Activity In Nuclear Extracts Research Articles

Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling.

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here, we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at histone H2B serine 6 or histone H3 serine 10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

Ubiquitin stimulated reversal of topoisomerase 2 DNA-protein crosslinks by TDP2.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface.

Vitamin D downregulates key genes of diabetes complications in cardiomyocyte.

Vitamin D deficiency has been reported to be associated with the incidence of type 1 and type 2 diabetes and worsening of diabetes complications. This study was designed to investigate the effect of vitamin D treatment on the expression of five key genes involved in the development of diabetic cardiomyopathy. Twenty-four male Sprague-Dawley rats were randomly divided into three groups. The first group served as control and the other two groups received an intraperitoneal injection of 45 mg/kg streptozotocin (STZ) to develop diabetes. Then groups were treated for 4 weeks either with placebo or vitamin D (two injections of 20,000 IU/kg). Serum levels of glucose, insulin, HbA1c, and advanced glycation end products (AGEs), as well as the gene expression of AGE cellular receptor (RAGE), glyoxalase, aldose reductase, O-GlcNAc transferase (OGT), and glutamine-fructose-6-phosphate aminotransferase (GFAT) and nuclear factor-kB (NF-kB) activity of nuclear extracts were assessed at the end of experiment. Increment in serum cholecalciferol could improve hyperglycaemia and hypoinsulinemia in diabetic rats. In addition, a significant reduction was observed in RAGE, OGT, and GFAT gene expression and NF-kB activity in cardiac myocytes. Vitamin D might contribute in reducing diabetic cardiomyopathy not only by improving blood glucose and insulin levels but also via downregulating AGE and hexosamine pathways and decreasing NF-kB activity in heart tissue.

Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis.

Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous in vitro studies with molecular approaches on AR42J cell showed that protein kinase D (PKD/PKD1) activation was required in NF-κB activation induced by cholecystokinin 8 (CCK) or carbachol (CCh) in pancreatic acinar cells. Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.

Torsional stress promotes trinucleotidic expansion in spermatids

Trinucleotide repeats are involved in various neurodegenerative diseases and are highly unstable both in dividing or non-dividing cells. In Huntington disease (HD), the age of onset of symptoms is inversely correlated to the number of CAG repeats within exon 1 of the HTT gene. HD shows paternal anticipation as CAG repeats are increased during spermatogenesis. CAG expansion were indeed found to be generated during the chromatin remodeling in spermatids where most histones are evicted and replaced by protamines. This process involves striking change in DNA topology since free supercoils must be eliminated. Using an in vitro CAG repeat reporter assay and a highly active nuclear extracts from spermatids, we demonstrate that free negative supercoils result in CAG TNR expansion at a stabilized hairpin. We also suggest a possible role for protamines in promoting localized torsional stress and consequently TNR expansion. The transient increase in torsional stress during spermiogenesis may therefore provide an ideal context for the generation of such secondary DNA structures leading to the paternal anticipation of trinucleotidic diseases.

In vitro neuroprotective potential of lichen metabolite fumarprotocetraric acid via intracellular redox modulation

The lichen-forming fungi Cetraria islandica has been largely used in folk medicines, and it has recently showed promising in vitro antioxidant effects in glial-like cells. Current work aimed at investigating the neuroprotective potential of its major isolated secondary metabolite: the depsidone fumarprotocetraric acid (FUM). H2O2 was used herein to induce oxidative stress (OS)-mediated cytotoxicity in two models of neurons and astrocytes cells (SH-SY5Y and U373-MG cell lines).We found that a pre-treatment with FUM significantly enhanced cell viability compared to H2O2-treated cells, and we selected the optimal concentrations in each model (1 and 25μg/ml, respectively) for assessing its cytoprotective mechanisms. FUM, which exerted effective peroxyl radical scavenging effect in the chemical oxygen radical antioxidant capacity (ORAC) assay, alleviated the alterations in OS markers provoked by H2O2. It attenuated intracellular ROS formation, lipid peroxidation and GSH depletion. At mitochondrial level, FUM prevented from the dissipation of mitochondrial membrane potential and the increase in mitochondrial calcium, implying a protective role against oxidative damage in mitochondrial membrane. Similarly, FUM pre-treatment diminished H2O2-induced apoptosis, as evidenced by the reduction in caspase-3 activity and expression; inmunoblot analysis also revealed a decrease in Bax and an increase in Bcl-2 proteins levels. Furthermore, FUM up-regulated the expression of the antioxidant enzymes catalase, superoxide dismutase-1, and hemeoxigenase-1. These findings and the activation of Nrf2 binding activity in nuclear extracts suggest a plausible involvement of Nrf2 signaling pathway in the cytoprotection by FUM. In conclusion, FUM emerges as a potential drug candidate in the therapy of OS-related diseases, such as the neurodegenerative disorders.

Pre-microRNA processing activity in nuclear extracts from Arabidopsis suspension cells.

MicroRNAs (miRNAs) play important roles in a variety of biological phenomena, such as development and responses to abiotic and biotic stresses, by regulating complementary target transcripts. Primary MIRNA transcripts (pri-miRNAs) are processed into miRNA/miRNA* duplexes via pre-miRNA intermediates by DICER family proteins. In plants miRNA/miRNA* duplexes are produced from pri-miRNAs exclusively by a complex containing DICER-LIKE1 (DCL1), Hyponastic leaves1 (HYL1) and SERREATE (SE) in the nucleus. Pri-miRNA or pre-miRNA processing activity has been detected using recombinant DCL1, HYL1 and SE proteins expressed in insect cells and immunopurified DCL1-HYL1-SE-containing complexes that transiently co-overexpressed in Nicotiana benthamiana. However, the processing of pre-miRNAs into miRNA/miRNA* using nuclear extracts has not been reported. Here I report the detection of pre-miRNA processing in nuclear extracts prepared from Arabidopsis suspension cells. In the nuclear extracts including DCL1, small RNAs that were 21 nucleotides in length were excised from a part of miRNA/miRNA* regions on pre-miRNAs. This system is potentially useful for in vitro analyses of pre-miRNA processing.

Non-canonical NF-κB pathway activation predicts outcome in borderline oestrogen receptor positive breast carcinoma.

Background:NF-κB signalling appears deregulated in breast tumours. The purpose of this study was to determine whether the non-canonical NF-κB pathway, is activated in oestrogen receptor positive (ER+) breast cancer, to identify any correlation between its activity and the clinico-pathological phenotype and to explore whether NF-κB2 and RelB subunits and/or any of their target genes might be used as a predictive marker.Methods:Two independent cohorts of ER+ early breast cancer patients treated with adjuvant endocrine therapy were included in the study. Activation of RelB and NF-κB2 subunits was determined in a training set of 121 patients by measuring DNA-binding activities in nuclear extracts from fresh frozen specimens by an ELISA-based assay. Samples of 15 ER− breast cancer patients were also included in the study. In a large validation cohort of 207 patients, nuclear immunostaining of RelB and NF-κB2 on formalin-fixed paraffin-embedded specimens was performed. Statistical correlation within clinico-pathological factors, disease-free survival (DFS) and overall survival (OS) was evaluated. Publicly available gene expression and survival data have been interrogated aimed to identify target genes.Results:Activation of NF-κB2 and RelB was found in 53.7 and 49.2% of the 121 ER+ tumours analysed, with similar levels to ER− breast tumours analysed in parallel for comparisons. In the validation cohort, we obtained a similar proportion of cases with activation of NF-κB2 and RelB (59.9 and 32.4%), with a 39.6% of co-activation. Multiplexing immunofluorescence in breast cancer tissue confirmed an inverse spatial distribution of ER with NF-κB2 and RelB nuclear expression in tumour cells. Interestingly, NF-κB2 and RelB mRNA expression was inversely correlated with ER gene (ESR1) levels (P<0.001, both) and its activation was significantly associated with worse DFS (P=0.005 and P=0.035, respectively) in ER+ breast cancer. Moreover, the co-activation of both subunits showed a stronger association with early relapse (P=0.002) and OS (P=0.001). Finally, higher expression of the non-canonical NF-κB target gene myoglobin was associated with a poor outcome in ER+ breast cancer (DFS, P<0.05).Conclusions:The non-canonical NF-κB pathway activation is inversely associated with oestrogen receptor expression in ER+ breast cancer and predicts poor survival in this subgroup. The myoglobin gene expression has been identified as a possible surrogate marker of the non-canonical NF-κB pathway activation in these tumours.

New strategy to address DNA-methyl transferase activity in ovarian cancer cell cultures by monitoring the formation of 5-methylcytosine using HPLC-UV.

Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). Aberrant expression and activity of these enzymes has been reported to play an important role in the initiation and progression of tumors and its response to chemotherapy. Therefore, there is a great interest in developing strategies to detect human DNMTs activity. We propose a simple, antibody-free, label-free and non-radioactive analytical strategy in which methyltransferase activity is measured trough the determination of the 5-methylcytosine (5mC) content in DNA by a chromatographic method (HPLC-UV) previously developed. For this aim, a correlation between the enzyme activity and the concentration of 5mC obtained by HPLC-UV is previously obtained under optimized conditions using both, un-methylated and hemi-methylated DNA substrates and the prokaryotic methyltransferase M.SssI as model enzyme. The evaluation of the methylation yield in un-methylated known sequences (a 623bp PCR-amplicon) turned to be quantitative (110%) in experiments conducted in-vitro. Methylation of hemi-methylated and low-methylated sequences could be also detected with the proposed approach. The application of the methodology to the determination of the DNMTs activity in nuclear extracts from human ovarian cancer cells has revealed the presence of matrix effects (also confirmed by standard additions) that hampered quantitative enzyme recovery. The obtained results showed the high importance of adequate sample clean-up steps.

Phytochemicals of Aristolochia tagala and Curcuma caesia exert anticancer effect by tumor necrosis factor-α-mediated decrease in nuclear factor kappaB binding activity.

Rationale:The active compounds or metabolites of herbal plants exert a definite physiological action on the human body and thus are widely used in human therapy for various diseases including cancer. Previous studies by our group have reported the anticarcinogenic properties of the two herbal plants extracts (HPE) of Aristolochia tagala (AT) Cham. and Curcuma caesia (CC) Roxb. in diethylnitrosamine-induced mouse liver cancer in vivo. The anticarcinogenic properties of these extracts may be due to the active compounds present in them.Objectives:Our objective was to analyze the phytochemical constituents present in AT and CC, to assay their antioxidant properties and to determine their role in a possible intervention on tumor progression.Materials and Methods:Qualitative and quantitative analysis of constituent with anticancer properties present in the crude methanol extract of the two plants CC and AT was carried out following standard methods. Separation of the phytochemical compounds was done by open column chromatography. The extracts were eluted out with gradients of chloroform-methanol solvents. Ultraviolet-visible spectra of individual fractions were recorded, and the fractions were combined based on their λmax. The free radical scavenging activity of crude extracts and fractions obtained was also determined; the radical scavenging activity was expressed as IC50. High-performance thin layer chromatography (HPTLC) analysis of fractionated compounds was carried out to identify partially the phytochemical compounds. The anti-inflammatory and anticancer activity of AT and CC extracts was studied in DEN induced BALB/c mice by analyzing the tumor necrosis factor-α (TNF-α) levels in serum and the nuclear factor kappaB (NF-κB) binding activity in nuclear extracts of the liver.Results:It was observed that both AT and CC contained compounds such as phenolics, tannins, flavonoids, terpenoids, etc., and both extracts exhibited antioxidant capacity. HPTLC analysis revealed the presence of phenolic compounds in CC and indicated the presence of anthocynidin 3-glycosides, 6-hydroxylated flavonols, some flavones and chalcone glycosides in AT and also confirmed the presence of compounds such as terpenes, phenols, steroids, and other organic compounds in CC and presence of flavonoids in AT. In vivo studies carried out in BALB/c mice showed that exposure to DEN caused an increase in TNF-α and NF-κB binding activity. The HPE (CC or AT) was seen to revert this effect.Conclusions:The current paper documents the antioxidant, anti-inflammatory, and anticancer activity of the two extracts probably through TNF-α-mediated decrease in NF-κB binding activity. The active components of AT and CC may act as the potential anticancer agents in hepatocellular carcinoma and warrants further investigation.

20-HETE Activates the Transcription of Angiotensin-Converting Enzyme via Nuclear Factor-κB Translocation and Promoter Binding.

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury, and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that, in human microvascular endothelial cells, 20-HETE increases angiotensin-converting enzyme (ACE) mRNA, protein, and ACE activity via an epidermal growth factor receptor (EGFR)/tyrosine kinase/mitogen-activated protein kinase (MAPK)/inhibitor of κB kinase (IKK)β-mediated signaling pathway. In this work, we show that, similar to epidermal growth factor (EGF), 20-HETE (10 nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of nuclear factor κB (NF-κB) activation prevented the 4.58-fold (±0.78; P < 0.05) 20-HETE-mediated induction of ACE. The 20-HETE increased NF-κB-binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (±0.18; P < 0.05) and 2.53-fold (± 0.24; P < 0.05), respectively. The 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-hydroxy-6,15-eicosadienoic acid and by inhibitors of EGFR, MAPK, IKKβ, and NF-κB activation. Sequence analysis demonstrated the presence of two and one putative NF-κB binding sites on the human somatic and germinal ACE promoters, respectively. Chromatin immunoprecipitation assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-κB to each of the putative binding sites (S1, 3.43 ± 0.3-fold enrichment versus vehicle; S2, 3.72 ± 0.68-fold enrichment versus vehicle; S3, 3.20 ± 0.18-fold enrichment versus vehicle; P < 0.05). This is the first study to identify NF-κB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-κB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Poly(ADP-Ribosyl)ation Affects Histone Acetylation and Transcription.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARylation regulates a wide variety of biological processes in most eukaryotic cells including energy metabolism and cell death, maintenance of genomic stability, chromatin structure and transcription. Inside the nucleus, cross-talk between PARylation and other epigenetic modifications, such as DNA and histone methylation, was already described. In the present work, using PJ34 or ABT888 to inhibit PARP activity or over-expressing poly(ADP-ribose) glycohydrolase (PARG), we show decrease of global histone H3 and H4 acetylation. This effect is accompanied by a reduction of the steady state mRNA level of p300, Pcaf, and Tnfα, but not of Dnmt1. Chromatin immunoprecipitation (ChIP) analyses, performed at the level of the Transcription Start Site (TSS) of these four genes, reveal that changes in histone acetylation are specific for each promoter. Finally, we demonstrate an increase of global deacetylase activity in nuclear extracts from cells treated with PJ34, whereas global acetyltransferase activity is not affected, suggesting a role for PARP in the inhibition of histone deacetylases. Taken together, these results show an important link between PARylation and histone acetylation regulated transcription.

Abstract 024: 20-HETE Activates the Transcription of Endothelial Angiotensin Converting Enzyme (ACE) via NF-kB Translocation and Promoter Binding

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite, promotes vascular dysfunction, injury and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) increases angiotensin converting enzyme (ACE) mRNA, protein, and cellular and extracellular ACE activity via EGFR-tyrosine kinase-MAPK-IKKβ- signaling pathway. Here we show that treatment of HMVEC with EGF (100 ng/ml) alone or with 20-HETE (10 nM) did not induce or further increase ACE mRNA levels. Pretreatment of cells with a neutralizing antibody against EGF did not prevent the 20-HETE- or 20-HETE+EGF-mediated ACE induction, indicating that the induction of ACE by 20-HETE is EGF-independent. The NF-kB inhibitor JSH-23 prevented the 4.58-fold (±0.78; p&lt;0.05) 20-HETE (10nM)-mediated induction of ACE. EMSA of 20-HETE-treated cells showed increased NF-kB binding activity in nuclear extracts. In cells transfected with luciferase promoter-reporter constructs for the somatic or germinal ACE promoter regions, 20-HETE (10 nM) increased promoter activity by 4.37-fold (±0.18; p&lt;0.05) and 2.53-fold (± 0.24; p&lt;0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two (Chr 17: S1: 61554061-61554075 , S2: 61554157-61554171) and one (Chr 17: S3: 61562250-61562264) putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. CHIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs vehicle; p&lt;0.05). In vivo, increased vascular 20-HETE levels are associated with increased phospho-IKK and ACE levels that are prevented by treatment with 20-HETE antagonists. This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Oligonucleotides designed to inhibit TLR9 block Herpes simplex virus type 1 infection at multiple steps

Herpes simplex virus type 1 (HSV-1) is an important human pathogen which requires activation of nuclear factor-kappa B (NFκB) during its replication cycle. The persistent nature of HSV-1 infection, and the emergence of drug-resistant strains, highlights the importance of research to develop new antiviral agents. Toll-like receptors (TLRs) play a prominent role during the early antiviral response by recognizing viral nucleic acid and gene products, activating NFκB, and stimulating the production of inflammatory cytokines. We demonstrate a significant effect on HSV-1 replication in ARPE-19 and Vero cells when oligonucleotides designed to inhibit TLR9 are added 2h prior to infection. A greater than 90% reduction in the yield of infectious virus was achieved at oligonucleotide concentrations of 10–20μM. TLR9 inhibitory oligonucleotides prevented expression of essential immediate early herpes gene products as determined by immunofluorescence microscopy and Western blotting. TLR9 oligonucleotides also interfered with viral attachment and entry. A TLR9 inhibitory oligonucleotide containing five adjacent guanosine residues (G-ODN) exhibited virucidal activity and inhibited HSV-1 replication when added post-infection. The antiviral effect of the TLR9 inhibitory oligonucleotides did not depend on the presence of TLR9 protein, suggesting a mechanism of inhibition that is not TLR9 specific. TLR9 inhibitory oligonucleotides also reduced NFκB activity in nuclear extracts. Studies using these TLR inhibitors in the context of viral infection should be interpreted with caution.

Function of alternative NF-κB activity in B-cell chronic lymphocytic leukemia cells

To investigate the function of alternative NF-κB activity in B-cell chronic lymphocytic leukemia cells (B-CLL). The mRNA expression of individual NF-κB subunits in CD5⁺CD19⁺ cells (CLL B-cells) from bone marrow (BM) of 56 patients with B-CLL was analyzed by quantitative RT-PCR. An ELISA-based NF-κB family transcription factor activity assay was performed to quantify the κB DNA-binding activity in nuclear extracts from CLL B-cells. Cell death of CLL-B cells was determined by PI staining, RelA and RelB expression at protein level of CLL B-cells by Western blot analyses. The expression levels of RelA, p50, RelB and p52 mRNA in CLL B-cells were all higher than that of normal B cells with statistical significance (P<0.05). RelA was activated in almost all the patients detected while RelB activity was induced in part of samples. The average RelA activity in CLL B-cells was increased compared to that in normal B cells while the average RelB activity was similar to that of normal B cells. When cultured in vitro for 24, 48 and 72 hours, the frequencies of cell death of CLL B-cells from RelA⁺/RelB⁻ group were(35.54±4.43)%,(50.92±8.44)%, and(49.24±8.16)%, respectively; that of the RelA⁺/RelB⁺ group were (20.65±2.37)%, (18.17±1.36)%, and (26.55±4.08)%, respectively. When the cells from RelA+/RelB⁻ group were co-cultured with bone marrow stromal cells (hBMSCs), the frequencies of cell death of CLL B-cells were decreased compared to that of the cells cultured alone, while the frequencies of cell death of RelA⁺/RelB⁻ CLL B-cells were higher than that of CLL B-cells from RelA⁺/RelB⁺ group when co-cultured with hBMSCs. RelA and RelB expression in CLL-B cells from the RelA⁺/RelB⁻ group was induced after co-cultured with hBMSCs for 48 h. RelB was reduced in the cytoplasm and increased in the nucleus in CLL-B cells from the RelA+/RelB+ group. The alternative NF-κB was indeed activated and presented heterogeneous in CLL B-cells from BM. Activation of RelB combined with RelA activity could provide the survival advantage to CLL B-cells from BM. Co-culture with hBMSCs could protect CLL-B cells through the induction of RelA and Rel B expressions.

Des-acyl ghrelin protects microvascular endothelial cells from oxidative stress-induced apoptosis through sirtuin 1 signaling pathway

ObjectiveGhrelin is a stomach-derived hormone. Acylation of ghrelin has been essential for its biological activities such as stimulating appetite. On the other hand, the function of des-acyl ghrelin (Des-G) has not been fully elucidated. The aim of the present study is to examine the anti-apoptotic effect of Des-G on endothelial cells. Materials/MethodsAfter human retinal microvascular endothelial cells (RMECs) were pretreated with or without 100nmol/L Des-G, apoptosis was induced with 0.1mmol/L hydrogen peroxide (H2O2). For pharmacological inhibition of surtuin 1 (SIRT1) catalytic activity, the cells were treated with 10μmol/L Ex-527. Inhibition of SIRT1 with siRNA was also performed. The quantitative estimation of DNA fragmentation was used as a marker of apoptosis. Furthermore, total SIRT activity in nuclear extracts, mRNA and protein levels of SIRT1, manganese superoxide dismutase (MnSOD) and catalase were determined. ResultsDes-G pretreatment protected RMECs from oxidative stress-induced apoptosis and increased SIRTs deacetylase activity in nuclear extracts. On the other hand, both pharmacological and siRNA mediated inhibition of SIRT1 attenuated the anti-apoptotic effect of Des-G. Moreover, Des-G increased mRNA and protein levels of SIRT1 and antioxidant enzymes such as MnSOD and CAT, which are downstream targets of SIRT1. Although the treatment of Ex-527 did not alter mRNA expression levels of SIRT1, it decreased mRNA expression levels of antioxidant enzymes in the cells with Des-G pretreatment. ConclusionsOur results suggest that SIRT1 signaling pathway contributes to protective effect of Des-G against oxidative stress-induced apoptosis.

Preparation of Nuclear Extracts from HeLa Cells

HeLa cells are the archetypal tissue culture cell line for the preparation of mammalian cell-free systems. These cells, derived from a cervical carcinoma, have been maintained in culture since the late 1940s. They grow with a doubling time of ∼24 h and can be cultured in medium containing fetal bovine serum (optimal serum for growth) or medium containing horse serum (in which they grow more slowly, but this serum is considerably less expensive). Nuclear extracts prepared from these cells have been used to determine the mechanisms of splicing and polyadenylation, and such extracts have been characterized extensively. HeLa cells are usually the cell type of choice for initiating cell-free analysis of nearly any aspect of mammalian gene expression. In some instances (e.g., analysis of tissue-specific alternative splicing), it is necessary to use nuclei from a different cell type. We have found that the protocol described here can be used successfully to prepare active nuclear extracts from a wide variety of tissue culture cells, including Drosophila S2 cells.

ACTIVATION AND EXPRESSION OF NF-κB IN CIRCULATING MONONUCLEAR CELLS IN APPARENTLY HEALTHY PEOPLE

ObjectivesJUPITER trial demonstrated that administering Rosuvastatin to ‘apparently healthy people’(with normal lipid levels, but hsCRP3 2 mg/L) could decrease the incidences of cardiovascular diseases significantly. Whereas, little is known about...

Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor

Gemcitabine is indicated in combination with cisplatin as first-line therapy for solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as an anti-metabolite. Structurally, however, gemcitabine has similarities to 5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor (DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with decitabine and gemcitabine. Reactivation of epigenetically silenced genes was examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and recombinant proteins was measured using a DNA methyl-transferase assay, and alterations in DNA methylation status were examined using methylation-specific PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine functionally inhibited DNA methyltransferase activity in both nuclear extracts and recombinant proteins. Gemcitabine dramatically destabilised DNMT1 protein. However, DNA CpG methylation was for the most part unaffected by gemcitabine. In conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates epigenetically silenced genes having activity equivalent to decitabine at concentrations significantly lower than those achieved in the treatment of patients with solid tumours. This property may contribute to the anticancer activity of gemcitabine.

Magnesium deficiency promotes secretion of high-mobility group box 1 protein from lipopolysaccharide-activated macrophages in vitro

BackgroundHigh-mobility group box 1 (HMGB1) is a critical mediator of sepsis that is closely related to sepsis lethality. Magnesium deficiency predisposes to worse outcomes from endotoxin challenge by promoting the production of cytokines. However, whether magnesium deficiency affects the expression and release of HMGB1 is not currently known. In the present study, we explored the effect of magnesium deficiency on the expression and secretion of HMGB1 in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. MethodsRAW264.7 cells were incubated with LPS in normal magnesium (1 mmol/L magnesium sulfate) or low magnesium (0.1 mmol/L magnesium sulfate) in Roswell Park Memorial Institute 1640 medium. An enzyme-linked immunosorbent assay was used to detect HMGB1 levels in the culture supernatant. Real-time polymerase chain reaction was used to assess the HMGB1 mRNA levels. A nuclear/cytoplasm extraction kit was used to extract the nuclear and cytoplasmic proteins. Western blotting was used to observe the changes in translocation of HMGB1 from the nucleus to the cytoplasm. A nuclear factor κ-light chain enhancer of activated B cells (NF-κB) p50/p65 transcription factor assay kit was used to analyze the NF-κB activity in nuclear extracts. ResultsMagnesium deficiency promoted translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular secretion in LPS-activated macrophages, while enhancing the expression of HMGB1 mRNA. Furthermore, magnesium deficiency promoted the translocation of NF-κB from the cytoplasm to the nucleus in LPS-activated macrophages. ConclusionsMagnesium deficiency promotes the translocation of HMGB1 from the nucleus to the cytoplasm and the expression of HMGB1 mRNA. Magnesium deficiency also activates the NF-κB signaling pathway.