@article{%a1.%Yb_110,

title = {Single Cell Determination of 7,8-dihydro-8-oxo-2′-deoxyguanosine by Fluorescence Techniques: Antibody vs. Avidin Labeling},

author = {Maraventano G and Ticli G and Cazzalini O and Stivala LA and Ramos-Gonzalez M and Rodríguez JL and Prosperi E},

url = {https://www.mdpi.com/1420-3049/28/11/4326},

doi = {10.3390/molecules28114326},

year  = {2023},

date = {2023-08-08},

journal = {Molecules},

volume = {28},

issue = {11},

pages = {4326},

abstract = {An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). Although several methods are available for the biochemical analysis of this molecule, its determination at the single cell level may provide significant advantages when investigating the influence of cell heterogeneity and cell type in the DNA damage response. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detection with the glycoprotein avidin has also been proposed because of a structural similarity between its natural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliability and sensitivity is not clear. In this study, we compared the immunofluorescence determination of 8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugated with the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different cell types by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species (ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, our results indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greater than that attained with avidin-AF488. These findings suggest that immunofluorescence techniques are best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1.%Ybt,

title = {In Vitro Neurotoxicity of Flumethrin Pyrethroid on SH-SY5Y Neuroblastoma Cells: Apoptosis Associated with Oxidative Stress},

author = {Barrios-Arpi L and Arias Y and Lopez-Torres B and Ramos-Gonzalez M and Ticli G and Prosperi E and Rodríguez J-L},

url = {https://www.mdpi.com/2305-6304/10/3/131#cite},

doi = {10.3390/toxics10030131},

year  = {2022},

date = {2022-07-20},

journal = {Toxics},

volume = {10},

issue = {3},

pages = {131},

abstract = {Pyrethroids are neurotoxicants for animals, showing a pattern of toxic action on the nervous system. Flumethrin, a synthetic pyrethroid, is used against ectoparasites in domestic animals, plants, and for public health. This compound has been shown to be highly toxic to bees, while its effects on other animals have been less investigated. However, in vitro studies to evaluate cytotoxicity are scarce, and the mechanisms associated with this effect at the molecular level are still unknown. This study aimed to investigate the oxidative stress and cell death induction in SH-SY5Y neuroblastoma cells in response to flumethrin exposure (1–1000 µM). Flumethrin induced a significant cytotoxic effect, as evaluated by MTT and LDH leakage assays, and produced an increase in the biomarkers of oxidative stress as reactive oxygen species and nitric oxide (ROS and NO) generation, malondialdehyde (MDA) concentration, and caspase-3 activity. In addition, flumethrin significantly increased apoptosis-related gene expressions (Bax, Casp-3, BNIP3, APAF1, and AKT1) and oxidative stress and antioxidative (NFκB and SOD2) mediators. The results demonstrated, by biochemical and gene expression assays, that flumethrin induces oxidative stress and apoptosis, which could cause DNA damage. Detailed knowledge obtained about these molecular changes could provide the basis for elucidating the molecular mechanisms of flumethrin-induced neurotoxicity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1.%Yb_38,

title = {Revisiting the Function of p21CDKN1A in DNA Repair: The Influence of Protein Interactions and Stability},

author = {Ticli G and Cazzalini O and Stivala LA and Prosperi E},

url = {https://www.mdpi.com/resolver?pii=ijms23137058},

doi = {10.3390/ijms23137058},

year  = {2022},

date = {2022-08-19},

journal = {International journal of molecular sciences},

volume = {23},

issue = {13},

pages = {7058},

abstract = {The p21 protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Ybv,

title = {A functional in vitro cell-free system for studying DNA repair in isolated nuclei},

author = {Guardamagna I and Bassi E and Savio M and Perucca P and Cazzalini O and Prosperi E and Stivala LA},

url = {https://journals.biologists.com/jcs/article/133/11/jcs240010/224737/A-functional-in-vitro-cell-free-system-for},

doi = {10.1242/jcs.240010},

year  = {2021},

date = {2021-08-25},

journal = {Journal of cell science},

volume = {133},

number = {11},

abstract = {Assessment of DNA repair is an important endpoint measurement when studying the biochemical mechanisms of the DNA damage response and when investigating the efficacy of chemotherapy, which often uses DNA-damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, such methods have some limitations, which are mainly due to the lack of chromatin organization in the DNA templates used. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the corresponding damage-activated cytosolic extracts, containing biotinylated dUTP, nuclei were able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 and DDB1, stimulated UVC-induced dUTP incorporation. In contrast, a DDB2PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different types of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.This article has an associated First Person interview with the first author of the paper.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Ybv,

title = {Transcriptional stress induces chromatin relocation of the nucleotide excision repair factor XPG},

author = {Scalera C and Ticli G and Dutto I and Cazzalini O and Stivala LA and Prosperi E},

url = {http://www.scopus.com/record/display.url?eid=2-s2.0-85108105818&origin=inward},

doi = {10.3390/ijms22126589},

year  = {2021},

date = {2021-08-25},

journal = {International journal of molecular sciences},

volume = {22},

pages = {6589},

abstract = {Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-?-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (?-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_453,

title = {A Functional in vitro Cell-Free System for Studying DNA Repair in Isolated Nuclei},

author = {Guardamagna I and Bassi E and Savio M and Perucca P and Cazzalini O and Prosperi E and Stivala LA},

url = {https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00681},

doi = {10.1242/jcs.240010},

year  = {2020},

date = {2020-01-01},

journal = {Journal of cell science},

volume = {133},

number = {11},

pages = {1-12},

abstract = {Assessing DNA repair is an important endpoint to study the DNA damage response for investigating the biochemical mechanisms of this process and the efficacy of chemotherapy, which often uses DNA damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, they show some limitations mainly due to the lack of chromatin organization. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the correspondent damage-activated cytosolic extracts, containing biotin-16-dUTP, nuclei are able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 or DDB1, stimulated UVC induced dUTP incorporation. In contrast, a DDB2PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different type of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_476,

title = {Dynamics of protein binding to sites of nascent unscheduled DNA repair synthesis in non-proliferating cells},

author = {Scalera C and Dutto I and Barbazza F and Abou Khouzam R and Ticli G and Cazzalini O and Stivala LA and Prosperi E},

url = {https://www.biorxiv.org/content/10.1101/2020.03.06.979039v1 },

doi = {10.1101/2020.03.06.979039},

year  = {2020},

date = {2020-01-01},

journal = {bioRxiv - Cold Spring Harbor Laboratory},

abstract = {The analysis of DNA repair mechanisms is of fundamental importance to understand how cells remove DNA damage and maintain their genome stability. Investigating the dynamic association of proteins at sites of active DNA synthesis has been successfully performed at DNA replication forks, providing important information on the process, and allowing the identification of new players acting at these sites. However, the applicability of these studies to DNA repair events at sites of nascent unscheduled DNA synthesis (UDS) in non-proliferating cells has been never tested. Here, we describe the analysis of dynamics association of protein participating in nucleotide excision repair (NER), and in other DNA repair processes, at sites of nascent UDS in non-proliferating cells, to avoid interference by DNA replication. Labeling with 5-ethynyl-2'-deoxyuridine (EdU) after DNA damage, followed by click reaction to biotinylate these sites, permits the analysis of dynamic association of proteins, such as DNA polymerases ? and ?, as well as PCNA, to active DNA repair synthesis sites. The suitability of this technique to identify new factors present at active UDS sites is illustrated by two examples of proteins previously unknown to participate in the UV-induced DNA repair process.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_447,

title = {Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells.},

author = {Dutto I and Scalera C and Tillhon M and Ticli G and Passaniti G and Cazzalini O and Savio M and Stivala LA and Gervasini C and Larizza L and Prosperi E},

url = {https://academic.oup.com/carcin/advance-article-abstract/doi/10.1093/carcin/bgz149/5556479?redirectedFrom=fulltext},

doi = {10.1093/carcin/bgz149},

year  = {2020},

date = {2020-01-01},

journal = {Carcinogenesis},

volume = {41},

number = {3},

pages = {257-266},

abstract = {Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency, and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase β and PCNA, together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y%_32,

title = {Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells.},

author = {Dutto I and Scalera C and Tillhon M and Ticli G and Passaniti G and Cazzalini O and Savio M and Stivala LA and Gervasini C and Larizza L and Prosperi E},

url = {https://academic.oup.com/carcin/advance-article-abstract/doi/10.1093/carcin/bgz149/5556479?redirectedFrom=fulltext},

doi = {10.1093/carcin/bgz149},

year  = {2020},

date = {2020-05-14},

journal = {Carcinogenesis},

abstract = {Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency, and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase β and PCNA, together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_92,

title = {Targeting Proliferating Cell Nuclear Antigen (PCNA) as an Effective Strategy to Inhibit Tumor Cell Proliferation.},

author = {Cardano M and Tribioli C and Prosperi E},

url = {http://www.eurekaselect.com/178337/article},

doi = {10.2174/1568009620666200115162814},

year  = {2020},

date = {2020-01-01},

journal = {Current cancer drug targets},

volume = {20},

number = {4},

pages = {240-252},

abstract = {Targeting highly proliferating cells is an important issue for many type aggressive tumors. Proliferating Cell Nuclear Antigen (PCNA) is an essential protein that participates in a variety of processes of DNA metabolism, including DNA replication and repair, chromatin organization and transcription, sister chromatid cohesion. In addition, PCNA is involved in cell survival, and possibly in pathways of energy metabolism, such as glycolysis. Thus, the possibility of targeting this protein for chemotherapy against highly proliferating malignancies is under active investigation. Currently, approaches to treat cells with agents targeting PCNA rely on the use of small molecules or on peptides that either bind to PCNA, or act as a competitor of interacting partners. Here, we describe the status of the art in the development of agents targeting PCNA and discuss their application in different types of tumor cell lines and in animal model systems. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y%g,

title = {Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells.},

author = {Bassi E and Perucca P and Guardamagna I and Prosperi E and Stivala LA and Cazzalini O.},

url = {https://bmccancer.biomedcentral.com/articles/10.1186/s12885-019-6258-0},

doi = {10.1186/s12885-019-6258-0},

year  = {2019},

date = {2019-10-29},

journal = {BMC cancer},

volume = {19},

number = {1},

pages = {1013},

abstract = {BACKGROUND: The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro. METHODS: In this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2Wt or DDB2PCNA-, were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy. RESULTS: The results have shown that DDB2Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2PCNA-, as also confirmed by the different extent of co-localization of DDB2Wt and some NER proteins (such as XPG), vs the DDB2 mutant form. CONCLUSION:The HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_64,

title = {In situ analysis of DNA-protein complex formation upon radiation-induced DNA damage},

author = {Ticli G and Prosperi E},

url = {https://www.mdpi.com/1422-0067/20/22/5736},

doi = {10.3390/ijms20225736},

year  = {2019},

date = {2019-11-15},

journal = {International journal of molecular sciences},

volume = {20},

pages = {5736},

abstract = {The importance of determining at the cellular level the formation of DNA-protein complexes after radiation-induced lesions to DNA is outlined by the evidence that such interactions represent one of the first steps of the cellular response to DNA damage. These complexes are formed through recruitment at the sites of the lesion, of proteins deputed to signal the presence of DNA damage, and of DNA repair factors necessary to remove it. Investigating the formation of such complexes has provided, and will probably continue to, relevant information about molecular mechanisms and spatiotemporal dynamics of the processes that constitute the first barrier of cell defense against genome instability and related diseases. In this review, we will summarize and discuss the use of in situ procedures to detect the formation of DNA-protein complexes after radiation-induced DNA damage. This type of analysis provides important information on the spatial localization and temporal resolution of the formation of such complexes, at the single-cell level, allowing the study of heterogeneous cell populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_168,

title = {A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage.},

author = {Perucca P and Mocchi R and Guardamagna I and Bassi E and Sommatis S and Nardo T and Prosperi E and Stivala LA and Cazzalini O},

url = {https://www.sciencedirect.com/science/article/pii/S0167488918300569?via%3Dihub},

doi = {10.1016/j.bbamcr.2018.03.012},

year  = {2018},

date = {2018-02-15},

journal = {Biochimica et Biophysica Acta (BBA) - Molecular Cell Research},

volume = {1865},

number = {6},

pages = {898-907},

abstract = {n mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2Wt and DDB2PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_134,

title = {CREBBP and p300 lysine acetyl transferases in the DNA damage response.},

author = {Dutto I and Scalera C and Prosperi E},

url = {https://link.springer.com/article/10.1007%2Fs00018-017-2717-4},

doi = {10.1007/s00018-017-2717-4},

year  = {2018},

date = {2018-02-16},

journal = {Cellular and molecular life sciences: CMLS},

volume = {75},

number = {8},

pages = {1325-1338},

abstract = {The CREB-binding protein (CREBBP, or in short CBP) and p300 are lysine (K) acetyl transferases (KAT) belonging to the KAT3 family of proteins known to modify histones, as well as non-histone proteins, thereby regulating chromatin accessibility and transcription. Previous studies have indicated a tumor suppressor function for these enzymes. Recently, they have been found to acetylate key factors involved in DNA replication, and in different DNA repair processes, such as base excision repair, nucleotide excision repair, and non-homologous end joining. The growing list of CBP/p300 substrates now includes factors involved in DNA damage signaling, and in other pathways of the DNA damage response (DDR). This review will focus on the role of CBP and p300 in the acetylation of DDR proteins, and will discuss how this post-translational modification influences their functions at different levels, including catalytic activity, DNA binding, nuclear localization, and protein turnover. In addition, we will exemplify how these functions may be necessary to efficiently coordinate the spatio-temporal response to DNA damage. CBP and p300 may contribute to genome stability by fine-tuning the functions of DNA damage signaling and DNA repair factors, thereby expanding their role as tumor suppressors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_108,

title = {Generation of the Rubinstein-Taybi syndrome type 2 patient-derived induced pluripotent stem cell line (IAIi001-A) carrying the EP300 exon 23 stop mutation c.3829A > T, p.(Lys1277*).},

author = {Alari V and Russo S and Rovina D and Gowran A and Garzo M and Crippa M and Mazzanti L and Scalera C and Prosperi E and Giardino D and Gervasini C and Finelli P and Pompilio G and Larizza L},

url = {https://www.sciencedirect.com/science/article/pii/S1873506118301570?via%3Dihub},

doi = {Stem cell research},

year  = {2018},

date = {2018-07-31},

journal = {Stem cell research},

volume = {30},

pages = {175-179},

abstract = {"

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder characterized by growth retardation, skeletal anomalies and intellectual disability, caused by heterozygous mutation in either the CREBBP (RSTS1) or EP300 (RSTS2) genes. We generated an induced pluripotent stem cell line from an RSTS2 patient's blood mononuclear cells by Sendai virus non integrative reprogramming method. The iPSC line (IAIi001RSTS2-65-A) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was stable by karyotyping. Mutation and western blot analyses demonstrated in IAIi001RSTS2-65-A the patient's specific non sense mutation in exon 23 c.3829A > T, p.(Lys 1277*) and showed reduced quantity of wild type p300 protein."},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y%b,

title = {Generation of three iPSC lines (IAIi002, IAIi004, IAIi003) from Rubinstein-Taybi syndrome 1 patients carrying CREBBP non sense c.4435G>T, p.(Gly1479*) and c.3474G>A, p.(Trp1158*) and missense c.4627G>T, p.(Asp1543Tyr) mutations.},

author = {Alari V and Russo S and Rovina D and Garzo M and Crippa M and Calzari L and Scalera C and Concolino D and Castiglioni E and Giardino D and Prosperi E and Finelli P and Gervasini C and Gowran A and Larizza L},

url = {https://www.sciencedirect.com/science/article/pii/S1873506119301837?via%3Dihub},

doi = {10.1016/j.scr.2019.101553},

year  = {2018},

date = {2018-10-04},

journal = {Stem cell research},

volume = {40},

pages = {101553},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_211,

title = {An improved method for the detection of nucleotide excision repair factors at local UV DNA damage sites.},

author = {Dutto I and Cazzalini O and Stivala LA and Prosperi E},

url = {http://www.sciencedirect.com/science/article/pii/S1568786417300034},

doi = {dx.doi.org/10.1016/j.dnarep.2017.01.005},

year  = {2017},

date = {2017-02-22},

journal = {DNA Repair},

volume = {51},

pages = {79-84},

abstract = {Among different DNA repair processes that cells use to face with DNA damage, nucleotide excision repair (NER) is particularly important for the removal of a high variety of lesions, including those generated by some antitumor drugs. A number of factors participating in NER, such as the TFIIH complex and the endonuclease XPG are also involved in basal processes, e.g. transcription. For this reason, localization of these factors at DNA damage sites may be difficult. Here we have applied a mild digestion of chromatin with DNase I to improve the in situ extraction necessary to detect chromatin-bound proteins by immunofluorescence. We have compared this method with different extraction protocols and investigated its application on different cell types, and with different antibodies. Our results show that a short DNase I treatment before the immunoreaction, enhances the fluorescence signal of NER proteins, such as XPG, DDB2 and XPC. In addition, our findings indicate that the antibody choice is a critical factor for accurate localization of DNA repair proteins at DNA damage sites. In conclusion, a mild DNA digestion with DNase I improves the immunofluorescence detection of the recruitment of NER factors at local DNA damage sites by enhancing accessibility to the antibodies, independently of the cell type.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_271,

title = {Assessing Cell Cycle Independent Function of the CDK Inhibitor p21(CDKN1A) in DNA Repair.},

author = {Dutto I and Tillhon M and Prosperi E},

url = {https://link.springer.com/protocol/10.1007%2F978-1-4939-2926-9_11},

doi = {10.1007/978-1-4939-2926-9_11},

year  = {2016},

date = {2016-06-09},

journal = {Methods in Molecular Biology - Cyclin-Dependent Kinase (CDK) Inhibitors},

volume = {1336},

pages = {123-139},

abstract = {The cyclin-dependent kinase (CDK) inhibitor p21(CDKN1A) is a small protein that is able to regulate many important cell functions, often independently of its activity of CDK inhibitor. In addition to cell cycle, this protein regulates cell transcription, apoptosis, cell motility, and DNA repair. In particular, p21 may participate in different DNA repair processes, like the nucleotide excision repair (NER), base excision repair (BER), and double-strand breaks (DSB) repair, because of its ability to interact with DNA repair proteins, such as proliferating cell nuclear antigen (PCNA), a master regulator of many DNA transactions. Although this role has been debated for a long time, the influence of p21 in DNA repair has been now established. However, it remain to be clarified how this role is coupled to proteasomal degradation that has been shown to occur after DNA damage. This chapter describes procedures to study p21 protein recruitment to localized DNA damage sites in the cell nucleus. In particular, we describe a technique based on local irrradiation with UV light through a polycarbonate filter with micropores; an in situ lysis procedure to detect chromatin-bound proteins by immunofluorescence; a cell fractionation procedure to study chromatin association of p21 by Western blot analysis, and p21 protein-protein interactions by an immunoprecipitation assay.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_270,

title = {p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates.},

author = {Dutto I and Sukhanova M and Tillhon M and Cazzalini O and Stivala LA and Scovassi AI and Lavrik O and Prosperi E},

url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146031},

doi = {10.1371/journal.pone.0146031.},

year  = {2016},

date = {2016-01-05},

journal = {Plos One},

volume = {11},

number = {1},

pages = {e0146031},

abstract = {The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_327,

title = {A DDB2 mutant protein unable to interact with PCNA promotes cell cycle progression of human transformed embryonic kidney cells.},

author = {Perucca P and Sommatis S and Mocchi R and Prosperi E and Stivala LA and Cazzalini O},

url = {https://www.tandfonline.com/doi/full/10.1080/15384101.2015.1120921},

doi = {10.1080/15384101.2015.1120921},

year  = {2015},

date = {2015-02-18},

journal = {Cell Cycle},

volume = {14},

number = {24},

pages = {3920-3928},

abstract = {DNA damage binding protein 2 (DDB2) is a protein involved in the early step of DNA damage recognition of the nucleotide excision repair (NER) process. Recently, it has been suggested that DDB2 may play a role in DNA replication, based on its ability to promote cell proliferation. We have previously shown that DDB2 binds PCNA during NER, but also in the absence of DNA damage; however, whether and how this interaction influences cell proliferation is not known. In this study, we have addressed this question by using HEK293 cell clones stably expressing DDB2(Wt) protein, or a mutant form (DDB2(Mut)) unable to interact with PCNA. We report that overexpression of the DDB2(Mut) protein provides a proliferative advantage over the wild type form, by influencing cell cycle progression. In particular, an increase in the number of S-phase cells, together with a reduction in p21(CDKN1A) protein level, and a shorter cell cycle length, has been observed in the DDB2(Mut) cells. These results suggest that DDB2 influences cell cycle progression thanks to its interaction with PCNA.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_340,

title = {Biology of the cell cycle inhibitor p21CDKN1A: molecular mechanisms and relevance in chemical toxicology.},

author = {Dutto I and Tillhon M and Cazzalini O and Stivala LA and Prosperi E},

url = {https://link.springer.com/article/10.1007/s00204-014-1430-4},

doi = { 10.1007/s00204-014-1430-4},

year  = {2015},

date = {2015-02-14},

journal = {Archives of Toxicology},

volume = {89},

pages = {155–178},

abstract = {The cell cycle inhibitor p21(CDKN1A) is a protein playing multiple roles not only in the DNA damage response, but also in many cellular processes during unperturbed cell growth. The main, well-known function of p21 is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases. In addition, p21 is involved in the regulation of transcription, apoptosis, DNA repair, as well as cell motility. However, p21 appears to a have a dual-face behavior because, in addition to its tumor suppressor functions, it may act as an oncogene, depending on the cell type and on the cellular localization. As a biomarker of the cell response to different toxic stimuli, p21 expression and functions have been analyzed in an impressive number of studies investigating the activity of several types of chemicals, in order to determine their possible harmful effects on human cells. Here, we review these studies in order to highlight the different roles p21 may play in the cell response to chemical exposure and to better evaluate the information provided by this biomarker.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{%a1:%Y_352,

title = {Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.},

author = {Necchi D and Pinto A and Tillhon M and Dutto I and Serafini MM and Lanni C and Govoni S and Racchi M and Prosperi E},

url = {https://www.sciencedirect.com/science/article/pii/S0027510715300282?via%3Dihub},

doi = {10.1016/j.mrfmmm.2015.07.009},

year  = {2015},

date = {2015-02-12},

journal = {Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis},

volume = {780},

pages = {15-23},

abstract = {Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase beta, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors. Copyright 2015 Elsevier B.V. All rights reserved.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}