Article

Identification of differentially expressed proteins in senescent human embryonic fibroblasts

Laboratory of Molecular & Cellular Ageing, Institute of Biological Research & Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., Athens 11635, Greece.
Mechanisms of Ageing and Development (Impact Factor: 3.51). 02/2006; 127(1):88-92. DOI: 10.1016/j.mad.2005.08.009
Source: PubMed

ABSTRACT Normal human fibroblasts undergo a limited number of divisions in culture, a process known as replicative senescence (RS). Although several senescence-specific genes have been identified, analysis at the level of protein expression can provide additional insights into the mechanisms that regulate RS. We have performed a proteomic comparison between young and replicative senescent human embryonic WI-38 fibroblasts and we have identified 13 proteins, which are differentially expressed in senescent cells. Some of the identified proteins are components of the cellular cytoskeleton, while others are implicated in key cellular functions including metabolism and energy production, Ca(2+) signalling, nucleo-cytoplasmic trafficking and telomerase activity regulation. In summary, our analysis contributes to the list of senescence-associated proteins by identifying new biomarkers and provides novel information on functional protein networks that are perturbed during replicative senescence of human fibroblast cultures.

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    • "In human diploid fibroblasts, several genes including inflammatory genes, cell cycle regulatory genes, cytoskeletal genes, and metabolic genes were differentially expressed [3] during replicative senescence and modifiable by dietary components such as antioxidants [4]. Human aging can be studied in vitro, specifically by using normal human diploid fibroblasts (HDFs) which undergo a limited number of cellular divisions in culture and progressively reached a state of irreversible growth arrest, a process termed as replicative senescence [1]. Senescent fibroblast cells are resistant to mitogen-induced proliferation, expressed senescence-associated í µí»½-galactosidase (SA í µí»½-gal), exhibited enlarged and flattened morphology, and showed altered gene expression [5]. "
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    ABSTRACT: The effect of γ -tocotrienol, a vitamin E isomer, in modulating gene expression in cellular aging of human diploid fibroblasts was studied. Senescent cells at passage 30 were incubated with 70 μ M of γ -tocotrienol for 24 h. Gene expression patterns were evaluated using Sentrix HumanRef-8 Expression BeadChip from Illumina, analysed using GeneSpring GX10 software, and validated using quantitative RT-PCR. A total of 100 genes were differentially expressed (P < 0.001) by at least 1.5 fold in response to γ -tocotrienol treatment. Amongst the genes were IRAK3, SelS, HSPA5, HERPUD1, DNAJB9, SEPR1, C18orf55, ARF4, RINT1, NXT1, CADPS2, COG6, and GLRX5. Significant gene list was further analysed by Gene Set Enrichment Analysis (GSEA), and the Normalized Enrichment Score (NES) showed that biological processes such as inflammation, protein transport, apoptosis, and cell redox homeostasis were modulated in senescent fibroblasts treated with γ -tocotrienol. These findings revealed that γ -tocotrienol may prevent cellular aging of human diploid fibroblasts by modulating gene expression.
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    • "Cellular senescence was first described by Hayflick and Moorfield in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrested (Hayflick and Moorhead, 1961; Campisi and d'Adda di Fagagna, 2007; Sedivy et al., 2007). The majority of senescent cells assume a characteristic flattened and enlarged morphology, and over the years a large number of molecular phenotypes have been described, such as changes in gene expression, protein processing and chromatin organization (Gonos et al., 1998; Shelton et al., 1999; Schwarze et al., 2002; Semov et al., 2002; Narita et al., 2003; Zhang et al., 2003; Yoon et al., 2004; Pascal et al., 2005; Xie et al., 2005; Zhang et al., 2005; Cong et al., 2006; Funayama et al., 2006; Trougakos et al., 2006; Zdanov et al., 2006; Zhang et al., 2007). The growth arrest occurs mostly in G1 phase (Pignolo et al., 1998). "
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    ABSTRACT: Cellular senescence, first observed and defined using in vitro cell culture studies, is an irreversible cell cycle arrest which can be triggered by a variety of factors. Emerging evidence suggests that cellular senescence acts as an in vivo tumor suppression mechanism by limiting aberrant proliferation. It has also been postulated that cellular senescence can occur independently of cancer and contribute to the physiological processes of normal organismal aging. Recent data have demonstrated the in vivo accumulation of senescent cells with advancing age. Some characteristics of senescent cells, such as the ability to modify their extracellular environment, could play a role in aging and age-related pathology. In this review, we examine current evidence that links cellular senescence and organismal aging.
    Mechanisms of Ageing and Development 07/2008; 129(7-8):467-74. DOI:10.1016/j.mad.2008.04.001 · 3.51 Impact Factor
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    ABSTRACT: Cellular senescence is an end point of a signal transduction programme leading to irreversible cell cycle arresst accompanied by characteristic alterations to cell morphology, biochemical properties and gene expression profile. This phenotype can be triggered by a variety of stimuli including telomere shortening, DNA damage or activated oncogenes. Senescence is now recognised as a tumour suppressor mechanism mediated by p53 and pRB pathways which act to prevent the proliferatio of cells that are at risk of tumourigenic transformation. RUNX1 is a transcription factor essential for definitive hematopoiesis and is frequently targeted in human leukaemias by chromosomal rearrangements. RUNX1 has been also demonstrated to act as a dominant oncogene in mice and the ectopic expression of RUNX1 in murine embryonic fibroblasts has been shown to cause senescence. The central aim of this study was to investigate the mechanism of senescence induction by RUNX1 and its fusion derived leukaemogenic oncoproteins in primary fibroblasts. My work showed that RUNX1 induces a strong senescence-like response in murine and human primary fibroblasts that requires intact DNA binding, CBFB interaction and C-terminal transcriptional activation/repression domains. However, surprising differences were found between the major RUNX1 fusion oncoprotein derivatives. The N-terminal fusion protein TEL-RUNX1 fails to induce senescence despite retention of a virtually full-lenght RUNX1 moiety, while the senescence-inducing potential is exaggerated in the truncated C-terminal fusion protein RUNX1-ETO (AML1-ETO). The potential to drive senescence is retained by the deletion mutant RUNX1-ETO[]469 which lacks critical corepressor binding sites suggesting that the repression of target genes may be a primary mechanism implicated in RUNX1-ETO induced senescence. Interestingly, CBFB-MYH11 fusion oncoprotein that affects RUNX1 indirectly by targeting CBFB cn also induce senescence when ectopically expressed in human primary cells. The RUNX1 and RUNX1-ETO induced senescent phenotypes differ from archetypal H-Ras [superscript v12] as arrest occurs without a preliminary phase of proliferation and the arrested cells lack prominent foci of DNA strand breaks and chromatin condensation. Notably however, RUNX1 and RUNX1-ETO display differences in their potency and the extent of engagement of p53 and Rb effector pathways. RUNX1-ETO is highly dependent on p53 function and unlike RUNX1 drives senescence in cells lacking intact p16Ink4a. RUNX1-ETO appears to exert its unique effects through potent induction of reactive oxygen species and p38MAPK phosphorylation. These findings illustrate the heterogeneous manifestations of senescence-like growth arrest and elucidate the distinctive biology and oncogenic properies of RUNX1 and its fusion derivatives.
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