Histone deacetylase inhibitors target diabetes via chromatin remodeling or as chemical chaperones?

Globally, obesity and diabetes (particularly type 2 diabetes) represents a major challenge to world health. Despite decades of intense research efforts, the genetic basis involved in diabetes pathogenesis & conditions associated with obesity are still poorly understood. Recent advances have led to exciting new developments implicating epigenetics as an important mechanism underpinning diabetes and obesity related disease. One epigenetic mechanism known as the "histone code" describes the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as lysine acetyltransferases or KATs and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. Some of the known inhibitors of HDACs (HDACi) have also been shown to act as "chemical chaperones" to alleviate diabetic symptoms. In this review, we discuss the available evidence concerning the roles of HDACs in regulating chaperone function and how this may have implications in the management of diabetes. © 2009 Bentham Science Publishers Ltd.

Time course-dependent changes in the transcriptome of human skeletal muscle during recovery from endurance exercise: From inflammation to adaptive remodeling

Re-programming of gene expression is fundamental for skeletal muscle adaptations in response to endurance exercise. This study investigated the time-course dependent changes in the muscular transcriptome following an endurance exercise trial consisting of 1 h of intense cycling immediately followed by 1 h of intense running. Skeletal muscle samples were taken at baseline, 3 h, 48 h, and 96 h post-exercise from eight healthy, endurance-trained, male individuals. RNA was extracted from muscle. Differential gene expression was evaluated using Illumina microarrays and validated with qPCR. Gene set enrichment analysis identified enriched molecular signatures chosen from the Molecular Signatures Database. Three h post-exercise, 102 gene sets were up-regulated [family wise error rate (FWER), P < 0.05]; including groups of genes related with leukocyte migration, immune and chaperone activation, and cyclic AMP responsive element binding protein (CREB) 1-signaling. Forty-eight h post-exercise, among 19 enriched gene sets (FWER, P < 0.05), two gene sets related to actin cytoskeleton remodeling were up-regulated. Ninety-six h post-exercise, 83 gene sets were enriched (FWER, P < 0.05), 80 of which were up-regulated; including gene groups related to chemokine signaling, cell stress management, and extracellular matrix remodeling. These data provide comprehensive insights into the molecular pathways involved in acute stress, recovery, and adaptive muscular responses to endurance exercise. The novel 96 h post-exercise transcriptome indicates substantial transcriptional activity, potentially associated with the prolonged presence of leukocytes in the muscles. This suggests that muscular recovery, from a transcriptional perspective, is incomplete 96 h after endurance exercise involving muscle damage.

The role of epigenetics in resistance to cisplatin chemotherapy in lung cancer

Non-small cell lung cancer (NSCLC) is the most common cause of cancer related death in the world. Cisplatin and carboplatin are the most commonly used cytotoxic chemotherapeutic agents to treat the disease. These agents, usually combined with drugs such as gemcitabine or pemetrexed, induce objective tumor responses in only 20-30% of patients. Aberrant epigenetic regulation of gene expression is a frequent event in NSCLC. In this article we review the emerging evidence that epigenetics and the cellular machinery involved with this type of regulation may be key elements in the development of cisplatin resistance in NSCLC. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

RanGTPase : a candidate for myc-mediated cancer progression

Background Ras-related nuclear protein (Ran) is required for cancer cell survival in vitro and human cancer progression, but the molecular mechanisms are largely unknown. Methods We investigated the effect of the v-myc myelocytomatosis viral oncogene homolog (Myc) on Ran expression by Western blot, chromatin immunoprecipitation, and luciferase reporter assays and the effects of Myc and Ran expression in cancer cells by soft-agar, cell adhesion, and invasion assays. The correlation between Myc and Ran and the association with patient survival were investigated in 14 independent patient cohorts (n = 2430) and analyzed with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. Results Myc binds to the upstream sequence of Ran and transactivates Ran promoter activity. Overexpression of Myc upregulates Ran expression, whereas knockdown of Myc downregulates Ran expression. Myc or Ran overexpression in breast cancer cells is associated with cancer progression and metastasis. Knockdown of Ran reverses the effect induced by Myc overexpression in breast cancer cells. In clinical data, a positive association between Myc and Ran expression was revealed in 288 breast cancer and 102 lung cancer specimens. Moreover, Ran expression levels differentiate better or poorer survival in Myc overexpressing breast (χ2 = 24.1; relative risk [RR] = 9.1, 95% confidence interval [CI] = 3.3 to 24.7, P <. 001) and lung (χ2 = 6.04; RR = 2.8, 95% CI = 1.2 to 6.3; P =. 01) cancer cohorts. Conclusions Our results suggest that Ran is required for and is a potential therapeutic target of Myc-driven cancer progression in both breast and lung cancers. © 2013 The Author.

DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana

DOUBLE-STRANDED RNA BIN DIN G (DRB) proteins have been functionally characterized in viruses, prokaryotes and eukaryotes and are involved in all aspects of RNA biology. Arabidopsis thaliana (Arabidopsis) encodes five closely related DRB proteins, DRB1 to DRB5. DRB1 and DRB4 are required by DICER-LIKE (DCL) proteins DCL1 and DCL4 to accurately and efficiently process structurally distinct double-stranded RNA (dsRNA) precursor substrates in the microRNA (miRNA) and trans-acting small-interfering RNA (tasiRNA) biogenesis pathways respectively. We recently reported that DRB2 is also involved in the biogenesis of specific miRNA subsets. Furthermore, the severity of the developmental phenotype displayed by the drb235 triple mutant plant, compared with those expressed by either drb2, drb3 and drb5 single mutants, or double mutant combinations thereof, indicates that DRB3 and DRB5 function in the same non-canonical miRNA pathway as DRB2. Through the use of our artificial miRNA (amiRNA) plant expression vector, pBlueGreen 2,3 we demonstrate here that unlike DRB2, DRB3 and DRB5 are not involved in the dsRNA processing stages of the miRNA biogenesis pathway, but are required to mediate RNA silencing of target genes of DRB2-associated miRNA s. © 2012 Landes Bioscience.

Fatty acid uptake and incorporation into phospholipids in the rat lens

PURPOSE. Phospholipids are a major component of lens fiber cells and influence the activity of membrane proteins. Previous investigations of fatty acid uptake by the lens are limited. The purpose of the present study was thus to determine whether exogenous fatty acids could be taken up by the rat lens and incorporated into molecular phospholipids. METHODS. Lenses were incubated with fluorescently labeled palmitic acid and then analyzed by confocal microscopy. Concurrently, lenses incubated with either fluorescently labeled palmitic acid or the more physiologically relevant (13)C(18)-oleic acid were sectioned into nuclear and cortical regions and analyzed by highly sensitive and structurally selective electrospray ionization tandem mass spectrometry techniques. RESULTS. The detection of fluorescently labeled palmitic acid, even after 16 hours of incubation, was limited to approximately the outer 25% to 30% of the rat lens. Mass spectrometry also revealed the presence of free (13)C(18)-oleic acid in the cortex but not the nucleus. No evidence could be found for incorporation of fluorescently labeled palmitic acid into phospholipids; however, a low level of (13)C(18)-oleic acid incorporation into phosphatidylethanolamine (PE), specifically PE (PE 16:0/(13)C(18) 18:1) was detected in the lens cortex after 16 hours. CONCLUSIONS. These data demonstrate that uptake of exogenous (e.g., dietary fatty acids) by the lens and their incorporation into phospholipids is minimal, most likely occurring only during de novo synthesis in the outermost region of the lens. This finding adds support to the hypothesis that once synthesized there is no active remodeling or turnover of fiber cell phospholipids.

A new plasmid display technology for the in vitro selection of functional phenotype–genotype linked proteins

Background: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype–genotype link between the displayed protein and the encoding gene. Results: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50–DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein–plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype–genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Induction of tumorigenicity by galectin-3 in a nontumorigenic human breast-carcinoma cell-line

The human galectin-3 is a galactoside-binding protein of 31 kDa which functions as a receptor for glycoproteins containing poly N-acetyllactosamine side chains and as a substrate for matrix metalloproteinases-2 and -9. We studied its expression by flow cytofluorimetry, Western, Northern and Southern analyses, in five cultured human breast carcinoma cell lines previously characterized as non-tumorigenic, poorly metastatic or metastatic in nude mice. The expression of galectin-3 correlated with the reported tumorigenicity of the cells. The introduction of recombinant galectin-3 into the null expressing non-tumorigenic BT-549 cells resulted in the acquisition of anchorage-independent growth properties in alland tumorigenicity in 3/4 sense transfected cell crones. The data indicate a relationship between galectin-3 expression and malignancy of human breast carcinoma cell lines.

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co‐transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA‐binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted β‐propeller‐forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C‐terminal domain (CTD) of pol II in vitro and in a two‐hybrid test in vivo. Furthermore, transcriptional run‐on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3′‐end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

KRAB zinc-finger proteins localise to novel KAP1-containing foci that are adjacent to PML nuclear bodies

The KRAB-zinc finger proteins (KRAB-ZFPs) represent a very large, but poorly understood, family of transcriptional regulators in mammals. They are thought to repress transcription via their interaction with KRAB-associated protein 1 (KAP1), which then assembles a complex of chromatin modifiers to lay down histone marks that are associated with inactive chromatin. Studies of KRAB-ZFP/KAP1-mediated gene silencing, using reporter constructs and ectopically expressed proteins, have shown colocalisation of both KAP1 and repressed reporter target genes to domains of constitutive heterochromatin in the nucleus. However, we show here that although KAP1 does indeed become recruited to pericentric heterochromatin during differentiation of mouse embryonic stem (ES) cells, endogenous KRAB-ZFPs do not. Rather, KRAB-ZFPs and KAP1 relocalise to novel nucleoplasmic foci that we have termed KRAB- and KAP1-associated (KAKA) foci. HP1s can also concentrate in these foci and there is a close spatial relationship between KAKA nuclear foci and PML nuclear bodies. Finally, we reveal differential requirements for the recruitment of KAP1 to pericentric heterochromatin and KAKA foci, and suggest that KAKA foci may contain sumoylated KAP1 - the form of the protein that is active in transcriptional repression.

Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing

Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing.

Molecular characterization of endocarditis-associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.