Feedback between p21 and reactive oxygen production is necessary for cell senescence

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Selenium chemoprevention by apoptosis has been well studied, but it is not clear whether selenium can activate early barriers of tumorigenesis, namely senescence and DNA damage response. To test this hypothesis, we treated normal and cancerous cells with a gradient concentration of sodium selenite, methylseleninic acid and methylselenocysteine for 48 h, followed by a recovery of 1-7 days. Here we show that selenium compounds at doses of </=LD(50) can induce cellular senescence, as evidenced by the expression of senescence-associated beta-galactosidase and 5-bromo-2-deoxyuridine incorporation, in normal but not cancerous cells. In response to clastogens, the ataxia telangiectasia mutated (ATM) protein is rapidly activated, which in turn initiates a cascade of DNA damage response. We found that the ATM pathway is activated by the selenium compounds, and the kinase activity is required for the selenium-induced senescence response. Pretreatment of the MRC-5 non-cancerous cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl suppresses the selenium-induced ATM activation and senescence. Taken together, the results suggest a novel role of selenium in the activation of early tumorigenesis barriers specific in non-cancerous cells, whereby selenium induces an ATM-dependent senescence response that depends on reactive oxygen species.

Cellular signaling pathways integrate multiple intra- and extracellular signals and thereby influence cell fate. Outcome of oncogene-targeted pharmacotherapies (cell cycle inhibition, apoptosis, senescence, autophagy) depends on variable factors (e.g. compound, dose, duration, cellular genetic background etc.). Oncogene-induced senescence is recognized as an important tumor-suppressive mechanism, bypassing of which may contribute to malignant transformation of the cells. On the other hand, senescence induction, in addition to apoptosis stimulation and proliferation suppression, is responsible for the cytostatic activity of many commonly used anticancer drugs. Therefore, senescence escape must be considered as an important drug-resistance factor. In our work we characterize,,DNA Damage-independent” senescence induced by tyrosine kinase inhibitors (TKI) and growth factor deprivation of non-small cell lung cancer (NSCLC) cell lines and other EGFR-dependent cells. Treatment of different cell lines with various concentrations of EGFR-TKIs was followed by the assessment of cell fate (i.e. apoptosis, cell cycle distribution, senescence and clonogenic survival) as well as intracellular signaling. We could show that treatment of EGFR-dependent cells with specific EGF receptor tyrosine kinase inhibitors (EGFR-TKI) can lead to generation of reactive oxygen species (ROS), activation of a DNA damage response (DDR) program and cellular senescence. Surprisingly, under certain experimental conditions DDR activation was found to be independent from detectable DNA damage. Key players of DDR signaling were shown to be essential for senescence induction. Molecular dissection of crucial signaling nodes and molecular switches involved in modulation of cell fate in oncogene dependent cells treated with specific EGFR-inhibitors is currently being performed. Furthermore, our work is focussed on the identification and characterization of cellular factors activating DNA damage response (DDR) pathways in the absence of DNA damage under certain experimental conditions. Understanding the interplay between theses decision making pathways either leading to proliferative arrest, cellular senescence or apoptosis will help improve current pharmacotherapies and develop new therapeutic approaches. Note: This abstract was not presented at the meeting. Citation Format: Boyka Markova, Frank Breitenbuecher, Martin Schuler. Alternative tumor suppressor mechanisms in lung carcinomas - modulation of senescence by growth factor-induced signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1033. doi:10.1158/1538-7445.AM2015-1033

Targeted protein degradation by the ubiquitin-proteasome system (UPS) has an essential role in carcinogenesis and cancer cell survival. Thus, the search for proteasome inhibitors is of great interest as a potential anticancer therapy.1 Recent studies by the Ferbeyre group suggest that certain UPS-targeted proteins play a role not just in carcinogenesis, but also in cellular senescence. The limited replicative potential of cells or tissues, called senescence, serves as a ‘mitotic clock’ for normal cell proliferation. During cancer development, it is often bypassed through inactivation of DNA damage checkpoints, and thus senescence is often considered a tumor suppressive mechanism.2 Senescence is induced by many causes such as telomere shortening, DNA damage, chromatin perturbations, oncogenes and reactive oxygen species (ROS).2 How these diverse triggers lead to cell cycle arrest and what common features define the senescent state is a topic under intensive investigation. The ubiquitin proteasome system (UPS) is a major protein degradation network in eukaryotic cells. One of the signaling networks that regulate the UPS is the Ras family of kinases. Ras is mutated in various cancer types and Ras signaling regulates cellular differentiation, survival, and proliferation. Extracellular signal regulated kinases (ERK) or MAP kinases (MAPK) are also commonly disrupted in cancer, and ubiquitinylation of Ras is essential to activate ERK/MAPK.3 Previously, Deschenes-Simard and colleagues uncovered a common feature of protein degradation in both Ras-induced and telomere dysfunction-induced senescence that they termed senescence-associated protein degradation (SAPD).4 In particular, they found that inhibition of ERK signaling attenuated Ras-induced senescence and permitted cells to adopt a transformed state.4 In order to model the specific proteome changes characteristic of SAPD, in the present study the authors compared the proteome of cells undergoing Ras-induced senescence in the absence or presence of the proteosome inhibitor MG132.5 The authors showed that Ras-induced senescence led to increased turnover of proteins whose functions are implicated in diverse cellular processes such as mitochondrion dysfunction (ATP5B, STAT3, TOM complex), proteotoxic stress (HSP27, HSP60, HSP90AB1), the DNA damage response, (CCDC6, SOD1, TOP2, Rap1), nucleolar and ribosome biogenesis dysfunction (NOLC1, NOP56/58, DDX1, NOL6, NOC2L, NCL, RPLP1, RSL1D1, NPM1), cell cycle arrest (YAP1, MCM2, ORCA, MYC, JUN, KAP1, TBX2), and impaired mRNA metabolism and translation (YBX1, SRRM1, SRRM2).5 Furthermore, some of these proteins, including RAP1, YAP1, MYC and STAT3, are also localized to PML nuclear bodies (PML-NBs) and are targets for sumoylation and phosphorylation followed by ubiquitylation, leading the authors to suggest that PML-NBs could be a site for nuclear SAPD. Figure 1. A schematic of the cellular processes affected by targeted protein degradation during Ras-induced senescence. Hyperactive ERK/MAPK (in red) leads to targeting of proteins to proteasomes for degradation (in blue). The authors suggest that PML nuclear bodies ... Other recently published findings support a role for the UPS as a trigger for the changes that accompany cellular aging; for example, proteosomal dysfunction can lead to mitochondrial ROS production, which is a common feature of the senescent state,6 and Mdm2-induced proteolysis of phosphoglycerate mutase is a hallmark of DNA damage-induced senescence.7 The precise mechanisms that link Ras, ERK and SAPD will be interesting to determine. For example, what are the key E3 ubiquitin ligases that trigger SAPD? The authors suggest CUL4A-DDB1 may be involved in Ras-induced SAPD5; if so, would this E3 ligase also be instrumental in DNA damage-induced or telomere dysfunction-induced senescence? An understanding of how these cellular networks become deranged will no doubt lead to further mechanistic insights not only in cellular senescence, but will hopefully also aid our understanding of aging and cancer.

Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNFα) and interferon gamma (IFNγ) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic β-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNFα and IFNγ induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFNγ and TNFα, short exposure to IFNγ alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFNγ-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGFβ) secretion and consequent autocrine/paracrine activation of the TGFβ/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFNγ contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFNγ/TNFα by DDR and senescence correlated with the lack of TGFβ and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGFβ/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFNγ/TNFα-evoked genotoxicity and cellular senescence.

A non-telomeric role of POT1a in regulating HSC metabolism via TIN2 localization

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. It is triggered as a stereotypic response to unrepaired nuclear DNA damage or to uncapped telomeres. In addition to a direct role of nuclear DNA double-strand breaks as inducer of a DNA damage response, two more subtle types of DNA damage induced by physiological levels of reactive oxygen species (ROS) can have a significant impact on cellular senescence: Firstly, it has been established that telomere shortening, which is the major contributor to telomere uncapping, is stress dependent and largely caused by a telomere-specific DNA single-strand break repair inefficiency. Secondly, mitochondrial DNA (mtDNA) damage is closely interrelated with mitochondrial ROS production, and this might also play a causal role for cellular senescence. Improvement of mitochondrial function results in less telomeric damage and slower telomere shortening, while telomere-dependent growth arrest is associated with increased mitochondrial dysfunction. Moreover, telomerase, the enzyme complex that is known to re-elongate shortened telomeres, also appears to have functions independent of telomeres that protect against oxidative stress. Together, these data suggest a self-amplifying cycle between mitochondrial and telomeric DNA damage during cellular senescence.

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium

Oxidative and nitrosative stress play an important role in the complications of diabetes mellitus. Free radicals are produced when there is an electron leak in the mito-chondria and a change in the mitochondrial membrane potential. The present study was undertaken to investigate the role of Semecarpus anacardium in protecting the mito-chondria by modulating the production of reactive oxygen species and reactive nitrogen species in diabetic rats. Diabetes was induced using streptozotocin at a dose of 50 mg/kg body weight and, starting 3 days after the induction, Semecarpus anacardium nut milk extract was administered for 21 days. The same duration of study was used for control, diabetes-induced and drug control groups, together with a group treated with metformin. After the experimental period, the animals were sacrificed and the levels of superoxide, hydrogen peroxide, nitrate and nitrite were estimated. Changes in mitochondrial membrane potential, intracellular reactive oxygen species and intracellular calcium were also determined. Confocal laser microscopic images were taken for mitochondria isolated from the liver and kidneys. The results of the study revealed that Semecarpus anacardium was able to decrease the production of reactive oxygen and nitrogen species, and reverse the changes in mitochondrial membrane potential and the influx of calcium into the mitochondria. The mitochondrial protective effect may be mediated by scavenging of free radicals and complexing of metal ions by virtue of the antioxidative effect of Semecarpus anacardium.

Molecular pathophysiology of Diamond-Blackfan anemia (DBA) involves disrupted erythroid-lineage proliferation, differentiation and apoptosis; with the activation of p53 considered as a key component. Recently, oxidative stress was proposed to play an important role in DBA pathophysiology as well. CRISPR/Cas9-created Rpl5- and Rps19-deficient murine erythroleukemia (MEL) cells and DBA patients’ samples were used to evaluate proinflammatory cytokines, oxidative stress, DNA damage and DNA damage response. We demonstrated that the antioxidant defense capacity of Rp-mutant cells is insufficient to meet the greater reactive oxygen species (ROS) production which leads to oxidative DNA damage, cellular senescence and activation of DNA damage response signaling in the developing erythroblasts and altered characteristics of mature erythrocytes. We also showed that the disturbed balance between ROS formation and antioxidant defense is accompanied by the upregulation of proinflammatory cytokines. Finally, the alterations detected in the membrane of DBA erythrocytes may cause their enhanced recognition and destruction by reticuloendothelial macrophages, especially during infections. We propose that the extent of oxidative stress and the ability to activate antioxidant defense systems may contribute to high heterogeneity of clinical symptoms and response to therapy observed in DBA patients.

Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species

Ischemia-reperfusion (I-R) injury represents a predominant etiology of acute kidney injury (AKI), for which effective treatments remain unavailable. In contrast, hibernating mammals exhibit notable resistance to cell death induced by I-R injury. However, the impact of I-R injury on cellular senescence—an important factor in AKI—has not been extensively studied in these species. Comparative biology may offer novel therapeutic insights. Renal proximal tubular epithelial cells (RPTECs) from the native hibernator Syrian hamster or mouse RPTECs were subjected to anoxia-reoxygenation. Proteins involved in DNA damage response (DDR) and cellular senescence were assessed using western blotting, reactive oxygen species (ROS) levels and cell death were quantified colorimetrically, and IL-6 with ELISA. Anoxia-reoxygenation induced oxidative stress in both mouse and hamster RPTECs; however, cell death was observed exclusively in mouse cells. While anoxia-reoxygenation elicited a DDR and subsequent senescence in mouse RPTECs, such responses were not detected in hamster RPTECs. Thus, RPTECs from the Syrian hamster exhibited increased ROS production upon reoxygenation but did not show DDR or cellular senescence. Further research is required to elucidate the specific protective molecular mechanisms in hibernators, which could potentially lead to the development of novel therapeutic approaches for I-R injury in non-hibernating species, including humans.

Reactive oxygen species (ROS) and signaling events are involved in the pathogenesis of endothelial dysfunction and represent a major contribution to vascular regulation. Molecular signaling is highly dependent on ROS. But depending on the amount of ROS production it might have toxic or protective effects. Despite a large number of negative outcomes in large clinical trials (e.g., HOPE, HOPE-TOO), antioxidant molecules and agents are important players to influence the critical balance between production and elimination of reactive oxygen and nitrogen species. However, chronic systemic antioxidant therapy lacks clinical efficacy, probably by interfering with important physiological redox signaling pathways. Therefore, it may be a much more promising attempt to induce intrinsic antioxidant pathways in order to increase the antioxidants not systemically but at the place of oxidative stress and complications. Among others, heme oxygenase (HO) has been shown to be important for attenuating the overall production of ROS in a broad range of disease states through its ability to degrade heme and to produce carbon monoxide and biliverdin/bilirubin. With the present review we would like to highlight the important antioxidant role of the HO system and especially discuss the contribution of the biliverdin, bilirubin, and biliverdin reductase (BVR) to these beneficial effects. The BVR was reported to confer an antioxidant redox amplification cycle by which low, physiological bilirubin concentrations confer potent antioxidant protection via recycling of biliverdin from oxidized bilirubin by the BVR, linking this sink for oxidants to the NADPH pool. To date the existence and role of this antioxidant redox cycle is still under debate and we present and discuss the pros and cons as well as our own findings on this topic.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

Albumin-bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells