Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors.
EZH2; Medulloblastoma; Polycomb repressive complex 2; histone lysine methylation; DZNEP
Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4+ T lymphocytes as well as in CD14+ monocytes. In normal naïve T cells and CD14+ monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5 expressing CD14+ monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non–CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14+ monocytes, this indicates a dominant role for AcH3 in CCR5 transcription.
We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.
Chromatin remodeling; histone modifications; DNA methylation; bivalent chromatin; poised chromatin; CCR5; T Cells; Monocytes
Pancreatic ductal adenocarcinoma (PDAC) is characterized by overexpression of Enhancer-of Zeste-Homolog-2 (EZH2), which plays a pivotal role in cancer-stem-cell (CSC) self-renewal through methylation of histone-H3-lysine-27 (H3K27m3). Against this background, EZH2 was identified as an attractive target and we investigated the interaction of the EZH2-inhibitor DZNeP with gemcitabine.
EZH2 expression was detected by quantitative-RT-PCR in 15 PDAC cells, including 7 primary cell cultures, showing expression values correlated with their originator tumors (Spearman-R2=0.89, P=0.01). EZH2 expression in cancer cells was significantly higher than in normal ductal pancreatic cells and fibroblasts.
DZNeP (5 μM, 72-hour-exposure) modulated EZH2 and H3K27m3 protein expression, and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.2 (PANC-1), 0.3 (MIA-PaCa-2) and 0.7 (LPC006). The drug combination reduced the percentages of cells in G2/M phase (e.g., from 27 to 19% in PANC-1, P<0.05), and significantly increased apoptosis compared to gemcitabine-alone. Moreover, DZNeP enhanced the mRNA and protein expression of the nucleoside transporters hENT1/hCNT1, possibly because of the significant reduction of deoxynucleotides content (e.g., 25% reduction of deoxycytidine-nucleotides in PANC-1), as detected by LC-MS/MS.
DZNeP decreased cell migration, which was additionally reduced by DZNeP/gemcitabine combination (-20% in LPc006, after 8-hour exposure, P<0.05), and associated with increased E-cadherin mRNA and protein expression. Furthermore, DZNeP and DZNeP/gemcitabine combination significantly reduced the volume of PDAC spheroids growing in CSC-selective-medium, and decreased the proportion of CD133+ cells.
All these molecular mechanisms underlying the synergism of DZNeP/gemcitabine combination support further studies on this novel therapeutic approach for treatment of PDAC.
Pancreatic ductal adenocarcinoma; EZH2; DZNeP; gemcitabine
Synthetic diacylglycerol lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG-lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α–pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG-lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N-alkyl chain in the R2 position of the DAG-lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively-charged DAG lactone derivatives play a central role in determining biological activity and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.
diacylglycerol (DAG)-lactones; PKC; plasma membrane; membrane anchoring; vesicles
Modified thrombin-binding aptamers (TBAs) carrying uridine (U), 2′-deoxy-2′-fluorouridine (FU) and North-methanocarbathymidine (NT) residues in the loop regions were synthesized and analyzed by UV thermal denaturation experiments and CD spectroscopy. The replacement of thymidines in the TGT loop by U and FU results in an increased stability of the antiparallel quadruplex structure described for the TBA while the presence of NT residues in the same positions destabilizes the antiparallel structure. The substitution of the thymidines in the TT loops for U, FU and NT induce a destabilization of the antiparallel quadruplex, indicating the crucial role of these positions. NMR studies on TBAs modified with uridines at the TGT loop also confirm the presence of the antiparallel quadruplex structure. Nevertheless, replacement of two Ts in the TT loops by uridine gives a more complex scenario in which the antiparallel quadruplex structure is present along with other partially unfolded species or aggregates.
Quadruplex; Thrombin-binding aptamer; NMR; Oligonucleotide synthesis; Uridine; 2′-Deoxy-2′-fluorouridine; North-methanocarbathymidine
Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.
DNA replication; polymerase; translesion DNA synthesis; X-ray crystallography
Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis. Expression array analysis of 23 SCLC cases and 42 normal tissues revealed that EZH2 and other PRC2 members were highly expressed in SCLC. ChIP-seq for H3K27me3 suggested that genes with H3K27me3(+) in SCLC were extended not only to PRC2-target genes in ES cells but also to other target genes such as cellular adhesion-related genes. These H3K27me3(+) genes in SCLC were repressed significantly, and introduction of the most repressed gene JUB into SCLC cell line lead to growth inhibition. Shorter overall survival of clinical SCLC cases correlated to repression of JUB alone, or a set of four genes including H3K27me3(+) genes. Treatment with EZH2 inhibitors, DZNep and GSK126, resulted in growth repression of SCLC cell lines. High PRC2 expression was suggested to contribute to gene repression in SCLC, and may play a role in genesis of SCLC.
N-methyl substituted diacylglycerol-indololactones (DAG-indololactones) are newly-synthesized effectors of protein kinase C (PKC) isoforms and exhibit substantial selectivity between RasGRP3 and PKC alpha. We present a comprehensive analysis of membrane interactions and biological activities of several DAG-indololactones. Translocation and binding activity assays underline significant variations between the PKC translocation characteristics affected by the ligands as compared to their binding activities. In parallel, the fluorescent properties of the ligands were employed for analysis of their membrane association profiles. Specifically, we find that a slight change in the linkage to the indole ring resulted in significant differences in membrane binding and association of the DAG-indololactones with lipid bilayers. Our analysis shows that seemingly small structural modifications of the hydrophobic regions of these biomimetic PKC effectors contribute to pronounced modulation of membrane interactions of the ligands
protein kinase C; phorbol ester; diacylglycerol-lactone; vesicles; FRET; tryptophane fluorescence
Incorporation of a bicyclo[3.1.0]hexane scaffold into the nucleoside sugar was devised to lock the embedded cyclopentane ring in conformations that mimic the furanose North and South sugar puckers. To analyze the effects of North-methanocarba-2'-deoxythymidine (N-MCdT) on the conformation of B-DNA, we crystallized d(CGCGAA[mcTmcT]CGCG) with two N-MCdTs. Instead of a duplex the 12mer forms a tetraloop-hairpin, whereby loop N-MCdTs adopt the C4'-exo pucker (NE; P=50°). This indicates that the bicyclic framework does not limit the sugar pucker to the anticipated C2'-exo range (NNW; P=−18°).
An enantioselective synthesis of suitably protected (1R,2S,4S,5S)-4-amino-1-(hydroxymethyl)bicyclo[3.1.0]hexan-2-ol, a key starting material for the synthesis of conformationally locked carbocyclic nucleosides, including the antiviral active North-methanocarba thymidine, is reported. Starting from 2-deoxyribose the target Boc-protected amine was prepared in 33% overall yield under condition that are ecologically friendlier than previous methods.
Conformationally locked carbocyclic nucleosides; North-methanocarbathymidine; antiviral; enantioselective synthesis
Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1–UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.
Beginning with a known 3-oxabicyclo[3.1.0]hexane scaffold (I), the relocation of the fused cyclopropane ring bond and the shifting of the oxygen atom to an alternative location engendered a new 2-oxabicyclo[3.1.0]hexane template (II) that mimics more closely the tetrahydrofuran ring of conventional nucleosides. The synthesis of this new class of locked nucleosides involved a novel approach that required the isocyanate II (B = NCO) with a hydroxyl-protected scaffold as a pivotal intermediate that was obtained in eleven steps from a known dihydrofuran precursor. The completion of the nucleobases was successfully achieved by quenching the isocyanate with the lithium salts of the corresponding acrylic amides that led to the uracil and thymidine precursors in a single step. Ring closure of these intermediates led to the target, locked nucleosides. The anti-HIV activity of 29 (uridine analogue), 31 (thymidine analogue), and 34 (cytidine analogue) was explored in human osteosarcoma (HOS) cells or modified HOS cells (HOS-313) expressing the herpes simplex virus 1 thymidine kinase (HSV-1 TK). Only the cytidine analogue showed moderate activity in HOS-313 cells, which means that the compounds are not good substrates for the cellular kinases.
Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb complex histone methytransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNAi-mediated knockdown or pharmacological inhibiton with 3-deazaneplanocin A (DZNep) increased CASZ1 expression, inhibited NB cell growth and induced neurite extension. Similarly, EZH2−/− mouse embryonic fibroblasts (MEFs) displayed 3-fold higher levels of CASZ1 mRNA compared to EZH2+/+ MEFs. In cells with increased expression of CASZ1, treatment with HDAC inhibitors decreased expression of EZH2 and the Polycomb complex component SUZ12. Under steady-state conditions H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors.
CASZ1; neuroblastoma; EZH2; NGFR; CLU; RUNX3
Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A1 and A3 receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A1, A2A, and A3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N6-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A1 or A3 receptors, respectively, were synthesized. The N6-cyclopentyl derivatives were A1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A1 receptors, as indicated by stimulation of binding of [35S]GTP-γ-S. The (N)-methanocarba-N6-(3-iodobenzyl)adenosine and its 2-chloro derivative had Ki values of 4.1 and 2.2 nM at A3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A3 receptors (EC50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.
Polycomb group (PcG) protein-dependent histone methylation and ubiquitination drives chromatin compaction leading to reduced tumor suppressor expression and increased cancer cell survival. Green tea polyphenols and S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors are important candidate chemopreventive agents. Previous studies indicate that (-)-epigallocatechin-3-gallate (EGCG), a potent green tea polyphenol, suppresses PcG protein level and skin cancer cell survival. Inhibition of AdoHcy hydrolase with 3-deazaneplanocin A (DZNep) inhibits methyltransferases by reducing methyl group availability. In the present study, we examine the impact of EGCG and DZNep cotreatment on skin cancer cell function. EGCG and DZNep, independently and in combination, reduce the level of PcG proteins including Ezh2, eed, Suz12, Mel18 and Bmi-1. This is associated with reduced H3K27me3 and H2AK119ub formation, histone modifications associated with closed chromatin. Histone deacetylase 1 level is also reduced and acetylated H3 formation is increased. These changes are associated with increased tumor suppressor expression and reduced cell survival and are partially reversed by vector-mediated maintenance of Bmi-1 level. The reduction in PcG protein level is associated with increased ubiquitination and is reversed by proteasome inhibitors, suggesting proteasome-associated degradation.
Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of P2Y1 receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem.
2000, 43, 829–842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y1 receptor and by using the radiolabeled antagonist [3H]2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y1 receptor was an (N)-methanocarba N6-methyl-2-iodo analogue 12, which displayed a Ki value in competition for binding of [3H]5 of 0.79 nM and a KB value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y1 receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y1 receptors. An enzymatic method of synthesis of the 3′,5′-bisphosphate from the corresponding 3′-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.
Cancer-testis antigens such as NY-ESO-1, MAGE-A1 and MAGE-A3 are immunogenic proteins encoded by genes, which are normally expressed only in male germ cells, but activated by ill-defined epigenetic mechanisms in human tumors including lung cancers. Previously we reported induction of these cancer-testis antigens in cancer cells, but not normal cells, by DNA demethylating agents and histone deacetylase inhibitors using clinically achievable exposure conditions. In the present study, we evaluated chromatin alterations associated with repression/activation of cancer-testis genes in lung cancer cells to further develop gene induction regimens for cancer immunotherapy. Repression of NY-ESO-1, MAGE-A1, and MAGE-A3 coincided with DNA hypermethylation, recruitment and binding of polycomb group proteins, and histone heterochromatin modifications within the promoters of these genes. De-repression coincided with DNA demethylation, dissociation of polycomb proteins, and presence of euchromatin marks within the respective promoters. ShRNAs were used to inhibit several histone methyl transferases (KMTs) and histone demethylases (KDMs) that mediate histone methylation and repress gene expression. Knockdown of KMT6, KDM1 or KDM5B markedly enhanced deoxyazacytidine (DAC)-mediated activation of these cancer-testis genes in lung cancer cells. DZNep, a pharmacologic inhibitor of KMT6 expression, recapitulated the effects of KMT6 knock-down. Following DAC-DZNep exposure, lung cancer cells were specifically recognized and lysed by allogeneic lymphocytes expressing recombinant T cell receptors recognizing NY-ESO-1 and MAGE-A3. Combining DNA demethylating agents with compounds such as DZNep that modulate histone lysine methylation may provide a novel epigenetic strategy to augment cancer-testis gene expression as an adjunct to adoptive cancer immunotherapy.
lung cancer; epigenetics; cancer-testis gene; KMT6; KDM1; KDM5B; SirT1; DZNep; immunotherapy
Two conformationally locked versions of L-deoxythreosyl phosphonate nucleosides (2 and 3) were synthesized to investigate the preference of HIV reverse transcriptase for a conformation displaying either a fully diaxial or fully diequatorial disposition of substituents. Synthesis of the enantiomeric 4-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexane-2-ol carbocyclic nucleoside precursors (diaxially disposed) proceeded straightforwardly from commercially available (1R,4S)-4-hydroxy-2-cyclopent-2-enyl-1-yl acetate employing a hydroxyl-directed Simmons-Smith cyclopropanation that culminated with a Mitsunobu coupling of the purine base. For the more complicated 1-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-3-ol carbocyclic nucleoside precursors (diequatorially disposed), the obligatory linear approach required the syntheses of key 1-aminobicyclo[3.1.0.]hexan-3-yl benzoate precursors that were assembled via the amide variant of the Kulinkovich reaction involving the intramolecular cyclopropanation of a substitued δ-vinylamide. Completion of the purine ring was achieved by conventional approaches but with much improved yields through the use of a microwave reactor. The syntheses of the phosphonates and the corresponding diphosphates were achieved by conventional means. None of the diphosphates, which were supposed to act as nucleoside triphosphate mimics, could compete with dATP even when present in a ten-fold excess.
Relatively new types of the modified nucleotides, namely carbocyclic sugars that are constrained to north or south (C2′ or C3′ exo) conformations, can be used for RNA nanoparticle design to control their structures and stability by rigidifying nucleotides and altering the helical properties of RNA duplexes. Two RNA structures, an RNA dodecamer and an HIV kissing loop complex where several nucleotides were replaced with north or south constrained sugars, were studied by molecular dynamics (MD) simulations. The substituted south constrained nucleotides in the dodecamer widened the major groove and narrowed and deepened the minor groove thus inducing local conformational changes that resemble a B-form DNA helix. In the HIV kissing loop complex, north and south constrained nucleotides were substituted into flanking bases and stems. The modified HIV kissing loop complex showed a lower RMSD value than the normal kissing loop complex. The overall twist angle was also changed and its standard deviation was reduced. In addition, the modified RNA dodecamer and HIV kissing loop complex were characterized by principal component analysis (PCA) and steered molecular dynamics (SMD). PCA results showed that the constrained sugars stabilized the overall motions. The results of the SMD simulations indicated that as the backbone δ angles were increased by elongation, more force was applied to the modified RNA due to the constrained sugar analogues.
RNA; North constrained sugar; South constrained sugar; Molecular dynamics simulations; RNA structure deformations; RNA nanodesign
Neuralized (Neurl) is a highly conserved E3 ubiquitin ligase, which in Drosophila acts upon Notch ligands to regulate Notch pathway signaling. Human Neuralized1 (NEURL1) was investigated as a potential tumor suppressor in medulloblastoma (MB). The gene is located at 10q25.1, a region demonstrating frequent loss of heterozygosity in tumors. In addition, prior publications have shown that the Notch pathway is functional in a proportion of MB tumors and that Neurl1 is only expressed in differentiated cells in the developing cerebellum. In this study, NEURL1 expression was downregulated in MB compared with normal cerebellar tissue, with the lowest levels of expression in hedgehog-activated tumors. Control of gene expression by histone modification was implicated mechanistically; loss of 10q, sequence mutation, and promoter hypermethylation did not play major roles. NEURL1-transfected MB cell lines demonstrated decreased population growth, colony-forming ability, tumor sphere formation, and xenograft growth compared with controls, and a significant increase in apoptosis was seen on cell cycle and cell death analysis. Notch pathway inhibition occurred on the exogenous expression of NEURL1, as shown by decreased expression of the Notch ligand, Jagged1, and the target genes, HES1 and HEY1. From these studies, we conclude that NEURL1 is a candidate tumor suppressor in MB, at least in part through its effects on the Notch pathway.
deazaneplanocin; Jagged1; Neuralized1; Notch; tumor suppressor
It is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3′-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4′-C-alkyl-deoxyadenosine (4′-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4′-C-methyl- and 4′-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4′-C-methyl- or 4′-C-ethyl-2′-deoxyadenosine. In vitro experiments with purified HIV-1 RT showed that 4′-C-methyl-2′-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4′-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.
How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified a novel inflammation-activated signalling in muscle stem (satellite) cells, by which the Polycomb Repressive Complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38alpha kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EzH2, the enzymatic sub-unit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. Anti-TNF antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signalling – p38alpha and EzH2 - invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signalling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signalling to Pax7 and satellite cell decision to proliferate or differentiate.
Pax7; p38; muscle stem (satellite) cells; regeneration; chromatin; Polycomb complex
DNA methylation, histone modifications, and nucleosomal occupancy collaborate to cause silencing of tumor-related genes in cancer. The development of drugs that target these processes is therefore important for cancer therapy. Inhibitors of DNA methylation and histone deacetylation have been approved by the Food and Drug Administration for treatment of hematologic malignancies. However, drugs that target other mechanisms still need to be developed. Recently, 3-deazaneplanocin A (DZNep) was reported to selectively inhibit trimethylation of lysine 27 on histone H3 (H3K27me3) and lysine 20 on histone H4 (H4K20me3) as well as reactivate silenced genes in cancer cells. This finding opens the door to the pharmacologic inhibition of histone methylation. We therefore wanted to further study the mechanism of action of DZNep in cancer cells. Western blot analysis shows that DZNep globally inhibits histone methylation and is not selective. Two other drugs, sinefungin and adenosine dialdehyde, have similar effects as DZNep on H3K27me3. Intriguingly, chromatin immunoprecipitation of various histone modifications and microarray analysis show that DZNep acts through a different pathway than 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor. These observations give us interesting insight into how chromatin structure affects gene expression. We also determined the kinetics of gene activation to understand if the induced changes were somatically heritable. We found that upon removal of DZNep, gene expression is reduced to its original state. This suggests that there is a homeostatic mechanism that returns the histone modifications to their “ground state” after DZNep treatment. Our data show the strong need for further development of histone methylation inhibitors
Histone methylation; EZH2; epigenetics; Polycomb; DZNep
Steric and electronic parameters such as the anomeric effect (AE) and gauche effect play significant roles in steering the North ⇆ South equilibrium of nucleosides in solution. Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in “fixed” relationships, either antiperiplanar where the AE is permanently “on”, or gauche where the AE is impaired. The consequences of these two alternatives were subject to high-level calculations and measured experimentally by x-ray crystallography, hydrolytic stability of the glycosyl bond, and pKa values.
Glioblastoma (GBM) is a malignant brain tumor with dismal prognosis. GBM patients have a median survival of less than 2 years. GBM is characterized by fast cell proliferation, infiltrative migration, and by the induction of angiogenesis. MicroRNAs and polycomb group (PcG) proteins have emerged as important regulators of gene expression.
Here we determined that miR-101 is down-regulated in GBM, resulting in overexpression of the miR-101 target PcG protein EZH2, a histone methyltransferase affecting gene expression profiles in an epigenetic manner.
Inhibition of EZH2 in vitro by pre-miR-101, EZH2 siRNA, or small molecule DZNep, attenuated GBM cell growth, migration/invasion, and GBM-induced endothelial tubule formation. In addition, for each biological process we identified ontology-associated transcripts that significantly correlate with EZH2 expression. Inhibition of EZH2 in vivo by systemic DZNep administration in a U87-Fluc-mCherry GBM xenograft mouse imaging model resulted in reduced tumor growth.
Our results indicate that EZH2 has a versatile function in GBM progression and that its overexpression is at least partly due to decreased miR-101 expression. Inhibition of EZH2 may be a potential therapeutic strategy to target GBM proliferation, migration, and angiogenesis.
cancer; microRNA; Policomb group; glioblastoma; angiogenesis