Qin M. Chen

Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints

Abstract: Normal human diploid fibroblasts (HDFs) undergo replicative senescence inevitably in tissue culture after a certain number of cell divisions. A number of molecular changes observed in replicative senescent cells occur in somatic cells during the process of aging. Genetic studies on replicative senescence indicate the control of tumor suppression mechanisms. Despite the significance of replicative senescence in aging and cancer, little is known about the central cause of the complex changes observed in replicative senescent cells. The interest in the phenomenon has intensified in recent years, since damaging agents, certain oncogenes and tumor suppressor genes have been found to induce features of senescence in early passage young HDFs or in immortalized tumor cells. The reported features of senescence are summarized here in order to clarify the concept of replicative senescence or premature senescence. The experimental results of extending the replicative life span by reducing ambient oxygen tension or by N‐tert‐butyl‐alpha‐phenylnitrone (PBN) argue a role of oxidative damage in replicative senescence. By inducing premature senescence with a pulse treatment of H2O2, we can study the role of the cell cycle checkpoint proteins p53, p21, p16 and Rb in gaining each feature of senescence. Although p53 and Rb control G1 arrest and Rb appears to control cell enlargement, activation of the senescent associate β‐galactosidase, loss of cell replication and multiple molecular changes observed in premature senescent or replicative senescent cells are likely controlled by mechanisms beyond the cell cycle checkpoints.

Translational Control of Nrf2 Protein in Activation of Antioxidant Response by Oxidants

Nf-E2 related factor-2 (Nrf2) is a basic leucine zipper transcription factor that binds and activates the antioxidant response element (ARE) in the promoters of many antioxidant and detoxification genes. We found that H2O2 treatment caused a rapid increase in endogenous Nrf2 protein level in rat cardiomyocytes. Semiquantitative or real-time reverse transcription-polymerase chain reaction failed to show an increase of Nrf2 mRNA level by H2O2 treatment. Measurements of Nrf2 protein stability excluded the possibility of Nrf2 protein stabilization. Although inhibiting protein synthesis with cycloheximide prevented H2O2 from elevating Nrf2 protein level, RNA synthesis inhibition with actinomycin D failed to do so. Measurements of new protein synthesis with [35S]methionine incorporation confirmed that H2O2 increased the translation of Nrf2 protein. Inhibitors of phosphoinositide 3-kinase were able to abolish the induction of Nrf2 protein by H2O2. Although H2O2 increased phosphorylation of p70 S6 kinase, rapamycin failed to inhibit H2O2 from elevating Nrf2 protein. H2O2 also induced phosphorylation of eukaryotic translation initiation factor (eIF) 4E and eIF2α within 30 and 10 min, respectively. Inhibiting eIF4E with small interfering siRNA or increasing eIF2α phosphorylation with salubrinal did not affect Nrf2 elevation by H2O2. Our data present a novel phenomenon of quick onset of the antioxidant/detoxification response via increased translation of Nrf2 by oxidants. The mechanism underlying such stress-induced de novo protein translation may involve multiple components of translational machinery.

p66Shc: at the crossroad of oxidative stress and the genetics of aging

The biology of aging has been mysterious for centuries. Removal of the p66(Shc) gene, which encodes an adaptor protein for cell signaling, extends lifespan by approximately 30% in mice and confers resistance to oxidative stress. The absence of p66(Shc) correlates with reduced levels of apoptosis. Oxidants induce phosphorylation of serine36 on p66(Shc), contributing to inactivation of members of the Forkhead transcription factor family, some of which appear to regulate the expression of antioxidant genes. The expression of p66(Shc) is regulated by the methylation status of its promoter. This leads us to hypothesize that increased methylation of the p66(Shc) promoter might contribute to the absence of its expression and therefore extended longevity in particular individuals.

Cystatin C increases in cardiac injury: a role in extracellular matrix protein modulation.

AIMSnNumerous lines of evidence suggest a role of oxidative stress in initiation and progression of heart failure. We identify novel pathways of oxidative stress in cardiomyocytes using proteomic technology.nnnMETHODS AND RESULTSnCardiomyocytes and cardiac fibroblasts isolated from rat hearts were treated with sublethal doses of H(2)O(2) for detection of secreted protein factors in the conditioned media by mass spectrometry-based proteomics. Comparison between the two cell types leads to the finding that H(2)O(2) caused an elevated cystatin C protein in the conditioned medium from cardiomyocytes. When cardiomyopathy was induced in mice by chronic administration of doxorubicin, elevated cystatin C protein was detected in the plasma. Myocardial ischaemia by left anterior descending coronary artery occlusion causes an increase in the level of cystatin C protein in the plasma. In myocardial tissue from the ischaemic area, an increase in cystatin C correlates with the inhibition of cathepsin B activity and accumulation of fibronectin and collagen I/III. Overexpressing cystatin C gene or exposing fibroblasts to cystatin C protein results in an inhibition of cathepsin B and accumulation of fibronectin and collagen I/III.nnnCONCLUSIONnOxidants induce elevated cystatin C production from CMCs. Cystatin C plays a role in cardiac extracellular matrix remodelling.

Far upstream element binding protein 1: a commander of transcription, translation and beyond.

The far upstream binding protein 1 (FBP1) was first identified as a DNA-binding protein that regulates c-Myc gene transcription through binding to the far upstream element (FUSE) in the promoter region 1.5u2009kb upstream of the transcription start site. FBP1 collaborates with TFIIH and additional transcription factors for optimal transcription of the c-Myc gene. In recent years, mounting evidence suggests that FBP1 acts as an RNA-binding protein and regulates mRNA translation or stability of genes, such as GAP43, p27Kip and nucleophosmin. During retroviral infection, FBP1 binds to and mediates replication of RNA from Hepatitis C and Enterovirus 71. As a nuclear protein, FBP1 may translocate to the cytoplasm in apoptotic cells. The interaction of FBP1 with p38/JTV-1 results in FBP1 ubiquitination and degradation by the proteasomes. Transcriptional and post-transcriptional regulations by FBP1 contribute to cell proliferation, migration or cell death. FBP1 association with carcinogenesis has been reported in c-Myc dependent or independent manner. This review summarizes biochemical features of FBP1, its mechanism of action, FBP family members and the involvement of FBP1 in carcinogenesis.

Apoptosis and heart failure: mechanisms and therapeutic implications.

A large volume of experimental data supports the presence of apoptosis in failing hearts. Apoptosis in many types of cells results from exposure to cytotoxic cytokines or damaging agents. Cytotoxic cytokines such as tumor necrosis factor (TNF)-α; or Fas ligand (FasL) bind to their receptors to activate caspase-8, while damaging agents can cause mitochondrial release of cytochrome c, which can initiate activation of caspase-9. Caspase-8 or -9 can activate a cascade of caspases. The p53 protein is often required for damaging agent-induced apoptosis. An imbalance of proapoptotic factors versus prosurvival factors in the bcl-2 family precedes the activation of caspases. Given these typical changes of apoptosis found in many cell types, the apoptotic pathway in cardiomyocytes is somewhat unconventional since in vivo experimental data reveal that apoptosis does not appear to be controlled by TNF-α;, FasL, p53 or decrease of bcl-2. In vitro and in vivo studies suggest the importance of mitochondria and activation of caspases in cell death occurring in failing hearts. Oxidants, excessive nitric oxide, angiotensin II and catecholamines have been shown to trigger apoptotic death of cardiomyocytes. Eliminating these inducers reduces apoptosis and reverses the loss of contractile function in many cases, indicating the feasibility of the pharmacological application of antioxidants, nitric oxide synthetase inhibitors, ACE inhibitors, angiotensin II receptor antagonists and adrenergic receptor antagonists. Most inducers of apoptosis initiate a cascade of signaling events, including activation of the p38 mitogen-activated protein kinase. Small molecule inhibitors of p38 have been shown to be capable of preventing apoptosis and loss of contractile function associated with ischemia and reperfusion. Although further experimental work is needed, several studies have already indicated the beneficial effect of caspase inhibitors against cell loss and features of heart failure in vitro and in vivo. These studies indicate the importance of inhibiting apoptosis in therapeutic interventions against heart failure.

Novel mechanisms of sublethal oxidant toxicity: induction of premature senescence in human fibroblasts confers tumor promoter activity

Aging is the highest risk factor for cancer. Although oxidants are thought to contribute to both aging and cancer, the interplay between oxidative stress, aging, and cancer has not been well studied. Human diploid fibroblasts (HDFs) undergo premature senescence in response to sublethal doses of H(2)O(2). To test the hypothesis that senescent or senescent-like HDFs function as a tumor promoter, we have employed an in vitro skin tumor promotion model, in which colony formation is measured using initiated mouse keratinocyte 308 cells seeded at clonal density. 308 cells form colonies when co-cultured with normal HDFs only in the presence of the tumor promoter phorbol 12-myristate 13-acetate (TPA), which induces an average of 5.75 colonies. When co-cultured with H(2)O(2)-treated HDFs, 308 cells form an average of 30.3 colonies. To understand the mechanism behind this phenomenon, we tested whether conditioned medium of HDFs, HDF extracellular matrix (ECM), density of HDFs, or the contact between keratinocytes and HDFs plays a role in 308 cell colony formation. The conditioned medium from prematurely senescent cells resulted in an average of eightfold more 308 cell colonies formed than the conditioned medium from normal HDFs, and the growth-promoting effect of the conditioned medium was trypsin sensitive. The ECM alone was not able to induce 308 cell colony formation. Increasing the density of normal HDFs or contact with normal HDFs but not senescent-like HDFs was inhibitory to the growth of 308 cells. Measurement of Connexin 43 indicated a decreased expression of the protein, which suggests an impaired gap junction communication in senescent-like HDFs. We conclude that H(2)O(2)-treated fibroblasts not only lose contact inhibition of the growth of initiated keratinocytes perhaps related to reduced gap junction communication but also increase production of secreted protein factors to enhance the growth of 308 keratinocytes.

Linking oxidative stress and genetics of aging with p66Shc signaling and forkhead transcription factors.

Genetics versus oxidative stress have been long-standing points of contention among theories seeking to explain the root of aging. Because aging is the highest risk factor for many diseases, it is to our advantage to better understand the biological mechanisms of this process. Caloric restriction has been the only reliable means of extending lifespan in mammalian models until recently. The discovery of mutant strains of mice with increased longevity could be a significant contributor to our understanding of the genetic and molecular basis of human aging. One genetic approach that increases the longevity of mice is the removal of the p66Shc gene, which encodes a protein belonging to a family of adaptors for signal transduction in mitogenic and apoptotic responses. Normally, p66Shc is tyrosine phosphorylated (activated) by various extracellular signals including EGF and insulin. However, serine phosphorylation of p66Shc can occur after oxidative stress either in association with or independently of tyrosine phosphorylation. p66Shc serine phosphorylation has been linked to inactivation of members of forkhead transcription factors, resulting in increased intracellular oxidant levels and increased sensitivity to apoptosis. Knocking out p66Shc allows moderately elevated activity of forkhead transcription factors and better-equipped antioxidant defenses at the cellular level. Recent reports have suggested that methylation of the p66Shc promoter has important implications in its expression regulation. This leads us to hypothesize that the methylation status of the p66Shc promoter may differ between individuals and therefore contribute to variations of longevity. We present evidence arguing that decreasing oxidative stress or increasing resistance to oxidative damage as a result of genetic variation or p66Shc knockout is likely contributing to individual differences in longevity.

Measurements of hydrogen peroxide induced premature senescence:senescence-associated β-galactosidase and DNA synthesis index in human diploid fibroblasts with down-regulated p53 or Rb

Human diploid fibroblasts (HDFs) inevitably undergo replicative senescence in culture after serial passages. Recent work indicates that early passage HDFs undergo irreversible growth arrest and develop features of senescence after being treated with oxidants and other agents. Senescence is usually measured by a decrease in DNA synthesis index and an increase in the activity of senescence-associated β-galactosidase (SA β-gal). We compared these two measurements here and found that IMR-90 HDFs lost the ability to synthesize DNA immediately but did not activate SA β-gal until 4 days after the treatment with 75 µM or 0.75 pmol/cell H2O2. Expression of human papillomaviral E6 or/and E7 genes results in reduction of p53 or/and Rb protein levels and increases in ED50 for DNA synthesis inhibition or SA β-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA β-gal one week following the treatment with H2O2 at doses lower than 150 µM or 1.5 pmol/cell. The dose response curve of SA β-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA β-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.

Cell cycle regulation in H2O2-induced premature senescence of human diploid fibroblasts and regulatory control exerted by the papilloma virusE6 and E7 proteins

Many biomarkers of replicative senescence appear in stress-induced premature senescence (SIPS) of human diploid fibroblasts (HDFs). The mRNA level of key cell cycle regulators was studied in H(2)O(2)-induced premature senescence of HDFs expressing or not the papillomavirus E6 and E7 proteins, which enhanced, respectively, the proteolysis of p53 and Rb. The CdKIs p21(waf-1) and p16(Ink-4a) were found overexpressed in H(2)O(2)-induced premature senescence, while p19(Ink-4d)and p27(Kip-1) were repressed. The results obtained in E6 HDFs suggest that p21(waf-1) and p16(Ink-4a) overexpressions are p53-independent, while p27(Kip-1) and p19(Ink-4d) down-regulations are p53-dependent.E6 regulated Rb, p130, p53 and p16(Ink-4a) mRNA level in non-stressing conditions, and regulated p130, p107, p53, p19(Ink-4d), p27(Kip-1) mRNA level in SIPS. SIPS modified the E6-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d) and p27(Kip-1) mRNA level, when compared to normal conditions.E7 regulated the mRNA level of all the genes studied, in all conditions, suggesting that the Rb family or other E7-interacting proteins might modify the expression of these genes. SIPS modified strongly the E7-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d), p27(Kip-1), p21(Waf-1) and Rb mRNA level, when compared to normal conditions. Further work is ongoing to test whether this E7-mediated regulatory control takes place through interactions with Rb or other E7-interacting proteins.