p66Shc Protein Research Articles

A novel role of Shc adaptor proteins in steroid hormone-regulated cancers

Tyrosine phosphorylation plays a critical role in growth regulation, and its aberrant regulation can be involved in carcinogenesis. The association of Shc (Src homolog and collagen homolog) adaptor protein family members in tyrosine phosphorylation signaling pathway is well recognized. Shc adaptor proteins transmit activated tyrosine phosphorylation signaling that suggest their plausible role in growth regulation including carcinogenesis and metastasis. In parallel, by sharing a similar mechanism of carcinogenesis, the steroids are involved in the early stage of carcinogenesis as well as the regulation of cancer progression and metastatic processes. Recent evidence indicates a cross-talk between tyrosine phosphorylation signaling and steroid hormone action in epithelial cells, including prostate and breast cancer cells. Therefore, the members of Shc proteins may function as mediators between tyrosine phosphorylation and steroid signaling in steroid-regulated cell proliferation and carcinogenesis. In this communication, we discuss the novel roles of Shc proteins, specifically p52(Shc) and p66(Shc), in steroid hormone-regulated cancers and a novel molecular mechanism by which redox signaling induced by p66(Shc) mediates steroid action via a non-genomic pathway. The p66(Shc) protein may serve as an effective biomarker for predicting cancer prognosis as well as a useful target for treatment.

Mitochondrial redox signaling by p66Shc is involved in regulating androgenic growth stimulation of human prostate cancer cells.

p66Shc is shown to negatively regulate the life span in mice through reactive oxygen species (ROS) production. Recent reports, however, revealed that p66Shc protein level is significantly elevated in several human cancer tissues and growth-stimulated carcinoma cells, suggesting a mitogenic and carcinogenic role for p66Shc. In this communication, we demonstrate for the first time that p66Shc mediates androgenic growth signals in androgen-sensitive human prostate cancer cells through mitochondrial ROS production. Growth stimulation of prostate cancer cells with 5alpha-dihydrotestosterone (DHT) is accompanied by increased p66Shc level and ROS production, which is abolished by antioxidant treatments. However, antioxidant treatments do not affect the transcriptional activity of androgen receptor (AR) as observed by its inability to block DHT-induced prostate-specific antigen expression, an AR-dependent correlate of prostate cancer progression. Elevated expression of p66Shc by cDNA transfection increases the basal cell proliferation and, thus, reduces additional DHT-induced cell proliferation. Furthermore, DHT increases the translocation of p66Shc into mitochondria and its interaction with cytochrome c. Conversely, both redox-negative p66Shc mutant (W134F), which is deficient in cytochrome c interaction, and p66Shc small interfering RNA decrease DHT-induced cell proliferation. These results collectively reveal a novel role for p66Shc-ROS pathway in androgen-induced prostate cancer cell proliferation and, thus, may play a role in early prostate carcinogenesis.

Apoptosis induced by oxidized lipids is associated with up-regulation of p66Shc in intestinal Caco-2 cells: protective effects of phenolic compounds

In this study, we investigated the alterations of the redox balance induced by the lipid fraction of oxLDL in Caco-2 intestinal cells, and the effects of tyrosol and protocatechuic acid, two dietary phenolic compounds. We found that oxidized lipids extracted from oxLDL (LipE) induced oxidative stress by determining, 6 h after treatment, ROS overproduction (about a 100% and a 43% increase of O 2 − and H 2O 2 production, respectively, P<.05: LipE vs. control) and, 12 h after treatment, GSH depletion (about a 26% decrease, P<.05: LipE vs. control), and by impairing the activities of superoxide dismutase, catalase and glutathione peroxidase. In response to the induced oxidative stress, we observed significant overexpression of glutathione peroxidase (6 h after treatment: P<.05), glutathione reductase and γ-glutamylcysteine synthetase (12 h after treatment: P<.05). Notably, when GSH depletion occurred, p66Shc protein expression increased by about 300% with respect to control ( P<.001; LipE vs. control). These effects were fully counteracted by dietary phenolics which inhibited ROS overproduction and GSH consumption, rendered the reactive transcription of glutathione-associated enzymes unnecessary and blocked the intracellular signals leading to the overexpression and rearrangement of p66Shc signalling molecule. Altogether, these results suggest that the impairment of the antioxidant system hijacks intestinal cells towards an apoptotic-prone phenotype via the activation of p66Shc molecule. They also propose a reappraisal of dietary polyphenols as intestinal protecting agents, indicating the antiapoptotic effect as a further mechanism of action of these antioxidant compounds.

Antagonism of p66shc by melanoma inhibitory activity

The p66shc protein governs oxidant stress and mammalian lifespan. Here, we identify melanoma inhibitory activity (MIA), a protein secreted by melanoma cells, as a novel binding partner and antagonist of p66shc. The N-terminal collagen homology-2 (CH2) domain of p66shc binds to the Src Homology-3 (SH3)-like domain of MIA in vitro. In cells, ectopically expressed MIA and p66shc colocalize and co-precipitate. MIA also co-precipitates with the CH2 domain of p66shc in vivo. MIA expression in vivo suppresses p66shc-stimulated increase in endogenous hydrogen peroxide (H(2)O(2)), and inhibits basal and H(2)O(2)-induced phosphorylation of p66shc on serine 36 and H(2)O(2)-induced death. In human melanoma cells expressing MIA, endogenous MIA and p66shc co-precipitate. Downregulation of MIA in melanoma cells increases basal and ultraviolet radiation (UVR)-induced phosphorylation of p66shc on serine 36, augments endogenous H(2)O(2) levels, and increases their susceptibility to UVR-induced death. These findings show that MIA binds to p66shc, and suggest that this interaction antagonizes phosphorylation and function of p66shc.

High levels of p66 shc and intracellular ROS in permanently arrested early embryos

A high incidence of permanent embryo arrest occurs during the first week of in vitro development. We hypothesize that this developmental arrest event is regulated by the stress adaptor protein p66 shc, a genetic determinant of life span in mammals, which regulates ROS metabolism, apoptosis, and cellular senescence. The aim of this study was to assess the relationship between intracellular oxidative stress levels with the incidence of embryo arrest and the expression of senescent-associated genes in embryos produced under different oxygen tensions. Embryos cultured under 20% oxygen conditions showed approximately 10-fold increase in oxidative stress, 2-fold increase in the percentage of 2- to 4-cell arrest, and significantly lower developmental capabilities compared to embryos cultured under a 5% oxygen environment. Quantification by real-time PCR and by semiquantitative immunofluorescence showed significantly higher p66 shc mRNA and protein levels, respectively, in embryos cultured in 20% versus those cultured in 5% oxygen atmosphere. No significant changes in p53 mRNA and protein levels were detected among embryos derived from both oxygen tensions. Taken together, these results demonstrate that p66 shc, but not p53, is significantly more abundant in an embryo population that exhibits higher frequencies of embryo arrest and quantities of intracellular ROS. These results further substantiate that p66 shc and oxidative stress are associated with a p53-independent embryonic arrest event for in vitro-produced embryos.

P66 Shc and tyrosine-phosphorylated Shc in primary breast tumors identify patients likely to relapse despite tamoxifen therapy

IntroductionShc adapter proteins are secondary messenger proteins involved in various cellular pathways, including those mediating receptor tyrosine kinase signaling and apoptosis in response to stress. We have previously reported that high levels of tyrosine-phosphorylated Shc (PY-Shc) and low levels of its inhibitory p66 Shc isoform are strongly prognostic for identifying both early node-negative and more advanced, node-positive, primary breast cancers with high risk for recurrence. Because aberrant activation of tyrosine kinases upstream of Shc signaling proteins has been implicated in resistance to tamoxifen – the most widely prescribed drug for treatment of estrogen receptor-positive breast cancer – we hypothesized that Shc isoforms may identify patients at increased risk of relapsing despite tamoxifen treatment.MethodsImmunohistochemical analyses of PY-Shc and p66 Shc were performed on archival primary breast cancer tumors from a population-based cohort (60 patients, 9 relapses) and, for validation, an independent external cohort (31 patients, 13 relapses) in which all patients received tamoxifen as a sole systemic adjuvant prior to relapse.ResultsBy univariate and multivariate analyses, the Shc proteins were very strong and independent predictors of treatment failure in both the population-based cohort (interquartile hazard ratio = 8.3, 95% confidence interval [CI] 1.8 to 38, P = 0.007) and the validating cohort (interquartile relative risk = 12.1, 95% CI 1.7 to 86, P = 0.013).ConclusionThese results suggest that the levels of PY-Shc and p66 Shc proteins in primary tumors identify patients at high risk for relapsing despite treatment with tamoxifen and therefore with further validation may be useful in guiding clinicians to select alternative adjuvant treatment strategies.

Expression in T-cells of the proapoptotic protein p66SHC is controlled by promoter demethylation

p66Shc plays a key role in oxidative stress-induced apoptosis. p66Shc gene expression is tissue-specific and controlled by promoter methylation. In T-cells p66Shc expression is induced by a variety of apoptotic stimuli. We have addressed the mechanisms regulating p66Shc expression in T-cells. We show that the increase in p66Shc protein following stimulation with a Ca 2+ ionophore results from enhanced gene expression, which is primarily dependent on DNA replication-independent promoter demethylation. Our data underline the role of CpG methylation in the control of p66Shc gene expression and provide evidence that Ca 2+ signaling may lead to epigenetic modifications in nondividing cells.

Distinct Signaling Functions for Shc Isoforms in the Heart

Thrombin activates protease-activated receptor-1 (PAR-1) and engages signaling pathways that influence the growth and survival of cardiomyocytes as well as extracellular matrix remodeling by cardiac fibroblasts. This study examines the role of Shc proteins in PAR-1-dependent signaling pathways that influence ventricular remodeling. We show that thrombin increases p46Shc/p52Shc phosphorylation at Tyr(239)/Tyr(240) and Tyr(317) (and p66Shc-Ser(36) phosphorylation) via a pertussis toxin-insensitive epidermal growth factor receptor (EGFR) transactivation pathway in cardiac fibroblasts; p66Shc-Ser(36) phosphorylation is via a MEK-dependent mechanism. In contrast, cardiac fibroblasts express beta(2)-adrenergic receptors that activate ERK through a pertussis toxin-sensitive EGFR transactivation pathway that does not involve Shc isoforms or lead to p66Shc-Ser(36) phosphorylation. In cardiomyocytes, thrombin triggers MEK-dependent p66Shc-Ser(36) phosphorylation, but this is not via EGFR transactivation (or associated with Shc-Tyr(239)/Tyr(240) and/or Tyr(317) phosphorylation). Importantly, p66Shc protein expression is detected in neonatal, but not adult, cardiomyocytes; p66Shc expression is induced (via a mechanism that requires protein kinase C and MEK activity) by Pasteurella multocida toxin, a Galpha(q) agonist that promotes cardiomyocyte hypertrophy. These results identify novel regulation of individual Shc isoforms in receptor-dependent pathways leading to cardiac hypertrophy and the transition to heart failure. The observations that p66Shc expression is induced by a Galpha(q) agonist and that PAR-1 activation leads to p66Shc-Ser(36) phosphorylation identifies p66Shc as a novel candidate hypertrophy-induced mediator of cardiomyocyte apoptosis and heart failure.

Apoptosis and Aging: Role of p66ShcRedox Protein

p66Shc was the first mammalian gene whose mutation was demonstrated to increase resistance to oxidative stress and to prolong life span. Many hypotheses have been formulated to explain the biochemical and molecular basis of mammalian aging. Among them the free radical theory of aging, which was first proposed half a century ago by Harman, has received much attention by biomedical scientists. This theory proposed that, because of their high reactivity, reactive oxygen species (ROS) would lead to unavoidable and potentially deleterious by-products, and such an increasingly damaging process could be responsible for degenerative diseases and aging. Recent reports suggest an important role of p66Shc protein in the regulation of cellular responses to oxidative stress, apoptosis, and aging. In this review we discuss what has been discovered about p66Shc in the past 10 years and we focus particularly on its role in ROS regulation, which appears to be extremely promising to define mammalian aging processes.

P66 Shc gene has a pro-apoptotic role in human cell lines and it is activated by a p53-independent pathway

p66 Shc protein has been proposed to be an indispensable factor for p53-dependent, mitochondria-mediated apoptosis in mice. Here, we show that p66 Shc plays a pro-apoptotic role also in cell lines of human origin such as SaOs-2 and HeLa, where p53 is either absent or inactivated, thus, suggesting that p66 Shc pro-apoptotic role is independent from the presence of a functional form of p53. The active form of p66 Shc is phosphorylated in Serine 36. We confirm the importance of Serine 36 phosphorylation for p66 Shc pro-apoptotic role, and our results suggest that the kinase involved in this process is activated independently from p53.

P53 and p66 Proteins Compete for Hypoxia-Inducible Factor 1 Alpha Stabilization in Young and Old Rat Hearts Exposed to Intermittent Hypoxia

Background: During development and aging, as well as under hypoxia, many cells can adapt to a stressful environment, while others are damaged and die by apoptosis. In particular, intermittent hypoxia, i.e., hypoxia followed by reoxygenation, determines different responses in young and adult myocardia. Objective: In the rat myocardium exposed to hypoxia, the roles played by p53 and p66 Shc proteins in matching, in an age-dependent mode, in stabilizing hypoxia-inducible factor-1α (HIF-1α), and in preventing its biological activity, which usually induces synthesis of rescue proteins against this stress, were investigated. Methods: Five animals from three groups, each consisting of 10 male Wistar rats, 8 days and 3 and 24 months old, were kept under physiological conditions; 5 young and 5 old rats were exposed to intermittent hypoxic challenges (12 h at 10% O₂ followed by 12 h at 21% O₂) for 8 days. Pregnant rats were kept for 3 days under hypoxic conditions before delivery, and 5 neonate rats were kept in intermittent hypoxia for 8 days. Left ventricles were excised and processed for TUNEL and Western blotting analyses. Results: HIF-1α stabilization by p53 along with decline in Bcl2, substantial caspase-3 expression, and a large number of apoptotic events make the hypoxic young myocardium the most damaged when compared to the neonatal one, in which HIF-1α is not stabilized. Moreover, high expression and activation of p66 in hypoxic young and in normoxic old myocardia suggests a pathological increase of the response to oxidative stress in the former and a physiological progressive increase in the latter. Conclusions: The different responses to hypoxic challenge during life show that the young seem the most reactive and damaged, as is well documented by p53-mediated HIF-1α stabilization. The neonate, not showing any modification in terms of HIF-1α expression and activation, seems ‘adapted’ to such an environment, since it was maintained in hypoxia 3 days before and 8 days after birth. In the old, increasing p66 expression and tyrosine phosphorylation, probably exerting a slight HIF-1α stabilization in the two experimental conditions, provide evidence of longevity and oxidative stress resistance, as suggested by the low number of apoptotic events seen upon hypoxic challenge, and this fact could be due to impairment of oxygen-sensing mechanisms or to adaptation of the cells to apoptosis.

Regulatory effects of the mitochondrial energetic status on mitochondrial p66Shc

p66(Shc) promotes apoptosis and controls the intracellular redox balance. A fraction of p66(Shc) exists within mitochondria, where it oxidizes cytochrome c to form hydrogen peroxide, which in turn induces mitochondrial permeability and apoptosis. However, cells tolerate p66(Shc) expression and accumulate oxidative damage under normal conditions, implying that the p66(Shc) functions must be tightly regulated. Here we review available knowledge on the regulation of p66(Shc) transcription, protein stabilization and post-translational modifications. In addition, we report novel investigations into the role of the mitochondrial import machinery on p66(Shc) activation, which highlight the energetic status of mitochondria as a crucial determinant of p66(Shc) function.

Rac1 Leads to Phosphorylation-dependent Increase in Stability of the p66shc Adaptor Protein: Role in Rac1-induced Oxidative Stress

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-induced apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.

Expression of p66Shc protein correlates with proliferation of human prostate cancer cells

p66(Shc), an isoform of Shc adaptor proteins, is shown to mediate various signals, including cellular stress. However, little is known about its involvement in carcinogenesis. We previously showed that p66(Shc) protein level is upregulated by steroid hormones in human carcinoma cells and is higher in prostate cancer (PCa) specimens than adjacent noncancerous cells. In this study, we investigated the role of p66(Shc) protein in PCa cell proliferation. Among different PCa cell lines tested, p66(Shc) protein level showed positive correlation with cell proliferation, that is, rapid-growing cells expressed higher p66(Shc) protein than slow-growing cells. Exposure of slow-growing LNCaP C-33 cells to epidermal growth factor (EGF) and 5alpha-dihydrotestosterone (DHT) led to upregulation of proliferation and p66(Shc) protein level. Conversely, growth suppression of fast-growing cells by cellular form of prostatic acid phosphatase (cPAcP) expression, a negative growth regulator, down-regulated their p66(Shc) protein level. Additionally, increased expression of p66(Shc) protein by cDNA transfection in LNCaP C-33 cells resulted in increased cell proliferation. Cell cycle analyses showed higher percentage of p66(Shc)-overexpressing cells at S phase (24%) than control cells (17%), correlating with their growth rates. On the other hand, transient knock-down of p66(Shc) expression by RNAi in rapidly growing cells decreased their proliferation as evidenced by the reduced cell growth as well as S phase in p66(Shc)-knocked down cells. The p66(Shc) signaling in cell growth regulation is apparently mediated by extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK). Thus, our results indicate a novel role for p66(Shc) in prostate carcinogenesis, in part, promoting cell proliferation.

Maladapted for Oxidative Stress

Alzheimer's disease is a neurodegenerative disease, and the accumulation of a toxic amyloid β peptide (Aβ) contributes to cell death. Smith et al . show that in cultured cell lines or primary cortical neurons, exposure to Aβ caused cell death and caused prolonged Ser 36 phosphorylation of the adaptor protein p66Shc, which had been previously implicated in the cellular response to oxidative stress. Using PC12 cells overexpressing either wild-type p66Shc or a Ser36 to Ala mutant (p66ShcS36A), the authors showed that cells with the dominant-negative mutant were less susceptible to Aβ-induced apoptosis. Furthermore, overexpression of p66ShcS36A also reduced the abundance of reactive oxygen species (ROS) in cells exposed to Aβ compared with the amounts detected in cells overexpressing p66Shc or nontransfected cells. The SH-SY5Y neuroblastoma cell line was used to investigate the role of the c-Jun N-terminal kinase (JNK) pathway in Aβ-stimulated p66Shc phosphorylation and cell death. Overexpression of a dominant-negative SEK1 (the kinase upstream of JNK) or pharmacological inhibition of SEK1 protected cells from Aβ-induced cell death and inhibited phosphorylation of JNK and p66Shc. Finally, transcription factors of the Forkhead (FKH) family, which are known to stimulate the expression of genes encoding ROS scavenging enzymes, were phosphorylated in response to Aβ exposure in SH-SY5Y and primary cortical neuron cultures. Phosphorylation of these transcription factors results in their sequestration in the cytosol and inhibition of expression of their target genes. Overexpression of p66ShcS36A in PC12 cells inhibited phosphorylation of FKHR, which suggests a link between p66Shc and regulation of ROS scavenging enzymes through FKH. Thus, the authors suggest that Aβ mediates its toxicity through an increase in ROS that in turn activates the JNK pathway, which results in phosphorylation of p66Shc and FKH, which decreases the cell's capacity to detoxify ROS. W. W. Smith, D. D. Norton, M. Gorospe, H. Jiang, S. Nemoto, N. J. Holbrook, T. Finkel, J. W. Kusiak, Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity. J. Cell. Biol. 169 , 331-339 (2005). [Abstract] [Full Text]

Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity

Excessive accumulation of amyloid β-peptide (Aβ) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aβ induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aβ toxicity. Treatment of cells and primary neuronal cultures with Aβ caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aβ-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aβ induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aβ-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aβ toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

P66shc is highly expressed in fibroblasts from centenarians

p66 shc−/− mice exhibit prolonged lifespan and increased resistance to oxidative and hypoxic stress. To investigate p66 shc involvement in human longevity, p66 shc mRNA and protein were evaluated in fibroblasts from young people, elderly and centenarians, exposed to oxidative or hypoxic stress. Unexpectedly, centenarians showed the highest basal levels of p66 shc . Oxidative stress induced p66 shc in all samples. At variance, hypoxic stress caused p66 shc reduction only in cells from centenarians. These changes occurred in absence of any modification of p66 shc promoter methylation pattern. Intriguingly, in cells from centenarians, p66 shc induction was affected by p53 codon 72 polymorphism. Thus, cells from centenarians present a peculiar regulation of p66 shc , suggesting that its role in mammalian longevity is more complex than previously thought.

Role of the p66Shc Isoform in Insulin-like Growth Factor I Receptor Signaling through MEK/Erk and Regulation of Actin Cytoskeleton in Rat Myoblasts

To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype.

Expression profiles of p53 and p66 shc during oxidative stress-induced senescence in fetal bovine fibroblasts

Somatic cells undergo a permanent cell cycle arrest, called cellular senescence, after a limited number of cell divisions in vitro. Both the tumor suppressor protein p53 and the stress-response protein p66 shc are suggested to regulate the molecular events associated with senescence. This study was undertaken to investigate the effect of different oxygen tensions and oxidative stress on cell longevity and to establish the role of p53 and p66 shc in cells undergoing senescence. As a model of cellular senescence, primary fetal bovine fibroblasts were cultured in either 20% O 2 or 5% O 2 atmospheres until senescence was reached. Fibroblasts cultured under 20% O 2 tension underwent senescence after 30 population doublings (PD), whereas fibroblasts cultured under 5% O 2 tension did not exhibit signs of senescence. Oxidative stress, as measured by protein carbonyl content, was significantly elevated in senescent cells compared to their younger counterparts and to fibroblasts cultured under 5% O 2 at the same PD. p53 mRNA gradually decreased in 20% O 2 cultured fibroblasts until senescence was reached, whereas p53 protein levels were significantly increased as well as p53 phosphorylation on serine 20, suggesting that p53 might be stabilized by posttranslational modifications during senescence. Senescence was also associated with high levels of p66 shc mRNA and protein levels, while the levels remained low and stable in dividing fibroblasts under 5% O 2 atmosphere. Taken together, our results show an effect of oxidative stress on the replicative life span of fetal bovine fibroblasts as well as an involvement of p53, serine 20-p53 phosphorylation and p66 shc in senescence.

3P66SHC, BUT NOT P53, IS INVOLVED IN EARLY ARREST OF IN VITROPRODUCED BOVINE EMBRYOS

High embryo losses occur in the first week of bovine embryo development, before the activation of the embryonic genome, with a high percentage of embryo death and arrest. Arrested embryos appear morphologically normal and do not exhibit any characteristic sign of apoptosis, including DNA fragmentation. We hypothesized that these embryos enter a senescence-like state and that both the cell cycle regulatory protein p53 and the stress-related protein p66shc, which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest of development. The aim of this study was to characterize the expression of p53 and p66shc in 2–4 cell-arrested bovine embryos. Our experimental model consists of in vitro-produced bovine embryos, co-cultured with oviductal cells. In our in vitro production system 86.8±41.4% of embryos cleave, 13.5±0.5% arrest at the 2–4 cell stage and 24.5±0.7% develop to the blastocyst stage. Cleavage occurs between 26 hours post-insemination (hpi) and 48 hpi. Embryos that cleave by 28hpi show only 0.6±0.3% of 2–4 cell arrest and 41.2±2.1% of blastocyst development, whereas 14.2±0.9% of later-cleaving embryos arrest at the 2–4 cell stage and only 26.5±1.7% develop into blastocysts. We compared 2–4 cell embryos collected at 28hpi with those arrested at the 2–4 cell stage and collected at Day 8 post-insemination. Quantification by Real Time PCR showed significantly higher p66shc mRNA levels (P&amp;lt;0.001), but no changes in p53 mRNA levels (P=0.860) in arrested embryos v. 28-hpi embryos (n=3 pools of 100 embryos each). We obtained the same pattern of p53 and p66shc mRNA expression when we compared 28-hpi embryos with later-cleaving embryos (28hpi to 48hpi, n=3 pools of 100 embryos each), and higher p66shc mRNA levels (P&amp;lt;0.050), or similar p53 mRNA levels (P=0.960). We also confirmed higher p66shc protein levels (P&amp;lt;0.001), but no changes in p53 protein levels (P=1.000), in later cleaving embryos compared with 28hpi-cleaving embryos by semi-quantitative immunocytochemistry (n=70). Statistical analysis was carried out using 2-sample t-test or the equivalent nonparametric test (Mann-Whitney test). Taken together, these results demonstrate that the developmental potential of in vitro-produced embryos is related to the time of first cleavage and that p66shc, but not p53, plays a role in early developmental arrest of in vitro-produced bovine embryos. Further experiments are required to investigate the functional role of p66shc in early embryo arrest. Funded by NSERC, CIHR, OGS and OMAFRA.