Association of increased autophagic inclusions labeled for β-galactosidase with fibroblastic aging
Introduction
Replicative senescence is defined as an irreversible state of growth arrest during G1 phase of the cell cycle, occurring after a finite number of cell divisions (Hayflick and Moorehead, 1961). At that time, a majority of cells presents specific senescent characteristics. Most of these features have been determined on fibroblast cellular models. They include morphological modifications such as increased size and transparence, rough and hairy appearance (Hayflick, 1965), specific senescence-associated-β-galactosidase expression at pH 6 (Dimri et al., 1995), inability to divide and to re-enter the cell cycle after stimulation by physiological mitogens (Hayflick, 1965) and reduction of telomere length (Allsopp and Harley, 1995).
Lipofuscin was found to accumulate during aging in several cell types (Glees and Hasan, 1976) and experimentally an inhibition of lysosomal enzymes induces such an accumulation (Ivy et al., 1984). Brunk and Terman (2002) suggested that, in aging cells, lysosomal enzymes are in short supply leading to an accumulation of lipofuscin which could be involved in cellular death.
Cultured senescent cells do not die immediately (Linskens et al., 1995), and the mechanisms of their death remain incompletely understood. While following serum deprivation, senescent human fibroblasts were found to be resistant to the induction of apoptotic death (Wang, 1995), p53-independent apoptosis and necrosis were observed in senescent human fibroblasts after several genotoxic stresses (Seluanov et al., 2001). The best-known types of cell death are necrosis, and two programmed cell deaths, i.e. apoptosis and autophagy. Autophagy or type II programmed cell death (Bursch et al., 1994) is a physiological mechanism occurring in many cell types whereby intracellular organelles and cytosol are first sequestered away from the remaining cytoplasm and then degraded within lysosomes via a specific vacuole formation leading to degradative vacuole, or autolysosome, in which lipofuscin may be found (Dunn, 1990a, Dunn, 1990b).
Autophagy is regulated by a protein system, first described in yeasts, made up of more than 14 proteins (Apg: autophagy defective genes). Various groups of genes have been identified. One group of genes is involved in the induction of autophagy and the formation of autophagosome (mTor, Apg 1, Apg 6, Apg 13, Apg 17: kinase signaling system and Apg 5, Apg 7, Apg 10, Apg 12, Apg 16: Apg protein conjugation system). Another group of genes is involved in size regulation of the autophagosome. AUT2 and AUT7 corresponding to the MAP LC3 human gene belong to this group (Lang et al., 1998). Beclin-1 human gene is structurally similar to the yeast autophagy gene, Apg 6 (Kametaka et al., 1998). It encodes a 60 kDa protein that interacts with the prototypic apoptosis inhibitor Bcl-2 (Liang et al., 1998) and promotes autophagy in autophagy-defective yeast with a targeted disruption of Apg 6 and in human MCF7 cell line (Liang et al., 1999). Thus its study was favoured.
The aim of this study was to determine the emergence of subcellular modifications corresponding to autophagic vacuoles, leading to lipofuscin accumulation during replicative senescence. For this purpose, we analyzed the cellular morphology and autophagy-associated gene expression in both young and old MRC5 fibroblasts, chosen as a model for replicative senescence study. As cells acquired senescent characteristics, we detected (i) a significant increase in autophagic inclusions, (ii) an unchanged level of autophagy regulating gene expression and (iii) a β-galactosidase activity clustered in autophagic vacuoles. These data suggest an accumulation of degradative autolysosomes with fibroblast aging.
Section snippets
Cellular material
Twelve population doubling (PD) normal human diploid fibroblasts MRC5 (5×106) were cultivated in a 75 cm3 flask with DMEM (Gibco-BRL, Cergy Pontoise, France), supplemented with 10% fetal calf serum (Gibco-BRL, Cergy Pontoise, France), 200 mM l-glutamine (Seromed, Berlin, Germany), 100 IU/ml penicillin, 50 μg/ml streptomycin (Gibco-BRL, Cergy Pontoise, France). These were kept in a humid incubator with 5% CO2. Fibroblasts continuously underwent approximately 67 passages. For electron microscopy
Results
The study of autophagic events was performed on both young and senescent MRC5 fibroblasts in order to detect possible modifications induced by in vitro cell aging.
Discussion
These data represent, to our knowledge, the first demonstration of an accumulation of autophagic vacuoles related to an enhanced β-galactosidase activity in senescent cells, meaning an increase of lysosomal mass and an accumulation of degradative vacuoles and lipofuscin with aging.
After labeling of MRC5 fibroblasts by monodansylcadaverine, a specific marker of autophagic vacuoles (Biederbick et al., 1995, Munafó and Colombo, 2001), an increase of autophagic inclusions was observed with the
Acknowledgements
We wish to thank C. Magba for technical assistance and D. Clausen for his help with the English version.
Grant numbers and sources of support: Region Rhône-Alpes: contract no. 00 81 60 45; Hospices Civils de Lyon; Ligue contre le Cancer du Rhône; Association pour la Recherche contre le Cancer.
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