All organisms respond to breaks in the DNA by promptly launching the DNA-damage response (DDR). This involves the recruitment of DNA repair proteins to sites of DNA damage and the activation of “checkpoint” activities that slow down or arrest cell-cycle progression, thus delaying key cell cycle transitions until the damage has been removed. In multicellular organisms, the inability to repair DNA damage and/or prolonged checkpoint activation can lead to programmed cell death or cause the cell to enter a permanent cell cycle arrest known as cellular senescence. The goal of my PhD period was to understand the mechanisms that lead to cellular senescence as opposite to transient checkpoint activation and to discover the role of novel modulators of the senescence condition, always in relation to DDR functions. It has been previously shown that telomere-initiated cellular senescence is associated with the activation of DDR. In my PhD thesis, I have shown that a DDR is persistently active during cellular senescence and it is necessary for its maintenance. I also investigated how the DDR signalling pathway remains active up to years after initial senescence establishment. My results support the conclusion that the presence of persistent DDR foci in senescent cells is due to their intrinsic irreparability. I therefore believe that the difference between transient checkpoint activation and senescence is the presence, in the latter, of irreparable DNA damage leading to protracted DDR signalling. I next attempted to probe the molecular features of irreparable DNA breaks. I discovered that exposing human normal fibroblasts to ionizing radiation can lead to the generation of DNA damage, a small portion of which, is not repairable. Therefore not all DNA breaks are efficiently repaired in the human genome. I also observed that persistent DDR is preferentially located at the telomeres or in their proximities. My present working hypothesis is that if DNA damage occurs at a telomeric site, it is not efficiently repaired and this leads to a chronic DDR activation. I also extended these observations to DNA damaging agents commonly used in cancer therapy and known to induce cellular senescence. I observed that also these compounds lead to the preferential generation of irreparable DNA damage in close proximity to telomeric tracts. Thus, my data indicate that telomeres are sinks of irreparable DNA damage and that are a genomic locus that, once damaged, enforces senescence. In this thesis, I also discuss my contribution to another project being pursued in my group, namely the study of the mechanisms that cause DDR activation in oncogene-induced senescence (OIS). In particular, I studied the contribution of reactive oxygen species (ROS) production following oncogenic Ras expression. I found that ROS are mitogenic mediators of oncogenic Ras and play a causative role in OIS by sustaining the hyperproliferation phase preceding OIS establishment. My results therefore highlight an unanticipated role of ROS in mediating senescence. In addition to their cell-autonomous functions, senescent cells can alter the tissue microenvironment and affect neighbouring cells through paracrine signalling. This phenomenon is mediated by the secretion of various chemokines and chemokine receptors. In this thesis, I describe the results obtained in a collaborative project with the Cell Proliferation Group (MRC Clinical Sciences Centre, Imperial College, London, UK) lead by Jesus Gil, in which I studied the contribution of the chemokine receptor CXCR2 in OIS. I discovered that CXCR2 control DDR functions both in transient checkpoint assays and in senescent cells. My results therefore indicate that DDR is controlled also by extracellular cues. Finally, in the appendix of my PhD thesis, I will present some results that I obtained during the first two years of my PhD program, but despite further investment in terms of time and efforts, we decided not to purse any further. These data point to a potential link between ROS, senescence and DDR activation. Indeed, it is already known that cellular senescence and aging are associated with an increase of cellular oxidative stress. In my study, I highlighted a signalling active role of ROS in maintenance of cellular senescence, in the absence of activated oncogenes: I found that the mitochondrial ROS increase observed both in telomere dysfunctional-induced and in irradiation-induced cellular senescence, and ROS play a role in mediating the signalling cascade that sustains DDR activation, therefore positively controlling the maintenance of the senescence state.
|Titolo:||Characterization of the mechanisms controlling the DNA damage response in human cellular senescence|
|Data di pubblicazione:||2008|
|Parole Chiave:||telomere-induced cellular senescence ; persistent DNA damage response ; checkpoint ; ROS ; chemokines ; oncogene-induced cellular senescence ; DNA hyper-replication|
|Settore Scientifico Disciplinare:||Settore MED/04 - Patologia Generale|
|Citazione:||Characterization of the mechanisms controlling the DNA damage response in human cellular senescence ; Tutor. P.G. Pelicci, supervisore: F. d'Adda di Fagagna, A. Musacchio, J. Bartek. - Milano : Università degli studi di Milano. FACOLTA' DI MEDICINA E CHIRURGIA, 2008. ((19. ciclo, Anno Accademico 2006/2007.|
|Appare nelle tipologie:||13 - Tesi di dottorato discussa entro ottobre 2010|