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  • The novel deacetylase inhibitor AR-42 demonstrates pre-clinical activity in B-cell malignancies in vitro and in vivo. 20532179

    While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.In mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC(50) (50% growth inhibitory concentration) of AR-42 is 0.61 microM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC(50) (concentration lethal to 50%) of AR-42 is 0.76 microM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Chromatin remodeling is required for gene reactivation after decitabine-mediated DNA hypomethylation. 20713525

    The DNA hypomethylating drug decitabine (DAC) reactivates silenced gene expression in cancer and is approved for the treatment of the myelodysplastic syndrome. Gene reactivation after DAC is variable and incompletely understood. Here, we established a cell line system (YB5) derived from the SW48 colon cancer cell line to study DAC-induced reactivation. YB5 contains a hypermethylated cytomegalovirus promoter driving green fluorescent protein (GFP), and the locus is transcriptionally silent. GFP reexpression can be achieved by DAC treatment, but the expression level of individual cells is heterogeneous. DAC-treated YB5 cells were separated into GFP-positive and GFP-negative subpopulations. By comparing DAC-treated sorted GFP-positive and GFP-negative cells, we found that their methylation levels were similarly decreased but that histone modifications and histone H3 densities were remarkably different. Despite a similar degree of (incomplete) DNA hypomethylation, GFP-positive cells reverted to an active chromatin structure marked by higher H3K9 acetylation, lower H3K27 trimethylation, and lower promoter nucleosome density. GFP-negative cells had histone modifications and promoter nucleosome density, similar to parental cells. On DAC withdrawal, gradual resilencing and remethylation occurred in both GFP-positive and GFP-negative cells, and the resilencing correlated with a gradual increase in nucleosome occupancy in GFP-positive cells. These data show that hypomethylation alone after DAC is insufficient for gene expression induction, and that chromatin resetting to an active state including nucleosome eviction is required for activation of protein expression. Our findings suggest that gene expression is the key in optimizing DAC treatment strategies in the clinic.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • p21(WAF1/CIP1) induction by 5-azacytosine nucleosides requires DNA damage. 18223691

    Decitabine (DAC) and 5-azacitidine have recently been approved for the treatment of myelodysplastic syndrome. The pharmacodynamic effects of DAC and 5-azacitidine outside their known activity as inhibitors of DNA methyltransferases (DNMTs) require further investigation. The purpose of this study was to investigate the effect of DAC on the expression of p21(WAF1/CIP1), a gene with a putative CpG island surrounding its promoter region. Promoter methylation analysis of p21(WAF1/CIP1) in leukemia cells revealed the absence of CpG methylation. However, DAC upregulated p21(WAF1/CIP1) expression in a dose-dependent manner (ED(50)=103.34 nM) and induced G2/M cell cycle arrest in leukemia cells. Sequential application of DAC followed by different histone deacetylase inhibitors induced expression of p21(WAF1/CIP1) synergistically. Upregulation of p21(WAF1/CIP1) paralleled DAC-induced apoptosis (ED(50)=153 nM). Low doses of DAC induced gamma-H2AX expression (ED(50)=16.5 nM) and upregulated p21(WAF1/CIP1) in congenic HCT 116 colon cancer cells in a DNMT-independent and p53-dependent fashion. Inhibition of p53 transactivation by pifithrin-alpha or the kinase activity of ATM by either the specific ATM inhibitor KU-5593 or caffeine abrogated p21(WAF1/CIP1) upregulation, indicating that DAC upregulation of p21(WAF1/CIP1) was p53- and ATM-dependent in leukemia cells. In conclusion, DAC upregulates p21(WAF1/CIP1) in DNMT-independent manner via the DNA damage/ATM/p53 axis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • 5-Aza-2'-deoxycytidine sensitizes busulfan-resistant myeloid leukemia cells by regulating expression of genes involved in cell cycle checkpoint and apoptosis. 19732952

    Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we examined the cytotoxic activity of combined 5-aza-2'-deoxycytidine (DAC) and Bu. Exposure of Bu-resistant B5/Bu250(6) ML cells to 0.5muM DAC resulted in G2-arrest and apoptosis. The observed G2-arrest was associated with hypomethylation and subsequent expression of epigenetically controlled genes including p16(INK4A), activation of the p53 pathway, and phosphorylation of CDC2. The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were suppressed. Combination of 0.05muM DAC and 5mug/ml Bu resulted in synergistic cytotoxicity, which was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an apoptotic response. P53 inhibition in B5/Bu250(6) cells using pifithrin-alpha alleviated these effects, suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-null K562 cells to [DAC+Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-AML3 cell line. We conclude that the synergistic effects of [DAC+Bu] are p53-dependent and involve cell cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve the cytoreductive efficacy of Bu-based pretransplant regimen in patients with ML.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Chromatin-modifying agents increase transcription of CYP46A1, a key player in brain cholesterol elimination. 20930312

    The major mechanism of brain cholesterol elimination is the conversion of cholesterol into 24S-hydroxycholesterol by CYP46A1, a neuron-specific cytochrome P450. Since increasing evidence suggests that upregulation of CYP46A1 may be relevant for the treatment of Alzheimer's disease, we aim to identify the molecular mechanisms involved in CYP46A1 transcription. Our previous studies demonstrated the role of Sp transcription factors in basal expression and histone deacetylase (HDAC) inhibitor-dependent derepression of CYP46A1. Here, we show that the demethylating agent 5'-Aza-2'-deoxycytidine (DAC) is a CYP46A1 inducer and that pre-treatment with DAC causes a marked synergistic activation of CYP46A1 transcription by trichostatin A. Surprisingly, bisulfite sequencing analysis revealed that the CYP46A1 core promoter is completely unmethylated in both human brain and non-neuronal human tissues where CYP46A1 is not expressed. Therefore, we have investigated Sp expression levels by western blot and real-time PCR, and their binding patterns to the CYP46A1 promoter, by electrophoretic mobility shift assay and chromatin immunoprecipitation assays, after DAC treatment. Our results showed that DAC decreases not only Sp1 and Sp3 protein levels, but also the binding activity of Sp3 to the +1 region of the CYP46A1 locus. Concomitantly, HDAC1 and HDAC2 were also significantly dissociated from the promoter. In conclusion, DAC induces CYP46A1 gene expression, in a DNA methylation-independent mechanism, decreasing Sp3/HDAC binding to the proximal promoter. Furthermore, by affecting the expression of the Sp3 transcription factor in neuronal cells, DAC might affect not only brain cholesterol metabolism, but also the expression of many other neuronal genes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-100

  • Demethylation of the coding region triggers the activation of the human testis-specific PDHA2 gene in somatic tissues. 22675509

    Human PDHA2 is a testis-specific gene that codes for the E(1)α 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.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-623
    Nombre del producto:
    Anti-RNA polymerase II Antibody, clone CTD4H8

  • Three epigenetic drugs up-regulate homeobox gene Rhox5 in cancer cells through overlapping and distinct molecular mechanisms. 19679824

    Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2'-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-371
    Nombre del producto:
    EZ-ChIP™

  • Epigenetic regulation of MDR1 gene through post-translational histone modifications in prostate cancer. 24344919

    Multidrug resistance 1 (MDR1) gene encodes for an ATP binding cassette transporter--P-glycoprotein (P-gp)-- involved in chemoresistance to taxanes. MDR1 promoter methylation is frequent in prostate carcinoma (PCa), suggesting an epigenetic regulation but no functional correlation has been established. We aimed to elucidate the epigenetic mechanisms involved in MDR1 deregulation in PCa.MDR1 promoter methylation and P-gp expression were assessed in 121 PCa, 39 high-grade prostatic intraepithelial neoplasia (HGPIN), 28 benign prostatic hyperplasia (BPH) and 10 morphologically normal prostate tissue (NPT) samples, using quantitative methylation specific PCR and immunohistochemistry, respectively. PCa cell lines were exposed to a DNA methyltransferases inhibitor 5-aza-2'deoxycytidine (DAC) and histone deacetylases inhibitor trichostatin A (TSA). Methylation and histone posttranscriptional modifications status were characterized and correlated with mRNA and protein expression. MDR1 promoter methylation levels and frequency significantly increased from NPTs, to HGPIN and to PCa. Conversely, decreased or absent P-gp immunoexpression was observed in HGPIN and PCa, inversely correlating with methylation levels. Exposure to DAC alone did not alter significantly methylation levels, although increased expression was apparent. However, P-gp mRNA and protein re-expression were higher in cell lines exposed to TSA alone or combined with DAC. Accordingly, histone active marks H3Ac, H3K4me2, H3K4me3, H3K9Ac, and H4Ac were increased at the MDR1 promoter after exposure to TSA alone or combined with DAC.Our data suggests that, in prostate carcinogenesis, MDR1 downregulation is mainly due to histone post-translational modifications. This occurs concomitantly with aberrant promoter methylation, substantiating the association with P-gp decreased expression.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Long non-coding RNA DBCCR1-003 regulate the expression of DBCCR1 via DNMT1 in bladder cancer. 27777512

    Many long non coding RNAs have been identified as key modulators in cancer development. A lncRNA, DBCCR1-003, derived from the locus of tumor suppressor gene DBCCR1 (deleted in bladder cancer chromosome region 1), has unknown function. In the present study, we explored function and molecular mechanism of DBCCR1-003 in bladder cancer (BC) development.We evaluated the expression levels of DBCCR1-003 in tissues and cells with western blot and quantitative real-time polymerase chain reaction. Multiple approaches including chromatin immunoprecipitation assay and RNA immunoprecipitation were used to confirm the direct binding of DBCCR1-003 to DNMT1. The recombinant vector overexpressing DBCCR1-003 was constructed. Cell proliferation assay, colony formation assay and flow cytometric analysis were employed to measure the role of DBCCR1-003 in regulation of cell proliferation, cycle and apoptosis.Firstly we detected the expression of DBCCR1-003, DBCCR1, DNMT1 (DNA methyltransferase 1) and DNA methylation in the promoter of DBCCR1. We found low expression of DBCCR1-003, same as DBCCR1, while high expression of DNMT1 and hypermethylation of DBCCR1 gene promoter in BC tissues and T24 cells line. Further studies revealed that treatment of DNMT inhibitor, 5-aza-2-deoxycytidine(DAC), or overexpression of DBCCR1-003 led to increased DBCCR1 expression by reversion of promoter hypermethylation and DNMT1 binding to DBCCR1 promoter in T24 cells. Importantly, RNA immunoprecipitation (RIP) showed that DBCCR1-003 physically associates with DNMT1. The binding of them was increased with the inhibition of DBCCR1 promoter methylation, indicating that DBCCR1-003 may bind to DNMT1 and prevent DNMT1-mediated the methylation of DBCCR1. Furthermore, overexpression of DBCCR1-003 resulted in significant inhibition of T24 cells growth through the inducing G0/G1 arrest and apoptosis.Taken together, these findings demonstrated that a novel tumor suppressor DBCCR1-003 regulates the expression of DBCCR1 via binding to DNMT1 and preventing DNMT1-mediated the methylation of DBCCR1 in BC. LncRNA DBCCR1-003 may serve as a novel biomarker and therapeutic target for BC in future cancer clinic.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-10086
    Nombre del producto:
    EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit