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Although senescence has long been implicated in aging-associated pathologies, it is not clearly understood how senescent cells are linked to these diseases. To address this knowledge gap, we profiled cellular senescence phenotypes and mRNA expression patterns during replicative senescence in human diploid fibroblasts. We identified a sequential order of gain-of-senescence phenotypes: low levels of reactive oxygen species, cell mass/size increases with delayed cell growth, high levels of reactive oxygen species with increases in senescence-associated β-galactosidase activity (SA-β-gal), and high levels of SA-β-gal activity. Gene expression profiling revealed four distinct modules in which genes were prominently expressed at certain stages of senescence, allowing us to divide the process into four stages: early, middle, advanced, and very advanced. Interestingly, the gene expression modules governing each stage supported the development of the associated senescence phenotypes. Senescence-associated secretory phenotype-related genes also displayed a stage-specific expression pattern with three unique features during senescence: differential expression of interleukin isoforms, differential expression of interleukins and their receptors, and differential expression of matrix metalloproteinases and their inhibitory proteins. We validated these phenomena at the protein level using human diploid fibroblasts and aging Sprague-Dawley rat skin tissues. Finally, disease-association analysis of the modular genes also revealed stage-specific patterns. Taken together, our results reflect a detailed process of cellular senescence and provide diverse genome-wide information of cellular backgrounds for senescence. [ABSTRACT FROM AUTHOR]

Abstract: Gene expression patterns vary dramatically in a tissue-specific and age-dependent manner. RNA-binding proteins that regulate mRNA turnover and/or translation (TTR-RBPs) critically affect the subsets of expressed proteins. Although many proteins implicated in age-related processes are encoded by mRNAs that are targets of TTR-RBPs, very little is known regarding the tissue- and age-dependent expression of TTR-RBPs in humans. Recent analysis of TTR-RBPs expression using human tissue microarray has provided us interesting insight into their possibly physiologic roles as a function of age. This analysis has also revealed striking discrepancies between the levels of TTR-RBPs in senescent human diploid fibroblasts (HDFs), widely used as an in vitro model of aging, and the levels of TTR-RBPs in tissues from individuals of advancing age. In this article, we will review our knowledge of human TTR-RBP expression in different tissues as a function of age. [Copyright &y& Elsevier]

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated β-galactosidase (SA-β-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-β-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest. [Copyright &y& Elsevier]

Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated β-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-β (TGF-β), in view of the high levels of TGF-β isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-β1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-β/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts. [Copyright &y& Elsevier]

Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-α, or okadaic acid. The most profound effects were induced by TNF-α and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: β-galactosidase, α-galactosidase A, β-glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of β-galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (β-galactosidase, α-galactosidase A, and β-glucoronidase) are constitutively expressed at high levels. TNF-α was the only agent that induced lysosomal activity in senescent fibroblasts, of which only α-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction. [Copyright &y& Elsevier]