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Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis
Publication . Pereira, Pedro D.; Caetano, Ana Serra; Cabrita, Marisa; Bekman, Evguenia; Braga, José; Rino, José; Santus, Renè; Filipe, Paulo L.; Sousa, Ana E.; Ferreira, João A.
We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2'-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases.Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics.
Cellular responses to topoisomerase ll-mediated DNA lesions
Publication . Pereira, Pedro Martins Didelet, 1983-; Ferreira, João António Augusto, 1958-
DNA Topoisomerases are an important family of enzymes that catalyze the introduction of topological changes at the level of the DNA molecule and are required for several vital cellular processes such as replication, transcription, DNA recombination and chromosome segregation. The activity of Topoisomerases type II (Topo2) relies on the introduction of a transient DSB in the DNA strand by formation of a covalent bond between the enzyme and the nucleic acid molecule. This reversible covalent interaction promotes unwinding of topological events by allowing the passage of another strand through the formed gap, followed by ligation of DNA ends. The ability of Topo2 to relax positively supercoiled DNA defines its role as a determinant factor in both replication and transcription. Topo2 is the target for several clinically relevant anti-cancer drugs, such as Etoposide and Idarubicin, commonly referred to as Topo2 poisons, which stabilize the cleavage complex formed between the enzyme and DNA during its catalytic activity, thus preventing religation of broken DNA ends. When a DNA-tracking system, such as replication and transcription complexes, collides with the cleavage complex they leave a permanent double-strand break (DSB) in its place. If these breaks are not properly repaired, they can lead to chromosome translocations, increased genomic instability and even trigger apoptotic cell death. Most studies focusing on DSB repair have used ionizing radiation (IR) as the lesion inducing agent. However, DSBs introduced by IR are intrinsically more complex to repair because of the base modifications and sequence deletions that they often involve, whereas Topo2-mediated DSBs are stabilized by an enzyme and cleavage is performed in a precise manner, allowing for DNA end homology to be preserved. It is known from studies using irradiation that heterochromatin (HC) and euchromatin (EC) represent separate entities with respect to both damage sensitivity and repair. The high degree of compaction present in heterochromatin is thought to protect DNA from damage although, when lesions do occur, this compaction further restricts the capability of DNA damage response proteins to access the site to properly signal and mediate repair. Indeed, DNA damage introduced by IR in HC has been shown to be refractory to repair and resolved with slower kinetics than in EC. However, not much is known about how these repair kinetics are affected by the particular nature of Topo2- induced lesions and the restrictions imposed by chromatin structure on its enzymatic activity. Eukaryotic cells have evolved two major conserved pathways to repair DSBs in order to prevent transmission of genomic defects to their offspring: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). HR only functions in later S or G2 phases of the cell cycle since it requires an available sister chromatid to use as template for faithful repair. NHEJ is active throughout the entire cell cycle and is the only DSB repair pathway available in G1 when there are no twin templates for HR. However, since it joins broken DNA ends regardless of sequence homology, there is a risk of introducing sequence errors during repair, such as deletions and translocations. In the present thesis we aimed to characterize how each of the two main DSB repair pathways, NHEJ and HR, contributes to repair of Topo2-mediated DSBs. This was done for separate cell cycle phases using a protocol for cell cycle synchronization based on a double-thymidine block. Cells with DSBs introduced by a short pulse of the Topo2 poison Etoposide were monitored for their cell cycle progression and usage of repair factors over a period of 24h after Etoposide exposure. We found a diverging pattern of DSB repair system usage between lesions introduced in different cell cycle stages. We also used cell lines deficient for either BRCA1, the major determinant of HR pathway initiation, or DNAPKcs, the catalytic core unit of the complex that initiates NHEJ, to investigate whether behavior of DNA damage checkpoints is dependent on the choice of repair system. Loss of DNAPKcs dramatically sensitized HCT116 cells to Topo2-mediated DSBs, whereas similar loss of BRCA1 did not induce a dose-dependent cell viability decline much beyond spontaneous levels, highlighting the importance of NHEJ as the system that handles the bulk of these lesions. Overall, our results highlight G2 as a critical “workstation” phase for DSB repair, particularly for lesions introduced in heterochromatin. These lesions were predominantly repaired by HR, therefore leading to an increase in Chk1 recruitment and prolongation of G2/M arrest. DSBs introduced in G2, by contrast, did not induce sufficient activation of HR to sustain a stable checkpoint arrest, leading to slippage of cells with unrepaired DSBs into mitosis which is associated with an increased risk of genomic instability. We also found that cells damaged in late S phase, when heterochromatin is the preferential target for Topo2, trigger a strong HR activation, whereas for cells damaged in early S, when Topo2 is focused on euchromatin, this was not observed. We conclude therefore that HR in G2 preferentially targets a specific subset of DSBs that are located in heterochromatin regions. We propose a model where slippage through checkpoint arrest is also a major determinant of repair system usage, particularly for DSBs arising in G1 and G2 phases since escaping arrest and passing to the following cell cycle phase will change the availability of repair pathways. Because of intrinsic limitations of the checkpoints operating at these stages, we conclude that a significant number of DSBs introduced in G1 are repaired by HR in S and G2 phases, while DSBs induced in G2 are mostly repaired by NHEJ in both G2 and G1. In this thesis we also provide evidence that generalized disruption of heterochromatin epigenetic marks sensitizes cells to the DNA damaging action of Etoposide-bound Topo2. By using an inhibitor of histone methylation, DZNep, prior to Etoposide, we could robustly determine synergistic interactions between these two drugs. This highlights the potential for use of DZNep in combination with existing drugs targeting Topo2 in the chemotherapy of cancer. Finally, we also present a new published methodology for accurate quantification of cell cycle dynamics by flow cytometry yielding absolute values (in units of time) based on the unique stoichiometric properties of the thymidine analogue EdU (5-ethynyl-2’- deoxyuridine).
Anthracyclines induce DNA damage response-mediated protection against severe sepsis
Publication . Figueiredo, Nuno; Chora, Ângelo Ferreira; Raquel, Maria Helena; Pejanovic, Nadja; Pereira, Pedro; Hartleben, Björn; Neves-Costa, Ana; Moita, Catarina; Pedroso, Dora; Pinto, Andreia; Marques, Sofia; Faridi, Hafeez; Costa, Paulo M.; Gozzelino, Raffaella; Zhao, Jimmy L.; Soares, Miguel P.; Gama-Carvalho, Margarida; Martinez, Jennifer; Zhang, Qingshuo; Döring, Gerd; Grompe, Markus; Simas, J Pedro; Huber, Tobias B.; Baltimore, David; Gupta, Vineet; Green, Douglas R.; Ferreira, João; Moita, Luis
Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.
DNA damage induced by hydroquinone can be prevented by fungal detoxification
Publication . Pereira, Pedro; Enguita, Francisco J.; Ferreira, João; Leitão, Ana Lúcia
Hydroquinone is a benzene metabolite with a wide range of industrial applications, which has potential for widespread human exposure; however, the toxicity of hydroquinone on human cells remains unclear. The aims of this study are to investigate the cytotoxicity and genotoxicity of hydroquinone in human primary fibroblasts and human colon cancer cells (HCT116). Low doses of hydroquinone (227-454 μM) reduce the viability of fibroblasts and HCT116 cells, determined by resazurin conversion, and induce genotoxic damage (DNA strand breaks), as assessed by alkaline comet assays. Bioremediation may provide an excellent alternative to promote the degradation of hydroquinone, however few microorganisms are known that efficiently degrade it. Here we also investigate the capacity of a halotolerant fungus, Penicillium chrysogenum var. halophenolicum, to remove hydroquinone toxicity under hypersaline condition. The fungus is able to tolerate high concentrations of hydroquinone and can reverse these noxious effects via degradation of hydroquinone to completion, even when the initial concentration of this compound is as high as 7265 μM. Our findings reveal that P. chrysogenum var. halophenolicum efficiently degrade hydroquinone under hypersaline conditions, placing this fungus among the best candidates for the detoxification of habitats contaminated with this aromatic compound.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/45502/2008