François Eliaers

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-β1 signaling pathway

Premature senescence of human diploid fibroblasts (HDFs) can be induced by exposures to a variety of oxidative stress and DNA damaging agents. In this study we developed a robust model of UVB-induced premature senescence of skin HDFs. After a series of 10 subcytotoxic (non-proapoptotic) exposures to UVB at 250 mJ/cm2, the so-called biomarkers of senescence were markedly expressed: growth arrest, senescence-associated β-galactosidase activity, senescence-associated gene overexpression, deletion in mitochondrial DNA. A set of 44 stress- and senescence-associated genes were found to be differentially expressed in this model, among which clusterin/apolipoprotein J (apo J) and transforming growth factor-β1 (TGF-β1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-β1 or its receptor II (TβRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-β1 in UVB-induced premature senescence. Both the latent and active forms of TGF-β1 were increased with time after the last UVB stress. Proteasome inhibition was ruled out as a potential mechanism of UVB-induced stress-induced premature senescence (SIPS). This model represents an alternative in vitro model in photoaging research for screening potential anti-photoaging compounds.

Protection of hypoxia-induced ATP decrease in endothelial cells by ginkgo biloba extract and bilobalide

Due to their localization at the interface between blood and tissue, endothelial cells are the first target of any change occurring within the blood, and alterations of their functions can seriously impair organs. During hypoxia, which mimics in vivo ischemia, a cascade of events occurs in the endothelial cells, starting with a decrease in ATP content and leading to their activation and release of inflammatory mediators. EGb 761 and one of its constituents, bilobalide, were shown to inhibit the hypoxia-induced decrease in ATP content in endothelial cells in vitro. Under these conditions, glycolysis was activated, as evidenced by increased glucose transport, as well as increased lactate production. Bilobalide was found to increase glucose transport under normoxic but not hypoxic conditions. In addition, EGb and bilobalide prevented the increase in total lactate production observed after 60 min of hypoxia. However, after 120 min of hypoxia, the total lactate production was similar under normoxic and hypoxic conditions, and both compounds increased this production. These results indicate that glycolysis slowed down between the 60th and 120th minute of hypoxia, while EGb and bilobalide delayed the onset of glycolysis activation. In another experimental model, both compounds were shown to increase the respiratory control ratio of mitochondria isolated from liver of rats treated orally. Since ischemia is known to uncouple mitochondria, the protection of ATP content and the delay in glycolysis activation observed during hypoxia in the presence of EGb 761 or bilobalide is best explained by a protection of mitochondrial respiratory activity, at least during the first 60 min of hypoxia incubation. Both products retain the ability to form ATP, thereby reducing the cells need to induce glycolysis, probably by preserving ATP regeneration by mitochondria as long as oxygen is available.

Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide

Abstract Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated β-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence–related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.

Stress-induced premature senescence. Essence of life, evolution, stress, and aging.

The stress syndrome was discovered accidentally by Hans Selye while searching for new hormones in the placenta.1 After injecting rats with crude preparations, Selye found adrenal enlargements and involution of thymus and lymph nodes, which he thought were specific for a particular hormone. It occurred to Selye that these symptoms might represent a nonspecific response to noxious agents. Indeed, this was found to be the case when he injected rats with diverse agents. Selye defined the stress response as the “general adaptation syndrome.”2,3 According to this theory, the initial reaction to stress is shock, it is followed by a countershock phase, and gradually resistance develops to the stressor. This resistance may turn into exhaustion, however, if the stressor persists, and death may ensue. Both specific and nonspecific resistance develops during stress.4 In his last scientific book, Selye defined biologic stress as “the non-specific response of the body to any demand made upon it.”5 Beside the transfer of the word “stress” from physics to biology, Selye also coined the words corticosteroids, glucocorticoids, and mineralocorticoids.6 Nowadays, the concept of stress has invaded most fields of the biologic, medical, and social sciences. Cellular and molecular biology has become interested in the study of the stress response of human, animal, and plant cells, the consensus being that “any environmental factor potentially unfavourable to living organism” is stress.7 It is also generally agreed that “if the limits of tolerance are exceeded and the adaptive capacity is over-worked, the result may be permanent damage or even death.”8 Three phases of the stress response have been defined based on experimental observations: (1) the response phase of alarm reaction with deviation of functional norm, decline of vitality, and excess of catabolic processes over anabolism, (2) the restitution phase or stage of resistance with adaptation processes and repair processes, and (3) either the end phase, that stage of exhaustion or long-term response when stress intensity is too high, leading to overcharge of the adaptation capacity, damage, chronic dis-

UVB-induced premature senescence of human diploid skin fibroblasts

In this work, we show that repeated stresses with UVB (290-320 nm) induce stress-induced premature senescence (SIPS) of skin human diploid fibroblasts (HDFs). HDFs at early cumulative population doublings were exposed three or five times to increasing subcytotoxic doses of UVB with one stress per day. After 2 days of recovery, several biomarkers of replicative senescence were established. First, there was an increase in the proportion of cells positive for senescence-associated beta-galactosidase activity. Second, there was a loss of replicative potential as assessed by a very low level of [3H]-thymidine incorporation. Third, the steady-state level of the mRNA of three senescence-associated genes, i.e. fibronectin, osteonectin and SM22, was increased in HDFs at 72 h after three and five exposures to UVB. In conclusion, these results suggest that it is possible to induce SIPS in HDFs after repeated exposures to subcytotoxic doses of UVB. This model could be used to test whether HDFs in UVB-induced premature senescence are able to promote epithelial cell growth and tumorigenesis in skin, as shown recently with HDFs in H(2)O(2)-induced premature senescence.

Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence

WI‐38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 μM tert‐butylhydroperoxide (t‐BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 μM H2O2 stress or five repeated 30 μM t‐BHP stresses along the same PD, respectively a 322±55 and 380±129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

Stress-induced premature senescence as alternative toxicological method for testing the long-term effects of molecules under development in the industry.

No alternative in vitro method exists fordetecting the potential long-term genotoxic effects ofmolecules at subcytotoxic concentrations, in terms ofdays and weeks after exposure(s) to the moleculetested. A theoretical model of cellular senescence ledto the concept that subcytotoxic stresses under anymolecules at subcytotoxic doses, such as moleculesunder development in the pharmaceutical, cosmetics andfood industry, might lead human fibroblasts into a stateclosely related to in vitro senescence. Thisconcept was then experimentally confirmed invitro: many biomarkers of replicative senescence ofhuman fibroblasts were found 72 h after theirexposure to various kinds of stressors used at non-cytotoxic concentrations. This phenomenon has beentermed stress-induced premature senescence (SIPS).Moreover, proteomics studies have revealed that,besides their effects on the appearance of thebiomarkers of senescence, sublethal stresses under avariety of stressors also lead to long-term specificchanges in the expression level of proteins which arestress-specific. These changes have been coined themolecular scars of stress. The proteins correspondingto these molecular scars may be identified using thelatest developments in mass spectrometry. This modelof stress-induced premature senescence may be appliedto the toxicological sciences when testing for thepotential irreversible long-term effects of moleculeson the cell fate.

Role of the PLA2‐independent peroxiredoxin VI activity in the survival of immortalized fibroblasts exposed to cytotoxic oxidative stress

Peroxiredoxin VI (PrxVI) is a bifunctional enzyme with non‐selenium glutathione peroxidase and Ca2+‐independent acidic phospholipase A2 activities. We demonstrate that transfection‐mediated PrxVI overexpression protects immortalized human WI‐38 and murine NIH3T3 fibroblasts against cytotoxic doses of tert‐butylhydroperoxide and H2O2. Mutants for either glutathione peroxidase or phospholipase A2 activity show that glutathione peroxidase but not phospholipase A2 activity is required to promote cell survival after stress. Also, ectopic PrxVI overexpression does not protect telomerase‐stabilized WI‐38 fibroblasts against stress‐induced premature senescence.

Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia‐induced decrease in ATP content. In addition, they all induced a concentration‐dependent increase in respiratory control ratio (RCR) of liver mitochondria pre‐incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m‐chlorophenyl hydrazone (mCCP)‐induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.