinformatic and molecular genetic evaluation of a further 60 genes differentially expressed after demethylation. In addition to HAI-2/SPINT2, four genes (PLAU, CDH1, IGFB3 and MT1G) had previously been shown to undergo promoter methylation in RCC. After bioinformatic prioritisation, expression and/or methylation analysis of RCC cell lines±primary tumours was performed for 34 genes. KRT19 and CXCL16 were methylated in RCC cell lines and primary RCC; however, 22 genes were differentially expressed after demethylation but did not show primary tumour-specific methylation (methylated in normal tissue (n=1); methylated only in RCC cell lines (n=9) and not methylated in RCC cell lines (n=12)). Re-expression of CXCL16 reduced growth of an RCC cell line in vitro. In a summary, a functional epigenomic analysis of four RCC cell lines using microarrays representing 11 000 human genes yielded both known and novel candidate TSGs epigenetically inactivated in RCC, suggesting that this is valid strategy for the identification of novel TSGs and biomarkers

Paired Associative Stimulation of the Auditory System: A Proof-Of-Principle Study

Background Paired associative stimulation (PAS) consisting of repeated application of transcranial magnetic stimulation (TMS) pulses and contingent exteroceptive stimuli has been shown to induce neuroplastic effects in the motor and somatosensory system. The objective was to investigate whether the auditory system can be modulated by PAS. Methods Acoustic stimuli (4 kHz) were paired with TMS of the auditory cortex with intervals of either 45 ms (PAS(45 ms)) or 10 ms (PAS(10 ms)). Two-hundred paired stimuli were applied at 0.1 Hz and effects were compared with low frequency repetitive TMS (rTMS) at 0.1 Hz (200 stimuli) and 1 Hz (1000 stimuli) in eleven healthy students. Auditory cortex excitability was measured before and after the interventions by long latency auditory evoked potentials (AEPs) for the tone (4 kHz) used in the pairing, and a control tone (1 kHz) in a within subjects design. Results Amplitudes of the N1-P2 complex were reduced for the 4 kHz tone after both PAS(45 ms) and PAS(10 ms), but not after the 0.1 Hz and 1 Hz rTMS protocols with more pronounced effects for PAS(45 ms). Similar, but less pronounced effects were observed for the 1 kHz control tone. Conclusion These findings indicate that paired associative stimulation may induce tonotopically specific and also tone unspecific human auditory cortex plasticity

Global profiling of histone and DNA methylation reveals epigenetic-based regulation of gene expression during epithelial to mesenchymal transition in prostate cells

<p>Abstract</p> <p>Background</p> <p>Previously we reported extensive gene expression reprogramming during epithelial to mesenchymal transition (EMT) of primary prostate cells. Here we investigated the hypothesis that specific histone and DNA methylations are involved in coordination of gene expression during EMT.</p> <p>Results</p> <p>Genome-wide profiling of histone methylations (H3K4me3 and H3K27me3) and DNA methylation (DNAMe) was applied to three cell lines at different stages of a stepwise prostate cell model involving EMT and subsequent accumulation of malignant features. Integrated analyses of epigenetic promoter modifications and gene expression changes revealed strong correlations between the dynamic changes of histone methylations and gene expression. DNA methylation was weaker associated with global gene repression, but strongly correlated to gene silencing when genes co-modified by H3K4me3 were excluded. For genes labeled with multiple epigenetic marks in their promoters, the level of transcription was associated with the net signal intensity of the activating mark H3K4me3 minus the repressive marks H3K27me3 or DNAMe, indicating that the effect on gene expression of bivalent marks (H3K4/K27me3 or H3K4me3/DNAMe) depends on relative modification intensities. Sets of genes, including epithelial cell junction and EMT associated fibroblast growth factor receptor genes, showed corresponding changes concerning epigenetic modifications and gene expression during EMT.</p> <p>Conclusions</p> <p>This work presents the first blueprint of epigenetic modifications in an epithelial cell line and the progeny that underwent EMT and shows that specific histone methylations are extensively involved in gene expression reprogramming during EMT and subsequent accumulation of malignant features. The observation that transcription activity of bivalently marked genes depends on the relative labeling intensity of individual marks provides a new view of quantitative regulation of epigenetic modification.</p

A Genome-wide Linkage Analysis and Mutation Analysis of Hereditary Congenital Blepharoptosis in a Japanese Family

Hereditary congenital ptosis (PTOS) is defined as drooping of the upper eyelid without any other accompanying symptoms and distinguished from syndromic blepharoptosis.Two previous linkage analyses assigned a PTOS locus (PTOS1) to 1p32-p34.1 and another (PTOS2) to Xq24-q27.1. In addition, in a sporadic case with a balanced chromosomal translocation t(1;8)(p34.3;q21.12), the ZFHX4 (zinc finger homeodomain 4) gene was found to be disrupted at the 8q21.12 breakpoint, but there was no gene at the 1p34.3 breakpoint, suggesting the existence of the third PTOS locus (PTOS1) at 8q21.12. We carried out a genome-wide linkage analysis in a Japanese PTOS family and calculated two-point and multipoint LOD scores with reduced penetrance. Haplotype analysis gave three candidate disease-responsible regions, i.e., 8q21.11-q22.1, 12q24.32-q24.33 and 14q21.1-q23.2. Although the family size is too small to define one of them, 8q21.11-q22.1 is a likely candidate region, because it contains the previously reported translocation breakpoint above. We thus performed mutation, Southern-blot and methylation analyses of ZFHX4, but could not find any disease specific change in the family. Nevertheless, our data may support the localization of PTOS1.長崎大学学位論文 学位記番号:博（医歯薬）甲第153号 博士（医学）学位授与年月日:平成20年3月19

Cystinosin, MPDU1, SWEETs and KDELR Belong to a Well-Defined Protein Family with Putative Function of Cargo Receptors Involved in Vesicle Trafficking

Classification of proteins into families based on remote homology often helps prediction of their biological function. Here we describe prediction of protein cargo receptors involved in vesicle formation and protein trafficking. Hidden Markov model profile-to-profile searches in protein databases using endoplasmic reticulum lumen protein retaining receptors (KDEL, Erd2) as query reveal a large and diverse family of proteins with seven transmembrane helices and common topology and, most likely, similar function. Their coding genes exist in all eukaryota and in several prokaryota. Some are responsible for metabolic diseases (cystinosis, congenital disorder of glycosylation), others are candidate genes for genetic disorders (cleft lip and palate, certain forms of cancer) or solute uptake and efflux (SWEETs) and many have not yet been assigned a function. Comparison with the properties of KDEL receptors suggests that the family members could be involved in protein trafficking and serve as cargo receptors. This prediction sheds new light on a range of biologically, medically and agronomically important proteins and could open the way to discovering the function of many genes not yet annotated. Experimental testing is suggested

Characterisation of the GRAF gene promoter and its methylation in patients with acute myeloid leukaemia and myelodysplastic syndrome

We report the isolation of the 5′ flanking region of GRAF (GTPase regulator associated with the focal adhesion kinase), previously described as a putative tumour suppressor gene of acute myelogenous leukaemia and myelodysplastic syndrome, and demonstrate its promoter activity in reporter gene assays. Two putative protein-binding sites are identified of which one was sensitive to CpG methylation. The suppressed GRAF expression could be restored in leukaemia cell lines by treatment with a demethylating agent and an inhibitor of histone deacetylases. In contrast to normal tissues, which tested negative for GRAF promoter methylation, 11 of 29 (38%) bone marrow samples from patients with acute myeloid leukaemia or myelodysplastic syndrome were positive

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions