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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10990/583

Autori: Casarano , Elena
Supervisore afferente all'Università: TELL, GIANLUCA
Abstract (in inglese): DNA is constantly subjected to damages by several agents, exogenous or endogenous, threating both cell cycle progression and genome stability1–3. Although different repair pathways ensure the removal of various types of lesions restoring the correct sequences, some processes, as aging, neurodegeneration as well as cancers, could differently impair their efficacy thus promoting an altered cellular sensitivity to damaging agents4–7. Therefore, because of the essential physiological role of DNA repair proteins played in the maintenance of genome stability, targeting some of them could potentially represent an approach to modulate the effects of different cytotoxic compounds, particularly regulating both the effect of chemotherapeutics drugs and the progression of neurodegenerative processes8–10. In this context, given the great relevance of Base Excision Repair (BER) in the development of the above mentioned pathologies, this pathway could represent a good target to modulate these diseases8,11–13. In particular, the main mammalian AP endonuclease, the Apurinic/apyrimidinic endonuclease-1 (Ape1) could be a promising candidate to this purpose, thanks to its key role in the BER pathway, in which Ape1 is devoted to removal of the AP-site formed upon removal of the damaged base and also to its involvement in the regulation of cellular responses to oxidative stress conditions, by redox activating some transcriptional factors14,15. In fact, an increase of reactive oxygen species (ROS) has been reported in both pathologies16,17, as well as Ape1 alterations have been observed both in neurodegenerative tissues and in different types of tumors, particularly aggressive and resistant to chemotherapy12,18–20. Nevertheless, Ape1 role in nervous system, specifically in pathologies such as neurodegeneration and cancers, is not well studied, yet. A detailed comprehension of Ape1 activity and expression upon genotoxic damage is needed as well as a better understanding of the crosstalks existing within the different enzymes of the BER pathway during cell response to genotoxic damages. These informations will be helpful in understanding the BER role in human pathologies as well as demonstrate the reliability of targeting BER enzymes activity for cancer treatment. To these aims, in this work of Thesis, I analyzed the role of Ape1 functions in response to different toxic agents inducing oxidative stress conditions and related either with neurodegenerative and carcinogenic processes (i. e. cadmium) or with chemoresistance phenomena (i. e. cisplatin). SF-767, a glioblastoma cell line, was treated with Ape1 inhibitors, specifically blocking either the DNA repair (Methoxyamine18,21, compound #3 and compound #5222) or the redox (E333023,24) activities, with or without the addiction of different genotoxicants. Data collected highlight a dysregulation of the other BER proteins, in particular of DNA polymerase δ, associated with the inhibition of the Ape1 endonuclease activity. Although this effect potentially promotes the feasibility of these inhibitors to modulate the cytotoxic activity of genotoxic agents, further results obtained in combination with cadmium or cisplatin treatments underline a different role in modulating cell response to these genotoxicants. In fact, while the inhibition of Ape1 endonuclease activity causes the activation of potential resistance mechanisms toward cisplatin treatment, on the other hand the sensitizing effect observed in combination with cadmium could strengthen the BER impairment induced by this metal on the repair pathway. Notably, the down-regulation of Polδ expression levels observed with both agents could explain these phenomena, being a protein involved in both replication and BER pathways. Interestingly, despite polymerase δ levels results similarly altered by cadmium as well as by direct endonucleasic inhibitors of Ape1, data suggest that the regulatory mechanism responsible for this effect is different. Additional experiments should be performed to further define their specific molecular effect on Polδ dysregulation. Overall these data highlight a complex mechanism of crosstalks existing between BER enzymes and involved in cell response to genotoxicants that require further studies to better understand the role of BER in genome stability and in chemoresistance phenomena. 1. De Bont, R. & van Larebeke, N. Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis 19, 169–185 (2004). 2. Friedberg, E. C. Suffering in silence: the tolerance of DNA damage. Nat. Rev. Mol. Cell Biol. 6, 943–953 (2005). 3. Hoeijmakers, J. H. J. DNA damage, aging, and cancer. N. Engl. J. Med. 361, 1475–1485 (2009). 4. Christmann, M., Tomicic, M. T., Roos, W. P. & Kaina, B. Mechanisms of human DNA repair: an update. Toxicology 193, 3–34 (2003). 5. David, S. S., O’Shea, V. L. & Kundu, S. Base-excision repair of oxidative DNA damage. Nature 447, 941–950 (2007). 6. Wilson, D. M. & Bohr, V. A. The mechanics of base excision repair, and its relationship to aging and disease. DNA Repair 6, 544–559 (2007). 7. Fojo, T. Cancer, DNA Repair Mechanisms, and Resistance to Chemotherapy. J. Natl. Cancer Inst. 93, 1434–1436 (2001). 8. Dianov, G. L. Base excision repair targets for cancer therapy. Am. J. Cancer Res. 1, 845–851 (2011). 9. Sharma, R. A. & Dianov, G. L. Targeting base excision repair to improve cancer therapies. Mol. Aspects Med. 28, 345–374 (2007). 10. Targeting DNA Repair to Enhance Cancer Therapy. (2011). at <http://www.hematology.org/Thehematologist/Mini-Review/1209.aspx> 11. Audebert, M. et al. Alterations of the DNA Repair Gene OGG1 in Human Clear Cell Carcinomas of the Kidney. Cancer Res. 60, 4740–4744 (2000). 12. Wang, D. et al. APE1 overexpression is associated with cisplatin resistance in non-small cell lung cancer and targeted inhibition of APE1 enhances the activity of cisplatin in A549 cells. Lung Cancer Amst. Neth. 66, 298–304 (2009). 13. Wu, H. I., Brown, J. A., Dorie, M. J., Lazzeroni, L. & Brown, J. M. Genome-wide identification of genes conferring resistance to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. Cancer Res. 64, 3940–3948 (2004). 14. Tell, G., Quadrifoglio, F., Tiribelli, C. & Kelley, M. R. The many functions of APE1/Ref-1: not only a DNA repair enzyme. Antioxid. Redox Signal. 11, 601–620 (2009). 15. Thakur, S. et al. APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions. Exp. Mol. Med. 46, e106 (2014). 16. Andersen, J. K. Oxidative stress in neurodegeneration: cause or consequence? Nat. Med. 10 Suppl, S18–25 (2004). 17. Toyokuni, S., Okamoto, K., Yodoi, J. & Hiai, H. Persistent oxidative stress in cancer. FEBS Lett. 358, 1–3 (1995). 18. Jiang, Y. et al. Role of APE1 in differentiated neuroblastoma SH-SY5Y cells in response to oxidative stress: use of APE1 small molecule inhibitors to delineate APE1 functions. DNA Repair 8, 1273–1282 (2009). 19. Kelley, M. R. et al. Role of the DNA base excision repair protein, APE1 in cisplatin, oxaliplatin, or carboplatin induced sensory neuropathy. PloS One 9, e106485 (2014). 20. Silber, J. R. et al. The apurinic/apyrimidinic endonuclease activity of Ape1/Ref-1 contributes to human glioma cell resistance to alkylating agents and is elevated by oxidative stress. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 8, 3008–3018 (2002). 21. Taverna, P. et al. Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells. Mutat. Res. 485, 269–281 (2001). 22. Rai, G. et al. Synthesis, Biological Evaluation and Structure-Activity Relationships of a Novel Class of Apurinic/Apyrimidinic Endonuclease 1 Inhibitors. J. Med. Chem. 55, 3101–3112 (2012). 23. Kelley, M. R. et al. Functional analysis of novel analogues of E3330 that block the redox signaling activity of the multifunctional AP endonuclease/redox signaling enzyme APE1/Ref-1. Antioxid. Redox Signal. 14, 1387–1401 (2011). 24. Su, D. et al. Interactions of APE1 with a redox inhibitor: Evidence for an alternate conformation of the enzyme. Biochemistry (Mosc.) 50, 82–92 (2011).
Parole chiave: Base Excision Repair (BER); Genome stability; Ape1; Cadmium; Cisplatin; Neuronal cells
MIUR : Settore BIO/11 - Biologia Molecolare
Lingua: eng
Data: 10-apr-2015
Corso di dottorato: Dottorato di ricerca in Scienze biomediche e biotecnologiche
Ciclo di dottorato: 27
Università di conseguimento titolo: Università degli Studi di Udine
Luogo di discussione: Udine
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