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Triple-negative breast cancer (TNBC) is associated with an elevated risk of recurrence and poor prognosis. Historically, only chemotherapy was available as systemic treatment, but immunotherapy and targeted therapies currently offer prolonged benefits. TNBC is a group of diseases with heterogeneous treatment sensitivity, and resistance is inevitable and early for a large proportion of the intrinsic subtypes. Although senescence induction by anticancer therapy offers an immediate favorable clinical outcome once the rate of tumor progression reduces, these cells are commonly dysfunctional and metabolically active, culminating in treatment-resistant repopulation associated with worse prognosis. This heterogeneous response can also occur without therapeutic pressure in response to damage or oncogenic stress, playing a relevant role in the carcinogenesis. Remarkably, there is preclinical and exploratory clinical evidence to support a relevant role of senescence in treatment resistance. Therefore, targeting senescent cells has been a scientific effort in many malignant tumors using a variety of targets and strategies, including increasing proapoptotic and decreasing antiapoptotic stimuli. Despite promising results, there are some challenges to applying this technology, including the best schedule of combination, assessment of senescence, specific vulnerabilities, and the best clinical scenarios. This review provides an overview of senescence in TNBC with a focus on future-proofing senotherapy strategies.

Examples of “one-two punch” strategy in TNBC.

Examples of senolytics clinical performance in solid tumors and breast cancer.

Examples of senescence inducers in TNBC.

Triple-negative breast cancer (TNBC) is associated with an elevated risk of recurrence and poor prognosis. Historically, only chemotherapy was available as systemic treatment, but immunotherapy and targeted therapies currently offer prolonged benefits. TNBC is a group of diseases with heterogeneous treatment sensitivity, and resistance is inevitable and early for a large proportion of the intrinsic subtypes. Although senescence induction by anticancer therapy offers an immediate favorable clinical outcome once the rate of tumor progression reduces, these cells are commonly dysfunctional and metabolically active, culminating in treatment-resistant repopulation associated with worse prognosis. This heterogeneous response can also occur without therapeutic pressure in response to damage or oncogenic stress, playing a relevant role in the carcinogenesis. Remarkably, there is preclinical and exploratory clinical evidence to support a relevant role of senescence in treatment resistance. Therefore, targeting senescent cells has been a scientific effort in many malignant tumors using a variety of targets and strategies, including increasing proapoptotic and decreasing antiapoptotic stimuli. Despite promising results, there are some challenges to applying this technology, including the best schedule of combination, assessment of senescence, specific vulnerabilities, and the best clinical scenarios. This review provides an overview of senescence in TNBC with a focus on future-proofing senotherapy strategies.

Platinum-based chemotherapy is commonly used for non-small cell lung cancer (NSCLC) treatment, yet clinical outcomes remain poor. Cellular senescence and its associated secretory phenotype (SASP) can have multiple tumour-promoting activities, although these are largely unexplored in lung cancer. Here we show that cisplatin-derived SASP enhances the malignant phenotype of lung cancer cells. Using xenograft, orthotopic and KrasG12V-driven murine NSCLC models, we demonstrate that cisplatin-induced senescent cells strongly promote tumour progression. Mechanistically, we find that a TGF-β-enriched SASP drives pro-proliferative effects through TGFβR1 and Akt/mTOR pathway activation. We validate the translational relevance of chemotherapy-induced SASP using clinical NSCLC samples from patients who received neoadjuvant platinum-based chemotherapy. Importantly, TGFβR1 inhibition with galunisertib or senolytic treatment significantly reduces tumour promotion driven by cisplatin-induced senescence. Finally, we demonstrate, using distinct murine NSCLC models, that addition of TGFBR1 inhibitors to platinum-based chemotherapy reduces tumour burden and improves survival, providing pre-clinical proof-of-concept for future trial designs.

Aging is a major risk factor for cerebral infarction. Since cellular senescence is intrinsic to aging, we postulated that stroke-induced cellular senescence might contribute to neural dysfunction. Adult male Wistar rats underwent 60-minute middle cerebral artery occlusion and were grouped according to 3 reperfusion times: 24 hours, 3, and 7 days. The major biomarkers of senescence: 1) accumulation of the lysosomal pigment, lipofuscin; 2) expression of the cell cycle arrest markers p21, p53, and p16(INK4a); and 3) expression of the senescence-associated secretory phenotype cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) were investigated in brain samples. Lipofuscin accumulation was scarce at the initial stage of brain damage (24 hours), but progressively increased until it reached massive distribution at 7 days post-ischemia. Lipofuscin granules (aggresomes) were mainly confined to the infarcted areas, that is parietal cortex and adjacent caudate-putamen, which were equally affected. The expression of p21, p53, and p16(INK4a), and that of IL-6, TNF-alpha, and IL-1 beta, was significantly higher in the ischemic hemisphere than in the non-ischemic hemisphere. These data indicate that brain cell senescence develops during acute ischemic infarction and suggest that the acute treatment of ischemic stroke might be enhanced using senolytic drugs.

Cellular senescence is considered a crucial process for tumour suppression, which can be facilitated by immune surveillance. However, when senescent cells persist in tissues, they can also trigger a plethora of tumour‐promoting effects. Here, we discuss the main hallmarks, mechanisms and roles of senescence in cancer and provide a comprehensive revision of the available tools for its detection., Senescence refers to a cellular state featuring a stable cell‐cycle arrest triggered in response to stress. This response also involves other distinct morphological and intracellular changes including alterations in gene expression and epigenetic modifications, elevated macromolecular damage, metabolism deregulation and a complex pro‐inflammatory secretory phenotype. The initial demonstration of oncogene‐induced senescence in vitro established senescence as an important tumour‐suppressive mechanism, in addition to apoptosis. Senescence not only halts the proliferation of premalignant cells but also facilitates the clearance of affected cells through immunosurveillance. Failure to clear senescent cells owing to deficient immunosurveillance may, however, lead to a state of chronic inflammation that nurtures a pro‐tumorigenic microenvironment favouring cancer initiation, migration and metastasis. In addition, senescence is a response to post‐therapy genotoxic stress. Therefore, tracking the emergence of senescent cells becomes pivotal to detect potential pro‐tumorigenic events. Current protocols for the in vivo detection of senescence require the analysis of fixed or deep‐frozen tissues, despite a significant clinical need for real‐time bioimaging methods. Accuracy and efficiency of senescence detection are further hampered by a lack of universal and more specific senescence biomarkers. Recently, in an attempt to overcome these hurdles, an assortment of detection tools has been developed. These strategies all have significant potential for clinical utilisation and include flow cytometry combined with histo‐ or cytochemical approaches, nanoparticle‐based targeted delivery of imaging contrast agents, OFF‐ON fluorescent senoprobes, positron emission tomography senoprobes and analysis of circulating SASP factors, extracellular vesicles and cell‐free nucleic acids isolated from plasma. Here, we highlight the occurrence of senescence in neoplasia and advanced tumours, assess the impact of senescence on tumorigenesis and discuss how the ongoing development of senescence detection tools might improve early detection of multiple cancers and response to therapy in the near future.

This article belongs to the Special Issue Oxidative Stress and Inflammation in Deafness: Current and Future Therapy., Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), whose loss of function leads to sustained SAPK activation. Dusp1 gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in Dusp1−/− mice. RNA sequencing analysis (GSE176114) indicated that Dusp1−/− cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated Dusp1−/− mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL+ HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in Dusp1−/− mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation, This work was funded by FEDER/B2017/BMD-3688-MULTITARGET&VIEWCM and 0551-PSL-6-E NITROPROHEAR grants to I.V.-N., J.M.B.-M., Á.G.-M. and L.R.-d.l.R. held SAF2017-HEARCODE, FPU (FPU16/03308; MECD) and CIBERER (Institute of Health Carlos III) contracts, respectively, co-financed with FEDER funds. Work by D.M.-E. and M.S. was funded by grant SAF2017-82613-R from the Spanish Ministry of Science co-funded by the European Regional Development Fund (ERDF). Work in the laboratory of M.S. was also funded by the IRB and “laCaixa” Foundation, and by grants from the European Research Council (ERC-2014-AdG/669622), and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282. D.M.-E. laboratory is supported by the Cancer Research UK (CRUK) Cambridge Centre Early Detection Programme (RG86786), by a CRUK Programme Foundation Award (C62187/A29760), by a CRUK Early Detection OHSU Project Award (C62187/A26989), and by a Medical Research Council (MRC) New Investigator Research Grant (NIRG) (MR/R000530/1).

Background SARS-CoV-2 infection of the respiratory system can progress to a multi-systemic disease with aberrant inflammatory response. Cellular senescence promotes chronic inflammation, named as senescence-associated secretory phenotype (SASP). We investigated whether COVID-19 disease is associated with cellular senescence and SASP. Methods Autopsy lung tissue samples from 11 COVID-19 patients and 43 age-matched non-COVID controls with similar comorbidities were analysed by immunohistochemistry for SARS-CoV-2, markers of senescence and key SASP cytokines. Virally-induced senescence was functionally recapitulated in vitro, by infecting epithelial Vero-E6 cells and a three-dimensional alveosphere system of alveolar type 2 (AT2) cells with SARS-CoV-2 strains isolated from COVID-19 patients. Results SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in AT2 cells. Infected AT2 cells expressed the angiotensin-converting-enzyme 2 (ACE2) and exhibited increased senescence (p16INK4A and SenTraGorTM positivity) and IL-1β and IL-6 expression. In vitro, infection of Vero-E6 cells with SARS-CoV-2 induced senescence (SenTraGorTM), DNA damage (γ-H2AX) and increased cytokine (IL-1β, IL-6, CXCL8) and Apolipoprotein B mRNA-editing (APOBEC) enzyme expression. Next-generation-sequencing analysis of progenies obtained from infected/senescent Vero-E6 cells demonstrated APOBEC-mediated SARS-CoV-2 mutations. Dissemination of the SARS-CoV-2-infection and senescence was confirmed in extra-pulmonary sites (kidney and liver) of a COVID-19 patient. Conclusions We demonstrate that in severe COVID-19, AT2 cells infected by SARS-CoV-2 exhibit senescence and a proinflammatory phenotype. In vitro, SARS-CoV-2 infection induces senescence and inflammation. Importantly, infected senescent cells may act as a source of SARS-CoV-2 mutagenesis mediated by APOBEC enzymes. Therefore, SARS-CoV-2-induced senescence may be an important molecular mechanism of severe COVID-19, disease persistence and mutagenesis.

Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal β-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues.

[EN] Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal beta-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues., R.M. laboratory members thank the financial support from the Spanish Government (project RTI2018-100910-B-C41) and the Generalitat Valenciana (project PROMETEO 2018/024). B.L.-T. is grateful to the Spanish Ministry of Economy for their PhD grants (FPU15/02707). I.G. thanks her contract from IDM. J.F.-B. thanks to his postdoctoral fellowship (CD19/00038). J.A.L.-D. thanks the financial support from the Ministry of Science/MICINN (IJCI-2017-33047). Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy cofunded by the European Regional Development Fund (ERDF) (SAF201348256-R), the European Research Council (ERC-2014-AdG/669622), and the "laCaixa" Foundation. The D.M.-E. laboratory is supported by the Cancer Research U.K. (CRUK), Cambridge Centre Early Detection Programme, by a CRUK Early Detection OHSU project award (C62187/A26989), by a Medical Research Council (MRC) New Investigators Research Grant (NIRG, MR/R000530/1).

Cellular senescence is a physiological mechanism whereby a proliferating cell undergoes a stable cell cycle arrest upon damage or stress and elicits a secretory phenotype. This highly dynamic and regulated cellular state plays beneficial roles in physiology, such as during embryonic development and wound healing, but it can also result in antagonistic effects in age-related pathologies, degenerative disorders, ageing and cancer. In an effort to better identify this complex state, and given that a universal marker has yet to be identified, a general set of hallmarks describing senescence has been established. However, as the senescent programme becomes more defined, further complexities, including phenotype heterogeneity, have emerged. This significantly complicates the recognition and evaluation of cellular senescence, especially within complex tissues and living organisms. To address these challenges, substantial efforts are currently being made towards the discovery of novel and more specific biomarkers, optimized combinatorial strategies and the development of emerging detection techniques. Here, we compile such advances and present a multifactorial guide to identify and assess cellular senescence in cell cultures, tissues and living organisms. The reliable assessment and identification of senescence is not only crucial for better understanding its underlying biology, but also imperative for the development of diagnostic and therapeutic strategies aimed at targeting senescence in the clinic.

Cellular senescence, an age-related process in response to damage and stress, also occurs during normal development and adult life. The thymus is a central lymphoepithelial organ of the immune system that exhibits age-related changes termed thymic involution. Since the mechanisms regulating thymic involution are still not well elucidated, we questioned whether cellular senescence is implicated in this process. We demonstrate, for the first time in situ, that cellular senescence occurs during human thymic involution using SenTraGor™, a novel chemical compound that is applicable in archival tissue material, providing thus further insights in thymus histophysiology.

[EN] Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence-associated lysosomal beta-galactosidase (SA-beta-gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose-encapsulated nanoparticles within these cells. Here, we show that galacto-conjugation of the BCL-2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav-Gal), that can be preferentially activated by SA-beta-gal activity in a wide range of cell types. Nav-Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav-Gal enhances the cytotoxicity of standard senescence-inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav-Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto-conjugation reduces Navitoclax-induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities., Royal Society, Grant/Award Number: RG160806; Medical Research Council, Grant/Award Number: MR/R000530/1; Cancer Research UK, Grant/Award Number: C62187/A26989 and C62187/A29760; CRUK Cambridge Centre Early Detection Programme, Grant/Award Number: RG86786

Cellular senescence is an important anticancer mechanism that restricts proliferation of damaged or premalignant cells. Cellular senescence also plays an important role in tissue remodeling during development. However, there is a trade-off associated with cellular senescence as senescent cells contribute to aging pathologies. The naked mole rat (NMR) (Heterocephalus glaber) is the longest-lived rodent that is resistant to a variety of age-related diseases. Remarkably, NMRs do not show aging phenotypes until very late stages of their lives. Here, we tested whether NMR cells undergo cellular senescence. We report that the NMR displays developmentally programmed cellular senescence in multiple tissues, including nail bed, skin dermis, hair follicle, and nasopharyngeal cavity. NMR cells also underwent cellular senescence when transfected with oncogenic Ras. In addition, cellular senescence was detected in NMR embryonic and skin fibroblasts subjected to γ-irradiation (IR). However, NMR cells required a higher dose of IR for induction of cellular senescence, and NMR fibroblasts were resistant to IR-induced apoptosis. Gene expression analyses of senescence-related changes demonstrated that, similar to mice, NMR cells up-regulated senescence-associated secretory phenotype genes but displayed more profound down-regulation of DNA metabolism, transcription, and translation than mouse cells. We conclude that the NMR displays the same types of cellular senescence found in a short-lived rodent.

Summary Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence., Graphical Abstract, Highlights • Small extracellular vesicles (sEVs) mediate paracrine senescence • Inhibition of sEV biogenesis prevents paracrine senescence • Multivesicular bodies and CD63 are increased during senescence in vivo • MS analysis identifies IFITM3 as partially responsible for sEV-paracrine senescence, Borghesan et al. show that the soluble fraction and small extracellular vesicles (sEVs) mediate paracrine senescence. RNA sequencing and loxP reporter systems confirm sEV-mediated paracrine senescence, while preventing sEV release averts senescence. Mass spectrometry and functional analysis show that the IFN protein, IFITM3, is partially responsible for this phenotype.

Summary Protein-primed DNA replication constitutes a strategy to initiate viral DNA synthesis in a variety of prokaryotic and eukaryotic organisms. Although the main function of viral terminal proteins (TPs) is to provide a free hydroxyl group to start initiation of DNA replication, there are compelling evidences that TPs can also play other biological roles. In the case of Bacillus subtilis bacteriophage ϕ29, the N-terminal domain of the TP organizes viral DNA replication at the bacterial nucleoid being essential for an efficient phage DNA replication, and it contains a nuclear localization signal (NLS) that is functional in eukaryotes. Here we provide information about the structural properties of the ϕ29 TP N-terminal domain, which possesses sequence-independent DNA-binding capacity, and dissect the amino acid residues important for its biological function. By mutating all the basic residues of the TP N-terminal domain we identify the amino acids responsible for its interaction with the B. subtilis genome, establishing a correlation between the capacity of DNA-binding and nucleoid localization of the protein. Significantly, these residues are important to recruit the DNA polymerase at the bacterial nucleoid and, subsequently, for an efficient phage DNA replication.

Spanish Ministries of Economy and Competitiveness and Science and Innovation; Fundacion Ramon Areces

During evolution, viruses have optimized the interaction with host factors to increase the efficiency of fundamental processes such as DNA replication. Bacteriophage ϕ29 protein p1 is a membrane-associated protein that forms large protofilament sheets that resemble eukaryotic tubulin and bacterial filamenting temperature-sensitive mutant Z protein (FtsZ) polymers. In the absence of protein p1, phage ϕ29 DNA replication is impaired. Here we show that a functional fusion of protein p1 to YFP localizes at the medial region of Bacillus subtilis cells independently of other phage-encoded proteins. We also show that ϕ29 protein p1 colocalizes with the B. subtilis cell division protein FtsZ and provide evidence that FtsZ and protein p1 are associated. Importantly, the midcell localization of YFP-p1 was disrupted in a strain that does not express FtsZ, and the fluorescent signal was distributed all over the cell. Depletion of penicillin-binding protein 2B (PBP2B) in B. subtilis cells did not affect the subcellular localization of YFP-p1, indicating that its distribution does not depend on septal wall synthesis. Interestingly, when ϕ29 protein p1 was expressed, B. subtilis cells were about 1.5-fold longer than control cells, and the accumulation of ϕ29 DNA was higher in mutant B. subtilis cells with increased length. We discuss the biological role of p1 and FtsZ in the ϕ29 growth cycle.

[EN] A naphthalimide-based two-photon probe (AHGa) for the detection of cell senescence is designed. The probe contains a naphthalimide core, an L-histidine methyl ester linker, and an acetylated galactose bonded to one of the aromatic nitrogen atoms of the L-histidine through a hydrolyzable N-glycosidic bond. Probe AHGa is transformed into AH in senescent cells resulting in an enhanced fluorescent emission intensity. In vivo detection of senescence is validated in mice bearing tumor xenografts treated with senescence-inducing chemotherapy., The authors thank the financial support from the Spanish Government (Projects MAT2015-64139-C4-1-R) and the Generalitat Valenciana (Project PROMETEOII/2014/047). B.L-T. is grateful to the Spanish Ministry of Economy for her PhD grant. I.G. thanks the Generalitat Valenciana for her PhD grant. A.B. thanks the Spanish Government for the financial support "Juan de la Cierva-Incorporacion". M.R. is grateful to "La Caixa" foundation for his PhD scholarship. Work in the M.S. group was funded by the CNIO and by grants from Spanish Ministry of Economy, the European Regional Development Fund (SAF project) the European Research Council (ERC Advanced Grant), and the Botin Foundation and Banco Santander (Santander Universities Global Division). D.M.-E. was funded by a "Ramon y Cajal" Programme Senior Grant and the "RETOS" National Programme for Research (MINECO), Cancer Research UK, and the CRUK Cambridge Centre Early Detection Programme.

A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of ο29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of ο29 TP led us to identify a bona fide NLS within residues 1-37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of ο29 TP attached to the 5′ DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes., Spanish Ministry of Economy and Competitiveness; Fundación Ramón Areces; Spanish Ministry of Education

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage φ29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call “Pro cage.” Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.

The Bacillus subtilis phage phi29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and x-ray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled.

Published by Oxford University Press on behalf of Nucleic Acids Research. Phage φ29 DNA replication takes place by a proteinpriming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the φ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the Km for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the φ29 double-stranded DNA binding protein p6 in the reaction, which decreased the Km of the DNA polymerase for dATP about 130-190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification., Spanish Ministry of Economy and Competitiveness [BFU2011–23645] ; Consolider-Ingenio from the Spanish 2800 Nucleic Acids Research, 2015, Vol. 43, No. 5 Ministry of Science and Innovation [CSD2007–00015 to M.S.] ; Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular‘Severo Ochoa’. I.H. was holder of an FPU fellowship from the Spanish Ministry of Education. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2011- 23645]

Recent discoveries are redefining our view of cellular senescence as a trigger of tissue remodelling that acts during normal embryonic development and upon tissue damage. To achieve this, senescent cells arrest their own proliferation, recruit phagocytic immune cells and promote tissue renewal. This sequence of events - senescence, followed by clearance and then regeneration - may not be efficiently completed in aged tissues or in pathological contexts, thereby resulting in the accumulation of senescent cells. Increasing evidence indicates that both pro-senescent therapies and antisenescent therapies can be beneficial. In cancer and during active tissue repair, pro-senescent therapies contribute to minimize the damage by limiting proliferation and fibrosis, respectively. Conversely, antisenescent therapies may help to eliminate accumulated senescent cells and to recover tissue function.

Bacteriophage terminal proteins (TPs) prime DNA replication and become covalently linked to the DNA 5'-ends. In addition, they are DNA-binding proteins that direct early organization of phage DNA replication at the bacterial nucleoid and, unexpectedly, contain nuclear localization signals (NLSs), which localize them to the nucleus when expressed in mammalian cells. In spite of the lack of sequence homology among the phage TPs, these three properties share some common features, suggesting a possible evolutionary common origin of TPs. We show here that NLSs of three different phage TPs, Φ29, PRD1 and Cp-1, are mapped within the protein region required for nucleoid targeting in bacteria, in agreement with a previously proposed common origin of DNA-binding domains and NLSs. Furthermore, previously reported point mutants of Φ29 TP with no nuclear localization still can target the bacterial nucleoid, and Cp-1 TP contains two independent NLSs, only one of them required for nucleoid localization. Altogether, our results show that nucleoid and nucleus localization sequence requirements partially overlap, but they can be uncoupled, suggesting that conservation of both features could have a common origin but, at the same time, they have been independently conserved during evolution.

et al., Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence., D.M.-E. has been funded by the Juan de la Cierva Program. A.M. and M. Collado were funded by the Miguel Servet Program. S.M.-C. holds a CIBERER (ISCIII) postdoctoral contract. Work in the laboratory of M.S. is funded by the CNIO and by grants from the MICINN (SAF), the Regional Government of Madrid, the European Research Council (Advanced ERC Grant), the Botín Foundation, the Ramón Areces Foundation, and the AXA Foundation. Work in the I.V.-N. group is supported by grants from MINECO (SAF2011-24391), Fundación de Investigación Médica Mutua Madrileña (2012), and AFHELO (FP7, European Union).

Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. Unexpectedly, we have found functional eukaryotic nuclear localization signals (NLSs) within the TP sequences of bacteriophages from diverse families and hosts. Given the role of bacteriophages as vehicles for horizontal gene transfer (HGT), we postulated that viral genomes that have covalently linked NLS-containing terminal proteins might behave as vectors for HGT between bacteria and the eukaryotic nucleus. To validate this hypothesis, we profited from the in vitro Φ29 amplification system that allows the amplification of heterologous DNAs producing linear molecules of DNA with TP covalently attached to both 5' ends. Interestingly, these in vitrogenerated TP-DNA molecules showed enhanced gene delivery in mammalian cells, supporting a possible role in HGT by transferring genes between prokaryotes and eukaryotes. Moreover, these TP-DNA molecules are a useful tool to amplify and subsequently deliver genes efficiently into the eukaryotic nucleus. Here, we suggest various possible applications and further developments of the technique with biotechnological and therapeutic purposes, This work was supported by Grant BFU 2011-23645 and Consolider-Ingenio Grant 2010 24717 from the Spanish Ministry of Economy and Competitiveness (MINECO) and by an institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa.”

During the course of evolution, viruses have learned to take advantage of the natural resources of their hosts for their own benefit. Due to their small dimension and limited size of genomes, bacteriophages have optimized the exploitation of bacterial host factors to increase the efficiency of DNA replication and hence to produce vast progeny. The Bacillus subtilis phage φ29 genome consists of a linear double-stranded DNA molecule that is duplicated by means of a protein-primed mode of DNA replication. Its genome has been shown to be topologically constrained at the size of the bacterial nucleoid and, as to avoid generation of positive supercoiling ahead of the replication forks, the bacterial DNA gyrase is used by the phage. In addition, the B. subtilis actin-like MreB cytoskeleton plays a crucial role in the organization of φ29 DNA replication machinery in peripheral helix-like structures. Thus, in the absence of an intact MreB cytoskeleton, φ29 DNA replication is severely impaired. Importantly, MreB interacts directly with the phage membrane protein p16.7, responsible for attaching φ29 DNA at the cell membrane. Moreover, the φ29-encoded protein p56 inhibits host uracil-DNA glycosylase activity and has been proposed to be a defense mechanism developed by the phage to prevent the action of the base excision repair pathway if uracil residues arise in replicative intermediates. All of them constitute incoming examples on how viruses have profited from the cellular machinery of their hosts.

During the course of evolution, viruses have learned to take advantage of the natural resources of their hosts for their own benefit. Due to their small dimension and limited size of genomes, bacteriophages have optimized the exploitation of bacterial host factors to increase the efficiency of DNA replication and hence to produce vast progeny. The Bacillus subtilis phage φ29 genome consists of a linear double-stranded DNA molecule that is duplicated by means of a protein-primed mode of DNA replication. Its genome has been shown to be topologically constrained at the size of the bacterial nucleoid and, as to avoid generation of positive supercoiling ahead of the replication forks, the bacterial DNA gyrase is used by the phage. In addition, the B. subtilis actin-like MreB cytoskeleton plays a crucial role in the organization of φ29 DNA replication machinery in peripheral helix-like structures. Thus, in the absence of an intact MreB cytoskeleton, φ29 DNA replication is severely impaired. Importantly, MreB interacts directly with the phage membrane protein p16.7, responsible for attaching φ29 DNA at the cell membrane. Moreover, the φ29-encoded protein p56 inhibits host uracil-DNA glycosylase activity and has been proposed to be a defense mechanism developed by the phage to prevent the action of the base excision repair pathway if uracil residues arise in replicative intermediates. All of them constitute incoming examples on how viruses have profited from the cellular machinery of their hosts.

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage φ29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the φ29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA,which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria., Spanish Ministry of Science and Innovation; Spanish Ministries of Education; Fundación Ramón Areces

The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli , respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1–73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage φ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the φ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the φ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane., Spanish Ministry of Education and Science Grants BFU2005-00733 (to M.S.) and BFU2005-01878 (to W.J.J.M.), Spanish Ministry of Science and Innovation Grant BFU2008-00215 (to M.S.), Spanish Ministry of Education and Science Grant Consolider-Ingenio 2010 24717 (to M.S.) and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘‘Severo Ochoa

Remarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage 29 as a model system. Previously, we demonstrated that the 29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated 29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the 29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo 29 DNA replication is presented., This investigation was supported by National Institutes of Health Research Grant 2RO1 GM27242-23, Dirección General de Investigación Científica y Técnica Grant PB98-0645 and an Institutional grant from Fundación Ramón Areces to the Centro Biología Molecular ‘Severo Ochoa’. A.S.R and D.M. were holders of a predoctoral fellowship from ‘Gobierno Vasco’ and ‘Fondo de Investigaciones Sanitarias’, respectively. The ‘Ramón y Cajal’ program of the Spanish ministry of Science and Technology supported W.J.J.M.

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call "Pro cage." Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.

The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage 29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the 29 genome and the B. subtilis chromosome. Binding of Spo0A near the 29 DNA ends, constituting the two origins of replication of the linear 29 genome, prevents formation of 29 protein p6-nucleoprotein initiation complex resulting in inhibition of 29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA-binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage 29 development and the bacterial chromosome replication during the onset of sporulation are discussed., This investigation was supported by grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to MS and WJJM, respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular 'Severo Ochoa'. VC and DM are holders of predoctoral fellowships from the Spanish Ministry of Science and Technology and the Fondo de Investigación Sanitaria, respectively. WJJM was supported by means of the 'Ramón y Cajal' program from the Spanish Ministry of Science and Technology.

Prokaryotic DNA replication is compartmentalized at the cellular membrane. Functional and biochemical studies showed that the Bacillus subtilis phage ϕ29-encoded membrane protein p16.7 is directly involved in the organization of membrane-associated viral DNA replication. The structure of the functional domain of p16.7 in complex with DNA, presented here, reveals the multimerization mode of the protein and provides insights in the organization of the phage genome at the membrane of the infected cell., This work was supported by the Ministerio de Educación y Ciencia (Grants BMC2002-04011-C05-03 (to A. A.), CTQ2004-04994/BQU (to J. L. A.), and BMC2002-03818 (to M. S.)), a Ramon y Cajal contract to (W. J. J. M.), the Comunidad de Madrid (GR/SAL/0195/2004 to W. J. J. M.), the FIS (fellowship to D. M-E.), and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

The Bacillus subtilis phage phi29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and x-ray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled.

Background: The brain, despite the blood-brain barrier, does not escape to the highly variable host rejection response mediated by a very strong and complex immune reaction when rat glioma cells are transplanted into the adult animal. Methods:Crosses were performed among parents that are able or enable to reject a well-known brain tumor cell line (C6). Newborn animals were also challenged with rat glioma cells both in the brain and the side flanks. Results: The percentage of susceptibility or resistance to develop a lethal glioma can be estimated knowing the parental phenotypes. When both parents had rejected an induced tumor, 63% of the progeny will also reject it. Similarly, if both parents died as a consequence of the tumor, 70% of the progeny would also be unable to reject the challenge of glioma C6 cells. Newborn animals do not have a mature immune system and they tolerate transplanted cells much better than adults. We found no rejection to glioma C6, at both brain and side flank sites, in 1-day-old neonatal Wistar rats. Tumors were beginning to be eliminated if the cells are inoculated at day 3 from birth on the flanks, and at 1week from birth on the brain. Conclusions: There is a genetic component conferring susceptibility or resistance to the lethal effect of tumor development and progression depending on the parental phenotype of the adult rats. Neonatal rats represent a much more reliable model than adults to study experimental therapies against gliomas.