DNA is constantly subjected to damaging agents, both of endogenous and exogenous origin. If not properly repaired, DNA damages may lead to severe pathological conditions, such as chronic diseases and cancer. Moreover, they can cause structural changes of the DNA duplex, leading to cell disfunctions. To overcome this issue, cells have evolved mechanisms directed to repair DNA damages collectively known as DNA-Damage Response (DDR). Although DDR has been widely explored and studied, many processes are not fully elucidated yet, prompting the need for further studies. Importantly, recent discoveries point towards novel non-canonical roles for DDR enzymes, and new alternative DNA-repair pathways are emerging that allow cells to survive when they are not able to perform classic DNA-repair (e.g. cancer cells resistance to current antitumoral treatments). A particular class of DNA-repair pathways, which includes classical Non-Homologous End-Joining (c-NHEJ) and Homologous-Directed Repair (HDR), is specialized in the repair of double-strand breaks (DSBs), which are the most toxic lesions in eukaryotic cells that, if not repaired, can lead to cell death. Alternative End-Joining (a-EJ) pathways represent a class of heterogeneous DSBs-repair mechanisms whose activation is triggered in cancer cells by down-regulation/absence of canonical DNA-repair pathways. One of these pathways, not characterized yet, emerges in U2-OS and SAOS-2 osteosarcoma cell lines when c-NHEJ apical factors are down-regulated under treatment with etoposide, a DSBs-inducing agent. In this context, we showed that, upon silencing of NHEJ apical factor DNA-PKcs, U2-OS cells are resistant to etoposide, whereas the silencing of pol lambda, alone or in combination with DNA-PKcs, increases their sensitivity. On the other hand, we did not observe the same cellular behavior upon treatment with camptothecin, a single-ended DSBs (se- DSBs)-inducing agent. Indeed, these type of DNA damages are normally repaired by Break-Induced Replication (BIR), a special type of HDR. Silencing of pol lambda and DNA-PKcs, alone or in combination, in presence of camptothecin, did not cause a significative difference in cell survival with respect to scramble cells. These results suggest that the a-EJ pathway that we observe upon etoposide treatment may be specific to a subtype of DNA damage, alternative to c-NHEJ and exploited by some cancer cells to survive. A better characterization of this mechanism of DSBs repair could lead to a broader understanding of cancer cells resistance to DNA-damaging agents, with the aim of developing new targeted cancer therapies. Another important DNA repair mechanism is Base Excision Repair (BER), a DDR pathway specialized in repairing small base lesions resulting from oxidation, alkylation, methylation or deamination. DNA glycosylases initiate BER by recognizing and excising modified bases, therefore these enzymes are essential for this pathway to work properly and to restore the regular DNA structure. Nevertheless, we and others have uncovered that the function of human DNA glycosylases goes beyond DNA repair in BER. Particularly, human glycosylases NEIL1, NEIL2 and OGG1 seem to be involved in regulation of G-quadruplexes (G4) dynamics, which are secondary structures that are formed in guanine-rich regulatory regions of the genome. Stabilization of G4 structures translates into modulation of gene expression, for example in oncogenes, and thus it is currently being exploited as therapeutic option for cancer patients. Interestingly, we showed that NEIL1, NEIL2 and OGG1 glycosylases-deficient cancer cell lines (HAP1) display increased cell death upon treatment with selected G4 ligands. Our results envision important roles of the human DNA glycosylases in modulation of G4 dynamics and, consequently, of gene expression.
Il DNA è costantemente sottoposto all’azione di agenti dannosi, sia endogeni che esogeni. Se non riparati, i danni al DNA portano a gravi condizioni patologiche (es. cancro). A tal proposito, le cellule hanno evoluto una serie di meccanismi di riparazione del DNA collettivamente noti sotto il nome di risposta al danno al DNA (DNA Damage Response- DDR). Una classe di meccanismi di riparazione del DNA, che include la riparazione delle rotture a doppio filamento con estremità non omologhe (classical Non-Homologous End-Joining- c-NHEJ) e la riparazione per ricombinazione (Homologous-Directed Repair- HDR), è specializzata nel riparo di rotture al doppio filamento (double-strand breaks- DSBs), le lesioni più citotossiche nelle cellule eucariotiche, le quali, se non riparate, possono portare e a instabilità genetica e morte cellulare. I meccanismi alternativi di riparazione di DSBs (Alternative End-Joining- a-EJ) sono una classe di processi eterogenei che si attivano nelle cellule tumorali quando i meccanismi canonici di riparazione del DNA sono de-regolati. Uno di questi meccanismi sembra emergere nelle linee cellulari di osteosarcoma U2-OS e SAOS-2 quando i fattori apicali di c-NHEJ ( es. DNA-PKcs) sono sotto-espressi e in seguito al trattamento con etoposide, un agente inducente DSBs. In tale contesto, abbiamo mostrato che, silenziando DNA-PKcs, le cellule U2-OS divengono resistenti all’etoposide, mentre il silenziamento della polimerasi lambda (pol lambda), singolarmente o in combinazione con DNA-PKcs, aumenta la loro sensibilità. D’altra parte, non abbiamo osservato lo stesso comportamento cellulare dopo il trattamento con camptotecina, un agente che causa DSBs con singolo filamento protrudente (single-ended double strand breaks- se-DSBs). Tale danno è solitamente riparato dalla replicazione indotta da rottura (Break-Induced Replication- BIR), un tipo di HDR. In presenza di camptotecina, il silenziamento di pol lambda e DNA-PKcs, singolarmente o in combinazione, non ha causato differenza significativa nella sopravvivenza cellulare rispetto alle cellule non silenziate. Tali risultati suggeriscono che l’a-EJ da noi osservato in seguito a trattamento con etoposide potrebbe essere specifico per una sottocategoria di DSBs. Una migliore caratterizzazione di questo a-EJ potrebbe portare ad una migliore comprensione di come le cellule tumorali resistono ai trattamenti, con lo scopo di sviluppare nuove mirate terapie anticancro. Un altro importante meccanismo di riparazione del DNA è la riparazione per escissione di base (Base Excision Repair- BER), un processo specializzato nella riparazione di piccole lesioni di basi causate da ossidazione, alchilazione, metilazione o deaminazione. Le DNA glicosilasi iniziano il BER riconoscendo e rimuovendo la base lesionata. Perciò tali enzimi sono essenziali affinché il BER ripristini correttamente la regolare struttura del DNA. Ciononostante, noi ed altri abbiamo osservato come le DNA glicosilasi umane assolvano ad altri ruoli oltre alla riparazione del DNA nel BER. In particolare, le glicosilasi umane NEIL1, NEIL2 e OGG1 sembrano essere coinvolte nella regolazione delle dinamiche delle tetradi di guanina (G-quadruplexes- G4), strutture secondarie che si formano in regioni regolatorie del genoma ricche di guanina. È stato osservato, a livello di alcuni oncogeni, che la stabilizzazione delle strutture G4 si traduce nella modulazione dell’espressione genica. Perciò, la formazione di G4 viene vista come un possibile bersaglio in future terapie antitumorali. Interessantemente, abbiamo mostrato che linee cellulari (HAP1) deficienti per NEIL1, NEIL2 e OGG1 manifestano un aumento nella morte cellulare a seguito di trattamento con agenti che legano strutture G4. I nostri risultati suggeriscono un ruolo importante delle glicosilasi umane nella modulazione delle dinamiche dei G4 e, di conseguenza, dell’espressione genica.
Oltre i ruoli canonici: modulazione delle dinamiche dei G-quadruplex da parte delle DNA glicosilasi e caratterizzazione di un nuovo meccanismo alternativo di riparazione del DNA.
GRANIGLIA, DESIA
2019/2020
Abstract
DNA is constantly subjected to damaging agents, both of endogenous and exogenous origin. If not properly repaired, DNA damages may lead to severe pathological conditions, such as chronic diseases and cancer. Moreover, they can cause structural changes of the DNA duplex, leading to cell disfunctions. To overcome this issue, cells have evolved mechanisms directed to repair DNA damages collectively known as DNA-Damage Response (DDR). Although DDR has been widely explored and studied, many processes are not fully elucidated yet, prompting the need for further studies. Importantly, recent discoveries point towards novel non-canonical roles for DDR enzymes, and new alternative DNA-repair pathways are emerging that allow cells to survive when they are not able to perform classic DNA-repair (e.g. cancer cells resistance to current antitumoral treatments). A particular class of DNA-repair pathways, which includes classical Non-Homologous End-Joining (c-NHEJ) and Homologous-Directed Repair (HDR), is specialized in the repair of double-strand breaks (DSBs), which are the most toxic lesions in eukaryotic cells that, if not repaired, can lead to cell death. Alternative End-Joining (a-EJ) pathways represent a class of heterogeneous DSBs-repair mechanisms whose activation is triggered in cancer cells by down-regulation/absence of canonical DNA-repair pathways. One of these pathways, not characterized yet, emerges in U2-OS and SAOS-2 osteosarcoma cell lines when c-NHEJ apical factors are down-regulated under treatment with etoposide, a DSBs-inducing agent. In this context, we showed that, upon silencing of NHEJ apical factor DNA-PKcs, U2-OS cells are resistant to etoposide, whereas the silencing of pol lambda, alone or in combination with DNA-PKcs, increases their sensitivity. On the other hand, we did not observe the same cellular behavior upon treatment with camptothecin, a single-ended DSBs (se- DSBs)-inducing agent. Indeed, these type of DNA damages are normally repaired by Break-Induced Replication (BIR), a special type of HDR. Silencing of pol lambda and DNA-PKcs, alone or in combination, in presence of camptothecin, did not cause a significative difference in cell survival with respect to scramble cells. These results suggest that the a-EJ pathway that we observe upon etoposide treatment may be specific to a subtype of DNA damage, alternative to c-NHEJ and exploited by some cancer cells to survive. A better characterization of this mechanism of DSBs repair could lead to a broader understanding of cancer cells resistance to DNA-damaging agents, with the aim of developing new targeted cancer therapies. Another important DNA repair mechanism is Base Excision Repair (BER), a DDR pathway specialized in repairing small base lesions resulting from oxidation, alkylation, methylation or deamination. DNA glycosylases initiate BER by recognizing and excising modified bases, therefore these enzymes are essential for this pathway to work properly and to restore the regular DNA structure. Nevertheless, we and others have uncovered that the function of human DNA glycosylases goes beyond DNA repair in BER. Particularly, human glycosylases NEIL1, NEIL2 and OGG1 seem to be involved in regulation of G-quadruplexes (G4) dynamics, which are secondary structures that are formed in guanine-rich regulatory regions of the genome. Stabilization of G4 structures translates into modulation of gene expression, for example in oncogenes, and thus it is currently being exploited as therapeutic option for cancer patients. Interestingly, we showed that NEIL1, NEIL2 and OGG1 glycosylases-deficient cancer cell lines (HAP1) display increased cell death upon treatment with selected G4 ligands. Our results envision important roles of the human DNA glycosylases in modulation of G4 dynamics and, consequently, of gene expression.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/12735