ATF2 knockdown reinforces oxidative stress-induced apoptosis in TE7 cancer cells

Abstract Cancer cells showing low apoptotic effects following oxidative stress-induced DNA damage are mainly affected by growth arrest. Thus, recent studies focus on improving anti-cancer therapies by increasing apoptosis sensitivity. We aimed at identifying a universal molecule as potential target to enhance oxidative stress-based anti-cancer therapy through a switch from cell cycle arrest to apoptosis. A cDNA microarray was performed with hydrogen peroxide-treated oesophageal squamous epithelial cancer cells TE7. This cell line showed checkpoint activation via p21 WAF1 , but low apoptotic response following DNA damage. The potential target molecule was chosen depended on the following demands: it should regulate DNA damage response, cell cycle and apoptosis. As the transcription factor ATF2 is implicated in all these processes, we focused on this protein. We investigated checkpoint activation via ATF2. Indeed, ATF2 knockdown revealed ATF2-triggered p21 WAF1 protein expression, suggesting p21 WAF1 transactivation through ATF2. Using chromatin immunoprecipitation (ChIP), we identified a hitherto unknown ATF2-binding sequence in the p21 WAF1 promoter. p-ATF2 was found to interact with p-c-Jun, creating the AP-1 complex. Moreover, ATF2 knockdown led to c-Jun downregulation. This suggests ATF2-driven induction of c-Jun expression, thereby enhancing ATF2 transcriptional activity via c-Jun-ATF2 heterodimerization. Notably, downregulation of ATF2 caused a switch from cell cycle arrest to reinforced apoptosis, presumably via p21 WAF1 downregulation, confirming the importance of ATF2 in the establishment of cell cycle arrest. 1-Chloro-2,4-dinitrobenzene also led to ATF2-dependent G2/M arrest, suggesting that this is a general feature induced by oxidative stress. As ATF2 knockdown also increased apoptosis, we propose ATF2 as a target for combined oxidative stress-based anti-cancer therapies. Keywords: oxidative stress-induced DNA damage cell cycle arrest ATF2 knockdown increase in apoptosis sensitivities combined treatment p21 WAF

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions.

Funder: DFGFunder: Projekt DEALBACKGROUND: Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. METHODS: Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. RESULTS: Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45-/-/Csk-/- non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. CONCLUSIONS: Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. MAJOR FINDINGS: Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation. Video Abstract

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56 Lck regulates T-cell activation independently of Lck/CD45 interactions

Funder: DFGFunder: Projekt DEALAbstract: Background: Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods: Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results: Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions: Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings: Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation. FN7ZF7ULbDVsMsT6Sckwh1Video Abstrac

Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro

Weaning triggers an adaptation of the gut function including luminal lactate generation by lactobacilli, depending on gastrointestinal site. We hypothesized that both lactobacilli and lactate influence porcine intestinal epithelial cells. In vivo experiments showed that concentration of lactate was significantly higher in gastric, duodenal and jejunal chyme of suckling piglets compared to their weaned counterparts. In an in vitro study we investigated the impact of physiological lactate concentration as derived from the in vivo study on the porcine intestinal epithelial cells IPEC-1 and IPEC-J2. We detected direct adherence of lactobacilli on the apical epithelial surface and a modulated F-actin structure. Application of lactobacilli culture supernatant alone or lactate (25 mM) at low pH (pH 4) changed the F-actin structure in a similar manner. Treatment of IPEC cultures with lactate at near neutral pH resulted in a significantly reduced superoxide-generation in Antimycin A-challenged cells. This protective effect was nearly completely reversed by inhibition of cellular lactate uptake via monocarboxylate transporter. Lactate treatment enhanced NADH autofluorescence ratio (F-cytosol/F-nucleus) in non-challenged cells, indicating an increased availability of reduced nucleotides, but did not change the overall ATP content of the cells. Lactobacilli-derived physiological lactate concentration in intestine is relevant for alleviation of redox stress in intestinal epithelial cells.Peer reviewe

Signaling Signatures and Functional Properties of Anti-Human CD28 Superagonistic Antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4+ T cells but not in cynomolgus T lymphocytes. The sustained Ca++-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells

Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin(+)] cells, whereas no changes were observed in SW 13 [vimentin(−)] cells after microinjection of protease. Treatment of SW 13 [vimentin(−)] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin(−)] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis

Photophysics of Clomeleon by FLIM: Discriminating Excited State Reactions along Neuronal Development

In this work, fluorescence lifetime imaging microscopy in the time domain was used to study the fluorescence dynamics of ECFP and of the ratiometric chloride sensor Clomeleon along neuronal development. The multiexponential analysis of fluorophores combined with the study of the contributions of the individual lifetimes (decay-associated spectra) was used to discriminate the presence of energy transfer from other excited state reactions. A characteristic change of sign of the pre-exponential factors of lifetimes from positive to negative near the acceptor emission maxima was observed in presence of energy transfer. By fluorescence lifetime imaging microscopy, we could show that the individual conformations of CFP display differential quenching properties depending on their microenvironment. Suitability of Clomeleon as an optical indicator to obtain a direct readout of the intracellular chloride concentrations in living cells was verified by steady-state and time-resolved spectroscopy. The simultaneous study of the photophysical properties of Clomeleon, the calcium indicator Cameleon, and ECFP with neuronal development provided a kinetic model for the mechanism when competitive quenching effects as well as energy transfer occur in the same molecule. Simultaneous analysis of donor and acceptor kinetics was necessary to discriminate Försters resonance energy transfer along neuronal development due to the different cellular effects involved