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Browsing by Author "Damante, Giuseppe"
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Item Activation of APE1/Ref-1 is dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP(2005-07) Pines, Alex; Perrone, Lorena; Bivi, Nicoletta; Romanello, Milena; Damante, Giuseppe; Gulisano, Massimo; Kelley, Mark R.; Quadrifoglio, Franco; Tell, GianlucaApurinic apyrimidinic endonuclease redox effector factor-1 (APE1/Ref-1) is involved both in the base excision repair (BER) of DNA lesions and in the eukaryotic transcriptional regulation. APE1/Ref-1 is regulated at both the transcriptional and post-translational levels, through control of subcellular localization and post-translational modification. In response to stress conditions, several cell types release ATP, which exerts stimulatory effects on eukaryotic cells via the purinergic receptors (P2) family. By using western blot and immunofluorescence analysis on a human tumour thyroid cell line (ARO), we demonstrate that purinergic stimulation by extracellular ATP induces quick cytoplasm to nucleus translocation of the protein at early times and its neosynthesis at later times. Continuous purinergic triggering by extracellular ATP released by ARO cells is responsible for the control of APE1/Ref-1 intracellular level. Interference with intracellular pathways activated by P2 triggering demonstrates that Ca2+ mobilization and intracellular reactive oxygen species (ROS) production are responsible for APE1/Ref-1 translocation. The APE1/Ref-1 activities on activator protein-1 (AP-1) DNA binding and DNA repair perfectly match its nuclear enrichment upon ATP stimulation. The biological relevance of our data is reinforced by the observation that APE1/Ref-1 stimulation by ATP protects ARO cells by H2O2-induced cell death. Our data provide new insights into the complex mechanisms regulating APE1/Ref-1 functions.Item An 'environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation(2000-03) Tell, Gianluca; Zecca, Alessandro; Pellizzari, Lucia; Spessotto, Paola; Colombatti, Alfonso; Kelley, Mark R.; Damante, Giuseppe; Pucillo, CarloThe Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H2O2 induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse–chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.Item Genome-wide analysis and proteomic studies reveal APE1/Ref-1 multifunctional role in mammalian cells(2009-02) Vascotto, Carlo; Cesaratto, Laura; Zeef, Leo AH.; Deganuto, Marta; D'Ambrosio, Chiara; Scaloni, Andrea; Romanello, Milena; Damante, Giuseppe; Taglialatela, Giulio; Delneri, Daniela; Kelley, Mark R.; Mitra, Sankar; Quadrifoglio, Franco; Tell, GianlucaApurinic apyrimidinic endonuclease/redox effector factor 1 (APE1/Ref-1) protects cells from oxidative stress by acting as a central enzyme in base excision repair pathways of DNA lesions and through its independent activity as a redox transcriptional co-activator. Dysregulation of this protein has been associated with cancer development. At present, contrasting data have been published regarding the biological relevance of the two functions as well as the molecular mechanisms involved. Here, we combined both mRNA expression profiling and proteomic analysis to determine the molecular changes associated with APE1 loss-of-expression induced by siRNA technology. This approach identified a role of APE1 in cell growth, apoptosis, intracellular redox state, mitochondrial function, and cytoskeletal structure. Overall, our data show that APE1 acts as a hub in coordinating different and vital functions in mammalian cells, highlighting the molecular determinants of the multifunctional nature of APE1 protein.Item The Intracellular Localization of APE 1 /Ref-1: More than a Passive Phenomenon?(2005-02) Tell, Gianluca; Damante, Giuseppe; Caldwell, David; Kelley, Mark R.Human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a perfect paradigm of the functional complexity of a biological macromolecule. First, it plays a crucial role, by both redox-dependent and –independent mechanisms, as a transcriptional coactivator for different transcription factors, either ubiquitous (i.e., AP-1, Egr-1, NF-κB, p53, HIF) or tissue-specific (i.e., PEBP-2, Pax-5 and -8, TTF-1), in controlling different cellular processes such as apoptosis, proliferation, and differentiation. Second, it acts, as an apurinic/apyrimidinic endonuclease, during the second step of the DNA base excision repair pathway, which is responsible for the repair of cellular alkylation and oxidative DNA damages. Third, it controls the intracellular reactive oxygen species production by negatively regulating the activity of the Ras-related GTPase Rac1. Despite these known functions of APE1/Ref-1, information is still scanty about the molecular mechanisms responsible for the coordinated control of its several activities. Some evidence suggests that the expression and subcellular localization of APE1/Ref-1 are finely tuned. APE1/Ref-1 is a ubiquitous protein, but its expression pattern differs according to the different cell types. APE1/Ref-1 subcellular localization is mainly nuclear, but cytoplasmic staining has also been reported, the latter being associated with mitochondria and/or presence within the endoplasmic reticulum. It is not by chance that both expression and subcellular localization are altered in several metabolic and proliferative disorders, such as in tumors and aging. Moreover, a fundamental role played by different posttranslational modifications in modulating APE1/Ref-1 functional activity is becoming evident. In the present review, we tried to put together a growing body of information concerning APE1/Ref-1's different functions, shedding new light on present and future directions to understand fully this unique molecule.Item Prognostic role of Ape/Ref-1 subcellular expression in stage I-III breast carcinomas(2002-01) Puglisi, Fabio; Barbone, Fabio; Tell, Gianluca; Aprile, Giuseppe; Pertoldi, Barbara; Raiti, Concetta; Kelley, Mark R.; Damante, Giuseppe; Sobrero, Alberto; Beltrami, Carlo Alberto; Di Loreto, CarlaThe purpose of the study was to evaluate the prognostic role of the DNA repair protein APE/Ref-1 in breast carcinomas. Immunohistochemical analysis for APE/Ref-1 was performed in a series of 133 consecutive stage I-III breast carcinomas. The relationship between APE/Ref-1 and other prognostic and predictive factors such as tumor size, nodal status, histologic grade, p53 expression, hormonal receptor status, vascular invasion and necrosis was investigated. The prognostic value of APE/Ref-1 was studied by univariate and multivariate analysis. The predominant pattern of APE/Ref-1 immunohistochemical expression was nuclear, although cytoplasmic and mixed nuclear/cytoplasmic localization was also observed. The percentage of cells with APE/Ref-1 cytoplasmic stain directly correlated with the percentage of p53 positive cases (rho=0.28, p=0.013). The small group of women whose tumors showed mixed nuclear/cytoplasmic APE/Ref-1 localization (n=5) experienced a significantly poorer survival (p=0.014) and Cox proportional hazard model analysis identified APE/Ref-1 as an independent prognostic factor. The results suggest that subcellular localisation of APE/Ref-1 may influence the aggressiveness of breast carcinomas.