Identification of differentially expressed proteins in senescent human embryonic fibroblasts

Biomedical Sciences Research Center Alexander Fleming, Βάρη, Attica, 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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    • "About half of the senescence-induced changes were identified in proteins involved in cytoskeletal organization. These included two isotypes of actin and cytokeratin, key structural proteins with major biological roles in early and late cellular development phases and status202122. The actin-binding proteins cofilin and transgelin are described to be crucial regulators of actin dynamics [23] . "
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    ABSTRACT: . Senescence of peritoneal mesothelial cells represents a biological program defined by arrested cell growth and altered cell secretory phenotype with potential impact in peritoneal dialysis. This study aims to characterize cellular senescence at the level of global protein expression profiles and modification of proteins with O -linked N-acetylglucosamine ( O -GlcNAcylation). Methods . A comparative proteomics analysis between young and senescent human peritoneal mesothelial cells (HPMC) was performed using two-dimensional gel electrophoresis. O -GlcNAc status was assessed by Western blot under normal conditions and after modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease O -GlcNAcylation or O -(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N -phenyl carbamate (PUGNAc) to increase O -GlcNAcylation. Results . Comparison of protein pattern of senescent and young HPMC revealed 29 differentially abundant protein spots, 11 of which were identified to be actin (cytoplasmic 1 and 2), cytokeratin-7, cofilin-2, transgelin-2, Hsp60, Hsc70, proteasome β -subunits (type-2 and type-3), nucleoside diphosphate kinase A, and cytosolic 5′(3′)-deoxyribonucleotidase. Although the global level of O -GlcNAcylation was comparable, senescent cells were not sensitive to modulation by PUGNAc. Discussion . This study identified changes of the proteome and altered dynamics of O -GlcNAc regulation in senescent mesothelial cells. Whereas changes in cytoskeleton-associated proteins likely reflect altered cell morphology, changes in chaperoning and housekeeping proteins may have functional impact on cellular stress response in peritoneal dialysis.
    Full-text · Article · Nov 2015
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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.
    Full-text · Article · Mar 2013 · Oxidative Medicine and Cellular Longevity
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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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    Full-text · Article · Feb 2012 · Clinics (São Paulo, Brazil)
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