Identification of differentially expressed proteins in
senescent human embryonic fibroblasts
Ioannis P. Trougakosa, Aggeliki Saridakib, George Panayotoub,
Efstathios S. Gonosa,*
aLaboratory of Molecular & Cellular Ageing, Institute of Biological Research & Biotechnology,
National Hellenic Research Foundation, 48 Vas. Constantinou Ave., Athens 11635, Greece
bProtein Chemistry Laboratory, Biomedical Sciences Research Center ‘‘Alexander Fleming’’, Athens 16672, Greece
Received 15 July 2005; accepted 24 August 2005
Available online 5 October 2005
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, Ca2+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.
# 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: 2D PAGE; Ageing; Human fibroblasts; Mass spectrometry; Proteomics; Replicative senescence
Ageing is the outcome of complicated interactions between
genetic factors and the accumulation of a variety of deleterious
stochastic changes overtime (Kirkwood, 2002). Human ageing
can be studied in vitro. Specifically, normal human fibroblasts
undergo a limited number of divisions in culture and
progressively reach a state of irreversible growth arrest, a
process termed as replicative senescence (RS). Replicative
senescence occurs because, owing to the biochemistry of DNA
replication, cells acquire one or more critically short telomere
(Holliday, 1996). Senescent cells have recently been shown to
accumulate with age in human tissues and it, thus, has been
proposed that they contribute to organismal ageing (Campisi,
2000). Moreover, senescent cells acquire phenotypic changes
that may contribute to certain age-related diseases, including
late-life cancer (Campisi, 2005).
Several genes that are linked to senescence have been
successfully identified and cloned on the basis of mRNA
expression changes between young and senescent cells (Gonos
et al., 1998; Lee et al., 1999; Ly et al., 2000). However, these
studies do not consider theweakness of the correlation between
a given level of transcripts and the abundance of the
corresponding proteins. It is well established that analysis at
the level of protein expression can provide additional and
complementary information. The development of proteomic
analysis methods using high-resolution two-dimensional gel
electrophoresis(2DGE) and mass-spectrometry (MS) offers the
advantage of identifying directly changes at the protein level.
Regarding senescence, this methodology has been successfully
applied in identifying new biomarkers during stress-induced
premature senescence (SIPS) in human diploid fibroblasts
(HDFs) (Dierick et al., 2002), as well as in conditionally
immortalized rat embryo fibroblasts (Benvenuti et al., 2002). In
this study,we have performeda proteomic comparison between
Mechanisms of Ageing and Development 127 (2006) 88–92
* Corresponding author. Tel.: +30 210 7273756; fax: +30 210 7273677.
E-mail address: firstname.lastname@example.org (E.S. Gonos).
0047-6374/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.