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Research summary and technological platforms

Back in 1995, the possibility of interplay between the free radical theory and Replicative Senescence (RS) was in its infancy.  Several laboratories started to study the effects of short sublethal exposures to various stressors like ethanol, hydrogen peroxide, tert-butylhydroperoxide, or UV, on the proliferative life span of human diploid fibroblasts, the best model of cellular senescence of proliferative cell types, so far. The preliminary findings were that such stress triggered irreversible growth arrest and senescent morphogenesis. Nothing was known on the effect of such stress on other biomarkers of cellular senescence, nor were known the molecular mechanisms explaining how such stress triggers the appearance of a senescent-like phenotype. In year 2000, we introduced the concept of stress-induced premature senescence (SIPS) and molecular scars, as long-term effects of subcytotoxic exposures to various types of stress.

Since 2000, we showed that the most prominent cellular & molecular biomarkers of RS regulation exist in SIPS, senescence-associated changes in the molecular regulation of the cell cycle, deletions in the mitochondrial DNA, senescence-associated changes in the expression level of genes which protein products play a role in the extracellular matrix, in morphogenis or in cell defense against stress., etc. We figured out the signal transduction processes triggering SIPS. We focussed on the role of the stress-induced increase in the secretion rate of the inactive form of Transforming Growth Factor-beta I (TGF-ß1), cleaved in its active form in the extracellular matrix. The increase abundancy of this active form autocrinely sustains the activation of signaling kinases and transcription factors involved in the expression of most biomarkers of senescence. We discovered that TGF-ß1 plays a central role in the appearance of the senescent phenotype after stress with ROS or UVB. We investigated the extent of telomere shortening and proteasome in SIPS induced by acute subcytotoxic stress.

We identified, by mass spectrometry after proteomic comparison, 30 proteins involved in SIPS and/or RS, which gave further information on the metabolism of cells in premature senescence, as for instance an increase of the glycolytic pathway. We set up genomic platforms for highlighting gene expression changes occuring when SIPS establishes, based on an in-house developed technology of low density DNA arrays (‘Senechips’, ‘Generalchip’) which give reproducible and reliable data on the changes of genee expression at the transcript level, giving further clues on the molecular mechanisms involved in the appearance of the prematurely senescent phenotype. All the data accumulated alllowed the functional investigation of the role of proteins / genes with changed expression level in RS and/or SIPS, for intance by ectopically overexpressing, using retroviral infection, some of the genes of interest, like for instance clusterin. We created a bioinformatic databank on ageing, that allowed the identification of other possible mechanisms intervening in human ageing. Lastly we validated our experimental and technological approaches as tools suitable for in vitro toxicology anddeveloped bioethically safe protocols to collect skin biopsies, in order to expand our research to in vivo findings .

Our results, together with the advancements performed by the scientific community now allows to predict a role for stress induced premature senescence in vivo, in human ageing.

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