Modulazione della risposta cellulare al danno al DNA nelle sindromi ad instabilità genomica

La mia tesi di dottorato tratta problematiche inerenti alla risposta cellulare al danno al DNA in linee cellulari di pazienti affetti da Ataxia Telangiectasia (A-T), una sindrome genetica dovuta alla mutazione del gene ATM codificante per una proteina essenziale per il mantenimento della stabilità genomica. Questa malattia manifesta aspetti clinici molto gravi come la predisposizione al cancro e la neurodegenerazione. Ho affrontato la questione attraverso due punti di vista analizzando, cioè, la sensibilità di queste cellule sia alle radiazioni che allo stress ossidativo in termini di induzione di danno primario al DNA e formazione di aberrazioni cromosomiche. Nel primo caso ho anche saggiato il ruolo della proteina p53 (un target di ATM nella risposta cellulare al danno al DNA) nella riparazione delle rotture a doppia elica (DSBs). Nel secondo ho comparato l’effetto della LCarnitina, una molecola coinvolta nel processo di formazione di energia per la cellula, con quella del Mannitolo, un noto scavenger di radicali liberi ossidrilici, nel tentativo di ridurre gli effetti deleteri dello stress ossidativo nelle cellule A-T. Abbiamo dimostrato che la proteina p53 interviene nel processo di riparazione delle DSBs in cellule A-T, favorendo il processo di “rejoining” delle estremità rotte. Inoltre abbiamo riscontrato che il pre-trattamento con L-Carnitina riduce il livello di stress ossidativo in cellule A-T in modo più marcato rispetto al Mannitolo agendo a tempi precoci dopo la formazione della lesione.DNA damage is one of the most critical threats to cellular homeostasis and life. In order to activate multiple signalling pathways at once, sophisticated sensing and transduction systems evolved to convey the damage signal simultaneously to multiple effectors. The ATM (Ataxia telangiectasia mutated) protein plays a central role in such a system. This protein kinase, identified as the product of the gene that is mutated (lost or inactivated) in the human genetic disorder Ataxia telangiectasia (A-T), is responsible for sounding the alarm throughout the cell announcing the presence of one of the most cytotoxic DNA lesions, the double-strand break. Cells from A-T patients exhibit chromosomal instability and extreme sensitivity to DSB-inducing agents such as ionizing radiation and radiomimetic chemicals. Notwithstanding, several features of the clinical and cellular phenotypes of A-T, in particular neurodegeneration and premature aging, suggest that defective cells may chronically suffer from increased oxidative stress. Support for this hypothesis comes from the observation that ATM-deficient cells are unusually sensitive to the toxic effects of hydrogen peroxide, nitric oxide, and superoxide. In this thesis we try to elucidate the mechanisms of DNA damage response in A-T cell lines handling this issue from two point of view. First, we analysed the the modulating effect of pre-treatment with L-carnitine (L-C) on oxidative stress-induced DNA damage both in normal and ATM-deficient lymphoblastoid cell lines. To understand the mechanism of action of L-carnitine, its effect in reducing the extent of both primary DNA damage and chromosomal aberrations, was compared with that of mannitol, a well known scavenger of the free hydroxyl radical. On the other hand, using a chemical inhibitor of p53 (PFT-α), the role of this protein in radiation-induced DNA damage on normal and A-T cell lines exposed to ionising radiations, was investigated. Our results suggest that L-C enhanced the extent and the rate of DNA repair of oxidative stress-induced lesions in A-T cell lines, at the very early recovery time; L-C has an effect also in reducing the formation of oxidative stress-induced chromosomal aberrations. Furthermore, L-C pre-treatment has a more pronounced effect in enhancing DNA repair of oxidative damage as compared to Mannitol. This could represent a tool for further research on the possible pharmacological applications of L-C in the A-T therapy. In addition, we demonstrated a role of p53 protein in “double-strand breaks repair” in A-T cells. In particular, our results suggests that p53 has a direct role in the NHEJ pathway, attending the phase of “DNA free-ends rejoining”. Howewer, if the activation of p53 is defective as in one of the A-T cell line studied, PFT-α seems to stimulate HR that is the pre-eminent repair process in G2 phase of cell-cycle. This unexpected effect of PFT-α on HR was confirmed in experiment performed with a CHO cell line defective for NHEJ treated with mitomycin-C. These findings suggest that the differential radiosensitivity of ATM-deficient cells depends both on the different mutations carried by the ATM gene and the activation of its downstream targets.Dottorato di ricerca in Evoluzione biologica e biochimic