Sara Barozzi

A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis.

Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not influenced by p66Shc expression. In basal conditions, p66Shc−/− and p53−/− cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation

The DNA-damage response (DDR) arrests cell-cycle progression until damage is removed. DNA-damage-induced cellular senescence is associated with persistent DDR. The molecular bases that distinguish transient from persistent DDR are unknown. Here we show that a large fraction of exogenously induced persistent DDR markers is associated with telomeric DNA in cultured cells and mammalian tissues. In yeast, a chromosomal DNA double-strand break next to a telomeric sequence resists repair and impairs DNA ligase 4 recruitment. In mammalian cells, ectopic localization of telomeric factor TRF2 next to a double-strand break induces persistent DNA damage and DDR. Linear, but not circular, telomeric DNA or scrambled DNA induces a prolonged checkpoint in normal cells. In terminally differentiated tissues of old primates, DDR markers accumulate at telomeres that are not critically short. We propose that linear genomes are not uniformly reparable and that telomeric DNA tracts, if damaged, are irreparable and trigger persistent DDR and cellular senescence.

Bacteria-Induced Gap Junctions in Tumors Favor Antigen Cross-Presentation and Antitumor Immunity

Bacterial infection induces extra gap junctions in tumors, through which tumor antigens travel to antigen-presenting cells, triggering an effective antitumor immune response. Special Delivery: Immune Cells Receive a Package of Tumor-Specific Peptides Immune cells on patrol often recognize cancer cells as abnormal and eliminate them. But as tumors progress and proliferate, they can become invisible to these immune guardians of order. An injection of bacteria into the tumor can render them again visible to the immune system, thus promoting tumor-directed immune responses. If we understood how this reappearing act worked, we might be able to exploit it for cancer treatment. Saccheri et al. have discovered that the injected bacteria perform a key function: They reactivate connexin 43, a protein often suppressed in cancer cells that forms tiny communication channels—gap junctions—between cells. Tumor peptides escape through these channels and enter immune cells, which display the peptides on their surfaces, successfully triggering a specific immune response against the cancer. The authors showed that the bacteria Salmonella or its components elicited increased amounts of connexin 43 in melanoma cell lines from mice or humans. This connexin was used by the cells to form new gap junctions, allowing small molecules of the dye Lucifer yellow to pass between tumor cells or from tumor cells into antigen-presenting dendritic cells, which could also transfer dye among themselves. When the authors artificially inserted the protein ovalbumin into the melanoma cells in culture, it was broken down into peptides by the tumor proteasome, and these peptides passed through the gap junctions into cocultured dendritic cells, where they were displayed on the surface. Ovalbumin-specific T cells could be activated in response, and this activation depended on connexin 43. The authors then tested in mice whether this gap junction route for getting tumor peptides to dendritic cells operates in living animals. Although bacteria injected into tumors caused the eradication of that particular tumor independent of connexin and gap junctions, the regression of distant metastases, mediated by cytotoxic T cells, required the infection-induced gap junction mechanism. The delivery of tumor peptides to dendritic cells through gap junctions can be harnessed to generate tumor-specific dendritic cells ex vivo, a way to create tumor-specific immune cells for infusion into patients. Incubation of bacteria-infected melanoma cells with dendritic cells in vitro allowed loading of the immune cells with tumor peptides, conferring an effective antitumor response when the dendritic cells were injected into tumor-bearing mice. And this approach could have even more practical potential: These in vitro–loaded immune cells protected mice against developing tumors from seeded cancer cells, a “vaccination”-style preventive strategy. Antigen-presenting dendritic cells (DCs) trigger the activation of cytotoxic CD8 T cells that target and eliminate cells with the antigen on their surface. Although DCs usually pick up and process antigens themselves, they can also receive peptide antigens from other cells via gap junctions. We demonstrate here that infection with Salmonella can induce, in both human and murine melanoma cells, the up-regulation of connexin 43 (Cx43), a ubiquitous protein that forms gap junctions and that is normally lost during melanoma progression. Bacteria-treated melanoma cells can establish functional gap junctions with adjacent DCs. After bacterial infection, these gap junctions transferred preprocessed antigenic peptides from the tumor cells to the DCs, which then presented those peptides on their surface. These peptides activated cytotoxic T cells against the tumor antigen, which could control the growth of distant uninfected tumors. Melanoma cells in which Cx43 had been silenced, when infected in vivo with bacteria, failed to elicit a cytotoxic antitumor response, indicating that this Cx43 mechanism is the principal one used in vivo for the generation of antitumor responses. The Cx43-dependent cross-presentation pathway is more effective than standard protocols of DC loading (peptide, tumor lysates, or apoptotic bodies) for generating DC-based tumor vaccines that both inhibit existing tumors and prevent tumor establishment. In conclusion, we exploited an antimicrobial response present in tumor cells to activate cytotoxic CD8 T cells specific for tumor-generated peptides that could directly recognize and kill tumor cells.

Spatial control of pa-GFP photoactivation in living cells

Photoactivatable green fluorescent protein (paGFP) exhibits peculiar photo‐physical properties making it an invaluable tool for protein/cell tracking in living cells/organisms. paGFP is normally excited in the violet range (405 nm), with an emission peak centred at 520 nm. Absorption cross‐section at 488 nm is low in the not‐activated form. However, when irradiated with high‐energy fluxes at 405 nm, the protein shows a dramatic change in its absorption spectra becoming efficiently excitable at 488 nm. Confocal microscopes allow to control activation in the focal plane. Unfortunately, irradiation extends to the entire illumination volume, making impracticable to limit the process in the 3D (three‐dimensional) space. In order to confine the process, we used two advanced intrinsically 3D confined optical methods, namely: total internal reflection fluorescence (TIRF) and two‐photon excitation fluorescence (2PE) microscopy. TIRF allows for spatially selected excitation of fluorescent molecules within a thin region at interfaces, i.e. cellular membranes. Optimization of the TIRF optical set‐up allowed us to demonstrate photoactivation of paGFP fused to different membrane localizing proteins. Exploitation of the penetration depth showed that activation is efficiently 3D confined even if limited at the interface. 2PE microscopy overcomes both the extended excitation volume of the confocal case and the TIRF constraint of operating at interfaces, providing optical confinement at any focal plane in the specimen within subfemtoliter volumes. The presented results emphasize how photoactivation by non‐linear excitation can provide a tool to increase contrast in widefield and confocal cellular imaging.

Photoactivation of pa-GFP in 3D: optical tools for spatial confinement

Photoactivatable fluorescent proteins represent an innovative tool for the direct observation of time dependent macromolecular events in living systems. The possibility of switching on a selected and confined subset of the expressed target proteins allows to follow biological processes reaching high signal to noise ratios. In particular, use of non-linear interactions to bring the molecules in the activated fluorescent form make it possible to extend the advantages of photoactivation to events that requires 3D spatial localization. In this work, we show the possibility to realize confined activated volumes in living cells, by employing photoactivatable green fluorescent protein (paGFP) in two-photon microscopy. The analysis of the kinetics of two-photon paGFP activation in dependence of the wavelength, the laser intensity and the exposure time is provided. This study allowed to assess the optimal conditions to induce photoactivation in living samples and to track the behaviour of tagged histone H2B during cellular division. Furthermore we investigate paGFP photoactivation under evanescent wave illumination. Total internal reflection set-up has been used to selectively activate subresolved distribution of proteins localized in the basal membrane surroundings. These two photoactivation methods provide a suitable tool for many biological applications, combining subresolved surface and in-depth three-dimensionally confined investigations.

Blue-light (488nm)-irradiation-induced photoactivation of the photoactivatable green fluorescent protein

We experimentally demonstrate the photoactivatable green fluorescent protein (paGFP) photoactivation in a wavelength range where the molecule barely absorbs. The photoactivation is induced at the same wavelength used to visualize the activated form of paGFP. This can be an obstacle in the intensity evaluation in photoactivation experiments. Power and kinetics based characterization of the effect was performed in model and cell systems. This study shows an operative threshold in which paGFP is not subjected to significant photoconversion. 488nm photoactivation is in tune with the broadening of the paGFP two-photon activation spectrum, indicating that multiple interactions lead to modifications of the molecular structure and alterations of its photophysical properties.

3D localized photoactivation of pa-GFP in living cells using two-photon interactions.

We report about two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm. The aim in this work was to evaluate the use of two-photon interactions for confining the molecular switching of pa-GFP in the bright state. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation and two-photon mode demonstrates clearly the better three- dimensional (3D) confinement and the possibility of selection of cell volumes of interest. This enables molecular trafficking studies at high signal to noise ratio

Erratum: Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation (Nature Cell Biology (2012) 14 (355-365))

The DNA damage response (DDR) arrests cell-cycle progression until damage is removed. DNA damage-induced cellular senescence is associated with persistent DDR. The molecular bases that distinguish transient from persistent DDR are unknown. Here we show that a large fraction of exogenously-induced persistent DDR markers are associated with telomeric DNA in cultured cells and mammalian tissues. In yeast, a chromosomal DNA double-strand break (DSB) next to telomeric sequences resists repair and impairs DNA ligase 4 recruitment. In mammalian cells, ectopic localization of telomeric factor TRF2 next to a DSB induces persistent DNA damage and DDR. Linear telomeric DNA, but not circular or scrambled DNA, induces a prolonged checkpoint in normal cells. In terminally-differentiated tissues of old primates, DDR markers accumulate at telomeres which are not critically short. We propose that linear genomes are not uniformly 12Correspondence should be addressed to Fabrizio, d’Adda di Fagagna IFOM Foundation – The FIRC Institute of Molecular Oncology Foundation, via Adamello 16, 20139 Milan, Italy, Tel. +39 02 574303.227, Fax +39 02 574303.231, fabrizio.dadda@ifomieo-campus.it. 8Present Address: TTFactor Srl, Milan, Italy. 9Present Address: Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy. 10Present Address: Fresenius Medical Care, Turin, Italy. 11These authors contributed equally Author Contributions F.R. generated and assembled data in Figs. 5a–c, 5e–f, 5h–i, 7a–c, 8d, S1b–c, S3b, S4c, S5, S6, S7b–c, S8a; M.C. and M.P.L. generated data in Fig. 6; S.B. performed the microinjection experiments; D.C. performed the analysis of sequencing data and generated data in Figs. 3a–b; J. M. K. generated data in Figs. 8e and S8b; G.B. contributed to the pre-processing and analysis of sequencing data in Figs. 3a–b; M.D. provided technical assistance; V.M. generated data in Figs. 5d and 5g and provided technical assistance; C.M.B. provided irradiated mice brain sections; U.H. provided baboons sections and edited the ms; M.F. generated and assembled data of all remaining figures, performed ChIP assays in mammalian cells, contributed to experimental design and ms writing; F.d’A.d.F. planned and supervised the project and wrote the ms. The authors declare no conflict of interests. NIH Public Access Author Manuscript Nat Cell Biol. Author manuscript; available in PMC 2013 July 22. Published in final edited form as: Nat Cell Biol. ; 14(4): 355–365. doi:10.1038/ncb2466. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript reparable and telomeric DNA tracts, if damaged, are irreparable and trigger persistent DDR and cellular senescence.

T2P-GFP: two-photon photo-activation of PA-GFP in the 720-840 nm spectral region

We report about a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm1. The aim in this work was to evaluate the use of two-photon interactions for activation of the molecules2. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The latter objects were used to investigate the ability of different wavelengths to activate the paGFP due to the anticipated more homogeneous density distribution. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation with a single photon at 413 nm and two-photons demonstrates clearly the advantages using non linear processes: much smaller volume in the cell can be activated unlike to a whole cell activation in single photon excitation regime.

THE OXIDATIVE STRESS RESPONSE GENE P66 SHC AND THE TUMOR SUPPRESSOR GENE P53 INDUCE MITOCHONDRIAL DNA DAMAGES

INTRODUCTION. The p66 gene regulates the oxidative stress response and life span in mammals (1). The p53 gene regulates various cellular responses to environmental stresses, particularly those inducing DNA damage, such as cell cycle arrest, nuclear DNA repair, senescence and apoptosis (2-3). Both p66 and p53 increase the intracellular concentrations of reactive oxygen species (ROS), measured by DCFDA staining of wt and -/PEFs counterparts (our man. in prep). Oxidative stress is considered the principal proximal mechanism of ageing in mammals and lower organisms (4). ROS are also responsible for nuclear DNA mutagenesis and tumor formation.