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  • Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells. 22105179

    In vitro disease modeling using pluripotent stem cells can be a fast track screening tool for toxicological testing of candidate drug molecules. Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents in drug screening. In the present investigation, we exposed 14- to 21-day-old embryoid bodies (EBs) to three different concentrations of DMSO [0.01% (low dose), 0.1% (medium dose) and 1.0% (high dose)] to identify the safest dose that could effectively be used as solvent. We found that DMSO treatment substantially altered the morphology and attachment of cells in concurrence with a significant reduction in cell viability in a dose-dependent manner. Gene expression studies revealed a selective downregulation of key markers associated with stemness (Oct-4, Sox-2, Nanog and Rex-1); ectoderm (Nestin, TuJ1, NEFH and Keratin-15); mesoderm (HAND-1, MEF-2C, GATA-4 and cardiac-actin); and endoderm (SOX-17, HNF-3β, GATA-6 and albumin), indicating an aberrant and untimely differentiation trajectory. Furthermore, immunocytochemistry, flow cytometry and histological analyses demonstrated substantial decrease in the levels of albumin and CK-18 proteins coupled with a massive reduction in the number of cells positive for PAS staining, implicating reduced deposits of glycogen. Our study advocates for the first time that DMSO exposure not only affects the phenotypic characteristics but also induces significant alteration in gene expression, protein content and functionality of the differentiated hepatic cells. Overall, our experiments warrant that hESC-based assays can provide timely alerts about the outcome of widespread applications of DMSO as drug solvent, cryoprotectant and differentiating agent.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1637
    Product Catalog Name:
    Anti-Tubulin Antibody, beta III isoform, CT, clone TU-20 (Similar to TUJ1)

  • Induction of Apoptosis in Pancreatic Cancer Cells by CDDO-Me Involves Repression of Telomerase through Epigenetic Pathways. 25152840

    Reactivation of telomerase in cancers provides an attractive target for developing novel agents to selectively destroy tumor cells. Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a synthetic oleanane triterpenoid, inhibited cell proliferation and induced apoptosis in pancreatic cancer cells at very low concentrations. The antiproliferative and apoptosis-inducing effects of CDDO-Me were associated with the inhibition of human telomerase reverse transcriptase (hTERT) mRNA, hTERT protein and reduction in hTERT telomerase activity. CDDO-Me inhibited multiple transcription factors that regulate hTERT expression positively (Sp1, c-Myc and NF-κB) and negatively (CTCF, E2F-1 and MAD1). CDDO-Me inhibited protein levels of DNA methyl transferases DNMT1 and DNMT3a, which also resulted in hypomethylation of hTERT promoter. In addition, transcriptionally active chromatin markers, such as acetylated histone H3 (Lys 9), acetylated histone H4, di-methyl H3 (Lys 4) and tri-methyl H3 (Lys 9) were all reduced in pancreatic cancer cells treated with CDDO-Me. Chromatin immunoprecipitation analysis showed decreased histone deacetylation and histone demethylation at hTERT promoter. Collectively, these results indicate that down-regulation of telomerase through epigenetic mechanisms plays a critical role in induction of apoptosis in pancreatic cancer cells by CDDO-Me.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Hypermethylated in cancer 1 (HIC1), a tumor suppressor gene epigenetically deregulated in hyperparathyroid tumors by histone H3 lysine modification. 22544915

    Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance.The aim of the study was to investigate whether HIC1 may act as a tumor suppressor in the parathyroid glands and whether deregulated expression involves epigenetic mechanisms.Parathyroid tumors from patients with pHPT included single adenomas, multiple tumors from the same patient, and cancer. Hyperplastic parathyroid glands from patients with sHPT and hypercalcemia and normal parathyroid tissue specimens were included in the study. Quantitative RT-PCR, bisulfite pyrosequencing, colony formation assay, chromatin immunoprecipitation, and RNA interference was used.HIC1 was generally underexpressed regardless of the hyperparathyroid disease state including multiple parathyroid tumors from the same patient, and overexpression of HIC1 led to a decrease in clonogenic survival of parathyroid tumor cells. Only the carcinomas showed a high methylation level and reduced HIC1 expression. Cell culture experiments, including use of primary parathyroid tumor cells prepared directly after operation, the general histone methyltransferase inhibitor 3-deazaneplanocin A, chromatin immunoprecipitation, and RNA interference of DNA methyltransferases and EZH2 (enhancer of zeste homolog 2), supported a role of repressive histone H3 modifications (H3K27me2/3) rather than DNA methylation in repression of HIC1.The results strongly support a growth-regulatory role of HIC1 in the parathyroid glands and suggest that perturbed expression of HIC1 may represent an early event during tumor development. Repressive histone modification H3K27me2/3 is involved in repression of HIC1 expression in hyperparathyroid tumors.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Mitochondrial glutamate carrier GC1 as a newly identified player in the control of glucose-stimulated insulin secretion. 19584051

    The SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting beta-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E beta-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E beta-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5 mM glucose and stimulated either by intermediate 7.5 mM glucose or non-nutrient 30 mM KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mM glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5 mM dimethyl glutamate) fully restored the secretory response at 15 mM glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mM glucose, both during first (-25%, p < 0.05) and second (-33%, p < 0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier.
    Document Type:
    Reference
    Product Catalog Number:
    AP132F
    Product Catalog Name:
    Goat Anti-Rabbit IgG Antibody, FITC conjugate

  • Assessment of the integrin alpha5beta1 antagonist JSM6427 in proliferative vitreoretinopathy using in vitro assays and a rabbit model of retinal detachment. 19815730

    To explore the role of integrin alpha5beta1 in proliferative vitreoretinopathy (PVR) pathogenesis by evaluating the expression alpha5beta1 on ARPE-19 cells and patient proliferative membranes, quantifying the inhibitory effects of JSM6427 (a small molecule alpha5beta1 inhibitor) on ARPE-19 cell adhesion and migration, and assessing the therapeutic potential of JSM6427 in a rabbit retinal detachment model.Expression of alpha5beta1 was evaluated on activated ARPE-19 cells by flow cytometry and on PVR membranes by immunohistochemistry. ARPE-19 cells were used in fibronectin-dependent adhesion and migration assays with various concentrations of JSM6427; IC(50) was calculated. In the rabbit model, eyes were intravitreally injected with vehicle or JSM6427 on day 0 or 1 after retinal detachment; BrdU was administered intravitreally on day 3, and retinal tissues were harvested on day 3 (4 hours later) or 7. Retinal scarring, cellular proliferation, and inflammatory responses were quantified, and retinal morphology was analyzed in retinal sections.Activated ARPE-19 cells and PVR membranes expressed high levels of alpha5beta1; expression was low in control eyes. JSM6427 provided a dose-dependent blockade of ARPE-19 cell adhesion to fibronectin (IC(50), 7.1 +/- 2.5 microM) and inhibition of migration (IC(50), 6.0 +/- 4.5 microM). In the rabbit model, intravitreal injection of JSM6427 provided significant inhibition of proliferation of retinal cells (Müller cells, microglia, and macrophages) on days 3 and 7 after detachment and inhibition of inflammatory response and retinal scarring on day 7 after detachment.JSM6427 is a promising treatment for PVR, with data suggesting that inhibition of alpha5beta1-fibronectin interactions addresses multiple pathways involving retinal pigment epithelial, glial, and inflammatory cells.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Choline deficiency alters global histone methylation and epigenetic marking at the Re1 site of the calbindin 1 gene. 19752176

    Maternal choline availability is essential for fetal neurogenesis. Choline deprivation (CD) causes hypomethylation of specific CpG islands in genes controlling cell cycling in fetal hippocampus. We now report that, in C57BL/6 mice, CD during gestational days 12-17 also altered methylation of the histone H3 in E17 fetal hippocampi. In the ventricular and subventricular zones, monomethyl-lysine 9 of H3 (H3K9me1) was decreased by 25% (Pless than 0.01), and in the pyramidal layer, dimethyl-lysine 9 of H3 (H3K9me2) was decreased by 37% (Pless than 0.05). These changes were region specific and were not observed in whole-brain preparations. Also, the same effects of CD on H3 methylation were observed in E14 neural progenitor cells (NPCs) in culture. Changes in G9a histone methyltransferase might mediate altered H3K9me2,1. Gene expression of G9a was decreased by 80% in CD NPCs (Pless than 0.001). In CD, H3 was hypomethylated upstream of the RE1 binding site in the calbindin 1 promoter, and 1 CpG site within the calbindin1 promoter was hypermethylated. REST binding to RE1 (recruits G9a) was decreased by 45% (Pless than 0.01) in CD. These changes resulted in increased expression of calbindin 1 in CD (260%; Pless than 0.05). Thus, CD modulates histone methylation in NPCs, and this could underlie the observed changes in neurogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Characterization of Na(+)/H(+) antiporter activity in PC-Cl3 thyroid cells. 12876378

    Background/Aims: In thyroid cells, the intracellular pH plays a key role in the control of the iodide uptake, since iodide accumulation is associated with an intracellular acidification. In the present paper we studied the kinetic proprieties of the Na(+)/H(+) antiporter (NHE) and the molecular expression of different NHE isoforms in rat thyroid PC-Cl3 cells. In addition the intracellular buffer capacity was also evaluated. Methods: pHi was measured using the pH sensitive fluorescent dye BCECF-AM. Amiloride, 5-(N,N-Dimethyl) hydrochloride was used to inhibit NHE activity. RT-PCR and western blot analyses were used to study the expression of NHE mRNA and protein isoforms. Results: PC-Cl3 cells shown a resting pHi, in the absence of CO(2)/HCO(3)(-), of 6.94 +/- 0.1; after an acid load PC-Cl3 cells recovered toward resting pHi value, using a Na-dependent H(+) extrusion mechanisms which was amiloride sensitive (K(i) = 23 microM). The kinetic parameters were K((Na)app) = 10 +/- 2 mM and V(max) = 0.23 +/- 0.02 DeltapH/min x 10(5) cells. NHE1, NHE2 and NHE3 were expressed at the mRNA level as well as at the protein level. Conclusion: PC-Cl3 cells express a functional Na/H exchange activity and different isoforms (NHE1, NHE2 and NHE3) are expressed in the plasma membrane.
    Document Type:
    Reference
    Product Catalog Number:
    AB3083

  • Distribution and role of Na(+)/K(+) ATPase in endocardial endothelium. 11738066

    OBJECTIVE: In mammalian cardiomyocytes, alpha isoforms of Na(+)/K(+) ATPase have specific localisation and function, but their role in endocardial endothelium is unknown. METHODS: Different alpha isoforms in endocardial endothelium and cardiomyocytes of rabbit were investigated by measuring contractile parameters of papillary muscles, by RT-PCR, by Western blots and by immunocytochemistry. RESULTS: Inhibition of Na(+)/K(+) ATPase by decreasing external K(+) from 5.0 to 0.5 mmol/l caused biphasic inotropic effects. The maximal negative inotropic effect at external K(+) of 2.5 mmol/l was significantly larger in +EE muscles (with intact endocardial endothelium) than in -EE muscles (with endocardial endothelium removed) (-22.5+/-2.4% versus -5.9+/-4.0%, n=7, P0.05). Further decrease of K(+) to 0.5 mmol/l caused endothelium-independent positive inotropy (27.8+/-11.8% for +EE versus 18.6+/-11.3% for -EE, n=7, P>0.05). Inhibition of Na(+)/K(+) ATPase either by dihydro-ouabain (10(-9) to 10(-4) mol/l, n=4) or by K(+) decrease following inhibition of Na(+)-H(+) exchanger by dimethyl-amiloride (50 micromol/l, n=6) caused endothelium-independent positive inotropic effects only. RT-PCR and Western Blot demonstrated alpha(1) and alpha(2) Na-K-ATPase isoforms in cardiomyocytes, but only alpha(1) in cultured endocardial endothelial cells. Immunohistochemistry showed that alpha(1) in endocardial endothelium was predominantly present at the luminal side of the cell (n=7) and that alpha(1) and alpha(2) displayed different localisation in cardiomyocytes. CONCLUSIONS: These results suggested that negative and positive inotropic effects of Na(+)/K(+) ATPase inhibition in +EE muscles could be attributed to inhibition of endocardial endothelial alpha(1) and muscle alpha(2) isoform, respectively. Accordingly, the endocardial endothelial alpha(1) isoform of Na(+)/K(+) ATPase may contribute to blood-heart barrier properties of this endothelium and may control cardiac performance via endothelial Na(+)/H(+) exchange.
    Document Type:
    Reference
    Product Catalog Number:
    3140

  • Epigenetic modifications of RASSF1A gene through chromatin remodeling in prostate cancer. 17473182

    The RAS-association domain family 1, isoform A (RASSF1A) gene is shown to be inactivated in prostate cancers. However, the molecular mechanism of silencing of the RASSFIA gene is not fully understood. The present study was designed to investigate the mechanisms of inactivation of the RASSF1A gene through the analysis of CpG methylation and histone acetylation and H3 methylation associated with the RASSF1A promoter region.Methylation status of the RASSF1A gene was analyzed in 131 samples of prostate cancer, 65 samples of benign prostate hypertrophy (BPH), and human prostate cell lines using methylation-specific PCR. Histone acetylation (acetyl-H3, acetyl-H4) and H3 methylation (dimethyl-H3-K4, dimethyl-H3-K9) status associated with the promoter region in prostate cells were analyzed by chromatin immunoprecipitation (ChIP) assay.Aberrant methylation was detected in 97 (74.0%) prostate cancer samples and 12 (18.5%) BPH samples. The methylation frequency of RASSF1A showed a significant increase with high Gleason sum and high stage. The ChIP assays showed enhancement of histone acetylation and dimethyl-H3-K4 methylation on the unmethylated RASSF1A promoter. TSA alone was unable to alter key components of the histone code. However, after 5-aza-2'-deoxy-cytidine treatment, there was a complete reversal of the histone components in the hypermethylated promoter. Levels of acetyl-H3, acetyl-H4, and dimethyl-H3-K4 became more enriched, whereas H3K9me2 levels were severely depleted.This is the first report suggesting that reduced histone acetylation or H3K4me2 methylation and increased dimethyl-H3-K9 methylation play a critical role in the maintenance of promoter DNA methylation-associated RASSF1A gene silencing in prostate cancer.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Genistein depletes telomerase activity through cross-talk between genetic and epigenetic mechanisms. 19358274

    Genistein, a natural isoflavone found in soybean products, has been reported to down-regulate telomerase activity and that this prevents cancer and contributes to the apoptosis of cancer cells. However, the precise molecular mechanism by which genistein represses telomerase is not clear. Here, we show that genistein inhibits the transcription of hTERT (human telomerase reverse transcriptase), the catalytic subunit of the human telomerase enzyme, in breast MCF10AT benign cells and MCF-7 cancer cells in a time- and dose-dependent manner. Three major DNA methyltransferases (DNMT1, 3a and 3b) were also decreased in genistein-treated breast cancer cells suggesting that genistein may repress hTERT by impacting epigenetic pathways. Sequential depletion of the hTERT promoter revealed that the hTERT core promoter region is responsible for the genistein-induced repression of hTERT transcription. Using a newly developed technique of chromatin immunoprecipitation (ChIP)-related bifulfite sequencing analysis, we found an increased binding of E2F-1 to the hTERT promoter is due to the site-specific hypomethylation of the E2F-1 recognition site. In addition, we found that genistein can remodel chromatin structures of the hTERT promoter by increasing trimethyl-H3K9 but decreasing dimethyl-H3K4 in the hTERT promoter. The repression of hTERT was enhanced by combination with genistein and the DNMT inhibitor, 5-aza-2'-deoxycytidine (5-aza-dCyd). These findings collectively show that genistein is working, at least in part, through epigenetic mechanisms of telomerase inhibition in breast benign and cancer cells and may facilitate approaches to breast cancer prevention and treatment using an epigenetic modulator combined with genistein.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™