ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma

<p>Abstract</p> <p>Background</p> <p>Cancer cells display widespread changes in DNA methylation that may lead to genetic instability by global hypomethylation and aberrant silencing of tumor suppressor genes by focal hypermethylation. In turn, altered DNA methylation patterns have been used to identify putative tumor suppressor genes.</p> <p>Methods</p> <p>In a methylation screening approach, we identified <it>ECRG4 </it>as a differentially methylated gene. We analyzed different cancer cells for <it>ECRG4 </it>promoter methylation by COBRA and bisulfite sequencing. Gene expression analysis was carried out by semi-quantitative RT-PCR. The <it>ECRG4 </it>coding region was cloned and transfected into colorectal carcinoma cells. Cell growth was assessed by MTT and BrdU assays. ECRG4 localization was analyzed by fluorescence microscopy and Western blotting after transfection of an <it>ECRG4-eGFP </it>fusion gene.</p> <p>Results</p> <p>We found a high frequency of <it>ECRG4 </it>promoter methylation in various cancer cell lines. Remarkably, aberrant methylation of <it>ECRG4 </it>was also found in primary human tumor tissues, including samples from colorectal carcinoma and from malignant gliomas. <it>ECRG4 </it>hypermethylation associated strongly with transcriptional silencing and its expression could be re-activated <it>in vitro </it>by demethylating treatment with 5-aza-2'-deoxycytidine. Overexpression of <it>ECRG4 </it>in colorectal carcinoma cells led to a significant decrease in cell growth. In transfected cells, ECRG4 protein was detectable within the Golgi secretion machinery as well as in the culture medium.</p> <p>Conclusions</p> <p><it>ECRG4 </it>is silenced via promoter hypermethylation in different types of human cancer cells. Its gene product may act as inhibitor of cell proliferation in colorectal carcinoma cells and may play a role as extracellular signaling molecule.</p

Epigenetische Regulation im Wnt Signalweg

Der kanonische Wnt Signalweg reguliert wichtige zelluläre Prozesse wie die Zellproliferation und Differenzierung. Fehlregulationen des Wnt Signalwegs, die zu einer konstitutiven Aktivierung führen, werden häufig während der Krebsentstehung beobachtet. Epigenetische Regulationsmechanismen sind an der Kontrolle der Wnt-Zielgene beteiligt und können bei fehlerhafter Ausführung ebenfalls zur konstitutiven Aktivierungen des Wnt Signalwegs führen. Aus diesem Grund wurde in dieser Doktorarbeit die epigenetische Inaktivierung von Wnt-Inhibitoren in verschiedenen Tumorarten untersucht, wobei neue prognostische und diagnostische Marker für maligne Gliome identifiziert werden konnten. Darüber hinaus wurden in Kolonkarzinomzelllinien die Auswirkungen von Histon Deacetylase Inhibitoren (HDIs) auf den Wnt Signalweg analysiert, um die Wirksamkeit der HDIs bei der Therapie von soliden Tumoren zu überprüfen. Hierbei konnte ein möglicher Mechanismus für die HDI-Wirkung aufgestellt werden

Hepatic Stellate Cells Support Hematopoiesis and are Liver-Resident Mesenchymal Stem Cells

Background/Aims: Hematopoiesis can occur in the liver, when the bone marrow fails to provide an adequate environment for hematopoietic stem cells. Hepatic stellate cells possess characteristics of stem/progenitor cells, but their contribution to hematopoiesis is not known thus far. Methods: Isolated hepatic stellate cells from rats were characterized with respect to molecular markers of bone marrow mesenchymal stem cells (MSC) and treated with adipocyte or osteocyte differentiation media. Stellate cells of rats were further co-cultured with murine stem cell antigen-1+ hematopoietic stem cells selected by magnetic cell sorting. The expression of murine hematopoietic stem cell markers was analyzed by mouse specific quantitative PCR during co-culture. Hepatic stellate cells from eGFP+ rats were transplanted into lethally irradiated wild type rats. Results: Desmin-expressing stellate cells were associated with hematopoietic sites in the fetal rat liver. Hepatic stellate cells expressed MSC markers and were able to differentiate into adipocytes and osteocytes in vitro. Stellate cells supported hematopoietic stem/progenitor cells during co-culture similar to bone marrow MSC, but failed to differentiate into blood cell lineages after transplantation. Conclusion: Hepatic stellate cells are liver-resident MSC and can fulfill typical functions of bone marrow MSC such as the differentiation into adipocytes or osteocytes and support of hematopoiesis

Stellate cells from rat pancreas are stem cells and can contribute to liver regeneration.

The identity of pancreatic stem/progenitor cells is still under discussion. They were suggested to derive from the pancreatic ductal epithelium and/or islets. Here we report that rat pancreatic stellate cells (PSC), which are thought to contribute to pancreatic fibrosis, have stem cell characteristics. PSC reside in islets and between acini and display a gene expression pattern similar to umbilical cord blood stem cells and mesenchymal stem cells. Cytokine treatment of isolated PSC induced the expression of typical hepatocyte markers. The PSC-derived hepatocyte-like cells expressed endodermal proteins such as bile salt export pump along with the mesodermal protein vimentin. The transplantation of culture-activated PSC from enhanced green fluorescent protein-expressing rats into wild type rats after partial hepatectomy in the presence of 2-acetylaminofluorene revealed that PSC were able to reconstitute large areas of the host liver through differentiation into hepatocytes and cholangiocytes. This developmental fate of transplanted PSC was confirmed by fluorescence in situ hybridization of chromosome Y after gender-mismatched transplantation of male PSC into female rats. Transplanted PSC displayed long-lasting survival, whereas muscle fibroblasts were unable to integrate into the host liver. The differentiation potential of PSC was further verified by the transplantation of clonally expanded PSC. PSC clones maintained the expression of stellate cell and stem cell markers and preserved their differentiation potential, which indicated self-renewal potential of PSC. These findings demonstrate that PSC have stem cell characteristics and can contribute to the regeneration of injured organs through differentiation across tissue boundaries

Epigenetic Changes during Hepatic Stellate Cell Activation

<div><p>Background and Aims</p><p>Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during <i>in vitro</i> activation of HSC.</p><p>Methods and Results</p><p>The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.</p><p>Conclusions</p><p>In summary, <i>in vitro</i> activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.</p></div

Analysis of stem/progenitor cell-associated factors in PSC by Western blot.

<p>The expression of CD133 was analyzed in cell membrane fractions of PSC and UCBSC. PSC of three different cell isolations, which were cultured for 7 days (7d), and UCBSC of three different clones were taken for the immunoblot. To indicate cell membrane protein fractions, annexin II served as a control. The notch1 receptor was detected predominantly in freshly isolated PSC cultured for 1 day (1d) and to a lesser extent also in culture-activated PSC (7d) as well as UCBSC. Nuclear localization of β-catenin in PSC and UCBSC indicated active β-catenin-dependet Wnt signaling as investigated by Western blot of nuclear protein fractions. In line with this, the Wnt target gene <i>PITX2</i> was also detected in both cell types. The stem/progenitor cell-associated numb1/3 isoforms were detected in PSC and UCBSC, whereas numb2/4 remained undetectable in both cell types. Numb2/4 isoforms were found in lysates of whole liver.</p

PSC can differentiate into hepatocyte-like cells <i>in vitro</i>.

<p>PSC were expanded for 7 days in culture and subsequently cultured for additional 14 days in (<b>A</b>) IMDM supplemented with 10% FCS (control medium) or (<b>B</b>) IMDM containing cytokines (FGF₄, HGF), linoleic acid-albumin and ITS (hepatocyte differentiation medium). (<b>B</b>) Hepatocyte-like cells appeared in primary cultures of PSC during cytokine treatment. (<b>C</b>) Under control conditions primary cultured PSC did not express the hepatocyte marker cytokeratin 18 (CK18) as investigated by immunofluorescence (red), but (<b>D</b>) CK18 synthesis was induced after treatment with hepatocyte differentiation medium for 14 days. (<b>E</b>) The hepatocyte-associated bile salt export pump (BSEP; red) remained undetectable in vimentin-expressing PSC (green) of the control, whereas (<b>F</b>) BSEP and vimentin were detectable in PSC treated with hepatocyte differentiation medium. The cell nuclei were marked by DAPI (blue). (<b>G</b>) Molecular markers of stellate cells (GFAP, α-SMA) as well as of immature and mature hepatocytes (HHEX, CYP7A1, α-fetoprotein, albumin, CX32, HNF1α, HNF4α, HNF6, MRP2) were analyzed by RT-PCR in the control (IMDM and 10% FCS; left column) and cytokine treated PSC (FGF₄, HGF, linoleic acid-albumin and ITS; right column) after 14 days of culture. The induction of notch3 and genes typically expressed by liver parenchymal cells indicated cell differentiation. (<b>H</b>) This cell differentiation potential was also preserved by the PSC clones, which displayed a comparable expression pattern after cytokine treatment for 7 days (right column). In the control, clonally expanded PSC were cultured in medium without cytokines (IMDM and 10% FCS; left column). (<b>I</b>) The PSC clone displayed the morphology of myofibroblast-like cells (control), but (<b>J</b>) developed into hepatocyte-like cells after treatment with hepatocyte differentiation medium for 7 days. (<b>K</b>) In this experimental setup, the release of albumin was measured by a rat-specific albumin ELISA. High amounts of albumin were secreted by primary cultured PSC and the PSC clone 2F5 after treatment with hepatocyte differentiation medium, whereas rat albumin was not detected (n.d.) under control conditions or in experimental media without cells (n = 3).</p