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The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.

The classification of the fungicide captan (CAS Number: 133-06-2) as a carcinogen agent is presently under discussion. Despite the mutagenic effect detected by the Ames test and carcinogenic properties observed in mice, the genotoxicity of this pesticide in humans is still unclear. New information is needed about its mechanism of action in mammalian cells. Here, we show that Chinese Hamster Ovary (CHO) cells exposed to captan accumulate Fpg-sensitive DNA base alterations. In CHO and HeLa cells, such DNA lesions require the XRCC1-dependent pathway to be repaired. Captan also induces a replicative stress that activated the ATR signaling response and resulted in double-strand breaks and micronuclei. The replicative stress is characterized by a dramatic decrease in DNA synthesis due to a reduced replication fork progression. However, impairment of the XRCC1-related repair process did not amplify the replicative stress, suggesting that the fork progression defect is independent from the presence of base modifications. These results support the involvement of at least two independent pathways in the genotoxic effect of captan that might play a key role in carcinogenesis. Environ. Mol. Mutagen. 60:286-297, 2019. © 2018 Wiley Periodicals, Inc.

The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA damage response and induces inflammatory signatures in host cells, but the precise relationship between these outcomes has not been addressed so far. Here, we show that the cellular proinflammatory response and senescence induced by CDT depend on the cytoplasmic DNA sensor cGAS through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular inflammatory response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.

International audience; The Cytolethal Distending Toxin (CDT) is produced by many Gram-negative pathogenic bacteria responsible for major foodborne diseases worldwide. CDT induces DNA damage and cell cycle arrest in host-cells, eventually leading to senescence or apoptosis. According to structural and sequence comparison, the catalytic subunit CdtB is suggested to possess both nuclease and phosphatase activities, carried by a single catalytic site. However, the impact of each activity on cell-host toxicity is yet to be characterized. Here, we analyze the consequences of cell exposure to different CDT mutated on key CdtB residues, focusing on cell viability, cell cycle defects, and DNA damage induction. A first class of mutant, devoid of any activity, targets putative catalytic (H160A), metal binding (D273R), and DNA binding residues (R117A-R144A-N201A). The second class of mutants (A163R, F156-T158, and the newly identified G114T), which gathers mutations on residues potentially involved in lipid substrate binding, has only partially lost its toxic effects. However, their defects are alleviated when CdtB is artificially introduced inside cells, except for the F156-T158 double mutant that is defective in nuclear addressing. Therefore, our data reveal that CDT toxicity is mainly correlated to CdtB nuclease activity, whereas phosphatase activity may probably be involved in CdtB intracellular trafficking.

The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin that activates the DNA damage response and induces inflammatory signatures in host cells, but the precise relationship between these outcomes has not been addressed so far. CDT induces a singular time-dependent increase of DNA damage and cell cycle defects, questioning on possible impaired response to this toxin over the cell cycle. Here, we identify mitosis as a crucial phase during CDT intoxination. Despite active cell cycle checkpoints and in contrast to other DNA damaging agents, CDT-exposed cells reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation. These micronuclei are recognized by cGAS that elicits an inflammatory signature resulting in cell distention and senescence. Our results unravel for the first time the mitotic consequences of CDT genotoxic activity and relate them to pro-inflammatory cellular response. These findings may have important implications during bacterial infection regarding CDT-mediated immunomodulatory and tumorigenic processes.

ObjectiveCampylobacter jejuni produces a genotoxin, cytolethal distending toxin (CDT), which has DNAse activity and causes DNA double-strand breaks. Although C. jejuni infection has been shown to promote intestinal inflammation, the impact of this bacterium on carcinogenesis has never been examined.DesignGerm-free (GF) ApcMin/+mice, fed with 1% dextran sulfate sodium, were used to test tumorigenesis potential of CDT-producing C. jejuni. Cells and enteroids were exposed to bacterial lysates to determine DNA damage capacity via γH2AX immunofluorescence, comet assay and cell cycle assay. To examine the interplay of CDT-producing C. jejuni, gut microbiome and host in tumorigenesis, colonic RNA-sequencing and faecal 16S rDNA sequencing were performed. Rapamycin was administrated to investigate the prevention of CDT-producing C. jejuni-induced tumorigenesis.ResultsGF ApcMin/+mice colonised with human clinical isolate C. jejuni81–176 developed significantly more and larger tumours when compared with uninfected mice. C. jejuni with a mutated cdtB subunit, mutcdtB, attenuated C. jejuni-induced tumorigenesis in vivo and decreased DNA damage response in cells and enteroids. C. jejuni infection induced expression of hundreds of colonic genes, with 22 genes dependent on the presence of cdtB. The C. jejuni-infected group had a significantly different microbial gene expression profile compared with the mutcdtB group as shown by metatranscriptomic data, and different microbial communities as measured by 16S rDNA sequencing. Finally, rapamycin could diminish the tumorigenic capability of C. jejuni.ConclusionHuman clinical isolate C. jejuni 81–176 promotes colorectal cancer and induces changes in microbial composition and transcriptomic responses, a process dependent on CDT production.

Through diet, people are exposed simultaneously to a variety of contaminants (e.g. heavy metals, mycotoxins, pesticides) that could have combined adverse effects on human health. A previous study identified six main mixtures of food contaminants to which French adult consumers are exposed. These complex mixtures are comprised of 11 to 19 chemicals that have numerous toxic properties. In the present study, we investigated the genotoxic effects of these food contaminants, as single molecules and in mixtures that reflect their occurrence in the French diet, using the γH2AX assay in two human cell lines (HepG2, LS-174 T). Results of detailed analysis of the 49 individual contaminants (including 21 tested in this study) demonstrated a positive genotoxic response to 14 contaminants in HepG2 and 12 in LS-174 T cells. Next, our results indicated that two mixtures out of six triggered significant γH2AX induction after 24 hr of treatment, at concentrations for which individual compounds did not induce any DNA damage, suggesting more than additive interactions between chemicals. γH2AX positive mixtures were then tested for mutagenicity with the innovative in vitro PIG-A assay in HepG2 cells coupled with the soft agar colony formation assay. The two γH2AX positive mixtures led to a significant increase in the frequency of PIG-A GPI-deficient cells and in the number of colonies formed in soft agar. In conclusion, our study demonstrates that two mixtures of contaminants present in the French diet induce genotoxicity and mutagenicity, and that the combined effects of single molecules present in these mixtures are likely not additive, highlighting potential problems for hazard assessment of mixtures. Environ. Mol. Mutagen. 59:742-754, 2018. © 2018 Wiley Periodicals, Inc.

The classification of the fungicide captan (CAS Number: 133-06-2) as a carcinogen agent is presently under discussion. Despite the mutagenic effect detected by the Ames test and carcinogenic properties observed in mice, the genotoxicity of this pesticide in humans is still unclear. New information is needed about its mechanism of action in mammalian cells. Here, we show that Chinese Hamster Ovary (CHO) cells exposed to captan accumulate Fpg-sensitive DNA base alterations. In CHO and HeLa cells, such DNA lesions require the XRCC1-dependent pathway to be repaired. Captan also induces a replicative stress that activated the ATR signaling response and resulted in double-strand breaks and micronuclei. The replicative stress is characterized by a dramatic decrease in DNA synthesis due to a reduced replication fork progression. However, impairment of the XRCC1-related repair process did not amplify the replicative stress, suggesting that the fork progression defect is independent from the presence of base modifications. These results support the involvement of at least two independent pathways in the genotoxic effect of captan that might play a key role in carcinogenesis. Environ. Mol. Mutagen. 60:286-297, 2019. © 2018 Wiley Periodicals, Inc.

International audience

The cytolethal distending toxin (CDT) is produced by many pathogenic Gram-negative bacteria and is considered as a virulence factor. In human cells, CDT exposure leads to a unique cytotoxicity associated with a characteristic cell distension and induces a cell cycle arrest dependent on the DNA damage response (DDR) triggered by DNA double-strand breaks (DSBs). CDT has thus been classified as a cyclomodulin and a genotoxin. Whereas unrepaired damage can lead to cell death, effective, but improper repair may be detrimental. Indeed, improper repair of DNA damage may allow cells to resume the cell cycle and induce genetic instability, a hallmark in cancer. In vivo, CDT has been shown to induce the development of dysplastic nodules and to lead to genetic instability, defining CDT as a potential carcinogen. It is therefore important to characterize the outcome of the CDT-induced DNA damage and the consequences for intoxicated cells and organisms. Here, we review the latest results regarding the host cell response to CDT intoxication and focus on DNA damage characteristics, cell cycle modulation and cell outcomes.

The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity.

Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape., Summary: Chromatibody is a chromatin-binding single-domain antibody, derived from llama nanobodies, that can be used as a novel non-invasive molecular tool to explore and manipulate chromatin in living cells.

International audience; The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichlacoli possesses a variety of virulent factors, among them the Cytolethal Distending Toxin (CDT). CDT displays dual DNase and phosphatase activities and induces DNA double strand breaks, cell cycle arrest and apoptosis in a broad range of mammalian cells. As CDT could promote malignant transformation, we investigated the cellular outcomes induced by acute and chronic exposures to E. coli CDT in normal human colon epithelial cells (HCECs). Moreover, we conducted a comparative study between isogenic derivatives cell lines of the normal HCECs in order to mimic the mutation of three major genes found in CRC genetic models: APC, KRAS, and TP53. Our results demonstrate that APC and p53 deficient cells showed impaired DNA damage response after CDT exposure, whereas HCECs expressing oncogenic KRAS(V12) were more resistant to CDT. Compared to normal HCECs, the precancerous derivatives exhibit hallmarks of malignant transformation after a chronic exposure to CDT. HCECs defective in APC and p53 showed enhanced anchorage independent growth and genetic instability, assessed by the micronucleus formation assay. In contrast, the ability to grow independently of anchorage was not impacted by CDT chronic exposure in KRAS(V12) HCECs, but micronucleus formation is dramatically increased. Thus, CDT does not initiate CRC by itself, but may have promoting effects in premalignant HCECs, involving different mechanisms in function of the genetic alterations associated to CRC.

Summary The Cytolethal Distending Toxin (CDT) is a geno- toxin produced by several pathogenic bacteria. It is generally admitted that CDT induces double-strand breaks (DSB) and cell cycle arrest in G2/M-phase, in an ATM-dependent manner. Most of these results were obtained at high dose (over 1 m gm l -1 ) of CDT and late after treatment (8-24 h). We provide here evidence that the Escherichia coli CDT (EcCDT) - at low dose (50 pg ml -1 or LD50) and early after treatment (3-6 h) - progressively induces DNA DSB, mostly in S-phase. DSB forma- tion is related to the single-strand breaks induction by CDT, converted into DSB during the S-phase. We also show that homologous recombination is mobilized to these S-phase-associated DSB. This model unveils a new mechanism for CDT genoto- xicity that may play a role in cells partly deficient in homologous recombination.

International audience

In vivo comet assay is increasingly used although this assay has serious limitations as standard conditions does not allow the use of frozen samples nor the processing -at the same time- of an important number of samples generated by in vivo studies. Therefore, the aim of our work was firstly to optimize samples preparation and alkaline comet assay protocol for frozen tissue and also to increase the number of samples processed in one experiment; secondly it was to use this optimized protocol to evaluate the genotoxic effects of dietary heme iron on colon mucosa and colon epithelial cells. To optimize samples preparation and include a step of samples freezing for further experiments, different experimental parameters were tested (i.e. buffer composition, temperature of freezing/defreezing… ). The lack of induced DNA strand breaks by the freezing process of colon rat samples was assessed by comparing the results obtained between frozen and fresh samples from the same rat. The extend of DNA damage was determined by alkaline comet assay and quantified by median % Tail DNA calculation on 100 cells from 2 experimental points. By modifying different experimental parameters, we determined the optimal conditions to prepare samples and perform alkaline comet assay on cryopreserved blood and colon mucosa without DNA strand breaks induction. In order to perform middle throughput comet assays, we replaced standard glass microscope slides by 20 wells Trevigen® comet slides. Quantification of DNA breaks by median % tail DNA was undertaken by using the Komet 6.0 software (Andor Technology). Once the protocol was suitable, we used it to study the effects of dietary heme iron. Epidemiological studies revealed that high red meat intake, rich in heme iron, is associated with an increased colorectal cancer risk. In rats, heme iron-induced colorectal carcinogenesis is associated with increased fecal lipoperoxidation. In our previous study (Bastide, Chenni et al. 2015), we showed that luminal lipid peroxidation induced by the hemoglobin diet is associated with a higher anaphase bridge index in the epithelium of intestine. The genotoxic effect of the hemoglobin diet in C57BL/6J mice was confirmed with immunolabelling of gammaH2AX, a Ser-139 phosphorylation of the histone variant H2A, a well-known marker of the occurrence of DNA double strand breaks and/or replication fork arrest. The aim of our present study was to check, with alkaline comet assay, if the heme-induced lipoperoxidation increase is associated with an increment of genotoxicity in the rat colon mucosa. For this purpose, four groups of rats received either control diet or hemin diet, added or not with calcium carbonate for 3 weeks. Calcium carbonate is used to increase the level of proof between heme-induced peroxidation and genotoxicity, since it annihilates the heme-induced increase of peroxidation via heme chelation. As expected, heme iron intake increased fecal lipoperoxidation and also genotoxicity. When calcium carbonate was added to the hemin diet, these parameters were normalized. These in vivo results were completed with ex vivo experiments to determine if aldehydes from heme-induced lipoperoxidation are responsible for an increase of genotoxicity. We used fecal water, the soluble part of colonic content, from control or hemin fed rats. Fecal waters were used before and after treatment with a polymer resin (4-Fmoc-hydrazinobenzoyl AM NovaGel™) for aldehyde trapping. With this trapping of aldehydes from fecal water, the weight of aldehydes in the genotoxic activity of fecal water can be determined. Fecal waters were used on murine colonic epithelial cells and genotoxicity was assayed. Similarly to the results obtained from in vivo study, fecal waters from hemin-fed rats induced genotoxicity. When fecal waters were depleted in aldehydes, the genotoxicity was normalized. Therefore, using the resin to specifically trap fecal aldehydes, we demonstrated that aldehydes alone are responsible -at least in great part- for fecal water genotoxicity. In conclusion, our results offer a suitable protocol to evaluate genotoxicity on in vivo cryopreserved colon mucosa and on in vitro murine colonic cells, with a middle throughput capacity. This protocol confirms the increase of genotoxicity in rat colon mucosa after an heme-iron diet. Moreover, this protocol enables the demonstration that aldehydes from heme-induced lipoperoxidation are responsible for this increase of genotoxicity.

Purpose The effect of one pesticide spraying season on DNA damage was measured on B and T lymphocytes among open-field farmers and controls. Methods At least two peripheral blood samples were collected from each individual: one in a period without any pesticide application, several weeks after the last use (January, at period P0), and another in the intensive pesticide spraying period (May or June, at period P4). DNA damage was studied by alkaline comet assay on isolated B or T lymphocytes. Results Longitudinal comparison of DNA damage observed at both P0 and P4 periods revealed a statistically significant genotoxic effect of the pesticide spraying season in both B (P = 0.02) and T lymphocytes (P = 0.02) in exposed farmers. In contrast, non-farmers did not show any significant modifications. DNA damage levels in B and T lymphocytes were significantly higher in farmers than in non-farmers during the P4 period (P = 0.003 and P = 0.001 for B and T lymphocytes, respectively) but not during the P0 period. The seasonal effect observed among farmers was not correlated with either total farm area, farm area devoted to crops or recent solar exposure. On average, farmers used pesticides for 21 days between P0 and P4. Between the two time points studied, there was a tendency for a potential effect of the number of days of fungicide treatments (r2 = 0.43; P = 0.11) on T lymphocyte DNA damage. Conclusions A genotoxic effect was found in lymphocytes of farmers exposed to pesticides, suggesting in particular the possible implication of fungicides. Electronic supplementary material The online version of this article (doi:10.1007/s00420-015-1024-3) contains supplementary material, which is available to authorized users.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.

International audience; The exocyst complex is involved in the final stages of exocytosis, when vesicles are targeted to the plasma membrane and dock. The regulation of exocytosis is vital for a number of processes, for example, cell polarity, embryogenesis, and neuronal growth formation. Regulation of the exocyst complex in mammals was recently shown to be dependent upon binding of the small G protein, Ral, to Sec5, a central component of the exocyst. This interaction is thought to be necessary for anchoring the exocyst to secretory vesicles. We have determined the structure of the Ral-binding domain of Sec5 and shown that it adopts a fold that has not been observed in a G protein effector before. This fold belongs to the immunoglobulin superfamily in a subclass known as IPT domains. We have mapped the Ral binding site on this domain and found that it overlaps with protein-protein interaction sites on other IPT domains but that it is completely different from the G protein-geranyl-geranyl interaction face of the Ig-like domain of the Rho guanine nucleotide dissociation inhibitor. This mapping, along with available site-directed mutagenesis data, allows us to predict how Ral and Sec5 may interact.

International audience; RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Cerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a CFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to gamma-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.

Delivery of cytoplasmic vesicles to discrete plasma-membrane domains is critical for establishing and maintaining cell polarity, neurite differentiation and regulated exocytosis. The exocyst is a multisubunit complex required for vectorial targeting of a subset of secretory vesicles. Mechanisms that regulate the activity of this complex in mammals are unknown. Here we show that Sec5, an integral component of the exocyst, is a direct target for activated Ral GTPases. Ral GTPases regulate targeting of basolateral proteins in epithelial cells, secretagogue-dependent exocytosis in neuroendocrine cells and assembly of exocyst complexes. These observations define Ral GTPases as critical regulators of vesicle trafficking.

National audience; The Cytolethal Distending Toxin (CDT) is a virulence factor produced by many pathogenic bacteria (Escherichia coli, Helicobacter hepaticus ...) colonizing the oral cavity, the gastrointestinal tract, the liver etc. The presence in the gut of genotoxin producing bacteria, such as E. coli, could be a predisposing factor for the development of cancer, including colon cancer responsible of 500 000 deaths per year in the world. CDT was discovered in clinical isolates from patients with diarrhea caused by an E. coli infection. CDT is essential for H. hepaticus and C. jejuni to sustainable colonize the gastrointestinal tract and induce severe mucosal and liver inflammation, in mouse models. In addition, the expression of CDT by H. hepaticus leads to dysplasic liver nodules. It is therefore important to characterize the CDT mechanism of action in eukaryotic cells. CDT induces DNA double-strand breaks (DSB), leading to cell cycle arrest, activation of DNA repair mechanisms and increased cell death by apoptosis. The induction of DSB by CDT activates a signal pathway similar to the one observed in response to irradiation. However, we have shown that at low doses of CDT (1000 times lower than those used in the literature), single-strand breaks are induced in S phase, leading to a replicative stress before DSB production. These damages cause the activation of a pathway involving RPA, ATR and CHK1 proteins, characteristic of a replicative stress, before activation of the ATM kinase. Our future goals are to characterize 1- the cell sensitivity, according to the cell cycle phases, 2- the type of damage, and 3- (the) DNA repair mechanism(s) involved in response of such damages.

In the search for ways of sensitizing tumor cells to chemotherapy or radiotherapy, the inhibition of DNA repair has recently been proposed as a target of clinical interest. Ionizing radiations, as well as several antitumor drugs, induce the formation of DNA double-strand breaks (DSBs), that are highly damaging to the DNA, leading to cell death and genomic instability. DSBs are mainly repaired by the Nonhomologous End joining (NHEJ) process, in which DNA dependent protein kinase (DNA-PK) is the key complex. Consequently, specific DNA-PK inhibitors have been selected and evaluated for sensitizing cells to chemotherapy or radiotherapy. The choice of DNA-PK as a pharmacological target of interest in cancer treatment is discussed.

Poster presented at the 52nd Congress of the European-Societies-of-Toxicology (EUROTOX); A new in vitro micronucleus test in living cells associating biological tracers and high-content imaging

National audience; Lʼalpha-herpesvirus de la maladie de Marek, responsable du développement de lymphomes T chez la poule, engendre encore actuellement des pertes économiques importantes dans la filière avicole malgré une vaccination systématique des animaux. La dérégulation du cycle cellulaire est une stratégie communément adoptée par les virus afin de bénéficier dʼun environnement favorable à leur réplication. Il nous donc a semblé intéressant de déterminer lʼimpact de lʼinfection par le MDV sur la régulation du cycle cellulaire. Lʼinfection de cellules primaires de peau de poulet (CESC) initialement quiescentes (phase-G0) par le MDV, provoque non seulement leur réentrée dans le cycle cellulaire, mais induit également un arrêt de la progression du cycle en phase S (phase de réplication de lʼADN cellulaire). Nous avons identifié la protéine de tégument VP22, essentielle à la réplication du virus, comme un régulateur majeur du cycle cellulaire. En effet, La surexpression de cette protéine dans la lignée aviaire LMH induit un blocage intra-S dans plus de 90% des cellules, blocage qui dépend de la localisation nucléaire de la protéine et de sa capacité à se lier aux histones. Nous avons également démontré que cette nouvelle activité de la protéine VP22 est conservée chez 2 autres espèces de Mardivirus et 2 alpha-herpesvirus humains, HSV-1 et VZV. Le mécanisme de blocage du cycle cellulaire en phase S associé à la VP22 sera discuté ainsi que le rôle potentiel de cette dérégulation du cycle cellulaire dans la lymphomagénèse.

National audience

SH3 (Src homology-3) domains are involved in protein–protein interactions through proline-rich domains. Many SH3-containing proteins are implicated in actin cytoskeleton organization. The aim of our ongoing work is to study the functions of the SH3-containing proteins in actin cytoskeleton regulation. The yeast Saccharomyces cerevisiae proteome includes 29 SH3 domains distributed in 25 proteins. We have examined the direct involvement of these SH3 domains in actin polymerization using an in vitro polymerization assay on GST (glutathione S-transferase)–SH3-coated beads. As expected, not all SH3 domains show polymerization activity, and many recruit distinct partners as assessed by microscopy and pull-down experiments. One such partner, Las17p, the yeast homologue of WASP (Wiskott–Aldrich syndrome protein), was assayed because it stimulates actin nucleation via the Arp2/3 (actin-related protein 2/3) complex. Ultimately, proteins involved in specific biological processes, such as membrane trafficking, may also be recruited by some of these SH3 domains, shedding light on the SH3-containing proteins and actin cytoskeleton functions in these processes.

International audience; The formation of actin filaments is crucial for endocytosis and other interrelated cellular phenomena such as motility, polarized morpho-genesis, and cytokinesis. In this paper we have investigated the role of the WASP/Las17-interacting protein Bzz1p in endocytosis and trafficking to the vacuole. We and others have recently shown that Bzz1p is an actin patch protein that interacts directly with Las17p via a SH3-polyproline interaction. Bzz1p functions with type I myosins to restore polarity of the actin cytoskeleton after NaCl stress. In an in vitro bead assay, GST-Bzz1p fusion protein triggers a functional actin polymerization machinery through its two C-terminal SH3 domains. In this paper we implicate Bzz1p with the type I myosins both in fluid-phase and in the internalization step of receptor mediated endocytosis. As deduced from their localization as GFP fusions , the vacuolar delivery of endocytic and biosynthetic cargoes as well as the multivesicular body pathway appear unaffected. We further elucidate Bzz1p direct participation in actin polymerization by demonstrating that each of the SH3 domains of Bzz1p individually is able to trigger actin poly-merization in a cell-free system dependent on Arp2/3, Las17p, Vrp1p, and the type I myosins. Taken together, our results show that Bzz1p participates , essentially via its SH3 domains, in early steps of endocytosis together with known actin nucleation activators.

The phosphatase CDC25B is one of the key regulators that control entry into mitosis through the dephosphorylation and subsequent activation of the cyclin-dependent kinases. Here we study the phosphorylation of CDC25B at mitosis by the kinase pEg3, a member of the KIN1/PAR-1/MARK family. Using mass spectrometry analysis we demonstrate that CDC25B is phosphorylated in vitro by pEg3 on serine 169, a residue that lies within the B domain. Moreover, using phosphoepitope-specific antibodies we show that serine 169 is phosphorylated in vivo, that this phosphorylated form of CDC25B accumulates during mitosis, and is localized to the centrosomes. This labelling is abrogated when pEg3 expression is repressed by RNA interference. Taken together, these results support a model in which pEg3 contributes to the control of progression through mitosis by phosphorylation of the CDC25 phosphatases.

International audience; Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84.

International audience; Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. We have revisited the Ras/Rap-Ral connections in Drosophila melanogaster by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. We show that (i) the Ral-centered protein network appears to be extremely conserved in human and flies, (ii) in this network, RGL is a functional Drosophila orthologue of RalGEFs, and (iii) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. Our data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities.

We identified families of proteins characterized by the presence of a domain similar to human p23 protein, which include proteins such as Sgt1, involved in the yeast kinetochore assembly; melusin, involved in specific interactions with the cytoplasmic integrin beta1 domain; Rar1, related to pathogenic resistance in plants, and to development in animals; B5+B5R flavo-hemo cytochrome NAD(P)H oxidoreductase type B in humans and mice; and NudC, involved in nucleus migration during mitosis. We also found that p23 and the HSP20/alpha-crystallin family of heat shock proteins, which share the same three-dimensional folding, show a pattern of conserved residues that points to a common origin in the evolution of both protein domains. The p23 and HSP20/alpha-crystallin phylogenetic relationship and their similar role in chaperone activity suggest a common function, probably involving protein-protein interaction, for those proteins containing p23-like domains.

International audience; RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

International audience; Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two-hybrid assays, in vitro, and in pull-down experiments performed with SK-BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.

International audience; The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.

International audience; The occurrence of DNA double-strand breaks (DSBs) induced by ionizing radiation has been extensively studied by biochemical or cell imaging techniques. Cell imaging development relies on technical advances as well as our knowledge of the cell DNA damage response (DDR) process. The DDR involves a complex network of proteins that initiate and coordinate DNA damage signaling and repair activities. As some DDR proteins assemble at DSBs in an established spatio-temporal pattern, visible nuclear foci are produced. In addition, post-translational modifications are important for the signaling and the recruitment of specific partners at damaged chromatin foci. We briefly review here the most widely used methods to study DSBs. We also discuss the development of indirect methods, using reporter expression or intra-nuclear antibodies, to follow the production of DSBs in real time and in living cells.

National audience; La Cytolethal Distending Toxin (CDT) est un facteur de virulence produit par de nombreuses bactéries pathogènes Gram négatives. Sa production est associée à différentes pathologies, dont le développement de cancer. Un lien de causalité a été établi entre dommages à l’ADN, mutagénèse et cancérogenèse. Or, différentes études ont classé CDT dans la famille des génotoxines bactériennes. L’action génotoxique de CDT repose sur l’activité de sa sous-unité catalytique CdtB, connue pour induire des cassures double-brin (CDBs) de l’ADN génomique eucaryote. Cependant, des travaux réalisés dans l’équipe ont montré qu’à des doses 1000 fois plus faibles que celles utilisées dans la littérature, CDT induit des dommages primaires (probablement de type cassure simple-brin) qui dégénèrent en CDB lors de la phase S. Afin de mieux documenter ce modèle, nous avons étudié ici les systèmes de réparations impliqués dans la réponse aux dommages induits par CDT. Nous avons ainsi confirmé l’importance des voies de réparations des CDBs (Homologous Recombinaison et Non-Homologous End-Joining). Nous avons également montré que le Nucleotide Excision Repair, impliqué dans la réparation des adduits à l’ADN, ne semble pas être impliqué dans la prise en charge des dommages induits par CDT. En revanche, nous avons démontré, pour la première fois, une implication des systèmes de réparation de dommages plus précoces comme le Single-Strand Break Repair et la voie de l’Anémie de Fanconi. En conclusion, notre travail conforte les données selon lesquelles CDT induit des CSB, et non des CDB directes de l’ADN. De plus, notre travail a permis d’éclaircir les effets cellulaires activés suite aux dommages induits par CDT.