Inhibition Of Gene Transcription Research Articles

Upregulation of heat shock protein 27 confers resistance to actinomycin D‐induced apoptosis in cancer cells

Actinomycin D (Act D) is a general transcriptional inhibitor that is approved for the treatment of sarcomas, and Wilms, germ cell and trophoblastic tumors. Little is known about the molecular mechanisms that dictate the sensitivity of cancer cells to Act D. In this study, we investigated the effects of Act D on heat shock proteins (HSPs) and the expression and roles of HSP27 in Act D-induced cancer cell apoptosis. We show that Act D upregulates HSP27 and HSP70 expression in cancer cells, whereas it inhibits HSP90 expression. The upregulation of HSP27 by Act D is not attributable to changes in HSP27 transcription or HSP27 synthesis. HSP27 knockdown leads to an increase in Act D-induced caspase 3 and caspase 7 cleavage, and sensitizes rhabdosarcoma cells and breast cancer cells to Act D-induced apoptosis. We conclude that upregulation of HSP27 represents an adaptive response that compromises the anticancer activity of Act D.

Interaction of PACAP with Sonic hedgehog reveals complex regulation of the Hedgehog pathway by PKA

Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (cGCPs) and its aberrant activation causes a cerebellar cancer medulloblastoma. Pituitary adenylate cyclase activating polypeptide (PACAP) inhibits Shh-driven proliferation of cGCPs and acts as tumor suppressor in murine medulloblastoma. We show that PACAP blocks canonical Shh signaling by a mechanism that involves activation of protein kinase A (PKA) and inhibition of the translocation of the Shh-dependent transcription factor Gli2 into the primary cilium. PKA is shown to play an essential role in inhibiting gene transcription in the absence of Shh, but global PKA activity levels are found to be a poor predictor of the degree of Shh pathway activation. We propose that the core Shh pathway regulates a small compartmentalized pool of PKA in the vicinity of primary cilia. GPCRs that affect global PKA activity levels, such as the PACAP receptor, cooperate with the canonical Shh signal to regulate Gli protein phosphorylation by PKA. This interaction serves to fine-tune the transcriptional and physiological function of the Shh pathway.

Abstract B34: Acute depletion reveals novel divisions of labor among human DNA methyltransferases in cancer

Abstract DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation genome-wide. Hypomethylation of repetitive sequences and transposable elements coupled with gene-specific promoter hypermethylation events contribute to the genomic instability and loss of tumor suppressor gene transcription observed in cancer. Regulation of aberrant methylation in cancer remains poorly understood. The aim of our study was to identify unique and overlapping target sites for each of the DNMTs to better understand regulation of normal and aberrant DNA methylation. We hypothesized that acute depletion of the DNMTs (individual and combination) mediated by siRNA technology in NCCIT human embryonic carcinoma cells would result in both distinct and broad changes in DNA methylation patterning. Genome-wide methylation was assayed using the HumanMethylation450 Bead Chip (450K array) allowing for specific CpG site methylation status determination. Select target sites were verified by bisulfite genomic sequencing. DNMT1 knockdown samples (individual/combination) revealed genome-wide hypomethylation, with the strongest demethylation occurring in gene bodies, 3′UTR, and intergenic sequences. Interestingly, only the DNMT1 individual knockdown showed significant hypermethylation in gene promoters. DNMT3 knockdowns showed more specific changes in DNA methylation, but surprisingly, more hypermethylation events occurred than hypomethylation at CpG dinucleotides. Hypermethylation observed in DNMT3 knockdown occurred primarily in gene bodies and 3′UTR, and overlapped significantly with those genes that become hypomethylated in DNMT1 knockdown, indicating a potential cross-regulatory role for the DNMTs to maintain proper regulation of DNA methylation at specific gene termini. Conversely, gene promoters were targeted for hypomethylation in DNMT3 knockdown, and did not significantly overlap with genes that become hypermethylated in DNMT1 knockdown. Of particular interest was that DNMT3B knockdown resulted in widespread non-CpG hypomethylation. In contrast, DNMT3L knockdown showed non-CpG hypermethylation, indicating a potential mechanism for regulation of non-CpG methylation where DNMT3B is responsible for non-CpG methylation, and DNMT3L acts to restrict DNMT3B's activity at non-CpG dinucleotides. Our results reveal a complex view of DNA methylation regulation, in which DNMTs not only target specific sites for methylation, but also cooperate to establish and maintain proper levels of DNA methylation at CpG and non-CpG dinucleotides. Moving forward, we believe our results will provide the framework needed to define the regulatory mechanisms by which DNA methylation is conferred and ultimately develop therapeutic strategies to correct aberrant methylation events that occur in cancer. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion reveals novel divisions of labor among human DNA methyltransferases in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B34.

β-Adrenergic Blockade for Anthracycline- and Trastuzumab-Induced Cardiotoxicity

Breast cancer is the second leading cause of cancer-related death among women in the United States.1 Approximately 25% to 30% of breast cancers have overexpression of human epidermal growth factor receptor 2 (HER2), which is associated with an adverse prognosis.2 Adjuvant chemotherapy with trastuzumab, the humanized monoclonal antibody that directly targets HER2, significantly improves disease-free and overall survival in these patients.3 However, trastuzumab, especially when given in combination with anthracyclines, has been associated with asymptomatic and symptomatic left ventricular (LV) dysfunction3 that can lead to premature discontinuation of trastuzumab therapy and significant cardiac morbidity. Article see p 420 In this issue of Circulation: Heart Failure , Seicean et al4 retrospectively evaluated the incidence of new symptomatic heart failure (HF) in patients with breast cancer treated with anthracyclines, trastuzumab, or both, at the Cleveland Clinic between 2005 and 2010. Using 1:2 propensity matching, they compared patients on continuous β-blockers during cancer treatment to those not on β-blockade, and assessed the effect of incidental β-blocker use on new symptomatic HF and noncardiac mortality during a median follow-up of ≈3 years. The results of this study show that trastuzumab, when given alone or in combination with anthracyclines, substantially increased the risk of symptomatic HF compared with anthracyclines alone (hazard ratio, 9.5; 95% confidence interval, 3.9–23.1). Furthermore, incidental β-blocker use significantly reduced the incidence of symptomatic HF (hazard ratio, 0.2; 95% confidence interval, 0.1–0.7) but did not affect noncardiac mortality in patients with breast cancer treated with trastuzumab, anthracyclines, or both. This article highlights 2 important issues: (1) it emphasizes the magnitude of trastuzumab cardiotoxicity in the real world, and (2) it provides support for the hypothesis that prophylactic β-blocker use may be cardioprotective in patients receiving trastuzumab, anthracyclines, or both. Anthracyclines cause a dose-dependent toxicity that is felt …

Abstract 3265: PI3Kδ inhibitor, GS-1101, impacts transcription and translation in mantle cell lymphoma.

Abstract PI3Kα, β, γ and δ are the most ubiquitous and important PI3K family class I member isoforms that contribute to cell growth and survival processes in cancer. Among these isoforms, PI3Kα and β are broadly expressed, where PI3Kγ and δ are uniquely expressed in T- and B-cells, respectively, making them ideal candidates for targeted therapies in hematological malignancies. GS-1101, also known as CAL-101, is a potent and selective PI3Kδ inhibitor (IC50=2.5 nM) that is currently in Phase II/III clinical trials in B-cell diseases. Previous studies have elucidated disruption of survival factors and cytokine signaling mediated by PI3K/Akt pathways as major mechanism of activity in B-cell malignancies. It has been established that oncoprotein PI3Kα isoform deregulates transcription and translation processes in solid tumors, and based on the knowledge of PI3Kδ and GS-1101, we hypothesized that 1) GS-1101 treatment will inhibit gene transcription and protein translation, 2) will impact transcription and translation regulators that are downstream of PI3K/Akt pathways and 3) will reduce short-lived mRNA and proteins such as Mcl-1, c-Myc and cyclin D1, which are important in mantle cell lymphoma (MCL), a B-cell lymphoma. In JeKo-1 and Mino MCL cell lines, PI3Kδ is the most prominent isoform. In these cell lines and in primary MCL cells, the level of apoptosis was modest (less than 15%) when cells were treated with 5 μM GS-1101 for 24 hr. Consistent with Annexin/PI data, slight levels of PARP cleavage was detected. Of the two Akt phosphorylation sites, Thr308 and Ser473, phosphorylation of Akt (Thr308) showed dose-dependent decrease in both cell lines when treated by GS-1101 for 1 hr following serum starvation; however, there was no change in Akt (Ser347) levels following 24 hr of GS-1101 treatment in full serum conditions. Global RNA synthesis was substantially reduced in MCL primary samples, reaching 80% of control at 1 μM and 50% or less at 5 μM for 24 hr treatment, while only minor changes (80-90% of control) were observed in JeKo-1 and Mino. Global translation inhibition measured by leucine incorporation was observed in JeKo-1, Mino and primary MCL cells. Phosphorylation levels of translation regulators p70S6K (Ser9) and S6 (Ser235/236) showed dose-dependent decrease when treated with GS-1101 for 24 hr in JeKo-1. Meanwhile, minor decrease of 4E-BP1 (Ser65) phosphorylation level was observed in JeKo-1 under the same conditions. In Jeko-1 cells, early response genes such as short-lived MCL1 mRNA level was reduced following 24 hr of GS-1101 treatment, however, other (PIM2, MYC, CCND1) mRNA levels decreased to a lesser extent. At the protein level, both Mcl-1 and cyclin D1 expression decreased in JeKo-1 cells. In conclusion, similar to inhibition of other PI3K isoforms, PI3Kδ inhibition impacted transcription and translation, and reduced cyclin D1 and Mcl-1 levels which are important for MCL cell survival and proliferation. Citation Format: Qingshan Yang, Lisa S. Chen, Sattva S. Neelapu, Brian J. Lanutti, Varsha Gandhi. PI3Kδ inhibitor, GS-1101, impacts transcription and translation in mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3265. doi:10.1158/1538-7445.AM2013-3265

Inhibition of pacemaker activity in interstitial cells of Cajal by LPSviaNF-κB and MAP kinase

To investigate lipopolysaccharide (LPS) related signal transduction in interstitial cells of Cajal (ICCs) from mouse small intestine. For this study, primary culture of ICCs was prepared from the small intestine of the mouse. LPS was treated to the cells prior to measurement of the membrane currents by using whole-cell patch clamp technique. Immunocytochemistry was used to examine the expression of the proteins in ICCs. LPS suppressed the pacemaker currents of ICCs and this could be blocked by AH6809, a prostaglandin E2-EP2 receptor antagonist or NG-Nitro-L-arginine Methyl Ester, an inhibitor of nitric oxide (NO) synthase. Toll-like receptor 4, inducible NO synthase or cyclooxygenase-2 immunoreactivity by specific antibodies was detected on ICCs. Catalase (antioxidant agent) had no action on LPS-induced action in ICCs. LPS actions were blocked by nuclear factor κB (NF-κB) inhibitor, actinomycin D (a gene transcription inhibitor), PD 98059 (a p42/44 mitogen-activated protein kinases inhibitor) or SB 203580 [a p38 mitogen-activated protein kinases (MAPK) inhibitor]. SB 203580 also blocked the prostaglandin E2-induced action on pacemaker currents in ICCs but not NO. LPS inhibit the pacemaker currents in ICCs via prostaglandin E2- and NO-dependent mechanism through toll-like receptor 4 and suggest that MAPK and NF-κB are implicated in these actions.

DNA methylation of peroxisome proliferator-activated receptor γ promoter region and down-regulation of its mRNA expression in liver decrease insulin sensitivity of fetal growth restricted rats

Objective To explore the effect of methylation ot peroxlsome prolllerator-acnvatecl receptor 7 （PPART） gene promoter in liver and its mRNA expression changes on decreasing of insulin sensitivity in feta[ growth restriction （FGR） rats. Methods Twenty pregnant rats were randomly divided into two groups on their first day of pregnancy., normal-protein group （NP） and low-protein group （LP）, ten in each. During pregnancy the LP group rats were fed with low-protein diet （8.00%protein）, while the NP group rats were fed with normal-protein diet （20.00% protein）. The offspring rats were fed with standard feed after 21 days of birth. Male offsprings in NP group were as control offsprings, and male FGR offsprings in LP group ware as FGR offsprings. At day 3, 7, 14, 30, 60 and 90, fasting blood of offsprings was collected to measure fasting plasma glucose （FPG） and fasting insulin（FINS）. Then insulin resistance index of homeostasis model assessment （HOMA 1R） and insulin sensitivity index （ISI） were calculated to evaluate insulin sensitivity. At day 7 and 90, liver tissue of male offsprings was collected to extract DNA and total RNA. The methylation level of PPARy gene promoter and its mRNA expression were detected by methylation specific-polymerase chain reaction （MS-PCR） and reverse transcription-RCR, respectively. The relationships between methylation of PPARy gene promoter and mRNA expression and insulin sensitivity were analyzed by Pearson correlation and nonparametric test method. Results （1） The mean offspring birth-weight of LP group was （4.92±0.36） g, which was lower than that [（6.43±0.59） g] of control group （t= 14.73, P〈0.05）. In LP group, the incidence of FGR offspring was 88.2% （97/110） and the FGR incidence of male ones was 94.1± （48/51）. （2） At day 90, compared with control offsprings, FPG [（8.95±1.83） mmol/L vs （6.21±1.14） mmol/L, t=-3.291, P〈0.05], FINS [（59.57±9.89） mU/Lvs （36.10%7.32） mU/L, 1=-4.916, P〈0.059 and HOMA-IR （0.967±0.297 vs 0. 410±0. 135, t= -4. 472, P〈0.05） of FGR offsprings were significantly higher; while ISI of FGR offspring was lower than that of control offsprings （-3. 043±0. 294 vs 2. 172±0. 354, t=4. 774, P〈0.05）. （3） There was no significant difference in methylation degree of PPAR7 gene promoter in liver between FGR and control offsprings at day 7 （0/8 vs 2/8, Fisher exact test, P〉0. 05）. The methylation degree of PPAR±＇ gene promoter in liver in FGR offsprings was significantly higher than that of control offsprings at day 90 （8/8 vs 2/8, Fisher exact test, P〈0.05）. Compared with control offsprings, PPARy gene mRNA expression level of FGR offsprings decreased significantly at day 90 （43.07±7.51 vs 146.72± 40.66, t=7.09, P〈0.05）. mRNA expression of PPAR7 gene was the lowest in exhaustive methylation offsprings （27.2±1.6）, and then in partial methylation ones （47.3±33.0）, the highest in no methylation ones （144.6±121.2） （P〈0. 05）. （4） The correlation analysis showed that PPARy mRNA expression level negatively correlated to the level of FPG （r =-0. 819）, FINS （r = 0. 906） and HOMA IR （r=- 0. 860）, P〈0.05 respectively but positively correlated to ISI level （r= 0. 947, P〈0. 05）. Conclusions Hypermethylation in promoter region ofPPAR7 gene might inhibit gene transcription, and he involved in the occurrence of insulin resistance in FGR rats. Key words: Fetal growth retadation Insulin resistance ; PPAR gamma ; DNA methylation

Genome-Wide Methylation Analysis of Myeloid Progenitors in Normal Bone Marrow and in Acute Promyelocytic Leukemia At Diagnosis and in Hematological Remission

AbstractAbstract 4611The PML-RARα fusion protein plays a pivotal role in acute promyelocytic leukemia (APL) genesis and one of the mechanisms through which this fusion protein exerts its activity is the epigenetic suppression of gene transcription. PML-RARα physically interacts with epigenetic-modifying enzymes including DNA methyltransferases leading to hypermethylation and silencing of target genes. Nevertheless, the nature of the genes critical to APL oncogenesis is unclear, particularly in humans. We compared the methylation patterns at diagnosis and at remission in four patients with APL and four healthy controls. In order to isolate myeloid progenitors in the bone marrow (BM) samples from patients at remission and healthy controls, CD117 positive cells were sorted. Genomic DNA was sodium bisulfite converted and methylation analysis was performed using Illumina HumanMethylation27 BeadChip Platform (which analyzes methylation sites, including promoter and methylation hotspots of over 14,000 genes). DNA methylation values from the bead array hybridizations were scored as b-values. The high-variance features (> 0.05) were identified and filtered, and the three groups (APL at diagnosis, at remission and healthy controls) were analyzed in two-way comparisons. The functional categories of differentially methylated genes were determined using the Ingenuity Pathway Analysis (IPA, Ingenuity Systems). After filtering, 2,996 out of 27,578 features from the platform were selected. One hundred and eighteen hypermethylated loci were exclusively found in APL at diagnosis, and 72 were commonly found in healthy subjects and patients in remission, pointing out the similarity between the 2 groups. Clustering based on the filtered set of features showed distinct methylation patterns for three of the four samples at diagnosis, and for two of the samples of APL at remission and three in healthy samples. The genes found hypermethylated at diagnosis were mainly associated with cell death regulation (IPA score=31) and cancer development (IPA score=13). In order to correlate methylation and gene expression profiles, the number of transcripts of 214 genes was analyzed by real time PCR using three platforms of PCR Array (SABioscience-Qiagen). We analyzed the methylation status and gene expression of 20 genes detected hypermethylated in APL at diagnosis, of which nine were downregulated (EGF, ERBB2, FHIT, IL1A, JAK2, MLH1, MMP-7, TERT and WNT2) and two upregulated (RARA and WT1) when compared to samples of healthy controls (Table). The analysis of WT1 expression is of special relevance because it has been used for minimal residual disease detection in APL. Our results suggest that the aberrant expression of WT1 is not dependent on the epigenetic control by PML/RARa. Among the genes founded hypermethylated and downregulated, TERT, that encodes the catalytic subunit of telomerase and which transcription seems to be repressed in differentiating cells, has been demonstrated to be regulated by WT1. This is not the reflection of differentiation, since only CD117+ cells were analyzed. We also analyzed the expression of six genes detected hypermethylated in samples of healthy controls, of interest BRCA2 and HGF genes were hypermethylated but their expression was down and upregulated, respectively. Aberrant expression HGF is frequent in AML and leads to the autocrine activation of its receptor tyrosine kinase, MET, which promotes cell growth and survival of HGF-expressing AML cells. In conclusion, our results provide a first glimpse of the variation in DNA methylation among samples from APL patients at diagnosis and at remission compared to healthy controls, and identified potential target genes capable of conferring survival advantage to the leukemic cell.TableCorrelation between gene methylation and expression in APL at diagnosis (D), remission (R) and in healthy controls (HC)Gene SymbolMethylated GroupExpression (Fold Change) D × HCp valueBCL2HC/RNSBRCA2HC−3.20.02CD5DNSCDH1DNSCDKN1CDNSCDKN2BDNSEGFD−12.660.04ERBB2D−2.150.001FHITD−6.430.02GREB1HCNSHGFHC21.530.04IL1AD−11.030.04JAK2D−2.550.03LEPDNSMETDNSMLH1D−3.520.001MMP-7D−6.430.01FN1HCNSNF1ADNSRARAD3.720.002SFNDNSTERTD−12.380.03TESCDNSTNFHC/RNSWNT2D−7.320.03WT1D111.520.05HC, Healthy Controls; R, Remission; D, Diagnosis; NS, not significant. Disclosures:No relevant conflicts of interest to declare.

Transcriptional Silencing by Single-Stranded RNAs Targeting a Noncoding RNA That Overlaps a Gene Promoter

RNAi using single-strand RNA would provide new options for therapeutic development and for investigating critical questions of mechanism. Using chemically modified single-strands, we test the hypothesis that single-stranded RNAs can engage the RNAi pathway and silence gene transcription. We find that a chemically modified single-stranded silencing RNA (ss-siRNA) designed to be complementary to a long noncoding RNA (lncRNA) requires argonaute protein, functions through the RNAi pathway, and inhibits gene transcription. These data expand the use of single-stranded RNA to cell nuclei.

Activation of GABAA receptors suppresses ethanol‐induced upregulation of type 1 IP3 receptors

Although Type 1 inositol 1,4,5-trisphosphate receptors (IP(3) Rs-1) are one of the major calcium channels to regulate intracellular Ca(2+) concentration, there have been few available data how their expression is modified by long-term exposure to ethanol. The present study attempted to clarify mechanisms of modification of IP(3) R-1 expression during long-term ethanol exposure by γ-aminobutyric acid (GABA)A receptors using mouse cerebral cortical neurons. Long-term exposure to ethanol induced IP(3) R-1 protein upregulation following increased expression of its mRNA. Pretreatment with muscimol, a selective GABA(A) receptor agonist, significantly suppressed the ethanol-induced upregulation of IP(3) R-1 protein and its mRNA, which was significantly abolished by bicuculline, a selective GABA(A) receptor antagonist. These results indicate that GABA(A) receptors negatively regulate the ethanol-induced upregulation of IP(3) R-1 protein expression via the suppression of gene transcription.

A Novel Regulatory Factor Recruits the Nucleosome Remodeling Complex to Wingless Integrated (Wnt) Signaling Gene Promoters in Mouse Embryonic Stem Cells

The nucleosome remodeling and deacetylation (NuRD) complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered a key player in the formation of a functional NuRD complex. The recruitment of MBD3 to methylated promoters may require other prerequisite factors. We show that cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), an essential gene for early embryonic development, plays a role in pluripotency of ESC by engaging MBD3 to the promoter region of Wnt signaling genes. The occupancy of MBD3 on several promoters of Wnt genes was significantly lost in the absence of CDK2AP1, resulting in hyperactivation of Wnt. We propose that the transcriptional modulation of the Wnt pathway mediated by NuRD requires the presence of essential auxiliary components such as CDK2AP1, which may aid the association of the complex with specific focal regions of the target promoters.

Adenylate kinase 2 (AK2) promotes cell proliferation in insect development

BackgroundAdenylate kinase 2 (AK2) is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP) ↔ ATP(GTP) + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms.ResultsThis study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown.ConclusionThese results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development.

The H3K4me3 Histone Demethylase Fbxl10 Is a Regulator of Chemokine Expression, Cellular Morphology, and the Metabolome of Fibroblasts

Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC domain-containing histone demethylase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 consists of a CxxC domain, a PHD domain, and an Fbox domain. By purifying the JmjC and the PHD domain of Fbxl10 and using different approaches we were able to characterize the properties of these domains in vitro. Our results suggest that Fbxl10 is rather a H3K4me3 than a H3K36me2 histone demethylase. The PHD domain exerts a dual function in binding H3K4me3 and H3K36me2 and exhibiting E3 ubiquitin ligase activity. We generated mouse embryonic fibroblasts stably overexpressing Fbxl10. These cells reveal an increase in cell size but no changes in proliferation, mitosis, or apoptosis. Using a microarray approach we were able to identify potentially new target genes for Fbxl10 including chemokines, the noncoding RNA Xist, and proteins involved in metabolic processes. Additionally, we found that Fbxl10 is recruited to the promoters of Ccl7, Xist, Crabp2, and RipK3. Promoter occupancy by Fbxl10 was accompanied by reduced levels of H3K4me3 but unchanged levels of H3K36me2. Furthermore, knockdown of Fbxl10 using small interfering RNA approaches showed inverse regulation of Fbxl10 target genes. In summary, our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control of fibroblasts.

The effect of quercetin on human neutrophil elastase‐induced mucin5AC expression in human airway epithelial cells

Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. Mucus hypersecretion is a common pathological change in chronic inflammatory diseases of the airway. We investigated the effect of quercetin on mucin 5AC (MUC5AC) expression induced by human neutrophil elastase (HNE) in airway epithelial cells and its molecular mechanisms. Human airway epithelial (HBE16) cells were pretreated with quercetin and were treated with HNE. We found that HNE induced a significant increase in the levels of MUC5AC and EGFR in cells treated only with HNE. Quercetin suppressed gene transcription and protein expression of MUC5AC in a dose-dependent manner, with significant inhibition from 40μM. mRNA and protein expressions of EGFR decreased markedly when pretreated with quercetin. Among three MAPK proteins, only phosphorylated ERK1/2 protein expression increased significantly after treatment with HNE alone and decreased significantly after pretreatment with quercetin. HNE also activated phosphorylated PKC protein expression which was attenuated when pretreated with quercetin. These results suggest that quercetin can inhibit HNE-induced MUC5AC expression in human airway epithelial cells through PKC/EGFR/ERK signal transduction pathway. In the future, quercetin might be a valuable treatment for mucin hypersecretion in chronic inflammatory airway diseases in clinic.

Demethylation of the Coding Region Triggers the Activation of the Human Testis-Specific PDHA2 Gene in Somatic Tissues

Human PDHA2 is a testis-specific gene that codes for the E1α subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the −122 to −6 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-2′-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy.

Bisphenol A-Mediated Suppression of LPL Gene Expression Inhibits Triglyceride Accumulation during Adipogenic Differentiation of Human Adult Stem Cells

The endocrine disrupting chemical, bisphenol A (BPA), has been shown to accelerate the rate of adipogenesis and increase the amount of triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. The objective of this study was to investigate if that observation is mirrored in human primary cells. Here we investigated the effect of BPA on adipogenesis in cultured human primary adult stem cells. Continuous exposure to BPA throughout the 14 days of differentiation dramatically reduced triglyceride accumulation and suppressed gene transcription of the lipogenic enzyme, lipoprotein lipase (LPL). Results presented in the present study show for the first time that BPA can reduce triglyceride accumulation during adipogenesis by attenuating the expression of LPL gene transcription. Also, by employing image cytometric analysis rather than conventional Oil red O staining techniques we show that BPA regulates triglyceride accumulation in a manner which does not appear to effect adipogenesis per se.

Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src–Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs.

Epigenetic regulation of death-associated protein kinase expression in primary gastric cancers from Chinese patients

Death-associated protein kinase (DAPK) is a novel serine/threonine kinase involved in apoptosis and tumor suppression. Promoter methylation is an important mechanism by which tumor suppressor gene transcription is repressed in cancer cells. Although reduced expression and aberrant methylation of DAPK has been reported in various human cancers, including gastric cancer (GC), the results remain discrepant. We aimed to investigate DAPK mRNA and protein expression in primary GC tissues from Chinese patients and establish a possible relationship between the promoter methylation status and the decreased expression of DAPK. The mRNA level, protein expression, and promoter methylation of DAPK were examined, in the cancer tissues and the corresponding, adjacent nontumor tissues of the 62 GC cases, by RT-PCR, western blotting and methylation-specific PCR, respectively. DAPK mRNA and protein expression in GC tissues was significantly reduced compared with corresponding nontumor tissues (P<0.0001). The methylation frequency of the DAPK promoter in primary GC tissues is significantly higher than in the corresponding nontumor tissues (54.8 vs. 17.7%, P<0.0001). Furthermore, DAPK mRNA expression in tissues containing aberrant promoter methylation was significantly reduced compared with GC tissues with unmethylated DAPK promoter (P<0.0001). Moreover, a significant correlation was demonstrated between the TNM stage and the degree of DAPK promoter methylation in primary GCs (P=0.04). DAPK protein and mRNA expression was reduced in GC tissues of Chinese patients. Diminished expression of DAPK was associated with promoter methylation.

Docetaxel and 5-fluorouracil induce human p53 tumor suppressor gene transcription via a short sequence at core promoter element

The p53 tumor suppressor protein is involved in cellular defense against agents that can cause genetic damage. Induction of p53 gene expression at transcriptional and post-transcriptional levels by such agents results in p53-regulated gene activation or suppression. Docetaxel (DOC), a member of the taxanes family that is widely used in cancer chemotherapy, activates p53 at the transcriptional level. We demonstrated that p53 is induced by low dose DOC treatment, resulting in MDR-1 gene suppression in human lung cancer cells. To identify the cis-element of p53 promoter that responds to DOC, p53 promoter region was cloned and promoter activity was analyzed on luciferase gene reporter assay. Promoter region (−78 to +129) contained the highest basal p53 promoter activity and deletion of +86 to +129 severely reduced basal promoter activity. Basal promoter region included the 21-bp element (PE21) that determines UV-inducible expression of p53 and mediates DOC-inducible p53 expression. On site-specific mutagenesis of PE21 (−78 to −58), with mutation of ATTG (−62 to −59) to CGGT, completely diminished the response to DOC. The same mutations also inhibited 5-fluorouracil (5-FU)-inducible p53 expression. Our data revealed that a sequence located at PE21 of p53 core promoter regulates p53 induction by chemotherapeutic agents.

Induction of DNA Damage Signaling upon Rift Valley Fever Virus Infection Results in Cell Cycle Arrest and Increased Viral Replication

Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus infecting a wide range of vertebrate hosts. Of particular interest is the nonstructural NSs protein, which forms large filamentous fibril bundles in the nucleus. Past studies have shown NSs to be a multifaceted protein important for virulence through modulation of the interferon response as well acting as a general inhibitor of transcription. Here we investigated the regulation of the DNA damage signaling cascades by RVFV infection and found virally inducted phosphorylation of the classical DNA damage signaling proteins, ataxia-telangiectasia mutated (ATM) (Ser-1981), Chk.2 (Thr-68), H2A.X (Ser-139), and p53 (Ser-15). In contrast, ataxia-telangiectasia mutated and Rad3-related kinase (ATR) (Ser-428) phosphorylation was decreased following RVFV infection. Importantly, both the attenuated vaccine strain MP12 and the fully virulent strain ZH548 showed strong parallels in their up-regulation of the ATM arm of the DNA damage response and in the down-regulation of the ATR pathway. The increase in DNA damage signaling proteins did not result from gross DNA damage as no increase in DNA damage was observed following infection. Rather the DNA damage signaling was found to be dependent on the viral protein NSs, as an NSs mutant virus was not found to induce the equivalent signaling pathways. RVFV MP12-infected cells also displayed an S phase arrest that was found to be dependent on NSs expression. Use of ATM and Chk.2 inhibitors resulted in a marked decrease in S phase arrest as well as viral production. These results indicate that RVFV NSs induces DNA damage signaling pathways that are beneficial for viral replication.