DNA damage response (DDR) and cellular senescence
We study the physiological consequences of DNA damage at the cellular and organismal level mainly, but not exclusively, in mammals. Nuclear DNA damage triggers a signaling and effector pathway known as the DNA damage response (DDR) that coordinates cell-cycle arrest (checkpoint functions) and DNA repair. Persistent DDR signaling establishes cellular senescence, a condition in which cells remain alive but permanently unable to further proliferate (Campisi and d'Adda di Fagagna, 2007; d'Adda di Fagagna, 2008).
Figure 1. Mouse metaphase chromosomal spreads. DNA is blue, telomeric TTAGGG repeats are in yellow.
Cellular senescence is crucial both in the study of ageing and cancer. We have worked on both of these fundamental processes.
Most normal human cells can undergo only a limited number of cells divisions before reaching the so-called "Hayflick limit" and entering the non-proliferative condition known as replicative cellular senescence. This arrest is dictated by telomeres, the end of linear chromosomes, when they shorten below a critical length. We have demonstrated that replicative cellular senescence is the outcome of the direct recognition of critically short telomeres and the arrest is DDR dependent. This has firmly placed DDR as a causative mechanism of cellular senescence (d'Adda di Fagagna et al., 2003).
Figure 2. Fabrizio d'Adda di
Fagagna next to Leonard Hayflick.
Oncogene activation in normal human cells is not sufficient to transform them into cancerous cells. Rather it leads to oncogene-induced cellular senescence (OIS). We have demonstrated that oncogene activation is intrinsically a genotoxic event and that the consequent DDR activation is the causative molecular mechanism responsible for senescence establishment. We have shown that oncogene-induced DNA damage is strictly DNA replication-dependent and that activated oncogenes alter the normal DNA replication process. Thus, we have proposed that oncogenes by misregulating DNA replication activate a robust DDR and consequent OIS establishment. Consistent with this, we have observed DDR activation in human tumor samples (Di Micco et al., 2006; Nuciforo et al., 2007).
Figure 3. Fluorescence microscopy image of a senescent cell expressing oncogenic Ras: the phosphorylated form of histone H2AX (γ-H2AX) is stained in green and the DNA damage checkpoint protein 53BP1 is in red, DNA is in blue.
Cellular senescence is associated with the secretion of a complex mix of cytokines. We provided supporting evidence that some interleukins play a senescence-enforcing role by increasing DDR activation, thus establishing a link between DDR signaling and and immune factors (Acosta et al., 2008; Fumagalli and d'Adda di Fagagna, 2009).
Cellular senescence can also be associated with a global increase of heterochromatin. Recently we have shown that heterochromatin is preferentially induced by oncogene-driven DNA replication stress and that it is not lost upon bypass of OIS in vitro and in vivo in human tumors at different stages: these observations challenge its proposed role in the suppression of proliferative genes. Rather, we have shown that heterochromatin acts by restraining DDR induced by oncogenes and that treating oncogene-expressing cells with heterochromatin-disrupting drugs, some of which already in clinical use such as histone deacetylase inhibitors (HDACi), boosts DDR and selectively induces death by apoptosis in a DDR-dependent manner (Di Micco et al., 2011).
DNA damage and cellular senescence
Cellular senescence is associated with persistent DDR signaling. Why DDR persists and DNA damage is not repaired is unknown. We have identified genomic loci that, if damaged, resist repair. We have also identified a potentially evolutionary role for DNA repair resistance. Our model predicts that cellular senescence, caused by different genotoxic stimuli, is the consequence of DNA damage at irreparable genomic sites. Thus, our genome may not be uniformly repairable.
DNA damage accumulation and ageing
DNA damage and DDR activation markers increase during ageing. Although this is often interpreted as the consequence of telomere shortening in proliferating cells, ageing in non-proliferating, such as terminally differentiated, cells requires a different mechanistic model. We are testing whether DDR accumulates in primates also in non-proliferating cells during ageing and at which preferential loci. Thus, we are seeking a model of cellular ageing independent from telomere attrition.
Oncogenes and DNA replication
The full impact of oncogene activation on DNA replication is presently unclear. By molecular DNA combing, we are probing genome-wide the impact of activated oncogenes on the process of DNA replication. Quantitative data are being collected to build mathematical models (in collaboration with John Bechhoefer, Canada) to be in turn experimentally tested.
We are also specifically testing the impact of oncogene activation on telomeres replication, their stability and functions. Thus, oncogene-induced DNA replication alteration may be an underlying mechanism responsible for genome instability and telomeres dysfunction in cancer.
Oncogenes and heterochromatin formation
The mechanisms that lead to oncogene-induced heterochromatin formation are presently unclear. We are probing the potential pathways that may seed the chromatin changes triggered by oncogene-induced DNA replication stress.
Oncogene-induced reactive oxygen species (ROS)
Several oncogenes have been shown to induce a redox stress in the cell. This has been put in relation to both DNA damage generation and cellular senescence. However, the molecular pathways involved are unclear and their impact on genome instability unproven. We have identified a cellular path that leads to ROS accumulation upon oncogene activation and carachterised its role in cell proliferation and genome stability. Thus, ROS may control genome stability by modulating cell proliferation.
DDR regulation in germ stem cellsGerm cells can be clearly identified in the gonad of Caenorahbditis elegans. We have discovered that DDR signaling in these cells is modulated by an essential stemness pathway. We have extended these observations to human cells and we have gained evidence that the pathways identified in C.elegans are evolutionary conserved and operative in humans. We are working to identify the molecular players involved and their role in cancer.
DDR regulation in mammalian somatic stem cells
Whether DDR is differentially modulated in somatic stem cells compared to their more differentiated progenitors and the outcomes of its activation are unknown. By using neural stem cells capable of growing homogeneously as adherent cultures, we discovered that DNA damaging treatments in neural stem cells generate a cellular phenotype related to cellular senescence previously unreported in differentiated cell types. We are studying this novel outcome and the pathways involved. Thus, somatic stem cells have a novel response to DNA damage generation.
DDR regulation upon cellular differentiation
We have discovered both in worms and in mammalian systems that stem cells differentiation may have an impact on DDR regulation. This includes both the suppression of DDR signaling in some systems and its enhancement in others. Intriguingly, DDR signaling and DNA repair functions can be uncoupled. Thus, cell differentiation modulates DDR functions.
The role of RNA in DDR signaling
Presently, a functional role of RNA in the modulation of the DDR signaling cascade is unreported. We discovered that RNA can modulate DDR activities, including signaling and checkpoint functions. We have identified the RNA synthesis pathways involved in the direct modulation of DDR at sites of DNA damage and the RNA species involved. Thus, a novel layer of DDR regulation dictated by RNA molecules is being unveiled.
Is cellular senescence an example of antagonistic pleiotropy?
In collaboration with Foundations Of the Life Sciences And Their Ethical Consequences (FOLSATEC) PhD program in our Campus, we are probing the strength of the arguments in favor of the hypothesis that cellular senescence is a fitting example of the evolutionary theory of antagonistic pleiotropy, as often assumed.
(update: Mar 2016)
Simon Fraser University
8888 University Drive
V5A 1S6 / Burnaby
25, rue du Dr. Roux
75724 Paris Cedex 15
Omics Science Center
RIKEN Yokohama Institute
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama,
Kanagawa 230-0045 Japan
Medical School, University of Athens
75 Mikras Asias str., Goudi
M. Prakash Hande
Department of Physiology - Faculty of Medicine
National University of Singapore
Block MD9, 2 Medical Drive
SINGAPORE 117597 - Singapore
Michael O. Hengartner
Institute of Molecular Biology
University of Zurich
Room 55 L 22
Department of Microbiology and Molecular Genetics
NJMS-UH Cancer Center, Rm G1226
205 South Orange Ave.
Newark, NJ 07103
Prof. Mihaela Zavolan
University of Basel
Klingelbergstrasse 50 / 70
CH - 4056 Basel
(update: May 2012)
2015.03.25 | Stefano Rizzato