A comparative analysis of the proteins between the fibroblasts from Werner's syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis

Abstract

Werner's syndrome (WS) is an autosomal recessive disorder causing symptoms of premature aging. The fibroblasts of WS patients have a shorter life-span than normal fibroblasts. We analyzed the fibroblast proteins from three WS patients and from three age-matched normal individuals using two-dimensional gel electrophoresis and image processing. The expressions of 12 proteins were shown to be augmented or suppressed in WS fibroblasts compared with normal fibroblasts: 11 of 12 spots on the electrophoresis gel of WS fibroblasts were denser than the corresponding spots of normal individual fibroblasts, while the remaining one spot was fainter in WS fibroblasts than in normal fibroblasts. The abundance of these proteins were compared to those of the corresponding proteins from normal fibroblasts at various cell passages in vitro reported in the TMIG-2DPAGE database. The result shows that the change in the protein patterns in in vitro aging did not necessarily correspond to the change in WS fibroblasts, except for three proteins abundant in WS fibroblasts, which increased their abundance during in vitro aging. These results suggest that the premature aging process of WS fibroblasts shares only part of the in vitro aging process of normal fibroblasts.

Introduction

Werner's syndrome (WS) is a typical human segmental progeroid disorder accompanied by a number of symptom phenotypes of premature aging (Epstein et al., 1966, Goto et al., 1981a). These symptoms include atherosclerosis, ocular cataracts, type II diabetes mellitus, osteoporosis, early graying, loss of hair and an aged appearance. Some differences, however, exist between WS and normal aging, such as ulceration of the skin, severe osteoporosis of the limbs and rare cancers in WS patients (Epstein et al., 1966, Goto et al., 1981b). The WS gene, whose locus was placed on the short arm of chromosome 8 (Goto et al., 1992Schellenberg et al., 1992), and was recently cloned by Yu et al. (1996), encodes a protein with 1432 amino acid residues with a significant homology to the E. coli RecQ helicase. A total of 19 mutations have been found thus far by Yu et al., 1996, Werner's Syndrome Collaborative Group, 1997Oshima et al. (1996)and by us (Goto et al., 1997Matsumoto et al., 1997).

One of the features characteristic of WS is that the replicative life-span of the fibroblasts from WS patients is substantially shortened compared to that of the fibroblasts from age-matched normal individuals. Hayflick and Moorhead (1961)first described that human diploid fibroblasts had a limited life-span in in vitro replication and provided an in vitro model for cellular senescence. However, the molecular mechanism responsible for the cellular senescence still remains to be clarified.

Recent studies have shown a number of important molecules and mechanisms that may be associated with cellular senescence, such as p21/Waf1/Cip1/Sdi1 (El-Deiry et al., 1993, Xiong et al., 1993, Harper et al., 1993, Noda et al., 1994), p66/mortalin (Wadhwa et al., 1993) and telomere and/or telomerase (Harley et al., 1990). The p21/Waf1/Cip1/Sdi1 molecules interact with Cdk family proteins, leading to the inhibition of cell proliferation and they are expressed better in senescent cells than in younger cells. Telomere length likely determines the maximal replicative capacity of human cells without telomerase activity. Mortalin, belonging to the heat shock protein 70 family, inhibits proliferation in senescent cells. Thus, multiple changes in gene expression are likely to be involved in the process of normal cell senescence. A number of studies have been made on the gene expression by pre-senescent and senescent normal fibroblasts and by WS fibroblasts, which showed that normal senescent cells and WS cells share some over-expressed mRNAs, including those for extracellular matrix proteins (Murano et al., 1991). On the other hand, Oshima et al. (1995)reported that the levels of c-fos expression were different between normal senescent fibroblasts and WS fibroblasts. These results suggest that normal senescent cells and WS cells may not necessarily share all the mechanisms underlying cellular senescence.

Little is known about the protein metabolism of normal senescent cells and WS cells, although we know much about the transcription products, as mentioned above. In this regard, the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) technique established by O'Farrell (1975), has been accepted as a powerful tool to analyze cellular protein alteration during differentiation, immortalization and aging. Celis et al., 1989, Celis et al., 1990constructed a two-dimensional gel cellular protein database using MRC-5 human embryonic lung fibroblasts and used it to identify polypeptides whose relative abundance differs between quiescent, proliferating and SV40 transformed cells; their 2-D gel protein database, however, included little information about cellular aging. We have recently established a TMIG-2DPAGE protein database (Toda and Kimura, 1997) based on a new concept of the 2-D gel protein database mainly directed toward gerontology research, which is now open to the Internet computer network on our world wide web (WWW) home page. In this paper, we report an application of the TMIG-2DPAGE protein database to screen WS-associated proteins.