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Stabilization and activation of the p53 tumor suppressor are triggered in response to various cellular stresses, including DNA damaging agents and elevated Reactive Oxygen Species (ROS) like H2O2. When cells are exposed to exogenously added H2O2, ATR/CHK1 and ATM/CHK2 dependent DNA damage signaling is switched on, suggesting that H2O2 induces both single and double strand breaks. These collective observations have resulted in the widely accepted model that oxidizing conditions lead to DNA damage that subsequently mediates a p53-dependent response like cell cycle arrest and apoptosis. However, H2O2 also induces signaling through stress-activated kinases (SAPK, e.g., JNK and p38 MAPK) that can activate p53. Here we dissect to what extent these pathways contribute to functional activation of p53 in response to oxidizing conditions. Collectively, our data suggest that p53 can be activated both by SAPK signaling and the DDR independently of each other, and which of these pathways is activated depends on the type of oxidant used. This implies that it could in principle be possible to modulate oxidative signaling to stimulate p53 without inducing collateral DNA damage, thereby limiting mutation accumulation in both healthy and tumor tissues.

FOXO transcription factors are negatively regulated by the PI3K-PKB/AKT signaling pathway and have been mainly considered to be tumor suppressors due to their inhibitory effect on cancer cell growth and survival. However, FOXOs can also support tumor development and progression by maintaining cellular homeostasis, facilitating metastasis and inducing therapy resistance. In agreement with these opposing views on the role of FOXOs in cancer, studies using FOXO levels or activity as prognostic markers for cancer patient disease progression and survival came to contradicting results. While it is clear that FOXOs are involved in various aspects of cancer, it is debatable whether FOXOs function as tumor suppressors or supporters, or may be both depending on the context. In this review, we describe the role of FOXOs in signaling pathways and processes relevant to cancer and evaluate recent advances in understanding the role of FOXOs in cancer. Based on recent insights it becomes clear that FOXOs may not be classical tumor suppressors and that targeting FOXO activity might hold promise in cancer therapy.

Cytokines interact closely with each other and play a crucial role in the progression of sepsis. We focused on the associations of a cytokine network with IL-35 in sepsis. First, the retrospective study included 42 patients with sepsis and 23 healthy controls. Blood samples were collected from patients on days 1, 2, 4. Levels of IL-35, IL-1β, IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-γ were measured. They all increased to various extend on days 1, 2, 4, and strongly associated with markers of disease severity. Network analysis revealed a network formed by IL-35, with IL-6, IL-10, IL-17A, TNF-α and IFN-γ throughout the acute phase of sepsis(days 1, 2 and4). Then, the CLP-induced septic rats were used. The recombinant human IL-35(rIL-35) upregulated the levels of IL-10, but downregulated IL-4, IL-6, IL-17A, TNF-α and IFN-γ, while it had no significant effect on IL-1β, and upregulated the percentages of CD4

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Stalling or slowing of the replication fork as a result of replication stress leads to formation of aberrant single-stranded DNA (ssDNA) stretches and potentially DNA double-stranded-breaks (DSBs). Accumulation of ssDNA activates the ATR-dependent DNA replication stress checkpoint response that slows progression from S/G2- to M-phase to protect genomic integrity (1). However, whether mild replication stress restricts proliferation remains controversial (2–6). Here we identify a novel cell cycle exit mechanism, that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the CDH1 bound Anaphase Promoting Complex / Cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead Box O (FOXOs) mediated repression of its transcriptional regulator E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible, and thereby play an important role in maintaining genomic stability.

Summary Symmetry breaking is an essential step in cell differentiation and early embryonic development. However, the molecular cues that trigger symmetry breaking remain largely unknown. Here, we show that mitochondrial H2O2 acts as a symmetry-breaking cue in the C. elegans zygote. We find that symmetry breaking is marked by a local H2O2 increase and coincides with a relocation of mitochondria to the cell cortex. Lowering endogenous H2O2 levels delays the onset of symmetry breaking, while artificially targeting mitochondria to the cellular cortex using a light-induced heterodimerization technique is sufficient to initiate symmetry breaking in a H2O2-dependent manner. In wild-type development, both sperm and maternal mitochondria contribute to symmetry breaking. Our findings reveal that mitochondrial H2O2-signaling promotes the onset of polarization, a fundamental process in development and cell differentiation, and this is achieved by both mitochondrial redistribution and differential H2O2-production.

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

Members of the forkhead box O (FOXO) family of transcription factors have been postulated to be tumor suppressors because of their established roles in cell-cycle arrest, apoptosis, DNA-damage repair and scavenging of reactive oxygen species. Recently, several animal model studies have shown that the FOXO proteins are indeed tumor suppressors. Furthermore, FOXO proteins have recently been implicated in the negative regulation of signaling by the hypoxia-inducible factor 1 during vascular development, raising the possibility that the FOXO proteins suppress not only tumor formation but also tumor angiogenesis and, possibly, metastasis. Here, we discuss recent advances in the understanding of the roles of FOXO family members in tumor suppression.

Bax and Bak comprise the mitochondrial gateway for apoptosis induced by diverse stimuli. Loss of both bax and bak is necessary to block cell death induced by such stimuli, indicating a great degree of functional overlap between Bax and Bak. Apoptosis is the major intrinsic pathway that limits the oncogenic potential of Myc. Using a switchable mouse model of Myc-induced apoptosis in pancreatic beta cells, we have shown that Myc induces apoptosis in vivo exclusively through Bax but not Bak. Furthermore, blockade of Myc-induced apoptosis by the inactivation of Bax, but not Bak, eliminates all restraints to the oncogenic potential of Myc, allowing the rapid and synchronous progression of invasive, angiogenic tumors.

Objectives: To obtain an estimate of working times of Class II copy-milled ceramic inlays as an indication of their efficiency, and to analyse factors of influence. Methods: In a controlled clinical trial, 173 MO/DO or MOD ceramic inlays of the Celay system were constructed in 101 patients. Treatment was carried out by seven dentists. The inlays were replacements of existing amalgam restorations in both molar and premolar teeth. Net-times needed for the treatment and for the laboratory stage were registered. Variables of influence on the working time were assessed by using ln-working times in ANOVA. Differences of working times were expressed as the relative difference between the upper and lower value of each variable (Δ). Results: The mean clinical treatment time was 67(±22) min. The laboratory stage required 60(±17) min working time. ‘Cavity modification technique’ (composite basing/conventional, Δ =17%), ‘clinical operator’ (seven dentists, Δ =57%), and ‘size of the restoration’ (large/medium, Δ =16%) significantly influenced the clinical treatment time (p Although the dentists were familiar with inlay construction, they showed a 25% decrease in working time towards the end of the study. Conclusions: Class II copy-milled ceramic inlays in this time-and-motion study required about 125 min of working time. Working time increased by applying composite basing and making large restorations. The dentist influenced working times, while efficiency increased over time. Dentists needed more laboratory time to produce an inlay than the dental technician.

Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme involved in the biosynthesis of ether phospholipids. To localize the enzyme in human peroxisomal disorders, indirect immunofluorescence and immunoblot analysis was performed. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, alkyl-DHAP synthase protein levels on immunoblots were strongly decreased and residual immunofluorescence was diffusely localized throughout the cytoplasm. In a particular neonatal adrenoleukodystrophy cell line, characterized by the absence of a functional peroxisomal targeting signal 1 receptor, the precursor form of the enzyme was detected in Western blots at levels comparable to that of the mature enzyme in control fibroblasts. Similarly, fibroblasts from patients with a single deficiency in the activity of either alkyl-DHAP synthase or DHAP-acyltransferase showed normal levels of the mature alkyl-DHAP synthase protein on immunoblots. Immunofluorescence experiments revealed a peroxisomal localization of both the precursor and the mature form of the enzyme. Collectively, these results visualize the peroxisomal localization of alkyl-DHAP synthase, indicate that the enzyme is unstable outside its target organelle and explain that normal enzyme protein levels found in some peroxisomal disorders result from protection against cytoplasmic degradation through import into peroxisomes. Additionally, alkyl-DHAP synthase could be detected in rat mesangial cells and murine NIH-3R3 fibroblasts by immunofluorescence as well as immunoblot analysis. Immunoelectron microscopy showed that the enzyme is predominantly located on the lumenal side of the peroxisomal membrane in rat and guinea pig liver.

The forkheadbox O (FOXO) family of transcription factors regulates a variety of cellular programs, including cell cycle arrest, reactive oxygen species (ROS) scavenging, and apoptosis, and are of key importance in the decision over cell fate. In animal model systems it has been shown that FOXO is involved in the regulation of long lifespan. FOXO activity is tightly controlled by the insulin signaling pathway and by a multitude of ROS-induced posttranslational modifications. Over the past years my lab has discovered that cysteines in FOXO transcription factors become oxidized in response to redox signaling and that this leads to the formation of highly specific intermolecular disulfide formation with a number of regulators of FOXO activity. We have shown that ROS-induced Lysine acetylation on FOXO depends on the formation of an intermolecular disulfide with the p300 or CBP acetyltransferases. Furthermore, nuclear shuttling of FOXO is triggered by a shift in the cellular redox state towards more oxidizing conditions and this depends on intermolecular disulfide formation between FOXO and nuclear import receptors. By comparing cysteines present in human FOXO3 and FOXO4 isoforms by a quantitative proteomics approach we have identified paralog-specific redox signaling. We speculate that intermolecular disulfide formation could be a more common phenomenon in redox signaling and that it could serve to stabilize protein-protein interactions that otherwise would be of low affinity. By this means specific signaling cascades can be reversibly activated for as long as the cellular redox state remains more oxidizing.