Simeon Santourlidis

Crucial Role of DNA Methylation in Determination of Clonally Distributed Killer Cell Ig-like Receptor Expression Patterns in NK Cells

Human NK cells are characterized by the expression of surface receptors of the killer cell Ig-like receptor (KIR) family, which are involved in the specific recognition of pathogenic target cells. Each NK cell expresses and maintains an individual subset of inhibitory and stimulatory KIR and in this way contributes to a diversified NK cell repertoire. To date, the molecular basis for generation of clonally distributed KIR expression patterns has been elusive. Here, analyses of DNA methylation patterns of KIR genes in NK cell lines as well as in NK cells, freshly isolated from peripheral blood, demonstrated that a small CpG island surrounding the transcriptional start site of each KIR gene is consistently demethylated in expressed KIR and methylated in unexpressed KIR. DNA-demethylating treatment resulted in a rapid and stable induction of transcription and cell surface expression of all formerly unexpressed KIR in NK cell lines, NK cell clones, and freshly isolated NK cells, but not in other cell types. In vitro methylation of KIR CpG islands repressed reporter gene expression in NK cells. We conclude that clonal patterns of KIR expression are mainly epigenetically determined and maintained through DNA methylation.

Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells

BackgroundThe miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT) process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors.FindingsWe show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE) was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus.ConclusionsThe present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.

Three Structurally and Functionally Divergent Kinds of Promoters Regulate Expression of Clonally Distributed Killer Cell Ig-Like Receptors (KIR), of KIR2DL4, and of KIR3DL3

The generation of killer cell Ig-like receptor (KIR) expression patterns in NK cells involves variegated silencing of KIR genes by DNA methylation. To identify regulatory elements involved in KIR gene activation, upstream regions of KIR genes were functionally characterized in NK3.3 cells as well as in primary NK cells. Three kinds of KIR promoters were defined, controlling clonally expressed KIR genes, the constitutively active KIR2DL4, and the weakly expressed KIR3DL3. Upstream of a short core promoter common to all KIR genes, a region containing functionally divergent elements was characterized. Although this region had no impact on the activity of the KIR2DL3 promoter, an inhibitory element was identified in the KIR2DL4 promoter and an activating element was found in the KIR3DL3 promoter. Upon treatment with a methyltransferase inhibitor, KIR3DL3 expression could be readily induced showing that the low levels of KIR3DL3 expression in peripheral blood are due to sustained DNA methylation of an otherwise fully functional promoter. Analysis of transcription factor binding sites identified a functional acute myeloid leukemia (AML) site common to all three KIR promoters. Mutation of this site led to a substantial increase in activity of all KIR promoters. Among the different members of the AML family, AML-2 was identified as the predominant KIR binding factor. The present study suggests that AML-2 acts as a repressor of KIR expression in mature NK cells and opens the possibility that AML factors and associated cofactors are involved in regulation of KIR expression during NK cell development.

Bortezomib/proteasome inhibitor triggers both apoptosis and autophagy-dependent pathways in melanoma cells.

Generally, both endoplasmic reticulum (ER) stress and mitochondrial dysregulation are a potential therapeutic target of anticancer agents including bortezomib. The treatment of melanoma cells with bortezomib was found to induce apoptosis together with the upregulation of Noxa, Mcl-1, and HSP70 proteins, and the cleavage of LC3 and autophagic formation. Also, bortezomib induced ER-stress as evidenced by the increase of intracellular Ca(2+) release. In addition, bortezomib enhanced the phosphorylation of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), c-jun-N-terminal kinase (JNK) and p38, and the activation of the transcription factors AP-1, ATF-2, Ets-1, and HSF1. Bortezomib-induced mitochondrial dysregulation was associated with the accumulation of reactive oxygen species (ROS), the release of both apoptosis inducing factor (AIF) and cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of Poly (ADP-ribose) polymerase (PARP). The pretreatment of melanoma cells with the inhibitor of caspase-3 (Ac-DEVD-CHO) was found to block bortezomib-induced apoptosis that subsequently led to the increase of autophagic formation. In contrast, the inhibition of ASK1 abrogated bortezomib-induced autophagic formation and increased apoptosis induction. Furthermore, the inhibition of JNK, of HSP70 also increased apoptosis induction without influence of bortezomib-induced autophagic formation. Based on the inhibitory experiments, the treatment with bortezomib triggers the activation of both ER-stress-associated pathways, namely IRE1α-ASK1-p38-ATF-2/ets-1-Mcl-1, and IRE1α-ASK1-JNK-AP-1/HSF1-HSP70 as well as mitochondrial dysregulation-associated pathways, namely ROS-ASK1-JNK-AP-1/HSF1-HS70, and AIF-caspase-3-PARP and Cyt.c, and caspase-9-caspase-3-PARP. Taken together, our data demonstrates for the first time the molecular mechanisms, whereby bortezomib triggers both apoptosis and autophagic formation in melanoma cells.

Induction of pluripotency in human cord blood unrestricted somatic stem cells

OBJECTIVE Generation of induced pluripotent stem (iPS) cells from human cord blood (CB)-derived unrestricted somatic stem cells and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. MATERIALS AND METHODS Unrestricted somatic stem cells isolated from human CB were reprogrammed to iPS cells using retroviral expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC. The reprogrammed cells were analyzed morphologically, by quantitative reverse transcription polymerase chain reaction, genome-wide microRNA and methylation profiling, and gene expression microarrays, as well as in their pluripotency potential by in vivo teratoma formation in severe combined immunodeficient mice and in vitro differentiation. RESULTS CB iPS cells are very similar to human embryonic stem cells morphologically, at their molecular signature, and in their differentiation potential. CONCLUSIONS Human CB-derived unrestricted somatic stem cells offer an attractive source of cells for generation of iPS cells. Our findings open novel perspectives to generate human leukocyte antigen-matched pluripotent stem cell banks based on existing CB banks. Besides the obvious relevance of a second-generation CB iPS cell bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.

Molecular characterization of KIR3DL3

Killer-cell immunoglobulin-like receptors (KIRs) are a structurally and functionally diverse family of molecules expressed by natural killer (NK) cells and T-cell subsets. The most centromeric gene in the human KIR cluster is KIR3DL3, a framework gene that is present in all haplotypes. KIR3DL3 has only one immunoreceptor tyrosine-based inhibitory motif and lacks the exon encoding the stem between the Immunoglobulin domains and the transmembrane region. We have investigated expression of KIR3DL3 in blood and decidual NK cells by reverse transcriptase polymerase chain reaction (RT-PCR) and protein analysis using a KIR3DL3-specific monoclonal antibody, CH21. KIR3DL3 mRNA was only detected in the CD56bright subset in cells from peripheral blood and in CD56bright decidual NK cells. The CD56bright NK92 cell line was also positive. Quantitative RT-PCR indicated a trend for higher expression of KIR3DL3 in female peripheral blood mononuclear cells compared to that in male. Using a bisulphite conversion method, we found that the promoter of KIR3DL3 was strongly methylated. Surface protein expression was detectable after demethylation. Like other KIRs, KIR3DL3 is highly polymorphic, and we detected 14 variants in 25 unrelated individuals. Nucleotide substitutions were scattered throughout the sequence, with a cluster of alleles at the start of the transmembrane region at the site where the remnant of the linking stem present in other KIR is found. We conclude that the KIR3DL3 gene is not a pseudogene but encodes a protein that is not expressed in healthy individuals. Protein expression might be induced under certain developmental or pathological situations.

Lineage-Specific Transition of Histone Signatures in the Killer Cell Ig-Like Receptor Locus from Hematopoietic Progenitor to NK Cells

The clonal distribution and stable expression of killer cell Ig-like receptor (KIR) genes is epigenetically regulated. To assess the epigenetic changes that occur during hemopoietic development we examined DNA methylation and chromatin structure of the KIR locus in early hemopoietic progenitor cells and major lymphocyte lineages. In hemopoietic progenitor cells, KIR genes exhibited the major hallmarks of epigenetic repression, which are dense DNA methylation, inaccessibility of chromatin to Micrococcus nuclease digest, and a repressive histone signature, characterized by strong H3K9 dimethylation and reduced H4K8 acetylation. In contrast, KIR genes of NK cells showed active histone signatures characterized by absence of H3K9 dimethylation and presence of H4K8 acetylation. Histone modifications correlated well with the competence of different lymphocyte lineages to express KIR; whereas H4K8 acetylation was high in NK and CD8+ T cells, it was almost absent in CD4+ T cells and B cells and, in the latter case, replaced by H3K9 dimethylation. In KIR-competent lineages, active histone signatures were also observed in silent KIR genes and in this case found in combination with dense DNA methylation of the promoter and nearby regions. The study suggests a two-step model of epigenetic regulation in which lineage-specific acquisition of euchromatic histone marks is a prerequisite for subsequent gene-specific DNA demethylation and expression of KIR genes.

Nucleolin regulates gene expression in CD34-positive hematopoietic cells

CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution. Nucleolin is an abundant multifunctional phosphoprotein of growing eukaryotic cells, involved in regulation of gene transcription, chromatin remodeling, and RNA metabolism, whose transcripts are enriched in murine hematopoietic stem cells, as opposed to differentiated tissue. Here we show that, in human CD34-positive hematopoietic cells, nucleolin activates endogenous CD34 and Bcl-2 gene expression, and cell surface CD34 protein expression is thereby enhanced by nucleolin. Nucleolin-mediated activation of CD34 gene transcription results from direct sequence-specific interactions with the CD34 promoter region. Nucleolin expression prevails in CD34-positive cells mobilized into peripheral blood (PB), as opposed to CD34-negative peripheral blood mononuclear cells (PBMCs). Therefore, in intact CD34-positive mobilized PB cells, a recruitment of nucleolin to the CD34 promoter region takes place, accompanied by nucleosomal determinants of gene activity, which are absent from the CD34 promoter region in CD34-negative PBMCs. Our data show that nucleolin acts as a component of the gene regulation program of CD34-positive hematopoietic cells and provide further insights into processes by which human CD34-positive hematopoietic stem/progenitor cells are maintained.

Induction of indoleamine 2, 3-dioxygenase by death receptor activation contributes to apoptosis of melanoma cells via mitochondrial damage-dependent ROS accumulation.

Although the induction of indoleamine 2, 3-dioxygenase (IDO) by several agents is well established, the mechanisms of its transcriptional regulation and those regulating its function as apoptotic mediator seem to be complex, agent-dependent, and cell type-specific. Besides their pro-apoptotic activity in melanoma cells, both anti-Fas agonist antibody (CH11) and the tumor necrosis factor (TNF)-alpha were found to induce IDO gene expression, the activation of apoptosis signal-regulating kinase (ASK1), and the activation of both c-Jun N-terminal kinase (JNK) and NF-kappaB pathways. In addition, the treatment of melanoma cells with CH11 or TNF-alpha induced the loss of mitochondrial membrane potential (Deltapsim), the accumulation of reactive oxygen species (ROS), the phosphorylation of Fas-associated domain (FADD), the cleavage of caspase-8, and truncation of Bid. Using RNA interference and pharmacological inhibitors, we could confirm the pro-apoptotic activity of IDO and address the mechanisms, which are responsible for its transcriptional regulation and the modulation of its pro-apoptotic activity during death receptor activation in melanoma cells. Thus, our data confirm the pro-apoptotic activity of IDO and provide an insight into the molecular mechanism of TNF-alpha and CH11-induced IDO expression, and the mechanism whereby IDO induces apoptosis of melanoma cells.

Origin-Dependent Neural Cell Identities in Differentiated Human iPSCs In Vitro and after Transplantation into the Mouse Brain

The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34(+) hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.