Aspirin-induced nuclear translocation of NFκB and apoptosis in colorectal cancer is independent of p53 status and DNA mismatch repair proficiency

Substantial evidence indicates nonsteroidal anti-inflammatory drugs (NSAIDs) protect against colorectal cancer (CRC). However, the molecular basis for this anti-tumour activity has not been fully elucidated. We previously reported that aspirin induces signal-specific IκBα degradation followed by NFκB nuclear translocation in CRC cells, and that this mechanism contributes substantially to aspirin-induced apoptosis. We have also reported the relative specificity of this aspirin-induced NFκB-dependent apoptotic effect for CRC cells, in comparison to other cancer cell types. It is now important to establish whether there is heterogeneity within CRC, with respect to the effects of aspirin on the NFκB pathway and apoptosis. p53 signalling and DNA mismatch repair (MMR) are known to be deranged in CRC and have been reported as potential molecular targets for the anti-tumour activity of NSAIDs. Furthermore, both p53 and MMR dysfunction have been shown to confer resistance to chemotherapeutic agents. Here, we set out to determine the p53 and hMLH1 dependency of the effects of aspirin on NFκB signalling and apoptosis in CRC. We specifically compared the effects of aspirin treatment on cell viability, apoptosis and NFκB signalling in an HCT-116 CRC cell line with the p53 gene homozygously disrupted (HCT-116p53−/−) and an HCT-116 cell line rendered MMR proficient by chromosomal transfer (HCT-116+ch3), to the parental HCT-116 CRC cell line. We found that aspirin treatment induced apoptosis following IκBα degradation, NFκB nuclear translocation and repression of NFκB-driven transcription, irrespective of p53 and DNA MMR status. These findings are relevant for design of both novel chemopreventative agents and chemoprevention trials in CRC

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background: We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-kappa B is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-kappa B in mediating cell survival in response to cigarette smoke exposure in HBECs. Methods: Both the pharmacologic inhibitor of NF-kappa B, curcumin, and RNA interference targeting p65 were used to block NF-kappa B signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay. Results: Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-kappa B -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-kappa B by the pharmacologic inhibitor curcumin (20 mu M) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium. Conclusion: The current study demonstrates that CSE activates NF-kappa B and up-regulates Bcl-XL through NF-kappa B activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-kappa B regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000260432600001&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Respiratory SystemSCI(E)28ARTICLEnull

SplitAx:A novel method to assess the function of engineered nucleases

Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1-158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization

A Virgo Environmental Survey Tracing Ionised Gas Emission (VESTIGE): IX. The effects of ram pressure stripping down to the scale of individual HII regions in the dwarf galaxy IC 3476 (*.**)

We study the IB(s)m galaxy IC 3476 observed in the context of the Virgo Environmental Survey Tracing Ionised Gas Emission (VESTIGE), a blind narrow-band H alpha+[NII] imaging survey of the Virgo cluster carried out with MegaCam at the CFHT. The deep narrow-band image reveals a very pertubed ionised gas distribution that is characterised by a prominent banana-shaped structure in the front of the galaxy formed of giant HII regions crossing the stellar disc. Star-forming structures, at similar to 8 kpc from the edges of the stellar disc, are also detected in a deep far-ultraviolet ASTROSAT/UVIT image. This particular morphology indicates that the galaxy is undergoing an almost edge-on ram pressure stripping event. The same H alpha+[NII] image also shows that the star formation activity is totally quenched in the leading edge of the disc, where the gas has been removed during the interaction with the surrounding medium. The spectral energy distribution fitting analysis of the multi-frequency data indicates that this quenching episode is very recent (similar to 50 Myr), and roughly corresponds to an increase of the star formation activity by a factor of similar to 161% in the inner regions with respect to that expected for secular evolution. The analysis of these data, whose angular resolution enables the study of the induced effects of the perturbation down to the scale of individual HII regions (r(eq) similar or equal to 40 pc), also suggests that the increase of star formation activity is due to the compression of the gas along the stellar disc of the galaxy, which is able to increase its mean electron density and boost the star formation process producing bright HII regions with luminosities up to L(H alpha) similar or equal to 10(38) erg s(-1). The combined analysis of the VESTIGE data with deep IFU spectroscopy gathered with MUSE and with high spectral resolution Fabry Perot data also indicates that the hydrodynamic interaction has deeply perturbed the velocity field of the ionised gas component while leaving that of the stellar disc unaffected. The comparison of the data with tuned high-resolution hydrodynamic simulations accounting for the different gas phases (atomic, molecular, ionised) consistently indicates that the perturbing event is very recent (50-150 Myr), once again confirming that ram pressure stripping is a violent phenomenon that is able to perturb the evolution of galaxies in rich environments on short timescales

A Virgo Environmental Survey Tracing Ionised Gas Emission (VESTIGE) IV. A tail of ionised gas in the merger remnant NGC4424

We observed the late-type peculiar galaxy NGC4424 during the Virgo Environmental Survey Tracing Galaxy Evolution (VESTIGE), a blind narrow-band H alpha+ [NII] imaging survey of the Virgo cluster carried out with MegaCam at the Canada-French-Hawaii Telescope (CFHT). The presence of a similar to 110 kpc (in projected distance) HI tail in the southern direction indicates that this galaxy is undergoing a ram pressure stripping event. The deep narrow-band image revealed a low surface brightness (Sigma(H alpha) similar or equal to 4 x 10(-18) erg s(-1) cm(-2) arcsec(-2)) ionised gas tail similar to 10 kpc in length extending from the centre of the galaxy to the north-west, thus in the direction opposite to the HI tail. Chandra and XMM X-rays data do not show a compact source in the nucleus or an extended tail of hot gas, while IFU spectroscopy (MUSE) indicates that the gas is photo-ionised in the inner regions and shock-ionised in the outer parts. Medium-resolution (MUSE) (MUSE) indicates that the gas is photo-ionised in the inner regions and shock-ionised in the outer parts. Medium-resolution (MUSE) and high-resolution (Fabry-Perot) IFU spectroscopy confirms that the ionised gas is kinematically decoupled from the stellar component and indicates the presence of two kinematically distinct structures in the stellar disc. The analysis of the SED of the galaxy indicates that the activity of star formation was totally quenched in the outer disc similar to 250-280 Myr ago, while only reduced by similar to 80% in the central regions. All this observational evidence suggests that NGC4424 is the remnant of an unequal-mass merger that occurred less than or similar to 500 Myr ago when the galaxy was already a member of the Virgo cluster, and is now undergoing a ram pressure stripping event that has removed the gas and quenched the activity of star formation in the outer disc. The tail of ionised gas probably results from the outflow produced by a central starburst fed by the collapse of gas induced by the merging episode. This outflow is sufficiently powerful to overcome the ram pressure induced by the intracluster medium on the disc of the galaxy crossing the cluster. This analysis thus suggests that feedback can participate in the quenching process of galaxies in high-density regions

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

Artificial endonucleases consisting of a Fokl cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off- target sites. Here we describe a structure- based approach to reducing off- target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology