Senescence-associated heterochromatin foci are dispensable for cellular senescence,occur in a cell type- and insult-dependent manner and follow expression of p16ink4a

Cellular senescence, an irreversible proliferation arrest evoked by stresses such as oncogene activation, telomere dysfunction, or diverse genotoxic insults, has been implicated in tumor suppression and aging. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), nuclear DNA domains stained densely by DAPI and enriched for histone modifications including lysine9-trimethylated histone H3. While cellular senescence occurs also in premalignant human lesions, it is unclear how universal is SAHF formation among various cell types, under diverse stresses, and whether SAHF occur in vivo. Here, we report that human primary fibroblasts (BJ and MRC-5) and primary keratinocytes undergoing replicative senescence, or premature senescence induced by oncogenic H-Ras, diverse chemotherapeutics and bacterial cytolethal distending toxin, show differential capacity to form SAHF. Whereas all tested cell types formed SAHF in response to activated H-Ras, only MRC-5, but not BJ fibroblasts or keratinocytes, formed SAHF under senescence induced by etoposide, doxorubicin, hydroxyurea, bacterial intoxication or telomere attrition. In addition, DAPI-defined SAHF were detected on paraffin sections of Ras-transformed cultured fibroblasts, but not human lesions at various stages of tumorigenesis. Overall, our results indicate that unlike the widely present DNA damage response marker γH2AX, SAHF is not a common feature of cellular senescence. Whereas SAHF formation is shared by diverse cultured cell types under oncogenic stress, SAHF are cell-type-restricted under genotoxin-induced and replicative senescence. Furthermore, while the DNA/DAPI-defined SAHF formation in cultured cells parallels enhanced expression of p16^ink4a, such ‘prototypic’ SAHF are not observed in tissues, including premalignant lesions, irrespective of enhanced p16^ink4a and other features of cellular senescence.     

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.     

S-glutathionylation in protein redox regulation

Protein S-glutathionylation, the reversible formation of mixed disulfides between glutathione and low-pKa cysteinyl residues, not only is a cellular response to mild oxidative/nitrosative stress, but also occurs under basal (physiological) conditions. S-glutathionylation has now emerged as a potential mechanism for dynamic, posttranslational regulation of a variety of regulatory, structural, and metabolic proteins. Moreover, substantial recent studies have implicated S-glutathionylation in the regulation of signaling and metabolic pathways in intact cellular systems. The growing list of S-glutathionylated proteins, in both animal and plant cells, attests to the occurrence of S-glutathionylation in cellular response pathways. The existence of antioxidant enzymes that specifically regulate S-glutathionylation would emphasize its importance in modulating protein function, suggesting that this protein modification too might have a role in cell signaling. The continued development of proteomic and analytical methods for disulfide analysis will help us better understand the full extent of the roles these modifications play in the regulation of cell function. In this review, we describe recent breakthroughs in our understanding of the potential role of protein S-glutathionylation in the redox regulation of signal transduction.     

Glutathione regulates telomerase activity in 3T3 fibroblasts

Changes in telomerase activity have been associated either with cancer, when activity is increased, or with cell cycle arrest when it is decreased. We report that glutathione, a physiological antioxidant present at high intracellular concentrations, regulates telomerase activity in cells in culture. Telomerase activity increases in 3T3 fibroblasts before exponential cell growth. The peak of telomerase activity takes place 24 h after plating and coincides with the maximum levels of glutathione in the cells. When cells are treated with buthionine sulfoximine, which decreases glutathione levels in cells, telomerase activity decreases by 60%, and cell growth is delayed. Glutathione depletion inhibits expression of E2F4 and Id2, which regulate the cell cycle. When glutathione levels are restored after incubation with glutathione monoethylester, telomerase activity and the cell cycle-related proteins return to control values. To discover the effect of glutathione redox status on the telomerase multicomplex structure, we incubated protein extracts from fibroblasts with different glutathione redox buffers. Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity. These results underscore the main role of glutathione in the control of telomerase activity and of the cell cycle.     

Fluorescein-labeled glutathione to study protein S-glutathionylation

Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins.     

Ras activation induces expression of raet1 family NK receptor ligands

NK cells play a crucial role in innate immunity against tumors. In many human tumors, Ras is chronically active, and tumor cells frequently express ligands for the activating NK cell receptor NKG2D. In this study, we report that Ras activation upregulates the expression of Raet1 protein family members Rae1α and Rae1β in mouse and ULBP1-3 in human cells. In addition, Ras also induced MHC class I chain-related protein expression in some human cell lines. Overexpression of the constitutively active H-RasV12 mutant was sufficient to induce NKG2D ligand expression. H-RasV12-induced NKG2D ligand upregulation depended on Raf, MAPK/MEK, and PI3K, but not ATM or ATR, two PI3K-like kinases previously shown to induce NKG2D ligand expression. Analysis of the 5' untranslated regions of Raet1 family members suggested the presence of features known to impair translation initiation. Overexpression of the rate-limiting translation initiation factor eIF4E induced Rae1 and ULBP1 expression in a Ras- and PI3K-dependent manner. Upregulation of NKG2D ligands by H-RasV12 increased sensitivity of cells to NK cell-mediated cytotoxicity. In summary, our data suggest that chronic Ras activation is linked to innate immune responses, which may contribute to immune surveillance of H-Ras transformed cells.     

A proteomic approach for dissecting H-Ras signaling networks in NIH/3T3 mouse embryonic fibroblast cells

To elucidate an understanding into H-Ras protein network, we have established various oncogene H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which are expressing G12V H-Ras, G12R H-Ras, and G12V/T35S H-Ras proteins under the tight control of expression by an antibiotic doxycycline. Here we provide a catalog of proteome profiles in total cell lysate derived from the oncogenic and partial loss of function H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF-MS analysis both commonly in oncogenic and partial loss of function H-Ras expression system. Thus, we tried to dissect H-Ras signaling pathway, especially a downstream effector molecule, Raf in NIH/3T3 cells using proteomics tools. In this study, we centralized upon the proteome profile changes as common targets for oncogenic H-Ras and a partial loss of function H-Ras in the H-Ras-expressing cells. Thirteen protein spots were selected as what the staining intensities on the gels for 2-DE images from both kinds of cells were consistently changed in their protein expression level. Differentially regulated expression was further confirmed for some subsets of candidates by semiquantitative RT-PCR and Western blot analysis using specific antibodies. Taken together, our results obtained and present here show that the comparative analysis of proteome from oncogenic and partial loss of function H-Ras-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly.     

GSH depletion, protein S-glutathionylation and mitochondrial transmembrane potential hyperpolarization are early events in initiation of cell death induced by a mixture of isothiazolinones in HL60 cells

We recently described that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) induces apoptosis at low concentrations (0.001-0.01%) and necrosis at higher concentrations (0.05-0.1%). In this study, we show that glutathione (GSH) depletion, reactive oxygen species generation, hyperpolarization of mitochondrial transmembrane potential (DeltaPsim) and formation of protein-GSH mixed disulphides (S-glutathionylation) are early molecular events that precede the induction of cell death by CMI/MI. When the cells exhibit common signs of apoptosis, they show activation of caspase-9, reduction of DeltaPsim and, more importantly, decreased protein S-glutathionylation. In contrast, necrosis is associated with severe mitochondrial damage and maximal protein S-glutathionylation. CMI/MI-induced cytotoxicity is also accompanied by decreased activity of GSH-related enzymes. Pre-incubation with L-buthionine-(S,R)-sulfoximine (BSO) clearly switches the mode of cell death from apoptosis to necrosis at 0.01% CMI/MI. Collectively, these results demonstrate that CMI/MI alters the redox status of HL60 cells, and the extent and kinetics of GSH depletion and S-glutathionylation appear to determine whether cells undergo apoptosis or necrosis. We hypothesize that S-glutathionylation of certain thiol groups accompanied by GSH depletion plays a critical role in the molecular mechanism of CMI/MI cytotoxicity.     

S-glutathionylation: indicator of cell stress and regulator of the unfolded protein response

The specific posttranslational modification of protein cysteine residues by the addition of the tripeptide glutathione is termed S-glutathionylation. This process is promoted by oxidative and nitrosative stress but also occurs in unstressed cells. Altered levels of S-glutathionylation in some proteins have been associated with numerous pathologies, many of which have been linked to redox stress in the endoplasmic reticulum (ER). Proper protein folding is dependent upon controlled redox conditions within the ER, and it seems that ER conditions can in turn affect rates of S-glutathionylation. This article seeks to bring together the ways through which these processes are interrelated and considers the implications of these interrelationships upon therapeutic approaches to disease.     

Active Ras-induced effects on skeletal myoblast differentiation and apoptosis are independent of constitutive PI3-kinase activity

23A2 myoblasts expressing GAP-resistant, constitutively active G12V:H-Ras (A2:G12V:H-Ras myoblasts) display a transformed morphology and do not undergo mitogen-deprivation-induced differentiation or the associated apoptosis. To determine the phenotype induced by F156L:H-Ras, a constitutively active mutant with enhanced nucleotide exchange activity rather than impaired GAP-stimulated GTPase activity, myoblast cell lines were established that stably express F156L:H-Ras at levels of H-Ras comparable to the A2:G12V:H-Ras myoblasts. These A2:F156L:H-Ras myoblast cell lines do not possess a transformed morphology, and while differentiation and apoptosis are impaired, these processes are not abrogated as in the A2:G12V:H-Ras myoblasts. Surprisingly, while expression of either G12V:H-Ras or F156L:H-Ras results in constitutive signaling through PI3-kinase, only cells expressing G12V:H-Ras additionally possess constitutive signaling through MAPK, and NFkappaB. Pharmacological abrogation of the Ras-induced constitutive PI3-kinase signal, however, is not responsible for the impaired differentiation or apoptosis in either A2:G12V:H-Ras myoblasts or A2:F156L:H-Ras myoblasts. Thus, our data suggest that a pathway distinct from those that signals through MAPK, NFkappaB or PI3-kinase is responsible for the impaired differentiation and apoptosis in 23A2 skeletal myoblasts expressing constitutively active Ras.     

Regulation of ploidy and senescence by the AMPK‐related kinase NUAK1

Senescence is an irreversible cell‐cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss‐of‐function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK‐related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant‐negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.     

H-Ras signals to cytoskeletal machinery in induction of integrin-mediated adhesion of T cells

The adhesive function of integrins is regulated through cytoplasmic signaling. The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines. Adhesion of T cells induced by chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta was inhibited by pertussis toxin, wortmannin, and cytochalasin B, suggesting that both G protein-sensitive phosphatidylinositol (PI) 3-kinase activation and cytoskeletal assemblies are involved. The chemokine-induced T cell adhesion could be mimicked by expression of small G proteins, fully activated H-RasV12, or H-RasV12Y40C mutant, which selectively binds to PI 3-kinase, in T cells, inducing activated form of LFA-1alpha and LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced F-actin polymerization which colocalized with profilin in T cells. Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1, which depended on endogenous MIP-1alpha and MIP-1beta through activation of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI 3-kinase activation, also induced active configuration of LFA-1 and LFA-1-mediated adhesion of ATL cells, whereas expression of a dominant-negative H-Ras mutant failed to do. Profilin-dependent spontaneous polymerization of F-actin in ATL cells was reduced by PI 3-kinase inhibitors. In this paper we propose that H-Ras-mediated activation of PI 3-kinase can be involved in induction of LFA-1-dependent adhesion of T cells, which is relevant to chemokine-mediated signaling, and that profilin may form an important link between chemokine- and/or H-Ras-mediated signals and F-actin polymerization, which results in triggering of LFA-1 on T cells or leukemic T cells.     

Identification of 30 protein species involved in replicative senescence and stress-induced premature senescence

Exposure of human proliferative cells to subcytotoxic stress triggers stress-induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress-induced premature senescence indicates that, at the level of protein expression, stress-induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress-induced premature senescence. These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways.     

Gene dosage-dependent effects of bcl-2 expression on cellular survival and redox status

The human oncogene bcl-2 exerts protective functions in numerous models of apoptotic cell death and increased oxidative stress. We investigated the effects of inducible bcl-2 overexpression on cellular survival and redox status in dopaminergic rat pheochromocytoma PC 12 cells. Induction of high-level expression of bcl-2 in PC 12 cells resulted in generation of oxidative stress and cessation of growth by cell cycle arrest. Cell cycle arrest in bcl-2-overexpressing PC 12 cells was prevented by an inhibitor of extracellular signal-related kinase (ERK 1/2) activation. Protective effects of bcl-2 expression against L-DOPA neurotoxicity decreased with increasing amounts of bcl-2. Furthermore, high-level bcl-2 overexpression sensitized cells towards oxidative stress and glutathione depletion. Our data suggest that bcl-2 expression is beneficial only in a limited gene dosage range and that high-level expression of bcl-2 exerts potential deleterious effects.     

Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX⁺ foci), enhanced senescence-associated β-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the 'distended' morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.     

Evidence that sprouty 2 is necessary for sarcoma formation by H-Ras oncogene-transformed human fibroblasts

Sprouty 2 (Spry2) acts as an inhibitor of receptor tyrosine kinase signaling in various cellular contexts. Interestingly, Spry2 also prevents the c-Cbl-induced degradation of epidermal growth factor receptor (EGFR). We compared human fibroblasts malignantly transformed by overexpression of H-Ras(V12) oncogene to their nontransformed parental cells and found that the malignant cells express a high level of Spry2. These cells also exhibited an increase in the level of EGFR compared with their precursor cells. We found that intact EGFR was required if H-Ras-transformed cells were to grow in the absence of exogenous growth factors or form large colonies in agarose. When we decreased expression of Spry2, using a Spry2-specific shRNA, the H-Ras(V12)-transformed fibroblasts could no longer form large colonies in agarose, grow in reduced levels of serum, or form tumors in athymic mice. The level of active H-Ras in these cells remained unaltered. A similar, but less pronounced, effect in tumor formation was observed when Spry2 was down-regulated in human patient-derived fibrosarcoma cell lines. In H-Ras-transformed cells Spry2 sustained the level and the downstream signaling activity of EGFR. In the parental, non-H-Ras-transformed fibroblasts, expression of Spry2 resulted in the inhibition of H-Ras and ERK activation, suggesting that the positive effect of Spry2 in tumor formation is specific to H-Ras transformation. Co-immunoprecipitation studies with H-Ras-transformed cells revealed that Spry2 and H-Ras interact and that H-Ras interacts with Spry2-binding partners, c-Cbl and CIN85, in a Spry2-dependent manner. These data show that Spry2 plays a critical role in the ability of H-Ras-transformed cells to form tumors in athymic mice.