Montariello, Daniela (2014) Involvement of Poly(ADP-Ribose) polymerase inhibitors as adiuvants of Topoisomerase 1 poisons in the p53/p63-dependent threshold mechanism of the cell fate decision between growth arrest and apoptosis. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Involvement of Poly(ADP-Ribose) polymerase inhibitors as adiuvants of Topoisomerase 1 poisons in the p53/p63-dependent threshold mechanism of the cell fate decision between growth arrest and apoptosis.
Creators:
CreatorsEmail
Montariello, Danieladaniela.montariello@unina.it
Date: 22 March 2014
Number of Pages: 59
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Molecolare e Biotecnologie Mediche
Scuola di dottorato: Scienze biologiche
Dottorato: Biochimica e biologia cellulare e molecolare
Ciclo di dottorato: 26
Coordinatore del Corso di dottorato:
nomeemail
Arcari, Paolopaolo.arcari@unina.it
Tutor:
nomeemail
Quesada, PieraUNSPECIFIED
Date: 22 March 2014
Number of Pages: 59
Uncontrolled Keywords: TOP 1 inhibitors; PARP inhibitors; p53; p63; carcinoma cells
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Additional Information: Attività svolta presso: Dipartimento di Biologia - Università degli Studi di Napoli Federico II; Institute of Pharmacology and Toxicology - University of Zurich
Date Deposited: 09 Apr 2014 10:17
Last Modified: 28 Jan 2015 09:32
URI: http://www.fedoa.unina.it/id/eprint/9625

Abstract

Poly(ADP-ribose) polymerase (PARP) inhibitors are thought as breakthrough for cancer treatment in solid tumours such as breast cancer through their effects on PARP’s enzymatic activity. PARP enzymes are involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Our previous findings showed that the hydrophilic PARP inhibitor PJ34 enhances the sensitivity of p53 proficient MCF7 breast carcinoma cells to topotecan, a DNA Topoisomerase 1 (TOP 1) inhibitor. It is already known that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract TOP 1 poisons through non covalent but specific interaction of poly(ADP-ribose) (PAR) with some TOP 1 sites which results in inhibition of DNA cleavage and stimulation of the religation reaction. Moreover, repair of DNA strand breaks induced by poisoned TOP 1 is slower in the presence of PARP inhibitors, leading to increased toxicity. In a first section, we combined the classical TOP 1 poison camptothecin or its water-soluble derivative topotecan with PJ34 to investigate the potentiation of chemotherapeutic efficiency in MCF7 (p53WT), MDA-MB231 (p53mut) breast carcinoma cells and SCC022 (p53null) skin squamous carcinoma cells. We showed that, following TPT/PJ34 combined treatment, MCF7 cells exhibit apoptotic death while MDA-MB231 and SCC022 cells are more resistant to these agents. Specifically, in MCF7, (i) PJ34 in combination with TPT causes a G2/M cell cycle arrest followed by massive apoptosis; (ii) PJ34 addition reverts TPT-dependent PARP-1 auto-modification and triggers caspase-dependent PARP-1 proteolysis; (iii) TPT, used as a single agent, stimulates p53 expression while in combination with PJ34 increases also the level of the pro-apoptotic isoform of p63 protein namely TAp63. The identification of p63 proteins as new players involved in the cancer cell response to TPT/PJ34 is relevant for a better understanding of the PARP-1-dependent signaling of DNA damage. Furthermore, our data indicate that, in response to TPT-PJ34 combined chemotherapy, a functional cooperation between p53 and TAp63 proteins may occur and be essential to trigger apoptotic cell death. A yet unsolved problem is the discrimination between covalent and non-covalent poly(ADP-ribosyl)ation (PARylation) of several nuclear proteins, including p53. Therefore, in a second section of the research, we got inside into this mechanism by using MCF7 breast carcinoma cells treated with topotecan, as we have demonstrated that they respond to DNA damage induced by topotecan treatment with p53 accumulation and PARP-1 auto-modification. By using in vitro analyses we were able to identify the phosphorylated form of p53 at serine 15 residue as a target/acceptor of the PAR synthesized in the nuclei of damaged cells. Furthermore, by cells co-immunoprecipitation experiments, it was found that PAR linked to PARP-1 interacts with p53 and is crucial for its nuclear stabilization. In a third section, we focused on the role/influence of PAR either in p53/p63 physical and functional interaction. For this part of the project, we compared again MCF7 and SCC022 cells with a different status of p53 as they showed a different sensitivity to apoptosis induced by TPT-dependent DNA damage.

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