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.

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.

Stress-induced premature senescence and replicative senescence are different phenotypes, proteomic evidence.

In this paper, we illustrate how a proteomic analysis can be useful to approach complex biological problems, in this case the concept of stress-induced premature senescence (SIPS). According to the stochastic theories of ageing, damage that accumulate with time in the cellular components are responsible for cellular ageing. As a corollary, some sort of premature senescence should appear if the damage level is artificially increased due to the presence of stressing agents at subcytotoxic level. It has been shown, in several different models, that at a long-term after subcytotoxic stresses of many different natures, human diploid fibroblasts (HDFs) display biomarkers of replicative senescence (RS), which led to the concept of SIPS as compared to telomere-dependent RS. We compared RS and SIPS of HDFs by proteome analysis. SIPS was induced by two very different stressors: tert-butyhydroperoxide or ethanol. First, only a part of the protein expression changes observed in RS were also observed in SIPS. Second, HDFs in SIPS show changes specific either to the long-term effects of t-BHP or ethanol or independent of the nature of the stress. These changes have been termed "molecular scars" of subcytotoxic stresses. This work is also an excellent opportunity to discuss on important methodological issue in proteomics: the absolute requirement to start from reliable and reproducible models, which was the case in this study. We also focus on the data handling and statistical analysis allowing to use two-dimensional gel electrophoresis patterns in a semi-quantitative analysis.

Identification of the phospholipase A(2) isoforms that contribute to arachidonic acid release in hypoxic endothelial cells: limits of phospholipase A(2) inhibitors.

Changes in endothelium functions during ischemia are thought to be of importance in numerous pathological conditions, with, for instance, an increase in the release of inflammatory mediators like prostaglandins. Here, we showed that hypoxia increases phospholipase A(2) (PLA(2)) activity in human umbilical vein endothelial cells. Both basal PLA(2) activity and PG synthesis are sensitive to BEL and AACOCF3, respectively, inhibitors of calcium-independent PLA(2) (iPLA(2)) and cytosolic PLA(2) (cPLA(2)), while OPC, an inhibitor of soluble PLA(2) (sPLA(2)) only inhibited the hypoxia-induced AA release and PGF(2alpha) synthesis. Hypoxia does not alter expression of iPLA(2), sPLA(2) and cPLA(2) and cycloheximide did not inhibit PLA(2) activation, indicating that hypoxia-induced increase in PLA(2) activity is due to activation rather than induction. However, mRNA levels for sPLA(2) displayed a 2-fold increase after 2 hr incubation under hypoxia. BAPTA, an intracellular calcium chelator, partially inhibited the AA release in normoxia and in hypoxia. Direct assays of specific PLA(2) activity showed an increase in sPLA(2) activity but not in cPLA(2) activity after 2hr hypoxia. Taken together, these results indicate that the hypoxia-induced increase in PLA(2) activity is mostly due to the activation of sPLA(2).

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

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 beta-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.

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 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 beta-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-beta1 (TGF-beta1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-beta1 or its receptor II (TbetaRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-beta1 in UVB-induced premature senescence. Both the latent and active forms of TGF-beta1 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.