Histone deacetylase inhibitors: A new mode for inhibition of cholesterol metabolism

<p>Abstract</p> <p>Background</p> <p>Eukaryotic gene expression is a complex process involving multiple cis and trans activating molecules to either facilitate or inhibit transcription. In recent years, many studies have focused on the role of acetylation of histone proteins in modulating transcription, whereas deacetylation of these same proteins is associated with inactivation or repression of gene expression. This study explores gene expression in HepG2 and F9 cell lines treated with Trichostatin A (TSA), a potent histone deacetylase inhibitor.</p> <p>Results</p> <p>These experiments show that TSA treatment results in clear repression of genes involved in the cholesterol biosynthetic pathway as well as other associated pathways including fatty acid biosynthesis and glycolysis. TSA down regulates 9 of 15 genes in this pathway in the F9 embryonal carcinoma model and 11 of 15 pathway genes in the HepG2 cell line. A time course study on the effect of TSA on gene expression of various enzymes and transcription factors involved in these pathways suggests that down regulation of <it>Srebf2 </it>may be the triggering factor for down regulation of the cholesterol biosynthesis pathway.</p> <p>Conclusion</p> <p>Our results provide new insights in the effects of histone deacetylases on genes involved in primary metabolism. This observation suggests that TSA, and other related histone deacetylase inhibitors, may be useful as potential therapeutic entities for the control of cholesterol levels in humans.</p

ATM, ATR and DNA-PKcs expressions correlate to adverse clinical outcomes in epithelial ovarian cancers

Background: Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia mutated and rad3 related (ATR) and DNA-dependent protein kinase catalytic sub-unit (DNA-PKcs) play critical roles in DNA damage response (DDR) by linking DNA damage sensing to DDR effectors that regulate cell cycle progression and DNA repair. Our objective was to evaluate if ATM, ATR and DNA-PKcs expressions could predict response to therapy and clinical outcome in epithelial ovarian cancers. Methods: We investigated ATM, ATR, and DNA-PKcs expressions in ovarian epithelial cancers [protein expression (n = 194 patients), mRNA expression (n = 156 patients)] and correlated to clinicopathological outcomes as well as expression of X-ray repair cross-complementing protein 1 (XRCC1), cell division cycle-45 (CDC45), cyclin-dependent kinase 1(CDK1) and Ki-67 in tumours. Results: High ATM protein expression was associated with serous cystadenocarcinomas (p = 0.021) and platinum resistance (p = 0.017). High DNA-PKcs protein expression was associated with serous cystadenocarcinomas (p = 0.006) and advanced stage tumours (p = 0.018). High ATM protein (p = 0.001), high ATM mRNA (p = 0.018), high DNA-PKcs protein (p = 0.002), high DNA-PKcs mRNA (p = 0.044) and high ATR protein (p = 0.001) expressions are correlated with poor ovarian cancer specific survival (OCSS). In multivariate Cox model, high DNA-PKcs (p = 0.006) and high ATR (p = 0.043) protein expressions remain independently associated with poor OCSS. Conclusions: ATM, ATR and DNA-PKcs expressions may have prognostic and predictive significances in epithelial ovarian cancer. General significance: The data presented here provides evidence that ATM, ATR and DNA-PKcs involved in DDR are not only promising biomarkers but are also rational targets for personalized therapy in ovarian cancer

Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting

<p>Abstract</p> <p>Background</p> <p>Testicular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.</p> <p>Methods</p> <p>Human embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-<it>trans </it>retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.</p> <p>Results</p> <p>Canonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.</p> <p>Conclusion</p> <p>During induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.</p

Ruthenium-containing linear helicates and mesocates with tuneable p53 selective cytotoxicity in colorectal cancer cells

YesThe ligands L1 and L2 both form separable dinuclear double‐stranded helicate and mesocate complexes with RuII. In contrast to clinically approved platinates, the helicate isomer of [Ru2(L1)2]4+ was preferentially cytotoxic to isogenic cells (HCT116 p53−/−), which lack the critical tumour suppressor gene. The mesocate isomer shows the reverse selectivity, with the achiral isomer being preferentially cytotoxic towards HCT116 p53+/+. Other structurally similar RuII‐containing dinuclear complexes showed very little cytotoxic activity. This study demonstrates that alterations in ligand or isomer can have profound effects on cytotoxicity towards cancer cells of different p53 status and suggests that selectivity can be “tuned” to either genotype. In the search for compounds that can target difficult‐to‐treat tumours that lack the p53 tumour suppressor gene, [Ru2(L1)2]4+ is a promising compound for further development

Differential Requirement of Histone Acetylase and Deacetylase Activities for IRF5-Mediated Proinflammatory Cytokine Expression

Recent evidence indicates a new role for histone deacetylases (HDACs) in the activation of genes governing the host immune response. Virus, along with other pathogenic stimuli, triggers an antiviral defense mechanism through the induction of IFN, IFN-stimulated genes, and other proinflammatory cytokines. Many of these genes have been shown to be regulated by transcription factors of the IFN regulatory factor (IRF) family. Recent studies from IRF5 knockout mice have confirmed a critical role for IRF5 in virus-induced type I IFN expression and proinflammatory cytokines IL-6, IL-12, and TNF-α; yet, little is known of the molecular mechanism of IRF5-mediated proinflammatory cytokine expression. In this study, we show that both HDACs and histone acetyltransferases (HATs) associate with IRF5, leading to alterations in its transactivation ability. Using the HDAC inhibitor trichostatin A, we demonstrate that ISRE, IFNA, and IL6 promoters require HDAC activity for transactivation and transcription, whereas TNFα does not. Mapping the interaction of corepressor proteins (HDAC1, silencing mediator of retinoid and thyroid receptor/nuclear corepressor of retinoid receptor, and Sin3a) and HATs to IRF5 revealed distinct differences, including the dependence of IRF5 phosphorylation on HAT association resulting in IRF5 acetylation. Data presented in this study support a mechanism whereby virus triggers the dynamic conversion of an IRF5-mediated silencing complex to that of an activating complex on promoters of target genes. These data provide the first evidence, to our knowledge, of a tightly controlled transcriptional mechanism whereby IRF5 regulates proinflammatory cytokine expression in conjunction with HATs and HDACs

Genetic variants and disease-associated factors contribute to enhanced interferon regulatory factor 5 expression in blood cells of patients with systemic lupus erythematosus

OBJECTIVE: Genetic variants of the interferon (IFN) regulatory factor 5 gene (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). The contribution of these variants to IRF-5 expression in primary blood cells of SLE patients has not been addressed, nor has the role of type I IFNs. The aim of this study was to determine the association between increased IRF-5 expression and the IRF5 risk haplotype in SLE patients. METHODS: IRF-5 transcript and protein levels in 44 Swedish patients with SLE and 16 healthy controls were measured by quantitative real-time polymerase chain reaction, minigene assay, and flow cytometry. Single-nucleotide polymorphisms rs2004640, rs10954213, and rs10488631 and the CGGGG insertion/deletion were genotyped in these patients. Genotypes of these polymorphisms defined both a common risk haplotype and a common protective haplotype. RESULTS: IRF-5 expression and alternative splicing were significantly up-regulated in SLE patients compared with healthy donors. Enhanced transcript and protein levels were associated with the risk haplotype of IRF5; rs10488631 displayed the only significant independent association that correlated with increased transcription from the noncoding first exon 1C. Minigene experiments demonstrated an important role for rs2004640 and the CGGGG insertion/deletion, along with type I IFNs, in regulating IRF5 expression. CONCLUSION: This study provides the first formal proof that IRF-5 expression and alternative splicing are significantly up-regulated in primary blood cells of patients with SLE. Furthermore, the risk haplotype is associated with enhanced IRF-5 transcript and protein expression in patients with SLE