Methylation of NKG2D ligands contributes to immune system evasion in acute myeloid leukemia.

Engagement of the activating receptor NKG2D (natural killer group 2 member D) with its ligands (NKG2DL) major histocompatibility complex class I related-A and -B (MICA/B), UL-16 binding protein families (ULBPs 1-6) is important to ensure the innate immunity to tumor cells. However, these cells have developed strategies to downregulate NKG2DL expression and avoid immune recognition. We demonstrate that DNA methylation can contribute to the absence of NKG2DL expression during tumor progression. We analyzed the DNA methylation profiles for each NKG2DL by pyrosequencing in acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), hepatocellular carcinoma (HC), breast cancer and colon cancer cell lines. High levels of DNA methylation for NKG2DL were found in some tumor cell lines, mainly in AML cells. This hypermethylation was correlated with the absence of transcription for NKG2DL. Higher DNA methylation levels for MICA, ULBP1 and ULBP2 were observed in AML patients (n=60) compared with healthy donors (n=25). However, no DNA methylation for NKG2DL was found in colon cancer patients (n=44). Treatment with demethylating agents (5-azacytidine and 5-aza-2'-deoxycytidine) restored the expression of NKG2DL on the cell surface of AML cells, leading to an enhanced recognition by NKG2D-expressing cells. Our data suggest that NKG2DL may be aberrantly silenced by DNA methylation as a consequence of tumor development in AML patients.

LINE-1 methylation in leukocyte DNA, interaction with phosphatidylethanolamine N-methyltransferase variants and bladder cancer risk.

BACKGROUND: Aberrant global DNA methylation is shown to increase cancer risk. LINE-1 has been proven a measure of global DNA methylation. The objectives of this study were to assess the association between LINE-1 methylation level and bladder cancer risk and to evaluate effect modification by environmental and genetic factors. METHODS: Bisulphite-treated leukocyte DNA from 952 cases and 892 hospital controls was used to measure LINE-1 methylation level at four CpG sites by pyrosequencing. Logistic regression model was fitted to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Interactions between LINE-1 methylation levels and environmental and genetic factors were assessed. RESULTS: The risk of bladder cancer followed a nonlinear association with LINE-1 methylation. Compared with subjects in the middle tertile, the adjusted OR for subjects in the lower and the higher tertiles were 1.26 (95% CI 0.99-1.60, P=0.06) and 1.33 (95% CI 1.05-1.69, P=0.02), respectively. This association significantly increased among individuals homozygous for the major allele of five single-nucleotide polymorphisms located in the phosphatidylethanolamine N-methyltransferase gene (corrected P-interaction<0.05). CONCLUSIONS: The findings from this large-scale study suggest that both low and high levels of global DNA methylation are associated with the risk of bladder cancer.

Single cell-derived clones from human adipose stem cells present different immunomodulatory properties.

Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single-cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, five single-cell clones were isolated (generally called 1.X and 3.X) from two volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1·10 and 1·22 expressed the lowest amounts, while clones 3·10 and 3·5 expressed more CD105 than the rest and clone 1·7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of interleukin (IL)-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore, and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of proinflammatory cytokines such as IL-1ß, while clones 1·10 and 1·22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are, together, more potent inhibitors than clones 3.X for proliferation of total, CD3(+) T, CD4(+) T and CD8(+) T lymphocytes and natural killer (NK) cells. The results of this work indicate that the adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.

Effects of short-term high-fat overfeeding on genome-wide DNA methylation in the skeletal muscle of healthy young men.

AIMS/HYPOTHESIS: Energy-dense diets that are high in fat are associated with a risk of metabolic diseases. The underlying molecular mechanisms could involve epigenetics, as recent data show altered DNA methylation of putative type 2 diabetes candidate genes in response to high-fat diets. We examined the effect of a short-term high-fat overfeeding (HFO) diet on genome-wide DNA methylation patterns in human skeletal muscle. METHODS: Skeletal muscle biopsies were obtained from 21 healthy young men after ingestion of a short-term HFO diet and a control diet, in a randomised crossover setting. DNA methylation was measured in 27,578 CpG sites/14,475 genes using Illumina's Infinium Bead Array. Candidate gene expression was determined by quantitative real-time PCR. RESULTS: HFO introduced widespread DNA methylation changes affecting 6,508 genes (45%), with a maximum methylation change of 13.0 percentage points. The HFO-induced methylation changes were only partly and non-significantly reversed after 6-8 weeks. Alterations in DNA methylation levels primarily affected genes involved in inflammation, the reproductive system and cancer. Few gene expression changes were observed and these had poor correlation to DNA methylation. CONCLUSIONS/INTERPRETATION: The genome-wide DNA methylation changes induced by the short-term HFO diet could have implications for our understanding of transient epigenetic regulation in humans and its contribution to the development of metabolic diseases. The slow reversibility suggests a methylation build-up with HFO, which over time may influence gene expression levels.

Epigenetic regulation of the immune system in health and disease.

Epigenetics comprises various mechanisms that mold chromatin structures and regulate gene expression with stability, thus defining cell identity and function and adapting cells to environmental changes. Alteration of these mechanisms contributes to the inception of various pathological conditions. Given the complexity of the immune system, one would predict that a higher-order, supragenetic regulation is indispensable for generation of its constituents and control of its functions. Here, we summarize various aspects of immune system physiology and pathology in which epigenetic pathways have been implicated. Increasing knowledge in this field, together with the development of specific tools with which to manipulate epigenetic pathways, might form a basis for new strategies of immune function modulation, both to optimize immune therapies for infections or cancer and to control immune alterations in aging or autoimmunity.

Specific hypermethylation of LINE-1 elements during abnormal overgrowth and differentiation of human placenta.

In human post-natal somatic cells, low global levels of DNA methylation have been associated with the hypomethylation of several repetitive elements, a feature that has been proposed to be a surrogate epigenetic marker. These data, mainly derived from the analysis of cancer cells, suggest a potential association between loss of cell-growth control and altered differentiation with hypomethylation of repetitive sequences. Partial hydatidiform moles (PHMs) can be used as an alternative model for investigating this association in a non-tumorigenic context. This gestational disease is characterized by abnormal overgrowth and differentiation of the placenta and spontaneous abortion. Here, we comprehensively analyse the DNA methylation of these trophoblastic tissues in both PHM and normal placenta at global and sequence-specific levels. Analysis of the global 5-methylcytosine content and immunohistochemistry indicate that PHM and normal placenta have identical global levels of DNA methylation. In contrast, bisulfite genomic sequencing shows that, whereas Alu, NBL2 and satellite 2 repetitive elements are equally methylated, LINE-1 sequences are hypermethylated in PHM tissues ( approximately 2-fold relative to normal placenta). Interestingly, altered demethylation is also found in triploid diandric embryos that originate from dispermic fertilization of an oocyte, a common event responsible for most PHMs. In conclusion, alterations of DNA methylation do not seem to be randomly distributed in PHM, as several repeated elements remain unaltered, whereas LINE-1 sequences are hypermethylated. In addition, our findings suggest that the hypomethylation of repetitive elements in cancer is directly linked to the neoplasic process and not a simple consequence of loss of growth control observed in most of the cancer cells.

Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer.

Colorectal cancer is a major cause of cancer death worldwide. A number of key oncogenes and tumor suppressor genes have been proposed to drive progression from healthy colonic epithelia to malignant tumors, including members of the Wnt/beta-catenin pathway. Recently, CpG island promoter hypermethylation was shown to cause inactivation of two extracellular Wnt inhibitors in colon cancer: secreted frizzled-related proteins (sFRPs) and Wnt inhibitory factor-1 (WIF-1). Here, we show for the first time that another extracellular Wnt inhibitor, the DICKKOPF-1 (DKK-1) gene, is transcriptionally silenced by CpG island promoter hypermethylation in colon cancer cell lines (n=9), whereas treatment with the DNA-demethylating agent 5-aza-2-deoxycytidine restored DKK-1 expression. Restoration of DKK-1 function in non-expressing cells bearing a truncated APC (Adenomatous Polyposis Coli) gene had no effect on beta-catenin/T-cell factor-dependent transcription, but induced tumor suppressor-like features such as reduced colony formation density and tumor growth inhibition in nude mice. These results suggest additional functions for DKK-1 other than inhibiting canonical Wnt signaling. In primary colorectal tumors, DKK-1 was found hypermethylated in 17% (nine of 54) of cases. Furthermore, while for both SFRP-1 and WIF-1 methylation-associated silencing occurred across the whole spectrum of colorectal tumorigenesis, DKK-1 promoter was selectively hypermethylated in advanced colorectal neoplasms (Duke's C and D tumors).

The role of 5-hydroxymethylcytosine in development, aging and age-related diseases.

DNA methylation at the fifth position of cytosines (5mC) represents a major epigenetic modification in mammals. The recent discovery of 5-hydroxymethylcytosine (5hmC), resulting from 5mC oxidation, is redefining our view of the epigenome, as multiple studies indicate that 5hmC is not simply an intermediate of DNA demethylation, but a genuine epigenetic mark that may play an important functional role in gene regulation. Currently, the availability of platforms that discriminates between the presence of 5mC and 5hmC at single-base resolution is starting to shed light on the functions of 5hmC. In this review, we provide an overview of the genomic distribution of 5hmC, and examine recent findings on the role of this mark and the potential consequences of its misregulation during three fundamental biological processes: cell differentiation, cancer and aging.

Checkpoint kinase 1 (CHK1) protein and mRNA expression is downregulated in aggressive variants of human lymphoid neoplasms.

CHK1: gene encodes for a serine/threonine kinase involved in the regulation of cell cycle progression and DNA damage checkpoints. To determine the role of CHK1 in the pathogenesis of lymphoid neoplasms and its relationship to other DNA damage response genes, we have analyzed the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid tissues. CHK1 protein and mRNA expression levels were very low in both reactive tissues and resting lymphoid cells, whereas tumor samples showed a variable pattern of expression related to their proliferative activity. However, seven aggressive tumors showed a dissociate pattern of extremely low or negative protein expression in spite of a high proliferative activity. Four of these tumors were diffuse large B-cell lymphomas (DLCLs) with concordant reduced levels of mRNA, whereas one blastoid mantle cell lymphoma (B-MCL) and two DLCLs had relatively normal levels of mRNA. No gene mutations, deletions, or hypermethylation of the promoter region were detected in any of these cases. In all these tumors ATM, CHK2, and p53 genes were wild type. These findings suggest that CHK1 inactivation in NHLs occurs by loss of protein expression in a subset of aggressive variants alternatively to ATM, CHK2, and p53 alterations.

The effect of exposure to nanoparticles and nanomaterials on the mammalian epigenome.

Human exposure to nanomaterials and nanoparticles is increasing rapidly, but their effects on human health are still largely unknown. Epigenetic modifications are attracting ever more interest as possible underlying molecular mechanisms of gene-environment interactions, highlighting them as potential molecular targets following exposure to nanomaterials and nanoparticles. Interestingly, recent research has identified changes in DNA methylation, histone post-translational modifications, and noncoding RNAs in mammalian cells exposed to nanomaterials and nanoparticles. However, the challenge for the future will be to determine the molecular pathways driving these epigenetic alterations, the possible functional consequences, and the potential effects on health.

Liver X Receptor Agonist Modifies the DNA Methylation Profile of Synapse and Neurogenesis-Related Genes in the Triple Transgenic Mouse Model of Alzheimer's Disease.

The liver X receptor agonist, GW3965, improves cognition in Alzheimer's disease (AD) mouse models. Here, we determined if short-term GW3965 treatment induces changes in the DNA methylation state of the hippocampus, which are associated with cognitive improvement. Twenty-four-month-old triple-transgenic AD (3xTg-AD) mice were treated with GW3965 (50 mg/kg/day for 6 days). DNA methylation state was examined by modified bisulfite conversion and hybridization on Illumina Infinium Methylation BeadChip 450 k arrays. The Morris water maze was used for behavioral analysis. Our results show in addition to improvement in cognition methylation changes in 39 of 13,715 interrogated probes in treated 3xTg-AD mice compared with untreated 3xTg-AD mice. These changes in methylation probes include 29 gene loci. Importantly, changes in methylation status were mainly from synapse-related genes (SYP, SYN1, and DLG3) and neurogenesis-associated genes (HMGB3 and RBBP7). Thus, our results indicate that liver X receptors (LXR) agonist treatment induces rapid changes in DNA methylation, particularly in loci associated with genes involved in neurogenesis and synaptic function. Our results suggest a new potential mechanism to explain the beneficial effect of GW3965.

Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα.

B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.

Contribution of JAK2 mutations to T-cell lymphoblastic lymphoma development.

The JAK-STAT pathway has a substantial role in lymphoid precursor cell proliferation, survival and differentiation. Nonetheless, the contribution of JAK2 to T-cell lymphoblastic lymphoma (T-LBL) development remains poorly understood. We have identified one activating TEL-JAK2 translocation and four missense mutations accumulated in 2 out of 16 T-LBL samples. Two of them are novel JAK2 mutations and the other two are reported for the first time in T-LBL. Notably, R683G and I682T might have arisen owing to RNA editing. Mutated samples showed different mutated transcripts suggesting sub-clonal heterogeneity. Functional approaches revealed that two JAK2 mutations (H574R and R683G) constitutively activate JAK-STAT signaling in γ2A cells and can drive the proliferation of BaF3-EpoR cytokine-dependent cell line. In addition, aberrant hypermethylation of SOCS3 might contribute to enhance the activation of JAK-STAT signaling. Of utmost interest is that primary T-LBL samples harboring JAK2 mutations exhibited increased expression of LMO2, suggesting a mechanistic link between JAK2 mutations and the expression of LMO2, which was confirmed for the four missense mutations in transfected γ2A cells. We therefore propose that active JAK2 contribute to T-LBL development by two different mechanisms, and that the use of pan-JAK inhibitors in combination with epigenetic drugs should be considered in future treatments.

Disarticulation of the left upper extremity for treatment of giant primary lymphedema--case report.

This unusually rare case of giant primary lymphedema of the left upper extremity in a 21-year old woman, who had undergone five surgical procedures (two Charles' procedures and three partial resections) without lasting clinical improvement, demonstrates the difficulty in achieving satisfactory long-term results in patients with grotesquely huge extremities secondary to primary lymphedema. Disarticulation of the left upper extremity was felt to be the most appropriate surgical option in this severe congenital lymphedema patient.

Role of sirtuins in stem cell differentiation.

Sirtuins play an essential role in the cellular response to environmental stress, promoting DNA repair, telomere stability, cell cycle arrest, cellular senescence, and apoptosis. Much attention has been given to the role of sirtuins in aging and cancer development; however, less is known about their role in stem cell regulation. This review focuses in this topic and discusses the possible implications in adult stem cell aging.

Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment.

Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a gamut of transcription factors but also by epigenetic events, mainly histone deacetylation and promoter DNA methylation. The DNA demethylation agent 5'-aza-2'-deoxycytidine (AzadC) has been widely described as an effective promoter of cardiomyogenic differentiation in various stem cell types. However, its toxicity and instability complicate its use. Therefore, the purpose of this study was to examine the effects of zebularine (1-(ß-D-ribofuranosyl)-1,2-dihydropyrimidin-2-1), a stable and non-toxic DNA cytosine methylation inhibitor, on mouse ESC (mESC) differentiation. Herein, we report that treating embryoid bodies, generated from mESCs, with 30 µM zebularine for 7 days led to greater cell differentiation and induced the expression of several cardiac-specific markers that were detected using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunostaining and flow cytometry. Zebularine enhanced the expression of cardiac markers and the appearance of beating cells that responded to cardiac drugs, including ion channel blockers (diltiazem) and ß-adrenergic stimulators (isoproterenol). Gene promoter methylation status was assessed using methylation-specific PCR (MSP) and validated by bisulfite sequencing analysis. Global gene expression profiling using microarrays showed that zebularine-differentiated cells are distinct from control ESCs. Pathway analysis revealed an enhancement of cellular processes such as embryonic development, cardiovascular system development and function. In addition, the whole-cell proteins exhibited different profiles as analyzed by two-dimensional differential-in-gel-electrophoresis. Our results indicate that zebularine regulates mesodermal differentiation of mESCs, controls promoter methylation of crucial cardiac genes and may help to improve cardiomyogenic differentiation.

Aberrant epigenetic regulation of bromodomain BRD4 in human colon cancer.

The bromodomain protein BRD4 is involved in cell proliferation and cell cycle progression, primarily through its role in acetylated chromatin-dependent regulation of transcription at targeted loci. Here, we show that BRD4 is frequently downregulated by aberrant promoter hypermethylation in human colon cancer cell lines and primary tumors. Ectopic re-expression of BRD4 in these colon cancer cell lines markedly reduced in vivo tumor growth, suggesting a role of BRD4 in human colon cancer.

The possible role of epigenetics in gestational diabetes: cause, consequence, or both.

Gestational diabetes mellitus (GDM) is defined as the glucose intolerance that is not present or recognized prior to pregnancy. Several risk factors of GDM depend on environmental factors that are thought to regulate the genome through epigenetic mechanisms. Thus, epigenetic regulation could be involved in the development of GDM. In addition, the adverse intrauterine environment in patients with GDM could also have a negative impact on the establishment of the epigenomes of the offspring.

Identification of (1H)-pyrroles as histone deacetylase inhibitors with antitumoral activity.

Histone deacetylases (HDACs) play a key role in the regulation of gene expression and chromatin structure, and drugs targeting these enzymes might have an important impact in the treatment of human cancer. Herein, we report the characterization of (1H)-pyrroles as a new subfamily of HDAC inhibitors obtained by computational modeling of class-I human HDACs. From a functional standpoint, (1H)-pyrroles are powerful inductors of acetylation of histones H3 and H4, and restore the expression of growth-inhibitory genes. From a cellular view, these compounds cause a marked decrease in the viability of cancer cells in vitro and in vivo, associated with a cell-cycle arrest at G2/M and an inhibition of angiogenesis. Thus, (1H)-pyrroles emerge as a novel group of HDAC inhibitors with promising antitumoral features.