Thierry Pascal

Comparison of replicative senescence and stress-induced premature senescence combining differential display and low-density DNA arrays

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stress display many features of senescence. Using differential display RT‐PCR, gene expression of HDFs in premature senescence induced by tert‐butylhydroperoxide or ethanol and in replicative senescence was compared to gene expression of HDFs at early cumulative population doublings. Thirty genes of known function were identified from the 265 differentially displayed cDNA fragments. A customized low‐density array allowed to confirm the relative level of the corresponding 30 transcripts. We found differential expression of genes coding for proteins implicated namely in growth arrest (PTEN, IGFBP‐3, LRP‐1 and CAV1), senescent morphogenesis (TGF‐β1 and LOXL2) and iron metabolism (TFR and FTL).

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-

Stress-Induced Premature Senescence or Stress-Induced Senescence-Like Phenotype: One In Vivo Reality, Two Possible Definitions?

No consensus exists so far on the definition of cellular senescence. The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening counting cell generations (definition 1). Other authors gave an enlarged functional definition encompassing any kind of irreversible arrest of proliferative cell types induced by damaging agents or cell cycle deregulations after overexpression of proto-oncogenes (definition 2). As stress increases, the proportion of cells in “stress-induced premature senescence-like phenotype” according to definition 1 or “stress-induced premature senescence,” according to definition 2, should increase when a culture reaches growth arrest, and the proportion of cells that reached telomere-dependent replicative senescence due to the end-replication problem should decrease. Stress-induced premature senescence-like phenotype and telomere-dependent replicatively senescent cells share basic similarities such as irreversible growth arrest and resistance to apoptosis, which may appear through different pathways. Irreversible growth arrest after exposure to oxidative stress and generation of DNA damage could be as efficient in avoiding immortalisation as “telomere-dependent” replicative senescence. Probabilities are higher that the senescent cells (according to definition 2) appearing in vivo are in stress-induced premature senescence rather than in telomere-dependent replicative senescence. Examples are given suggesting these cells affect in vivo tissue (patho)physiology and aging.

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.

Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts.

Repeated exposures to sublethal concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of WI-38 human diploid fibroblasts. We found 16 replicative senescence-related genes with similar alterations in expression level in replicative senescence and two models of stress-induced premature senescence. Among these genes was IGFBP-3. Using a siRNA approach, we showed that IGFBP-3 regulates the appearance of several biomarkers of senescence after repeated exposures of WI-38 fibroblasts to tert-butylhydroperoxide and ethanol.

mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells

To explain why mitochondrial DNA (mtDNA)‐depleted or rho0 cells still keep a mitochondrial membrane potential (Δψm) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1‐ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase‐polymerase chain reaction on L929 mtDNA‐depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up‐regulated in mtDNA‐depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor α. Interestingly, up‐ or down‐regulation of mtCLIC protein expression changes Δψm whereas the chloride channel inhibitor NPPB reduces the Δψm in mtDNA‐depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA‐depleted cells incorporate, in a NPPB‐sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA‐depleted cells, a feature required to prevent apoptosis and to drive continous protein import into mitochondria.

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.

Human diploid fibroblasts display a decreased level of c-fos mRNA at 72 hours after exposure to sublethal H2O2 stress

PATRICK DUMONT,a,b MAGGI BURTON,b QIN M. CHEN,c CHRISTOPHE FRIPPIAT,b THIERRY PASCAL,b JEAN-FRANÇOIS DIERICK,b FRANÇOIS ELIAERS,b FLORENCE CHAINIAUX,b JOSÉ REMACLE,b AND OLIVIER TOUSSAINTa,b bThe University of Namur (FUNDP), Department of Biology, Laboratory of Cellular Biochemistry and Biology, Rue de Bruxelles, 61, B-5000 Namur, Belgium cUniversity of Arizona, Pharmacology & Toxicology, 1703 E. Mabel Street, Tucson, Arizona 85721, USA

Heme oxygenase-1 and interleukin-11 are overexpressed in stress-induced premature senescence of human WI-38 fibroblasts induced by tert -butylhydroperoxide and ethanol

Acute repeated exposures to subcytotoxic concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of human diploid fibroblasts. In the present work we found an increased mRNA and protein level of interleukin-11 and heme oxygenase-1 in premature senescence of WI-38 human diploid foetal lung fibroblasts induced by both tert-butylhydroperoxide and ethanol. We tested whether interleukin-11 and heme oxygenase-1 could protect against tert-butylhydroperoxide- or ethanol-induced premature senescence when stable overexpression was established using a retroviral vector-based transduction. No protective effect was found against the decrease of the proliferative potential, the increase of the proportion of senescence-associated ß-galactosidase positive cells and the increase of the mRNA levels of six senescence-associated genes.