Review
From the Hayflick mosaic to the mosaics of ageing.: Role of stress-induced premature senescence in human ageing

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Abstract

The Hayflick limit—senescence of proliferative cell types—is a fundamental feature of proliferative cells in vitro. Various human proliferative cell types exposed in vitro to many types of subcytotoxic stresses undergo stress-induced premature senescence (SIPS) (also called stress-induced premature senescence-like phenotype, according to the definition of senescence). The known mechanisms of appearance the main features of SIPS are reviewed: senescent-like morphology, growth arrest, senescence-related changes in gene expression, telomere shortening. Long before telomere-shortening induces senescence, other factors such as culture conditions or lack of ‘feeder cells’ can trigger either SIPS or prolonged reversible G0 phase of the cell cycle. In vivo, ‘proliferative’ cell types of aged individuals are likely to compose a mosaic made of cells irreversibly growth arrested or not. The higher level of stress to which these cells have been exposed throughout their life span, the higher proportion of the cells of this mosaic will be in SIPS rather than in telomere-shortening dependent senescence. All cell types undergoing SIPS in vivo, most notably the ones in stressful conditions, are likely to participate in the tissular changes observed along ageing. For instance, human diploid fibroblasts (HDFs) exposed in vivo and in vitro to pro-inflammatory cytokines display biomarkers of senescence and might participate in the degradation of the extracellular matrix observed in ageing.

Section snippets

The Hayflick limit today

In 1961 Hayflick and Moorhead reported that human diploid fibroblasts (HDFs) divide a finite number of times. Hayflick called ‘phase I’ the primary culture, ‘phase II’ the many cumulative populations doublings (CPDs) of luxuriant growth after the primary culture, and ‘phase III’ the subsequent growth arrest [1], [2]. In 1974 ‘phase III’ was termed ‘the Hayflick limit’ [3]. The phrase ‘replicative senescence’ is now widely used. Medline refers to more than 6100 papers with ‘replicative

Effects of culture conditions on senescence

The deregulation of the mitotic machinery of HDFs during in vivo ageing of middle-aged and late-aged donors was analysed with DNA μarrays. Many changes were found in the expression level of genes necessary for the cell cycle to proceed [14]. Most of these genes, however, are different from those undergoing expression changes in in vitro replicatively senescent HDFs. This suggests that in vivo senescence is somewhat different from in vitro replicative senescence.

When cultured under 3% O2, i.e.

The Hayflick mosaic

In vivo the proliferative capacity of HDFs is never completely exhausted. HDFs of centenarians are still able to divide in vitro, sometimes for a number of CPDs that renders them undiscriminable from explants of HDFs of young donors [43]. Stem cells are present in the connective tissues of dermis and skeletal muscle derived from geriatric humans. These cells contain lineage-committed myogenic, adipogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineage-uncommitted

The mosaics of ageing

The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening, which counts cell generations [11] (definition 1). Other authors enlarged this definition to a functional definition encompassing all kinds of irreversible arrests of proliferative cell types including that induced by damaging agents (definition 2) [6]. Irreversible growth arrest of proliferative cell types induced by damaging agents may be called stress-induced senescence-like phenotype,

SIPS in vitro

Many proliferative cell types (lung and skin HDFs, human melanocytes, endothelial cells, human retinal pigment epithelial cells, human erythroleukemia cells, etc.) exposed to subcytotoxic stress (UV, organic peroxides, H2O2, ethanol, mitomycin C, hyperoxia, γ-irradiations, homocysteine, hydroxyurea, tert-butylhydroperoxide (t-BHP) etc.) undergo SIPS. SIPS can be defined as the long term effects of subcytotoxic stress on proliferative cell types, including irreversible growth arrest of (a

Growth arrest

HDFs in H2O2-induced SIPS cannot launch a mitogenic response when stimulated with serum or usual growth factors [68]. Most of HDFs in H2O2-induced SIPS are growth arrested in the G1 phase of the cell cycle [65]. Hyperoxia under 40% O2 also leads to growth arrest of HDFs in the G1 phase [69].

The proportion of HDFs positive for SA β-gal activity correlates with CPDs. It also increases in SIPS, induced by tert-butylhydroperoxide (t-BHP) and H2O2 (for a review: [64]). SA β-gal activity is rather a

Is telomere shortening involved in SIPS?

WI-38 HDFs kept under 40% O2 for 3 CPDs undergo SIPS. An accelerated telomere restriction fragment (TRF) shortening (500 bp/PD) is observed [69]. Forty percent O2 induces single-strand breaks and accelerated TRF shortening of human retinal pigment epithelial cells [89]. WI-38 HDFs undergo accelerated TRF (490 bp/stress) and irreversible growth arrest after four exposures to subcytotoxic t-BHP stress, with a stress at every 2 CPDs. After these stresses, the cells stop proliferating. The control

Hayflick mosaic or mosaics of ageing?

From definition 1 of senescence, the fact that HDFs can endure more PDs at physiological low O2 partial pressures decreases the probabilities to find senescent cells based on the end-replication problem in vivo. Theoretically, starting from the two first telomerase-negative cells that appear during in vivo differentiation, and that will become fibroblasts (in the case of symmetric divisions) 250 cells (>1015 cells) must be produced before the first telomere-dependent replicatively senescent

Conclusions

The models of SIPS are already used in toxicology to seek possible long term effects of molecules in R&D. One can also detect possible anti-ageing effects of molecules in human cells in SIPS. Automation of the models of SIPS will lighten the budgetary and ethical burden of in vivo tests [120]. These systems give more useful mechanistic information compared to the information gained when using lower invertebrate animals as toxicological models.

The appearance of SIPS could be due to exacerbated

Acknowledgements

O. Toussaint is a research associate and F. Chainiaux a research assistant of the FNRS, Belgium. J.-F. Dierick, C. Frippiat, P. Dumont are FRIA fellows, Belgium. J. P. Magalhaes thanks FCT, Portugal. We thank the European Union, 5th Framework Programme, Quality of Life, R&D, ‘Protage’ (QLK6-CT-1999-02193) and ‘Functionage’ (QLK6-CT-2001-00310) and the Région Wallonne Project ‘Modelage’.

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