The p68 RNA Helicase

The p68 protein is immunologically related to large T antigen, the dominant transforming oncogene of simian virus 40. The deduced amino acid sequence of human p68 contains amino acid sequence motifs characteristic of a large family of putative RNA helicases called "DEAD box" proteins. The family derives its name from the sequence of the most conserved motif: aspartate- glutamate-alanine-aspartate. Human p68 protein was immunoprecipitated from Hela cells and shown to possess RNA-dependent ATPase activity. As expected for an RNA helicase, the ATPase activity is stimulated preferentially by RNA lacking secondary structure. Cell staining shows that the sub-nuclear location of mammalian p68 changes dramatically during the cell cycle. p68 is present in the nucleoplasm and excluded from the nucleoli in interphase. This pattern is reversed in telophase, when p68 appears to be concentrated in prenucleolar bodies. The human p68 cDNA was completed by anchor PCR and the human p68 gene was mapped to chromosome 17q23. Putative p68 homologues were cloned from yeast and named DBP2 in S.cerevisiae and dbp2 in S.pombe. The yeast genes were shown by gene disruption and tetrad analysis to be essential for viability. The p68, DBP2 and dbp2 genes contain an intron at the same site in helicase motif V. The intron is 1.2 kb long in p68, 1 kb long in DBP2, and 700 bp long in dbp2. The unusual length, position and conservation of the intron in DBP2 suggest that it may have a function. The possibility that the intron autoregulates DBP2 expression was examined by Northern and Western blotting in S.cerevisiae

Adenoviruses with tcf binding sites in multiple early promoters show enhanaced selectivity for tumour cells with constitutive activation of the Wnt signalling pathway.

Mutation of the adenomatous polyposis coli and g-catenin genes in colon cancer leads to constitutive activation of transcription from promoters containing binding sites for Tcf/LEF transcription factors. We have constructed adenoviruses with Tcf binding sites in the early promoters, in order to target viral replication to colon tumours. Tcf regulation of the E1A promoter confers a 100-fold selectivity for cells with activated wnt signalling in viral burst and cytopathic effect assays. p300 is a coactivator for beta-catenin, and E1A inhibits Tcf-dependent transcription through sequestration of p300, but mutation of the p300 binding site in E1A leads to a 10-fold reduction in cytopathic effect of all of the Tcf-regulated viruses. When Tcf sites are inserted in the E1A, E1B, E2 and E4 promoters the viruses show up to 100,000-fold selectivity for cells with activated wnt signalling.</p

Determinanats of interferon-stimulated gene induction by RNAi vectors

RNA interference is widely used to silence gene expression in mammalian cells. We recently reported that an shRNA expressed from the H1 promoter in a lentiviral vector could induce the expression of a large group of interferon-stimulated genes (ISGs). This response was unrelated to silencing of the gene targeted by the shRNA MORF4L1. In parallel, we constructed lentiviral vectors expressing shRNA from the U6 promoter and found that these too could induce expression of OAS1, a classic interferon target gene. The U6 vectors give a higher frequency of ISG induction than comparable lentiviral H1 vectors, suggesting that there might be a fundamental flaw in the vector design. We have characterized the U6 vectors in detail and report here that ISG induction is a consequence of the presence of an AA di-nucleotide near the transcription start site. A single nucleotide deletion in the siRNA sequence abolished OAS1 induction, suggesting that the mechanism underlying the response uses a sensor that can detect 19 bp RNA duplexes but not 14 bp duplexes. Adenoviral VA RNA I, which inhibits dsRNA-dependent protein kinase (PKR), was tested as a fusion partner to express shRNA on the grounds that it might prevent nonspecific off-target effects. Fusion of VA RNA I to a lamin shRNA was moderately effective in silencing lamin expression, but gave strong OAS1 induction by an shRNA that does not induce OAS1 when expressed from the U6 or H1 promoters. To avoid interferon induction by U6 vectors, we recommend preserving the wild-type sequence around the transcription start site, in particular a C/G sequence at positions -1/+1, and we describe a simple cloning strategy using the Gateway recombination system that facilitates this task.</p

Determinanats of interferon-stimulated gene induction by RNAi vectors

RNA interference is widely used to silence gene expression in mammalian cells. We recently reported that an shRNA expressed from the H1 promoter in a lentiviral vector could induce the expression of a large group of interferon-stimulated genes (ISGs). This response was unrelated to silencing of the gene targeted by the shRNA MORF4L1. In parallel, we constructed lentiviral vectors expressing shRNA from the U6 promoter and found that these too could induce expression of OAS1, a classic interferon target gene. The U6 vectors give a higher frequency of ISG induction than comparable lentiviral H1 vectors, suggesting that there might be a fundamental flaw in the vector design. We have characterized the U6 vectors in detail and report here that ISG induction is a consequence of the presence of an AA di-nucleotide near the transcription start site. A single nucleotide deletion in the siRNA sequence abolished OAS1 induction, suggesting that the mechanism underlying the response uses a sensor that can detect 19 bp RNA duplexes but not 14 bp duplexes. Adenoviral VA RNA I, which inhibits dsRNA-dependent protein kinase (PKR), was tested as a fusion partner to express shRNA on the grounds that it might prevent nonspecific off-target effects. Fusion of VA RNA I to a lamin shRNA was moderately effective in silencing lamin expression, but gave strong OAS1 induction by an shRNA that does not induce OAS1 when expressed from the U6 or H1 promoters. To avoid interferon induction by U6 vectors, we recommend preserving the wild-type sequence around the transcription start site, in particular a C/G sequence at positions -1/+1, and we describe a simple cloning strategy using the Gateway recombination system that facilitates this task.</p

5-Fluorocytosine increases the toxicity of Wnt-targeting replicating adenoviruses that expresss cytosine deaminase as a late gene

Clinical studies with oncolytic adenoviruses have shown that existing viruses are safe but lack efficacy. To selectively increase the toxicity of oncolytic adenoviruses targeting colon tumours, we have inserted the yeast cytosine deaminase gene (yCD) after the fibre gene in the major late transcript. yCD was expressed using either an internal ribosome entry site (IRES) or by alternative splicing of a new exon analogous to the Ad41 long fibre exon. The IRES-CD virus gave higher yCD expression on Western blots. Both approaches result in yCD expression restricted to the period after viral DNA replication. Viral burst size was reduced by less than similar to10-fold by 5-fluorocytosine (5-FC), showing that expression of yCD as a late gene is compatible with virus replication. Cytopathic effect assays in colon cancer cell lines showed that both yCD viruses have similar to10-fold increased toxicity in the presence of the prodrug 5-FC, which is converted to 5-fluorouracil (5-FU) by yCD. Toxicity was higher following addition of 5-FC immediately after infection. The largest gain in toxicity was seen in HT29 colon cancer cells, which are the least permissive colon cancer cells for the parental virus, indicating that the new 5-FC/yCD viruses may have broader applications for colon cancer therapy than their predecessors.</p

Actor-network theory. Synthesis and evaluation of Bruno Latour’s post-social drift

En este trabajo, inicialmente, se expone cómo la teoría del actor-red concibe: el fracaso del proyecto de la modernidad y de sus viejos dualismos, la muerte de lo social como un factor explicativo sólido y fundamental, y el nacimiento o el redescubrimiento del mundo postsocial o posthumanista. Después, se describe cómo esta teoría propone, como principal alternativa, que el principio de simetría propio de la sociología socioconstructivista sea generalizado, utilizando para ello una semiótica relacional que incluya a todos los actantes implicados en cada situación, sean humanos o no-humanos. A continuación, se examinan los más relevantes excesos, insuficiencias y ambigüedades de esta teoría, presentes tanto en sus diagnósticos como en sus propuestas. Con todo ello, se busca constatar que la sociología y otras ciencias sociales tienen motivos para escuchar e incluso aprender de esta original e innovadora teoría, pero que también los tienen, y muchos, para discrepar, reafirmarse y contradecirla.This study begins by describing how actor-network theory conceives: the failure of the project of modernity and its old dualisms, the death of the social as a solid and fundamental explanatory factor, and the birth or rediscovery of the post-social or post-humanist world. It goes on to describe how the theory proposes, as the main alternative, that the principle of symmetry of socio-constructivist sociology be generalised, using relational semiotics that includes all actants involved in each situation, whether human or non-human. It then discusses the most notable deficiencies, limitations and drawbacks of this theory, present in both its diagnoses and proposals. Hence the study seeks to confirm that sociology and other social sciences have reasons to understand and indeed learn from this very original and innovative theory, but that they also have many reasons to disagree, contradict and indeed to reaffirm their own positions

Chromatin immunoprecipitation anaylsis fails to support the latency model for regulation of p53 DNA binding activity in vivo

p53 can adopt two forms in vitro, a latent form that binds naked DNA poorly and an active form that binds DNA well. Conversion of the latent form to the active form is thought to occur by an allosteric mechanism induced by phosphorylation and acetylation. Despite the large differences in affinity produced by regulatory modifications in vitro, mutation of putative regulatory sites has not produced correspondingly large effects on transcription of p53 target genes in vivo. To determine whether genotoxic stress regulates DNA binding by p53 in vivo, we have performed quantitative chromatin immunoprecipitation (ChIP) assays on tumor and normal cell lines containing wildtype p53. ChIP recovers several hundredfold more p21 and MDM2 promoter DNA from p53 wild-type than p53-null cells, indicating that the assay is specific for p53. Genotoxic stress induces much smaller increases in chromatin precipitation, which are matched by changes in the p53 protein level. Thus, in the experimental systems tested, allosteric regulation of DNA binding is not a major level of regulation of p53 activity. The p53 target genes tested can be divided into a group showing high promoter occupancy in vivo (p21, MDM2, and PUMA) and a group giving substantially weaker or background p53 binding (bax, AIP1, and PIG3). Neither group shows selective recruitment of p53 to the promoter in cells undergoing apoptosis, indicating that the decision to undergo apoptosis or cell cycle arrest depends on other changes in the cell.</p

Promoter-specific p53-dependent histone acetylation following DNA damage.

We have used chromatin immunoprecipitation (ChIP) to measure p53-dependent histone acetylation at the p21, MDM2, and PUMA promoters. The pattern of histone acetylation was different at each promoter. H3 and H4 acetylation increased at both the p21 and PUMA promoters in response to p53 activation, whereas there was only a minimal increase in H4 acetylation and no increase in H3 acetylation at the MDM2 promoter. The high p53 occupancy of the p21, MDM2 and PUMA promoters has been attributed to the presence of two p53 binding sites in these promoters, but mutation of the p53 binding sites in integrated p21 promoter constructs showed that the two sites in the p21 promoter do not cooperate to stabilize p53 binding. Despite 10-fold higher p53 binding to the proximal than the distal site in the p21 promoter, both sites showed similar patterns of H3 and H4 acetylation. Mutation of the binding sites showed that acetylation of the proximal, low-affinity site requires p53 binding to that site but not to the distal, high-affinity site. Since low-affinity p53 binding sites can confer strong acetylation, the DNA binding affinity in vitro is an unreliable guide to the likely importance of p53 in regulating candidate target genes in vivo.</p

RAD001 (Everolimus) Improves the Efficacy of Replicating Adenoviruses that Target Colon Cancer.

Selectively replicating adenoviruses have the potential to cure cancer but have shown little efficacy in clinical trials. We have tested the ability of the mTOR kinase inhibitor RAD001 (everolimus) to enhance the response of xenografts to an oncolytic adenovirus. The virus has Tcf sites inserted in the early viral promoters and replicates selectively in cells with activation of the Wnt signaling pathway. To enhance tumor cell infection, an integrin targeting peptide (CDCRGDCFC) was inserted into the fiber gene of the virus. RAD001 combines three useful properties: it inhibits tumor cell growth directly, blocks angiogenesis, and suppresses the immune response. RAD001 does not block viral protein expression, DNA replication, or cytopathic effect in tumor cells in vitro. After 6 weeks of daily RAD001 treatment, ongoing viral DNA replication could be detected in tumor xenografts, showing that RAD001 does not inhibit virus replication in vivo. I.v. injection of virus alone produced a small delay in xenograft growth, whereas combination therapy substantially prolonged the survival of the mice. We suggest that collapsing the tumor vasculature after the initial infection traps the virus and facilitates local spread within the tumor. Unlike conventional drugs, which require continued access to the tumor through the vascular system, oncolytic viruses are in principle less sensitive to late reductions in perfusion because they are produced locally within the tumor.</p