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: 3.4). 02/2006; 127(1):88-92. DOI: 10.1016/j.mad.2005.08.009
Source: PubMed


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.
    Oxidative Medicine and Cellular Longevity 03/2013; 2013(5, supplement 1):454328. DOI:10.1155/2013/454328 · 3.36 Impact Factor
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    • "The free radical theory of aging suggests that the aging process involves the accumulation of oxidative damage to cells and tissues, which progressively increases morbidity and mortality (1). Human aging can be studied in vitro using normal human diploid fibroblasts (HDFs), which undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest; this process is termed replicative senescence (2). "
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    ABSTRACT: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. The cell cycle was arrested in the G(0)/G(1) phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G(0)/G(1) phase and increased cell populations in the G(2)/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.
    Clinics (São Paulo, Brazil) 02/2012; 67(2):135-43. DOI:10.6061/clinics/2012(02)08 · 1.19 Impact Factor
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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 · 3.40 Impact Factor
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