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Research Description

Stress-Induced Premature Senescence (SIPS) as long-term effects of subcytotoxic stress in human ageing

  1. Molecular mechanisms of SIPS

  2. Genes/proteins differentially expressed in SIPS. Longevity candidates.

  3. In vitro toxicology & SIPS induced by oxidative stress and UVB

  4. SIPS triggered by proinflammatory cytokines

  5. Theoretical models: cellular ageing, evolution & stress

  6. Role of proteasome in SIPS

  7. Energy metabolism against SIPS and cell death

  8. Reactive oxygen species and cell death (before 1996)

1. Molecular mechanisms of SIPS

Induction of biomarkers of senescence by subcytotoxic stress

Serial cultures of HDFs (human diploid fibroblasts) lead to replicative senescence. Oxidative stress and UVB stress just under the threshold of cytotoxicity precipitates the appearance of many biomarkers of replicative senescence. After stress, the cells are always given several days of recovery to discriminate between the short- and the long-term effects of stress. Several biomarkers of senescence were examined: Senescence-Associated b-galactosidase activity (SA b-gal, concentration-dependent shift from 'younger' to 'older' morphotype with a strong positive correlation between the morphotypes and the cells positive for SA b-gal, decreased ability to duplicate DNA, increase in mRNA steady-state level of cyclin-dependent kinase inhibitors, hypophosphorylation of the retinoblastoma protein, similar change of the steady-state level of mRNA of several genes in senescent HDFs and in HDFs in SIPS, common 4,977 bp deletion of mitochondrial (mt) DNA. A novel deletion was discovered in HDFs in SIPS and was sequenced. A sophisticated method of quantitative PCR was developed to quantify the ratio of deletions compared to the number of total mt DNA copies and to quantify the formation of heteroduplexes between the amplified target DNA and the internal standard, thereby allowing more accurate quantifications.

Research articles :70, 68, 67, 66, 65, 62, 61, 56, 42, 39, 36, 35, 33, 4

Conceptual & review articles :64, 63, 55, 57, 48, 38, 36, 32, 29, 18, 15

Role of TGF-b1

TGF-b1 was found to trigger the appearance of SA b-gal, senescent morphology and increase in the steady-state mRNA level of four senescence-associated genes. Subcytotoxic H2O2 stress induced an increase in TGF-ß1 steady-state mRNA level and an increased release in TGF-b1. Antibodies against TGF-b1 or TGF-ß1 receptor II prevented the appearance of SA-bgal, senescent morphology and increase in steady-state mRNA level of the 4 senescence-associated genes. It was also shown that the retinoblastoma protein controls the appearance of those biomarkers. The signaling cascade involved was studied: H2O2 activates p38 MAPK and ATF-2 which leads to TGF-b1 overexpression, which in turn locks the loop responsible for p38MAPK sustained activation. At 24 hrs after stress, hypophosphorylated Rb starts to interact with ATF-2, which allows the appearance of the biomarkers of senescence.

Research articles : 68, 56, 52, 40

Role of telomeres

The role of telomeres in the establishment of SIPS was also studied in hTERT-positive HDFs and in normal HDFs. SIPS also appeared in the telomerase positive HDFs. The telomere shortening observed after such subcytotoxic stress under H2O2 or UVB was not sufficient to reach the telomere length observed in replicative senescence.

Research articles : 66, 51

Review articles : 61, 60

2. Genes/proteins differentially expressed in SIPS. Longevity candidates

Proteome & transcriptome analyses are being performed in 2 projects of the European 5th Framework programme to identify proteins and mRNAs involved in SIPS in comparison with non-stressed cells and normally senescent cells (34).

Proteome analysis was based on high resolution 2D-gels, with 6 samples per situation (young cells, old cells, cells in SIPS, control cells) arising from independent cultures. In total 60 gels have been analysed. The results gave 64 proteins undergoing expression changes out of 2219 analysed in each gel. Using the latest developments of nano-electrospray and MALDI MS, we already identified 30 of these 64 proteins.

Transcriptome analysis is being performed in the same experimental conditions using DD RT-PCR and DNA microarrays with senescent cells, cells under SIPS and appropriate controls. Several mRNA species have been confirmed to be differentially expressed in SIPS when compared to young cells.

The roles of the genes / proteins found in these screening studies are being analysed (transfection studies, etc). For instance, overexpression of apolipoprotein J has been shown to protect against SIPS Other genes will be tested as longevity candidate in transgenic animals. The molecular mechanisms of protection will be studied. Unknown genes will be characterized.

Research articles : 72, 67, 65, 50, 34

Conceptual & review articles : 74, 60

3. In vitro toxicology and SIPS induced by oxidative stress and UVB

No in vitro method exists for testing the long-term effects (in terms of days and weeks) of non-cytotoxic concentrations of xenobiotics on cell fate. The need is urgent for the development of such methods using normal (nonmalignant) human cells. We proposed methods based on the demonstration that repeated exposures of HDFs to noncytotoxic concentrations of xenobiotics might trigger the appearance of biomarkers of senescence. For instance, antineoplasic molecules at concentrations as low as 10-7 M and repeated exposures to low levels of UVB were shown to induce SIPS after several incubations of 2 hrs. These methods allow a decrease of the budgetary and ethical burden of animal testing. All the experimental models of SIPS developed allow to test new compounds as for instance in UVB-induced SIPS. Microarrays bearing DNA of genes undergoing senescence or SIPS-specific expression changes are constructed. We shall not neglect the potential applications arising from the discovery of genes involved in SIPS.

Research articles : 62, 58

Conceptual & review articles : 74, 32

4. SIPS triggered by proinflammatory cytokines

The signalling pathways of IL-1a and TNFa are generally presented as generating transient increases in reactive oxygen species. After 3 to 5 repeated stimulations of HDFs for 1h/day followed by 3 days of recovery, a significant shift of the HDFs morphotypes to the 'older' morphotypes, and a significant increase in the proportion of HDFs positive for the SA b-gal activity were observed. The antioxidants vitamine E and N-acetyl-cysteine protected against these changes. These were among the first results suggesting a direct pro-ageing effect of pro-inflammatory cytokines after repeated stimulations (21, 41). Recent arguments have been produced in the literature in favor of the in vivo occurence of this phenomenon (61).

5. Theoretical models : cellular ageing, evolution & stress

Far-from-equilibrium thermodynamics allows a global systemic description of the cellular behaviour. This approach transcends the genetic and stochastic considerations on ageing as well as some evolutionary questions about ageing. The fundamental difference between the processes of development and ageing could reflect the intrinsic differences existing between biological systems where an increase in specific entropy production is respectively still possible or not. The increase of the potential of specific entropy production which probably occurred with evolution might explain in part why life span could increase. However this specific entropy production-driven increase in life span was possible only in those species which did not take advantage of their increased potential of specific entropy production to ameliorate their reproductive capacity at the expense of possible increases in repair capacity. The criteria of stability of far-from-equilibrium open systems and the theory of attractors also help to sort the possible types of cellular stress responses: normal ageing, hormesis, stress-induced premature senescence, apoptosis or necrosis. This approach also allowed to consider the role of evolution and calorie restriction in ageing.

Theoretical articles : 1, 18, 28, 36, 49, 58, 59

6. Role of proteasome in SIPS

In a project of the European 5th Framework programme with 6 other laboratories, we study the role of proteasome in SIPS (73, 31).

7. Energy metabolism against SIPS and cell death

We have shown that a decrease in the capabilities of the cells to regenerate ATP also affected the appearance of the biomarkers of SIPS such as morphotype transition and S-A b-gal activity (18, 36). Bilobalide, which stimulate the mitochondrial energy metabolism, was shown to be protective against SIPS (16).

Neuroblastoma cells differentiated with NGF (17) and HDFs were exposed to oxidative conditions together with partial uncoupling of the mitochondrial respiration, or decrease level of substrates of the energy metabolism, replacing for instance D-glucose with L-glucose (9). A synergistic effect of cell death took place between the impairment of energy metabolism and oxidative stress. Naftidrofuryl oxalate and bilobalide, which stimulate the mitochondrial energy metabolism, were shown to be protective against oxidative stress-induced cell death (9, 16, 23).

8. Reactive oxygen species and cell death

A comprehensive review on the work carried out by our laboratory in this field before 1994 is available (8). A mathematial model of the antioxidant system was generated that contained the main reactions of production and elimination of reactive oxygen species (3), which as tested experimentally (7). We also generated a mathematical model of oxidative stress-induced cell death which considers both the stressor concentration and the time necessary for the cells to resume mitosis (24).