EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation

<p>Abstract</p> <p>Background</p> <p>Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed <it>EFS </it>(<it>embryonal Fyn-associated substrate</it>) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated <it>EFS </it>promoter region CpG island in tumor classification and metastatic progression.</p> <p>Methods</p> <p><it>EFS </it>methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients.</p> <p>Results</p> <p>Analysis of 16 UM showed full methylation of the <it>EFS </it>CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with <it>EFS </it>methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with <it>EFS </it>methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of <it>EFS </it>mRNA expression with <it>EFS </it>methylation in UM. We further found that <it>EFS </it>methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. <it>EFS </it>methylation always affects both alleles in normal and tumor samples.</p> <p>Conclusions</p> <p>Biallelic <it>EFS </it>methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The <it>EFS </it>methylation of a UM may depend on which type of precursor cell the tumor originated from.</p

Investigating the effects of vitreous humour (crude extract) on growth and differentiation of rat mesenchymal stem cells (rMSCs) and human NTERA2 cells

It is very well documented that retinoic acid (RA) reduces growth rate by induction of cell differentiation in certain conditions and cell lines. On the other hand, hyaluronic acid (HA) is known for its growth induction on cultured cells. A natural source of HA, rabbit vitreous humour (VH), was previously shown to promote wound repair in model animals. In search for its possible mechanisms, VH extract was tested on the cultured mesenchymal stem cells and NTERA2 as human embryonal carcinoma cells in the presence of RA. Changes in some cellular and molecular markers (A2B5, Oct4, Sox2) showed that VH and possibly HA interfere with differentiating effects of RA. Therefore, this reagent may affect cell proliferation and tissue regeneration by inhibition of cell differentiation.Хорошо известно, что ретиноевая кислота (RA) снижает темпы роста, индуцируя дифференциацию клеточных линий в определенных условиях. Вместе с тем известно, что гиалуроновая кислота (HA) индуцирует рост культивируемых клеток. Ранее было показано, что естественный источник НА, стекловидное тело (VH) кролика, вызывает заживление ран у модельных животных. В поисках возможного механизма этого процесса экстракт стекловидного тела был исследован на культивируемых мезенхимальных стволовых клетках и клетках NTERA2 эмбриональной карциномы человека в присутствии RA. Изменения некоторых клеточных и молекулярных маркеров (A2B5, Oct4, Sox2) показали, что VH и, возможно, HA влияют на дифференцирующие эффекты RA. Таким образом, это вещество может влиять на пролиферацию клеток и регенерацию тканей, ингибируя дифференциацию клеток.Добре відомо, що ретиноєва кислота (RA) знижує темпи росту, індукуючи диференціацію кліткових ліній в певних умовах. Разом з тим відомо, що гіалуронова кислота (НА) індукує ріст культиво- ваних клітин. Раніше було показано, що природне джерело НА, склоподібне тіло (VH) кроля, викликає загоєння ран у модельних тварин. В пошуках можливого механізму цього процесу екстракт склоподібного тіла був досліджений на культивованих мезенхімальних стовбурових клітинах та клітинах NTERA2 ембріональної карциноми людини в присутності RA. Зміни деяких клітинних та молекулярних маркерів (A2B5, Oct4, Sox2) показали, що VH і, можливо, НА впливають на диференціюючі ефекти RA. Таким чином, ця речовина може впливати на проліферацію і регенерацію тканин, інгібуючи диференціацію клітин

Sequential DNA methylation of the Nanog and Oct-4 promoters in human NT-2 cells during neuronal differentiation.: J.Biol.Chem.

NRC publication: Ye

Glioblastoma-secreted factors induce IGFBP7 and angiogenesis by modulating Smad-2-dependent TGF-beta signaling

Insulin-like growth factor-binding protein 7 (IGFBP7) is a selective biomarker of glioblastoma (GBM) vessels, strongly expressed in tumor endothelial cells and vascular basement membrane. IGFBP7 gene regulation and its potential role in tumor angiogenesis remain unclear. Mechanisms of IGFBP7 induction and its angiogenic capacity were examined in human brain endothelial cells (HBECs) exposed to tumor-like conditions. HBEC treated with GBM cell (U87MG)-conditioned media (-CM) exhibited fourfold upregulation of IGFBP7 mRNA and protein compared to control cells. IGFBP7 gene regulation in HBEC was methylation independent. U87MG-CM analysed by enzyme-linked immunosorbent assay contained ~5\u2009pm transforming growth factor (TGF)-\u3b21, a concentration sufficient to stimulate IGFBP7 in HBEC to similar levels as U87MG-CM. Both pan-TGF-\u3b2-neutralizing antibody (1D11) and the TGF-\u3b21 receptor (activin receptor-like kinase 5, ALK5) antagonist, SB431542, blocked U87MG-CM-induced IGFBP7 expression in HBEC, indicating that TGF-\u3b21 is an important tumor-secreted effector capable of IGFBP7 induction in endothelial cells. HBEC exposed to either U87MG-CM or IGFBP7 protein exhibited increased capillary-like tube (CLT) formation in Matrigel. Both TGF-\u3b21- and U87MG-CM-induced Smad-2 phosphorylation and U87MG-CM-induced CLT formation in HBEC were inhibited by the ALK5 antagonist, SB431542. These data suggest that proangiogenic IGFBP7 may be induced in brain endothelial cells by TGF-\u3b2s secreted by GBM, most likely through TGF-\u3b21/ALK5/Smad-2 pathway.NRC publication: Ye

Epigenetic modifications of SOX2 enhancers, SRR1 and SRR2, correlate with in vitro neural differentiation: J.Neurosci.Res.

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2-D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell-type-specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell-type-specific epigenomes and suggest mechanisms by which they may be created and maintainedNRC publication: Ye

Dynamic DNA methylation and histone acetylation of Sox2 enhancers during the differenctiation of NT2 cells into neurons and astrocytes.

NRC publication: Ye

Involvement of NOS3 in RA-induced neural differentiation of human NT2/D1 cells

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5\u2032 regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter ( 12734 to 12989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.Peer reviewed: YesNRC publication: Ye