Differential Proteome Analysis of Replicative Senescence in Rat Embryo Fibroblasts

Abstract

Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and ␣-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4␤, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence.

Heat Shock Protein 27 Is the Major Differentially Phosphorylated Protein Involved in Renal Epithelial Cellular Stress Response and Controls Focal Adhesion Organization and Apoptosis

“…In the case of ProteinProspector searches, protein identifications were selected as significant hits by using criteria outlined in Ref. 39. A few MS/MS identifications were verified or established by manual data interpretation (de novo sequencing).…”

Differential protein synthesis and expression levels in normal and neoplastic human prostate cells and their regulation by type I and II interferons

“…Tryptic in-gel digestion was essentially performed as described earlier (Benvenuti et al, 2002;Naaby-Hansen et al, 2003). In brief, pieces of 2D gels were destained, dehydrated with 25 mM ammonium bicarbonate (ABC)/50% acetonitrile (ACN), and vacuum-dried.…”

Identification of novel candidates for replicative senescence by functional proteomics

“…The list of proteins that we found to be differentially expressed upon loss of proliferative potential in the conditionally immortalized tsa cell lines was then compared with a similar differential proteome study conducted on replicative senescence of primary REFs upon serial passaging (Benvenuti et al, 2002). This identified nine differentially expressed features, shown in Table 4, that were in common between the two studies even though they had been conducted entirely separately.…”

“…This analysis identified 43 spots whose expression changed more than 1.5-fold upon loss of proliferative potential. The study presented here has been carried out in parallel with another study where we have also used differential proteomics to directly examine replicative senescence in asynchronous cultures of serially passaged Sprague-Dawley REFs (Benvenuti et al, 2002).…”

“…Prior to mass spectrometry, tryptic in-gel digests were carried out on all samples using a protocol similar to already published procedures (e.g., http://donatello.ucsf.edu/ ingel.html). Details of the procedures for peptide mass mapping by matrix-assisted laser desorption/ionization and nano-HPLC electrospray ionization tandem mass spectrometry have been previously published (Benvenuti et al, 2002).…”