Roles of Coactivators in Hypoxic Induction of the Erythropoietin Gene

Hypoxia-inducible expression of the erythropoietin (EPO) gene is mediated principally by hypoxia-inducible factor 2alpha (HIF-2alpha) in Hep3B cells under physiologic conditions. How/whether p300/CBP and the members of p160 coactivator family potentiate hypoxic induction of endogenous EPO and other HIF-2alpha and hypoxia-inducible factor 1alpha (HIF-1alpha) target genes remains unclear.We demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that the histone acetyl transferase (HAT) coactivators p300, SRC-1 and SRC-3 are recruited to the 3' enhancer of the EPO gene upon hypoxic stimulation, and that each associates with the enhancer in a periodic fashion. Hypoxia induced acetylation of the EPO gene 5' promoter at histone 4 and lysine 23 of histone 3. Knocking down SRC-3, but not SRC-1 or SRC-2, using short interfering RNAs (siRNAs), reduced EPO transcriptional activity. Knocking down p300 resulted in dramatic down-regulation of hypoxic stimulation of EPO gene transcription, negated recruitment of RNA polymerase II to the gene's promoter, and eliminated hypoxia-stimulated acetylation at the promoter and recruitments of SRC-1 and SRC-3 to the enhancer. The inhibitory effects of knocking down p300 and the chromatin remodeling coactivator, Brm/Brg-1, on EPO transcription were additive, suggesting that p300 and Brm/Brg-1 act independently. p300 was also required for hypoxia induced transcription of the HIF-1alpha target gene, VEGF, but was dispensable for induction of two other HIF-1alpha target genes, PGK and LDHA. Knocking down CBP, a homolog of p300, augmented hypoxic induction of VEGF, LDHA and PGK. Different HIF target genes also exhibited different requirements for members of the p160 coactivator family.p300 plays a central coactivator role in hypoxic induction of EPO. The coactivators exhibit different specificities for different HIF target genes and each can behave differently in transcriptional regulation of different target genes mediated by the same transcription factor

Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.United States. National Institutes of Health (R01GM107536)Alex's Lemonade Stand FoundationHoward Hughes Medical InstituteBoston Children's Hospital. Manton Center for Orphan Disease ResearchNational Institute of General Medical Sciences (U.S.) (T32GM007753

1, 9-Pyrazoloanthrones Downregulate HIF-1α and Sensitize Cancer Cells to Cetuximab-Mediated Anti-EGFR Therapy

Cetuximab, a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR), is currently approved for the treatment of several types of solid tumors. We previously showed that cetuximab can inhibit hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis by inhibiting the activation of EGFR downstream signaling pathways including Erk, Akt, and mTOR. 1, 9-pyrazoloanthrone (1, 9 PA) is an anthrapyrazolone compound best known as SP600125 that specifically inhibits c-jun N-terminal kinase (JNK). Here, we report 1, 9 PA can downregulate HIF-1α independently of its inhibition of JNK. This downregulatory effect was abolished when the oxygen-dependent domain (ODD) of HIF-1α (HIF-1α-ΔODD, the domain responsible for HIF-1α degradation) was experimentally deleted or when the activity of HIF-1α prolyl hydroxylase (PHD) or the 26S proteasomal complex was inhibited, indicating that the 1, 9 PA downregulates HIF-1α by promoting PHD-dependent HIF-1α degradation. We found that the combination of 1, 9 PA and cetuximab worked synergistically to induce apoptosis in cancer cells in which cetuximab or 1, 9 PA alone had no or only weak apoptotic activity. This synergistic effect was substantially decreased in cancer cells transfected with HIF-1α-ΔODD, indicating that downregulation of HIF-1α was the mechanism of this synergistic effect. More importantly, 1, 9 PA can downregulate HIF-1α in cancer cells that are insensitive to cetuximab-induced inhibition of HIF-1α expression due to overexpression of oncogenic Ras (RasG12V). Our findings suggest that 1, 9 PA is a lead compound of a novel class of drugs that may be used to enhance the response of cancer cells to cetuximab through a complementary effect on the downregulation of HIF-1α

Are antifibrinolytic drugs equivalent in reducing blood loss and transfusion in cardiac surgery? A meta-analysis of randomized head-to-head trials

BACKGROUND: Aprotinin has been shown to be effective in reducing peri-operative blood loss and the need for re-operation due to continued bleeding in cardiac surgery. The lysine analogues tranexamic acid (TXA) and epsilon aminocaproic acid (EACA) are cheaper, but it is not known if they are as effective as aprotinin. METHODS: Studies were identified by searching electronic databases and bibliographies of published articles. Data from head-to-head trials were pooled using a conventional (Cochrane) meta-analytic approach and a Bayesian approach which estimated the posterior probability of TXA and EACA being equivalent to aprotinin; we used as a non-inferiority boundary a 20% increase in the rates of transfusion or re-operation because of bleeding. RESULTS: Peri-operative blood loss was significantly greater with TXA and EACA than with aprotinin: weighted mean differences were 106 mls (95% CI 37 to 227 mls) and 185 mls (95% CI 134 to 235 mls) respectively. The pooled relative risks (RR) of receiving an allogeneic red blood cell (RBC) transfusion with TXA and EACA, compared with aprotinin, were 1.08 (95% CI 0.88 to 1.32) and 1.14 (95% CI 0.84 to 1.55) respectively. The equivalent Bayesian posterior mean relative risks were 1.15 (95% Bayesian Credible Interval [BCI] 0.90 to 1.68) and 1.21 (95% BCI 0.79 to 1.82) respectively. For transfusion, using a 20% non-inferiority boundary, the posterior probabilities of TXA and EACA being non-inferior to aprotinin were 0.82 and 0.76 respectively. For re-operation the Cochrane RR for TXA vs. aprotinin was 0.98 (95% CI 0.51 to 1.88), compared with a posterior mean Bayesian RR of 0.63 (95% BCI 0.16 to 1.46). The posterior probability of TXA being non-inferior to aprotinin was 0.92, but this was sensitive to the inclusion of one small trial. CONCLUSION: The available data are conflicting regarding the equivalence of lysine analogues and aprotinin in reducing peri-operative bleeding, transfusion and the need for re-operation. Decisions are sensitive to the choice of clinical outcome and non-inferiority boundary. The data are an uncertain basis for replacing aprotinin with the cheaper lysine analogues in clinical practice. Progress has been hampered by small trials and failure to study clinically relevant outcomes

Natural Selection and Adaptive Evolution of Leptin in the Ochotona Family Driven by the Cold Environmental Stress

BACKGROUND: Environmental stress can accelerate the evolutionary rate of specific stress-response proteins and create new functions specialized for different environments, enhancing an organism's fitness to stressful environments. Pikas (order Lagomorpha), endemic, non-hibernating mammals in the modern Holarctic Region, live in cold regions at either high altitudes or high latitudes and have a maximum distribution of species diversification confined to the Qinghai-Tibet Plateau. Variations in energy metabolism are remarkable for them living in cold environments. Leptin, an adipocyte-derived hormone, plays important roles in energy homeostasis. METHODOLOGY/PRINCIPAL FINDINGS: To examine the extent of leptin variations within the Ochotona family, we cloned the entire coding sequence of pika leptin from 6 species in two regions (Qinghai-Tibet Plateau and Inner Mongolia steppe in China) and the leptin sequences of plateau pikas (O. curzonia) from different altitudes on Qinghai-Tibet Plateau. We carried out both DNA and amino acid sequence analyses in molecular evolution and compared modeled spatial structures. Our results show that positive selection (PS) acts on pika leptin, while nine PS sites located within the functionally significant segment 85-119 of leptin and one unique motif appeared only in pika lineages-the ATP synthase alpha and beta subunit signature site. To reveal the environmental factors affecting sequence evolution of pika leptin, relative rate test was performed in pikas from different altitudes. Stepwise multiple regression shows that temperature is significantly and negatively correlated with the rates of non-synonymous substitution (Ka) and amino acid substitution (Aa), whereas altitude does not significantly affect synonymous substitution (Ks), Ka and Aa. CONCLUSIONS/SIGNIFICANCE: Our findings support the viewpoint that adaptive evolution may occur in pika leptin, which may play important roles in pikas' ecological adaptation to extreme environmental stress. We speculate that cold, and probably not hypoxia, may be the primary environmental factor for driving adaptive evolution of pika leptin

OVERHEATED SECURITY? The Securitisation of Climate Change and the Governmentalisation of Security

Since the mid-2000s, climate change has become one of the defining security issues in political as well as academic debates and amongst others has repeatedly been discussed in the UN Security Council and countless high level government reports in various countries. Beyond the question whether the characterisation as ‘security issue’ is backed up by any robust empirical findings, this begs the question whether the ‘securitisation’ of climate change itself has had tangible political consequences. Moreover, within this research area there is still a lively discussion about which security conceptions apply, how to conceptualise (successful) securitisation and whether it is a (politically and normatively) desirable approach to deal with climate change. The aim of this dissertation is to shed light on these issues and particularly to contribute to a more thorough understanding of different forms or ‘discourses’ of securitisation and their political effects on a theoretical and empirical level. Theoretically, it conceptualises securitisation as resting on different forms of power, which are derived from Michel Foucault’s governmentality lectures. The main argument is that this framework allows me to better capture the ambiguous and diverse variants of securitisation and the ever-changing concept of security as well as to come to a more thorough understanding of the political consequences and powerful effects of constructing issues in terms of security. Empirically, the thesis looks at three country cases, namely the United States, Germany and Mexico. This comparative angle allows me to go beyond the existing literature on the securitisation of climate change that mostly looks at the global level, and to come to a more comprehensive and detailed understanding of different climate security discourses and their political consequences. Concerning the main results, the thesis finds that climate change has indeed been securitised very differently in the three countries and thus has facilitated diverse political consequences. These range from an incorporation of climate change into the defence sector in the US, the legitimisation of far-reaching climate policies in Germany, to the integration of climate change into several civil protection and agricultural insurance schemes in Mexico. Moreover, resting on different forms of power, the securitisation of climate change has played a key role in constructing specific actors and forms of knowledge as legitimate as well as in shaping certain identities in the face of the dangers of climate change. From a normative perspective, neither of these political consequences is purely good or bad but highly ambiguous and necessitates a careful, contextual assessment