HDACs and the senescent phenotype of WI-38 cells

BACKGROUND: Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells. RESULTS: Pre- and post-senescent WI-38 cells were treated with the HDAC inhibitors butyrate or trichostatin A (TSA). Following HDAC inhibitor treatment, pre-senescent cells increased p21(WAF1 )and β-galactosidase expression, assumed a flattened senescence-associated morphology, and maintained a lower level of proteasome activity. These alterations also occurred during normal replicative senescence of WI-38 cells, but were not accentuated further by HDAC inhibitors. We also found that HDAC1 levels decline during normal replicative senescence. CONCLUSION: Our findings indicate that HDACs impact numerous phenotypic changes associated with cellular senescence. Reduced HDAC1 expression levels in senescent cells may be an important event in mediating the transition to a senescent phenotype

RNAa Is Conserved in Mammalian Cells

Background: RNA activation (RNAa) is a newly discovered mechanism of gene activation triggered by small doublestranded RNAs termed ‘small activating RNAs ’ (saRNAs). Thus far, RNAa has only been demonstrated in human cells and is unclear whether it is conserved in other mammals. Methodology/Principal Findings: In the present study, we evaluated RNAa in cells derived from four mammalian species including nonhuman primates (African green monkey and chimpanzee), mouse, and rat. Previously, we identified saRNAs leading to the activation of E-cadherin, p21, and VEGF in human cells. As the targeted sequences are highly conserved in primates, transfection of each human saRNA into African green monkey (COS1) and chimpanzee (WES) cells also resulted in induction of the intended gene. Additional saRNAs targeting clinically relevant genes including p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2 were also designed and transfected into COS1 and WES cells. Of the nine genes, p53, PAR4, WT1, and NKX3-1 were induced by their corresponding saRNAs. We further extended our analysis of RNAa into rodent cell types. We identified two saRNAs that induced the expression of mouse Cyclin B1 in NIH/3T3 and TRAMP C1 cells, which led to increased phosphorylation of histone H3, a downstream marker for chromosome condensation and entry into mitosis. We also identified two saRNAs that activated the expression of CXCR4 in primary rat adipose–derived stem cells. Conclusions/Significance: This study demonstrates that RNAa exists in mammalian species other than human. Our finding

Formulation of Small Activating RNA Into Lipidoid Nanoparticles Inhibits Xenograft Prostate Tumor Growth by Inducing p21 Expression

Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21WAF1/ Cip1 (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-of-principle that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease

Upregulation of Cyclin B1 by miRNA and its implications in cancer

It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3′UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth

Socio-Emotional Competencies and School Performance in Adolescence: What Role for School Adjustment?

There is growing evidence in the literature of positive relationships between socio-emotional competencies and school performance. Several hypotheses have been used to explain how these variables may be related to school performance. In this paper, we explored the role of various school adjustment variables in the relationship between interpersonal socio-emotional competencies and school grades, using a weighted network approach. This network approach allowed us to analyze the structure of interrelations between each variable, pointing to both central and mediatory school and socio-emotional variables within the network. Self-reported data from around 3,400 French vocational high school students were examined. This data included a set of interpersonal socio-emotional competencies (cognitive and affective empathy, socio-emotional behaviors and collective orientation), school adjustment measures (adaptation to the institution, school anxiety, self-regulation at school, and self-perceived competence at school) as well as grades in mathematics and French language. The results showed that self-regulation at school weighted the most strongly on the whole network, and was the most important mediatory pathway. More specifically, self-regulation mediated the relationships between interpersonal socio-emotional competencies and school grades

RNA activation of haploinsufficient Foxg1 gene in murine neocortex

More than one hundred distinct gene hemizygosities are specifically linked to epilepsy, mental retardation, autism, schizophrenia and neuro-degeneration. Radical repair of these gene deficits via genome engineering is hardly feasible. The same applies to therapeutic stimulation of the spared allele by artificial transactivators. Small activating RNAs (saRNAs) offer an alternative, appealing approach. As a proof-of-principle, here we tested this approach on the Rett syndrome-linked, haploinsufficient, Foxg1 brain patterning gene. We selected a set of artificial small activating RNAs (saRNAs) upregulating it in neocortical precursors and their derivatives. Expression of these effectors achieved a robust biological outcome. saRNA-driven activation (RNAa) was limited to neural cells which normally express Foxg1 and did not hide endogenous gene tuning. saRNAs recognized target chromatin through a ncRNA stemming from it. Gene upregulation required Ago1 and was associated to RNApolII enrichment throughout the Foxg1 locus. Finally, saRNA delivery to murine neonatal brain replicated Foxg1-RNAa in vivo

The Public Repository of Xenografts enables discovery and randomized phase II-like trials in mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Regulation of proteasome activity and substrate recognition in colon cancer cells

The proteasome is a multicatalytic protease complex that plays a central role in regulating fundamental cellular processes. NF-κB is a transcription factor whose activity is critically dependent on the proteasome, and plays a central role in regulating cell proliferation, apoptosis, and inflammatory responses. Experiments were performed to obtain insight into the role the proteasome plays in regulating NF-κB in colon cancer cells. ^ NF-κB is activated by the signal-induced proteasome degradation of the IκBα inhibitory protein, which allows NF-κB to transmigrate into the nucleus and activate target genes. However, IκBα levels are only transiently lowered since one gene activated by NF-κB is IκBα. We found that in addition to the transcriptional up-regulation of IκB, NF-κB stimuli also increased the stability of newly synthesized IκBα. This stabilization pathway involved the p38 MAP kinase, which suppressed the destabilizing effects of the C-terminal domain of IκBα. We propose that this p38-mediated pathway serves to replenish IκBα levels after stimulation, as well as limit sequential rounds of NF-κB activation. ^ We also obtained evidence that the level of proteasome subunit expression can impact NF-κB regulation. Histone deacetylase (HDAC) inhibitors induce cell cycle arrest and differentiation of colon cancer cells. We found that HDAC inhibitor-induced differentiation decreased proteasome activity and reprogrammed the NF-κB response. Specifically, TNF-α activation was suppressed, while IL-1β activation was largely unaffected. The selective impact of HDAC inhibitors on TNF-α-induced NF-κB activation appeared to relate to the fact that TNF-α-induced activation was mediated by the proteasome, whereas NF-κB activation by IL-1β was proteasome independent. The drop in proteasome activity was the result of reduced expression of the catalytic β-type subunits of the proteasome. These findings indicate that cellular differentiation status can significantly impact proteasome activity and NF-κB responses. This conclusion was further supported by the fact that proteasome activity and subunit expression naturally declined upon senescence of normal human cells. Our studies also revealed that HDACs play a central role in regulating senescence in normal cells. ^ These results highlight a number important biological features of proteasome and NF-κB regulation in colon cancer cells. These findings may be exploitable for the development of novel cancer treatments and anti-inflammatory agents.