Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism

In cancer cells, global genomic hypomethylation is found together with localized hypermethylation of CpG islands within the promoters and regulatory regions of silenced tumor suppressor genes. Demethylating agents may reverse hypermethylation, thus promoting gene re-expression. Unfortunately, demethylating strategies are not efficient in solid tumor cells. DNA demethylation is mediated by ten-eleven translocation enzymes (TETs). They sequentially convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is associated with active transcription; 5-formylcytosine; and finally, 5-carboxylcytosine. Although α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, the major n-3 polyunsaturated fatty acids, have anti-cancer effects, their action, as DNA-demethylating agents, has never been investigated in solid tumor cells. Here, we report that EPA demethylates DNA in hepatocarcinoma cells. EPA rapidly increases 5hmC on DNA, inducing p21Waf1/Cip1 gene expression, which slows cancer cell-cycle progression. We show that the underlying molecular mechanism involves TET1. EPA simultaneously binds peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), thus promoting their heterodimer and inducing a PPARγ-TET1 interaction. They generate a TET1-PPARγ-RXRα protein complex, which binds to a hypermethylated CpG island on the p21 gene, where TET1 converts 5mC to 5hmC. In an apparent shuttling motion, PPARγ and RXRα leave the DNA, whereas TET1 associates stably. Overall, EPA directly regulates DNA methylation levels, permitting TET1 to exert its anti-tumoral function.-Ceccarelli, V., Valentini, V., Ronchetti, S., Cannarile, L., Billi, M., Riccardi, C., Ottini, L., Talesa, V. N., Grignani, F., Vecchini, A., Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism

Epigenetic reprogramming of breast cancer cells by valproic acid occurs regardless of estrogen receptor status

Estrogen receptors (ERs) are a recognized prognostic factor and therapeutic target in breast cancer. The loss of ER expression relates to poor prognosis, poor clinical outcome and impairs the use of anti-estrogenic treatment. Histone deacetylase inhibitors are candidate drugs for cancer therapy. Among them, valproic acid (VPA) is a long used and safe anti-epileptic drug. We studied the biological consequences of the chromatin remodeling action of VPA in a normal human mammary epithelial cell line and in ERα-positive and ERα-negative breast cancer cell lines. In these cells and regardless of their ER status, VPA-induced cell differentiation, as shown by increased milk lipids production, decreased expression of the CD44 antigen and growth arrest in the G0-G1 phase of the cell cycle. These effects were accompanied by decreased Rb phosphorylation, hyperacetylation of the p21WAF1/CIP1 gene promoter and increased p21 protein expression. Only in breast cancer cells, cyclin B1 expression was decreased and the cells accumulated also in G2. ERα expression decreased in ERα-positive, increased in ERα-negative and was unchanged in normal mammary epithelial cells, as did the expression of progesterone receptor, a physiological ERα target. VPA decreased the expression of the invasiveness marker pS2 in ERα-positive breast cancer cells, but did not cause its re-expression in ERα-negative cells. Overall, these data suggest that in both ERα-positive and -negative malignant mammary epithelial cells VPA reprograms the cells to a more differentiated and "physiologic" phenotype that may improve the sensitivity to endocrine therapy and/or chemotherapy in breast cancer patients. © 2008 Elsevier Ltd. All rights reserved

Transcriptional targeting by microRNA-Polycomb complexes A novel route in cell fate determination

Advances in the understanding of the epigenetic events underlying the regulation of developmental genes expression and cell lineage commitment are revealing novel regulatory networks. These also involve distinct components of the epigenetic pathways, including chromatin histone modification, DNA methylation, repression by polycomb complexes and microRNAs. Changes in chromatin structure, DNA methylation status and microRNA expression levels represent flexible, reversible and heritable mechanisms for the maintenance of stem cell states and cell fate decisions. We recently provided novel evidence showing that microRNAs, besides determining the post-transcriptional gene silencing of their targets, also bind to evolutionarily conserved complementary genomic seed-matches present on target gene promoters. At these sites, microRNAs can function as a critical interface between chromatin remodeling complexes and the genome for transcriptional gene silencing. Here, we discuss our novel findings supporting a role of the transcriptional chromatin targeting by polycomb-microRNA complexes in lineage fate determination of human hematopoietic cells

Targeting fusion protein/corepressor contact restores differentiation response in leukemia cells

Correction to: The EMBO Journal (2005) 24, 1232–1242. doi:10.1038/sj.emboj.760059

Timing, Thrusting Mode, and Negative Inversion Along the Circeo Thrust, Apennines, Italy: How the Accretion-To-Extension Transition Operated During Slab Rollback

The evolution of the Apennine wedge has seen the time-space migration of the forebulge, foredeep, thrust wedge, and back-arc extension phases in the wake of the Eastward rollback of the subducting Adria slab. In this framework, thrusting and post-orogenic extensional faulting have occurred in two parallel forelandward-migrating ribbons, with extensional deformation overprinting or partly exploiting anisotropies of the inherited thrust system. Here, we explore the tectonic framework and the timing of thrusting and subsequent negative inversion of the Circeo thrust, one of the major thrusts in the inner portion of the central Apennines, with the main aim to constrain the timing and mode of the compression to extension switch. Structural analysis, carbonate C and O and clumped isotopes analysis, X-ray diffraction of clay minerals, and U-Pb dating of calcite slickenfibers have been integrated with seismic interpretation, cross-section balancing, and 1D burial and thermal modeling. We show that the Circeo thrust developed during Langhian-Serravallian time. Its extensional reactivation is dated at the Serravallian, during the stacking of an underlying thrust slice, before the onset of Pliocene back-arc extension in the area. Combination of our data with the age of thrusts, extensional basins, and base of the foredeep infill of the central Apennines, demonstrates that forelandward migration of the foredeep-thrust system occurred at variable velocities. Accelerations and decelerations are synchronous, respectively, with the opening of the Liguro-Provençal and Tyrrhenian back-arc basins and with the interluding quiescent period.ISSN:1944-919

Effect of fatty acids on Ras, ERK1/2, and phospho-C/EBPβ protein levels.

<p>(A) U937 cells were treated with 100 µM fatty acids (OA, oleic; LA, linoleic; LNA, α-linolenic; AA, arachidonic; EPA, eicosapentaenoic; DHA, docosahexaenoic) for 24 hours. Total cell lysates or isolated non raft membrane fractions (50 µg protein) were subjected to Western blotting with the indicated antibodies. Pan-Ras Ab was used to detected all Ras isoforms. For each protein, one representative out of three experiments is reported. (B) Quantitative analysis. The chart shows normalized Western blot band densities, presented as fold induction with respect to U937 control cells. Images of independent blots were acquired using the Versadoc Imaging System and signals were quantified using Quantity One Software. Data are the means ± S.D. of three independent experiments. (*, p<0.05)</p

Influence of intron 1 CpG island B demethylation on RNAPII and p53 binding to H-Ras gene.

<p>(A) Schematic representation of H-Ras exon 1, intron 1, and exon 2. (B) ChIP was performed in control (<i>white bars</i>), OA (<i>gray bars</i>), and EPA treated (<i>black bars</i>) U937 cells, using RNAPII Ab. qRT-PCR was performed using specific primers for exon 1, intron 1 region C, CpG island B, intron 1 region D, and exon 2. The results shown are the mean ± SD of three independent experiments. (C) ChIP was performed as in (B) using a p53 Ab. The region within CpG island B containing the p53 element was amplified by qRT-PCR. The results shown are the mean ± SD of three independent experiments. (D) PCR of DNA from p53 Ab immunoprecipitated complex. Input, fragmented DNA before immunoprecipitation, negative controls rIgG. One representative out of three experiments is shown.</p

EPA increases H-Ras exon 2 transcription and demethylates intron 1 CpG island B.

<p>(A) mRNA content was evaluated for H-Ras exon 1 and exon 2 after 1-,3-, and 24-h treatment with 100 µM fatty acids using qRT-PCR. <i>White bars</i>, control U937 cells; <i>gray bars</i>, OA; <i>black bars</i>, EPA. Data are presented as relative expression by calculating 2^−ΔΔCt normalized to untreated U937 cells. The means ± S.D. of three separate experiments are shown (*, p<0.01). (B) H-Ras intron 1 CpG island B sequence. The underlined sequences indicate the forward and reverse primers utilized for cloning and sequencing after bisulfite reaction. The cloned fragment (242 bp) contains 26 CpGs (in bold). The nucleotide sequences outside the CpG island B are in italics. (C) Sequencing of individual clones generated by PCR after bisulfite reaction. Black and white square represent methylated and unmethylated CpGs, respectively.</p

Effect of EPA on Ras isoforms expression.

<p>mRNA content was evaluated for H-Ras, N-Ras, and K-Ras after 1-, 3-, and 24-h treatment with 100 µM fatty acids, using qRT-PCR. <i>White bars</i>, control U937; <i>gray bars</i>, OA; <i>black bars</i>, EPA. Data are presented as relative expression by calculating 2^−ΔΔCt normalized to untreated U937 cells. The means ± S.D. of three separate experiments are shown (*, p<0.01).</p