Arsenic Trioxide Inhibits DNA Methyltransferase and Restores TMS1 Gene Expression in K562 Cells

2014 ◽  
Vol 133 (1) ◽  
pp. 18-25 ◽  
Author(s):  
Hongli Li ◽  
Yan Wang ◽  
Wenwei Xu ◽  
Lin Dong ◽  
Yan Guo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Flow Cytometry ◽  
Western Blot ◽  
Arsenic Trioxide ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
K562 Cells ◽  
Leukemia Cell Line ◽  
Dose Dependent ◽  
Experimental Group

Background: Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor genes in human cancers. The demethylating, dose-dependent effect of arsenic trioxide (As2O3) on several tumor-related genes has already been postulated. However, whether such a demethylating effect also applies to the TMS1 gene in chronic myeloid leukemia cell line K562 cells has not been studied so far. The aim of the present study was to detect the methylation status of the TMS1 gene in K562 cells and the demethylation effect of As2O3 on TMS1 as well as TMS1 apoptosis-associated protein Bcl-2/Bax and DNA methyltransferase (DNMT) expression. Methods: TMS1 mRNA expression in K562 cells and normal bone marrow was determined by reverse transcription (RT) polymerase chain reaction (PCR), and the DNA methylation status of the TMS1 promoter in K562 cells treated with different concentrations of As2O3 for 48 h was determined by methylation-specific PCR. RT-PCR and Western blot were used to detect TMS1 and DNMT expression. We also assessed TMS1-associated apoptosis protein Bcl-2/Bax expression by Western blot and apoptosis rates by flow cytometry using annexin V/propidium iodide double staining. Results: In K562 cells, TMS1 was completely methylated and both TMS1 mRNA and protein showed a low expression, but 2 μmol/l As2O3 could significantly restore the expression of the TMS1 gene both at mRNA and protein level (p < 0.01) by fully reversing DNA methylation. As2O3 decreased mRNA and protein expression of DNMT1 (p < 0.05) in a dose-dependent manner. Flow cytometry showed that in the experimental group (2 μmol/l As2O3), cell apoptosis was significantly increased compared with the control group (no As2O3; p < 0.05). In the experimental group, Western blot showed that the expression of the anti-apoptotic protein Bcl-2 was significantly decreased; however, the proapoptotic protein Bax was markedly increased and the Bcl-2/Bax ratio was markedly reduced (p < 0.01). Conclusions: As2O3 could restore the expression of TMS1 by inhibiting DNMT to reverse the hypermethylation and induced apoptosis of K562 cells by downregulation of Bcl-2/Bax expression.

Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Jessilyn Dunn ◽  
Haiwei Qiu ◽  
Soyeon Kim ◽  
Daudi Jjingo ◽  
Ryan Hoffman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Western Diet ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

scholarly journals Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of the Gamma Interferon (IFN-γ) Promoter and Subsequent Downregulation of IFN-γ Production

1998 ◽  
Vol 18 (9) ◽  
pp. 5166-5177 ◽  
Author(s):  
Judy A. Mikovits ◽  
Howard A. Young ◽  
Paula Vertino ◽  
Jean-Pierre J. Issa ◽  
Paula M. Pitha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Methylation Status ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1 ◽  
De Novo Methylation

ABSTRACT The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-γ), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-γ, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-γ gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-γ gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-γ gene, and increased IFN-γ production, demonstrating a direct link between methyltransferase and IFN-γ gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-γ gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

Title: Promoter Methylation of Selected Genes and Response to Azacitidine (AZ) Therapy in Patients with Non-BCR-ABL Myeloproliferative Disorders (MPDs)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4625-4625
Author(s):  
Nicholas Achille ◽  
Laura Michaelis ◽  
Scott E. Smith ◽  
Eliza Germano ◽  
Nancy J. Zeleznik-Le ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Promoter Methylation ◽  
Research Funding ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Cpg Islands ◽  
Myeloproliferative Disorders ◽  
Response To Treatment ◽  
Promoter Regions ◽  
Days Of Therapy

Abstract Abstract 4625 Background: Gene silencing via methylation of CpG islands in the promoter regions of many genes but specifically of APAF1, p15INK4B, p16INK4A, RARB, and CDH1 appears to play a role in pathogenesis of myeloid malignancies. Azacitidine (AZ) causes demethylation by inhibiting DNA methyltransferase and has already been shown to be an effective therapy for myelodysplastic syndromes. The demethylation induced by AZ is detectable in about 48 hours and increases significantly after 5 days of therapy. After that, the effect tends to plateau. Methods: We initiated a Phase 2 study of patients with non-BCR-ABL MPDs to determine clinical response to AZ therapy and correlate it with promoter DNA methylation and gene re-expression. The protocol was approved by the institutional IRB. Patients received AZ 75mg/m2 s/c for days 1–7 and repeated every 28 days for a minimum of 4 cycles. Responders were allowed to continue treatment until disease progression. Pretreatment and D 7 peripheral blood samples were analyzed for promoter methylation status and expression of the 5 genes mentioned above. Bisulfite conversion of DNA was followed by quantitative PCR using primers specific for methylated or for unmethylated promoter regions. For gene re-expression analysis, quantitative RT-PCR was performed with RNA isolated from the same patient samples and the same time points as the DNA methylation analyses. Results: Seven patients were enrolled before the study closed due to lack of accrual. The diagnoses were: Myelofibrosis (MF) 4, essential thrombocythemia 1, unclassified MPD with dysplasia 2. One patient with MF and one with unclassified MPD responded, the latter with normalization of marrow karyotype. Both responses were accompanied by significant decrease in APAF1 promoter methylation and surprisingly, an increase in promoter methylation of RARB. In three of the non-responders, APAF1 methylation increased. In patients with decreased Apaf1 methylation, a statistically significant increase in mRNA expression was observed. Conclusions: Within its limitations, this small trial shows that the methylation status of selected genes, particularly of APAF1 and RARB (inversely) is associated with response to treatment with azacitidine in patients with MPDs. In non-responders, Apaf1 methylation appears to increase. A larger study will be necessary to confirm these preliminary observations. Disclosures: Smith: Seattle Genetics, Inc.: Research Funding; Cephalon: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Spectrum: Consultancy; GSK: Speakers Bureau. Nand:Celgene Corporation: Research Funding.

scholarly journals Naturally occurring methylation inhibitor: DNA hypomethylation and hemoglobin synthesis in human K562 cells.

1987 ◽  
Vol 7 (5) ◽  
pp. 1759-1763 ◽  
Author(s):  
S B Lyon ◽  
L Buonocore ◽  
M Miller
Keyword(s):  
Dna Methylation ◽  
Genomic Dna ◽  
Dna Methyltransferase ◽  
Globin Gene ◽  
K562 Cells ◽  
Gene Probe ◽  
Dna Hypomethylation ◽  
Naturally Occurring ◽  
Methylation Inhibitor

A naturally occurring methylation inhibitor isolated from rabbit liver and named methinin inhibits a number of methyltransferases. Methinin is a low-molecular-weight compound (1,400) that has an active amine group. This compound inhibits the DNA methyltransferase of human erythroleukemia cells (K562) in vitro. When the K562 cells were grown in medium containing methinin, fetal hemoglobin was produced. Small but detectable amounts of adult hemoglobin were also produced. Methinin was not toxic to these cells. The overall rate of genomic DNA methylation was reduced by 60% in cells grown in medium containing methinin. Southern blots of genomic DNA from methinin-treated cells and untreated cells hybridized to a 32P-labeled globin gene probe showed that one site in the globin gene region was hypomethylated. Methinin is a naturally occurring compound which inhibits DNA methylation both in vitro and in vivo.

scholarly journals Developmentally Regulated DNA Methylation in Dictyostelium discoideum

2006 ◽  
Vol 5 (1) ◽  
pp. 18-25 ◽  
Author(s):  
Mariko Katoh ◽  
Tomaz Curk ◽  
Qikai Xu ◽  
Blaz Zupan ◽  
Adam Kuspa ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genomic Dna ◽  
Dna Methyltransferase ◽  
Critical Role ◽  
Methylation Status ◽  
Cpg Islands ◽  
Developmentally Regulated ◽  
Unusual Distribution ◽  
Methyltransferase Gene ◽  
Morphological Defects

ABSTRACT Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure, and cellular differentiation of eukaryotes. Previous studies failed to detect 5-methylcytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila melanogaster, mouse, and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation-sensitive and -insensitive restriction endonucleases, we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development.

OS05.2.A MGMT promoter status in IDH1/2 mutant anaplastic astrocytoma patients assessed by DNA methylation profiling and qMS-PCR: a report from the EORTC Brain Tumor Group

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii6-ii7
Author(s):  
C M S Tesileanu ◽  
P J French ◽  
M Sanson ◽  
A A Brandes ◽  
W Wick ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Anaplastic Astrocytoma ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
High Grade Glioma ◽  
Phase Iii ◽  
Isocitrate Dehydrogenase 1 ◽  
Kappa Score ◽  
Dna Methylation Profiling ◽  
Methylation Profiling

Abstract BACKGROUND Temozolomide (TMZ) efficacy in high-grade glioma is related to O 6-methylguanine DNA methyltransferase promoter (MGMTp) methylation. We compared the prognostic and predictive effect of MGMTp between DNA methylation profiling (the MGMT-STP27 model) and quantitative methylation specific polymerase chain reaction (qMS-PCR) in isocitrate dehydrogenase 1 and 2 (IDH1/2) mutant (mt) anaplastic astrocytoma patients. MATERIAL AND METHODS The 2x2 factorial design phase III CATNON trial randomized 751 adult patients with newly diagnosed 1p/19q non-codeleted anaplastic glioma to 59.4 Gy radiotherapy (RT), RT with concurrent TMZ, RT with 12 cycles of adjuvant TMZ, or RT with concurrent and adjuvant TMZ. MGMTp methylation status was assessed with the MGMT-STP27 model using 850k EPIC data, and qMS-PCR. IDH1/2 mutation status was determined with a next-generation sequencing panel. Overall survival (OS) was measured from date of randomization. RESULTS We identified 444 IDH1/2mt anaplastic astrocytoma patients of which MGMT-STP27 data was available for 440 patients (99.1%), qMS-PCR data for 361 patients (81.3%), and both for 357 patients (80.4%). MGMTp was methylated in 365 patients (83.0%) for the MGMT-STP27 model, and 168 patients (46.5%) for qMS-PCR. The agreement between the MGMT-STP27 model and qMS-PCR is 59.9% with a Cohen’s Kappa score of 0.229. At database lock, 289 patients with MGMT-STP27 data were still alive and 236 patients with qMS-PCR data. The median OS of MGMTp methylated glioma patients was 9.1 yrs [95 % confidence interval (CI) 7.5-not reached] for the MGMT-STP27 model, and not reached [95 % CI 9.1-not reached] for the qMS-PCR data. For MGMTp unmethylated glioma patients, the median OS was 6.9 yrs [95% CI 6.2-not reached] for the MGMT-STP27 model, and 6.8 yrs [95% CI 6.2–9.7] for the qMS-PCR data. The hazard ratio (HR) for OS based on MGMTp methylation was 0.88 [95% CI 0.58–1.31] for the MGMT-STP27 data, and 0.72 [95% CI 0.50–1.03]) for the qMS-PCR data. The HR for OS after RT with any TMZ vs RT alone for the MGMT-STP27 model was 0.53 [95% CI 0.37–0.78] for MGMTp methylated, and 0.54 [95% CI 0.25–1.18] for MGMTp unmethylated glioma patients; and for the MS-PCR data was 0.34 [95% CI 0.19–0.61] for MGMTp methylated, and 0.53 [95% CI 0.33–0.85] for MGMTp unmethylated glioma patients. CONCLUSION MGMTp methylation, regardless of assay, was neither prognostic nor predictive for outcome to temozolomide in IDH1/2mt anaplastic astrocytoma patients.

Arsenic Trioxide Targets the Interleukin-6 Cascade in Multiple Myeloma by Promoting Lysosomal Degradation of the Interleukin-6 Receptor Complex.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5159-5159
Author(s):  
Wai Chung Cheung ◽  
Yok Lam Kwong
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Line ◽  
Arsenic Trioxide ◽  
Western Blotting ◽  
Interleukin 6 ◽  
Receptor Complex ◽  
Lysosomal Degradation ◽  
Dose Dependent

Abstract Introduction. Treatment of multiple myeloma (MM), a B-cell neoplasm characterized by clonal expansion of plasma cells in the bone marrow, remains unsuccessful in a significant proportion of patients, so that innovative strategies are needed. Arsenic trioxide (As2O3) has shown notable efficacy against MM in vitro and in clinical studies. Multiple cellular pathways in MM are targeted by As2O3. As cellular growth of MM cells is interleukin-6 (IL-6) dependent, we investigated if As2O3 also targeted the IL-6 cascade. Materials and methods. The IL-6-dependent MM cell line U266 was used as an in vitro model. Cell growth was measured by MTT assay, and apoptosis by flow cytometry. Protein phosphorylation was studied by Western blotting with specific antibodies. Expression of IL-6 receptor (IL-6R) was investigated by Western blotting and flow cytometry. Gene expression was detected by quantitative polymerase chain reaction (Q-PCR). Results. As2O3 showed a time and dose related inhibition of U266 cellular proliferation by induction of apoptosis. At clinically achievable concentrations (2 – 4 μmol/L), As2O3-induced apoptosis was associated with inhibition of constitutive tyrosine phosphorylation of JAK2 and STAT3, in a time and dose-dependent manner. Furthermore, pre-treatment of U266 cells with As2O3 prevented rescue of phosphorylation of JAK2 and STAT3 by exogenous IL-6, implying that the IL-6 cascade was targeted. Using Western blot analysis, we showed that As2O3 induced a time and dose-dependent down-regulation of both components of the IL-6R complex: IL-6R alpha subunit (IL-6Rα) and gp130 signal transducer. These results were confirmed by flow cytometry, showing that As2O3 treatment led to a down-regulation of surface expression of the IL-6Rα. Interestingly, Q-PCR did not reveal any change in the mRNA levels of the two genes with As2O3 treatment, suggesting that As2O3 downregulated IL-6R complex via a post-transcriptional mechanism. It is known that under physiological conditions, the IL-6R complex is internalized upon ligand binding and is targeted to lysosomes for degradation. Treatment of the U266 cell line with the lysosome inhibitor ammonium chloride totally abrogated As2O3-induced degradation of IL-6Rα and gp130. These results suggested that As2O3 might promote lysosomal degradation of IL-6Rα and gp130 by inducing a ligand-independent internalization of the receptor complex. Conclusion. Our results demonstrated that As2O3 suppressed IL-6-induced JAK/STAT3 signaling pathway in MM cells and this might be, at least partly, mediated by promoting ligand-independent internalization and lysosomal degradation of the IL-6R complex. These results have significant implications on the use of As2O3 in the treatment of patients with MM and other malignancies that are IL-6 dependent.

Hypermethylation of O6-Methylguanine-DNA Methyltransferase (MGMT) Predicts Response to Laromustine (Cloretazine®) in Patients with Acute Myelogenous Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4463-4463
Author(s):  
Ilan Bernstein ◽  
Francis Giles ◽  
Susan O’Brien ◽  
Elizabeth Sullivan ◽  
Norbert Vey ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Risk ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Single Agent ◽  
Epigenetic Silencing ◽  
Dna Hypermethylation ◽  
Mgmt Gene ◽  
Methylguanine Dna Methyltransferase ◽  
Encoding Gene

Abstract Laromustine (Cloretazine®) (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl] hydrazine), a sulfonylhydrazine prodrug producing chlorethylating and carbamoylating subspecies, has demonstrated clinical activity in patients with hematologic disorders (Giles et al., J Clin Oncol, 2007). The effect of laromustine is modulated primarily through the formation of hard chloroethylating electrophiles with preferential alkylating activity for the O6 position of guanine, ultimately resulting in the formation of interstrand cross-links which prevent DNA replication and transcription, leading to cell death. The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) plays a major role in repairing O6-chloroethylguanine alkylations, critical to the formation of interstrand crosslinks. Epigenetic silencing of the MGMT encoding gene related to DNA hypermethylation has been shown to participate in the pathogenesis of neoplastic disease (Hegi et al., NEJM 2005). Since the alkylating properties of laromustine target DNA sites normally repaired by MGMT, the absence of the enzyme may represent a unique cellular environment for specific susceptibility to laromustine (Ishiguro et al., Mol Cancer Ther, 2005). In vitro findings have supported the hypothesis that cellular content of MGMT may predict response. In vivo correlation between clinical response in patients treated with laromustine and the cellular evidence of epigenetic silencing of the encoding MGMT gene has yet to be demonstrated (Giles et al., Clin Cacner Res, 2004). Our research aimed to determine the DNA methylation status of MGMT isolated from the peripheral blood or bone marrow of patients with AML or high-risk MDS enrolled to a phase II, single-agent study of laromustine (600 mg/m2 IV over 30 minutes) (Giles et al. J Clin Oncol, 2007). We also aimed to establish a correlation between hypermethylation of MGMT and clinical response to laromustine. Combined bisulfite restriction analysis (COBRA) was used to determine the MGMT gene methylation status of patients treated with laromustine. Bone marrow or peripheral blood leukocyte samples from 76 patients enrolled in a phase II, single agent study of laromustine were coded and blinded to investigators. DNA from each sample was extracted and bisulfite treated. PCR was used to amplify the MGMT CpG Island promoter region (REF/NT_008818.15/Hs10_8975) from 58 patients, prior to methylation specific restriction enzyme digestion. Results were correlated with clinical data of response to laromustine. The DNA methylation status of MGMT was determined in 58 of the enrolled patients. DNA hypermethylation was found in 3 of the 58 patients (5%). Two of these 3 patients achieved a complete response (CR) (66%), compared to 11 of the 55 patients who achieved a CR, CR with platelet recovery &lt; 100,000/mm3 (CRp) or a partial response (PR) and did not exhibit MGMT hypermethylation (20%). Hypermethylation of the MGMT gene promoter is a rare event in AML (5%), however, epigenetic inactivation of MGMT may predict a subgroup of patients with a higher likelihood of response to laromustine (Odds Ratio=8). Clinical investigation of laromustine in patients with AML, high-risk MDS and other cancers, specifically those with a higher methylation frequency of the MGMT encoding gene is warranted.

Epor Over-Expression In TEL-AML1 Positive ALL: Critical Roles for DNA Methylation, GATA-2 or MicroRNAs?

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3619-3619
Author(s):  
Marie Breen ◽  
Vivien Hodges ◽  
Marketa Zaliova ◽  
Jan Trka ◽  
Terence R.J. Lappin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Western Blot ◽  
Cell Line ◽  
Methylation Status ◽  
Chromosomal Translocation ◽  
Over Expression ◽  
Cpg Sites ◽  
Reh Cells ◽  
Epor Expression

Abstract Abstract 3619 Erythropoietin (Epo) and its receptor (EpoR) play vital roles in regulating red blood cell production. However, aberrant over-expression of EpoR has been reported in a wide range of tumours and hematological malignancies, particularly acute lymphoblastic leukemia (ALL) with the chromosomal translocation TEL-AML1 (ETV6/RUNX1). Around 25% of B-cell ALLs contain this chromosomal translocation which is associated with a favourable, prognosis but the role of EpoR is unknown. We have investigated whether methylation, transcription factors or miRNA regulation are implicated in the mechanism of the specific EpoR up-regulation associated with the TEL-AML1 rearrangement using two human B precursor cell line models. The pre-B REH cell line is TEL-AML1 positive whereas the pre-B NALM-6 cell line is TEL-AML1 negative; in addition the erythroleukemia cell line UT-7, which constitutively over- expresses EpoR was used as a positive control. Sequencing confirmed that the EpoR in these cell lines was wild type. Quantitative RT-PCR (RQ-PCR) of EpoR mRNA levels showed ~25 fold higher expression in REH cells compared to NALM-6 cells, verified by western blot analysis. RQ-PCR analysis of 20 pediatric ALL patient samples confirmed a 14-fold up-regulation (p<0.05) of EpoR in TEL-AML1 positive patients compared to hyper-diploid ALL patients without the translocation. Furthermore, the EpoR is functional in REH cells as serum starvation followed by stimulation with 10 U/ml Epo clearly activated the JAK2/STAT5, PI3K/Akt and Ras/ERK downstream signaling pathways within 15 min. In contrast, NALM-6 cells showed no activation of the signaling pathways in response to Epo. DNA methylation is an epigenetic marker which can directly impact on gene transcription, with aberrant methylation reported in many cancers. The methylation status of 18 CpG sites over 342 bp within the EpoR promoter encompassing the GATA and Sp1 binding sites was analysed by pyrosequencing in REH and NALM-6 cells. Fifteen of the 18 CpG sites showed significantly higher (p<0.001) levels of methylation in the NALM-6 cells (13-71%) compared to REH (2.4-10%) and UT-7 (1.8-8.5%) cells. The 15 significantly different sites had an average of 4.1% methylation in REH and UT-7 cells and 36.6% methylation in NALM-6 cells, showing a difference of 32.5% between the REH and NALM-6 cells. Comparable levels of methylation were seen at three CpG sites (7, 8 and 18) located near the Sp1 binding site. Treatment of NALM-6 cells with the IC50 dose (0.5 μmol) of Decitabine resulted in a regional decrease in methylation but no overall increase in EpoR expression whereas treatment of REH cells with Decitabine had no effect on methylation or expression. This suggests that methylation status of the EpoR promoter is altered by the presence of the TEL- AML1 fusion protein but may not be the critical factor in the up-regulation of EpoR expression. The GATA family of zinc-finger proteins have well characterised roles in hematopoiesis and EpoR regulation. Measurement of the expression of all six GATA transcription factors, by RQ-PCR showed that only GATA-2 was significantly up-regulated in REH cells (p<0.001) compared to the TEL-AML1 negative NALM-6 cell line with a Ct difference of 8.6 (~388 fold change). The up-regulation of GATA-2 was recapitulated in 10 TEL-AML1 positive patients and 10 hyper-diploid ALL patients who showed an average 28-fold difference in expression (p<0.0001). An increase in GATA-2 protein levels was also evident in REH cells using western blot analysis. Furthermore, initial studies have indicated differential expression, between the REH and NALM-6 cell lines, in miRNA whose targets have been predicted (www.diana.pcbi.upenn.edu) to be EpoR and/or GATA-2. Further investigation of the modulation of EpoR expression by methylation, GATA-2 or miRNAs, will lead to a greater understanding of of the factors regulating the expression of EpoR and the potential for alternative forms of therapeutic intervention in patients with TEL-AML1 ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Suppression of Antimicrobial Peptide Expression by Ureaplasma Species

2014 ◽  
Vol 82 (4) ◽  
pp. 1657-1665 ◽  
Author(s):  
Li Xiao ◽  
Donna M. Crabb ◽  
Yuling Dai ◽  
Yuying Chen ◽  
Ken B. Waites ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Flow Cytometry ◽  
Host Defense ◽  
Methylation Status ◽  
Innate Immune ◽  
Gene Promoters ◽  
Defense Genes ◽  
Content Type ◽  
Cell Defense

ABSTRACTUreaplasmaspecies commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome ofUreaplasmainfections. THP-1 cells, a human monocytoid tumor line, were cocultured withUreaplasma parvumandU. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR.In vitroantimicrobial activities of synthetic AMPs againstUreaplasmaspp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation withU. parvumandU. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1,DEFA5,DEFA6, andCAMP), was significantly downregulated compared to that ofTNFAandIL-8, which were upregulated.In vitroflow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity againstUreaplasmaspp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation withU. parvuminfection. No DNA methylation status changes were detected uponUreaplasmainfection. In conclusion, AMPs havein vitroactivity againstUreaplasmaspp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization.

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