Effect of cell treatment procedures on in vitro genotoxicity assessment.

So far, the majority of in vitro toxicological experiments are conducted after an acute 24 h treatment that does not represent a realistic human chemical exposure. Recently, new in vitro approaches have been proposed to study the chemical toxicological effect over several days in order to be more predictive of a representative exposure scenario. In this study, we investigated the genotoxic potential of chemicals (direct or bioactived clastogen, aneugen and apoptotic inducer) with the γH2AX and pH3 biomarkers, in the human liver-derived HepaRP cell line. We used different treatment durations, with or without a three-day recovery stage (release period), before genotoxicity measurement. Data were analysed with the Benchmark Dose approach. We observed that the detection of clastogenic compounds (notably for DNA damaging agents) was more sensitive after three days of repeated treatment compared to one or three treatments over 24 h. In contrast, aneugenic chemicals were detected as genotoxic in a similar manner whether after a 24 h exposure or a three-day repeated treatment. Globally, the release period decreases the genotoxicity measurement substantially. For DNA damaging agents, after high concentration treatments, γH2AX induction was always observed after a three-day release period. In contrast, for DNA topoisomerase inhibitors, no effect could be observed after the release period. In conclusion, in the HepaRP cell line, there are some important differences between a one-day acute and a three-day repeated treatment protocol, indicating that different cell treatment procedures may differentiate chemical genotoxic mechanisms of action more efficiently.

Mutagenicity and genotoxicity assessment of the emerging mycotoxin Versicolorin A, an Aflatoxin B1 precursor.

Aflatoxin B1 (AFB1) is the most potent natural carcinogen among mycotoxins. Versicolorin A (VerA) is a precursor of AFB1 biosynthesis and is structurally related to the latter. Although VerA has already been identified as a genotoxin, data on the toxicity of VerA are still scarce, especially at low concentrations. The SOS/umu and miniaturised version of the Ames test in Salmonella Typhimurium strains used in the present study shows that VerA induces point mutations. This effect, like AFB1, depends primarily on metabolic activation of VerA. VerA also induced chromosomal damage in metabolically competent intestinal cells (IPEC-1) detected by the micronucleus assay. Furthermore, results from the standard and enzyme-modified comet assay confirmed the VerA-mediated DNA damage, and we observed that DNA repair pathways were activated upon exposure to VerA, as indicated by the phosphorylation and/or relocation of relevant DNA-repair biomarkers (γH2AX and 53BP1/FANCD2, respectively). In conclusion, VerA induces DNA damage without affecting cell viability at concentrations as low as 0.03 µM, highlighting the danger associated with VerA exposure and calling for more research on the carcinogenicity of this emerging food contaminant.

Dairy By-Products and Lactoferrin Exert Antioxidant and Antigenotoxic Activity on Intestinal and Hepatic Cells.

The dairy industry generates a large volume of by-products containing bioactive compounds that may have added value. The aim of this study was to evaluate the antioxidant and antigenotoxic effects of milk-derived products, such as whey, buttermilk, and lactoferrin, in two human cell lines: Caco-2 as an intestinal barrier model and HepG2 as a hepatic cell line. First, the protective effect of dairy samples against the oxidative stress caused by menadione was analyzed. All these dairy fractions significantly reversed the oxidative stress, with the non-washed buttermilk fraction presenting the greatest antioxidant effect for Caco-2 cells and lactoferrin as the best antioxidant for HepG2 cells. At concentrations that did not impact cell viability, we found that the dairy sample with the highest antigenotoxic power against menadione, in both cell lines, was lactoferrin at the lowest concentration. Additionally, dairy by-products maintained their activity in a coculture of Caco-2 and HepG2, mimicking the intestinal-liver axis. This result suggests that the compounds responsible for the antioxidant activity could cross the Caco-2 barrier and reach HepG2 cells on the basal side, exerting their function on them. In conclusion, our results show that dairy by-products have antioxidant and antigenotoxic activities, which would allow revaluing their use in food specialties.

Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium.

The whitening and opacifying agent titanium dioxide (TiO2) is used worldwide in various foodstuffs, toothpastes and pharmaceutical tablets. Its use as a food additive (E171 in EU) has raised concerns for human health. Although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO2 particles has not been documented. Here we analyzed E171 particle translocation in vivo through the pig buccal mucosa and in vitro on human buccal TR146 cells, and the effects on proliferating and differentiated TR146 cells. In the buccal floor of pigs, isolated TiO2 particles and small aggregates were observed 30 min after sublingual deposition, and were recovered in the submandibular lymph nodes at 4 h. In TR146 cells, kinetic analyses showed high absorption capacities of TiO2 particles. The cytotoxicity, genotoxicity and oxidative stress were investigated in TR146 cells exposed to E171 in comparison with two TiO2 size standards of 115 and 21 nm in diameter. All TiO2 samples were reported cytotoxic in proliferating cells but not following differentiation. Genotoxicity and slight oxidative stress were reported for the E171 and 115 nm TiO2 particles. These data highlight the buccal mucosa as an absorption route for the systemic passage of food-grade TiO2 particles. The greater toxicity on proliferating cells suggest potential impairement of oral epithelium renewal. In conclusion, this study emphasizes that buccal exposure should be considered during toxicokinetic studies and for risk assessment of TiO2 in human when used as food additive, including in toothpastes and pharmaceutical formulations.

Chronic exposure to Cytolethal Distending Toxin (CDT) promotes a cGAS-dependent type I interferon response.

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.

Cytolethal Distending Toxin Promotes Replicative Stress Leading to Genetic Instability Transmitted to Daughter Cells.

The cytolethal distending toxin (CDT) is produced by several Gram-negative pathogenic bacteria. In addition to inflammation, experimental evidences are in favor of a protumoral role of CDT-harboring bacteria such as Escherichia coli, Campylobacter jejuni, or Helicobacter hepaticus. CDT may contribute to cell transformation in vitro and carcinogenesis in mice models, through the genotoxic action of CdtB catalytic subunit. Here, we investigate the mechanism of action by which CDT leads to genetic instability in human cell lines and colorectal organoids from healthy patients' biopsies. We demonstrate that CDT holotoxin induces a replicative stress dependent on CdtB. The slowing down of DNA replication occurs mainly in late S phase, resulting in the expression of fragile sites and important chromosomic aberrations. These DNA abnormalities induced after CDT treatment are responsible for anaphase bridge formation in mitosis and interphase DNA bridge between daughter cells in G1 phase. Moreover, CDT-genotoxic potential preferentially affects human cycling cells compared to quiescent cells. Finally, the toxin induces nuclear distension associated to DNA damage in proliferating cells of human colorectal organoids, resulting in decreased growth. Our findings thus identify CDT as a bacterial virulence factor targeting proliferating cells, such as human colorectal progenitors or stem cells, inducing replicative stress and genetic instability transmitted to daughter cells that may therefore contribute to carcinogenesis. As some CDT-carrying bacterial strains were detected in patients with colorectal cancer, targeting these bacteria could be a promising therapeutic strategy.

Repeated exposure of Caco-2 versus Caco-2/HT29-MTX intestinal cell models to (nano)silver in vitro: Comparison of two commercially available colloidal silver products.

Colloidal silver products are sold for a wide range of disinfectant and health applications. This has increased the potential for human exposure to silver nanoparticles (AgNPs) and ions (Ag+), for which oral ingestion is considered to be a major route of exposure. Our objective was to evaluate and compare the toxicity of two commercially available colloidal silver products on two human intestinal epithelial models under realistic exposure conditions. Mesosilver™ and AgC were characterized and a concentration range between 0.1 and 12 µg/mL chosen. Caco-2 cells vs. co-culture of Caco-2 and mucus-secreting HT29-MTX cells (90/10) were used. Repeated exposure was carried out to determine cell viability over 18 days of cell differentiation in 24-well plates. Selected concentrations (0.1, 1, and 3 µg/mL) were tested on cells cultured in E-plates and Transwells with the same repeated exposure regimen, to determine cell impedance, and cell viability and trans-epithelial electrical resistance (TEER), respectively. Silver uptake, intracellular localisation, and translocation were determined by CytoViva™, HIM-SIMS, and ICP-MS. Genotoxicity was determined on acutely-exposed proliferating Caco-2 cells by γH2AX immunofluorescence staining. Repeated exposure of a given concentration of AgC, which is composed solely of ionic silver, generally exerted more toxic effects on Caco-2 cells than Mesosilver™, which contains a mix of AgNPs and ionic silver. Due to its patchy structure, the presence of mucus in the Caco-2/HT29-MTX co-culture only slightly mitigated the deleterious effects on cell viability. Increased genotoxicity was observed for AgC on proliferating Caco-2 cells. Silver uptake, intracellular localisation, and translocation were similar. In conclusion, Mesosilver™ and AgC colloidal silver products show different levels of gut toxicity due to the forms of distinct silver (AgNPs and/or Ag+) contained within. This study highlights the applicability of high-resolution (chemical) imaging to detect and localize silver and provides insights into its uptake mechanisms, intracellular fate and cellular effects.

Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B.

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.

Cytolethal Distending Toxin Subunit B: A Review of Structure-Function Relationship.

The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions.

Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not.

The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays-the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays.

Exposure to the Fungicide Captan Induces DNA Base Alterations and Replicative Stress in Mammalian Cells.

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.

Campylobacter jejuni promotes colorectal tumorigenesis through the action of cytolethal distending toxin.

OBJECTIVE: Campylobacter 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. DESIGN: Germ-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. RESULTS: GF 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. CONCLUSION: Human clinical isolate C. jejuni 81-176 promotes colorectal cancer and induces changes in microbial composition and transcriptomic responses, a process dependent on CDT production.

The Colibactin Genotoxin Generates DNA Interstrand Cross-Links in Infected Cells.

Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCE Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli, we showed that colibactins directly generate DNA cross-links in cellulo Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents.

Around and beyond 53BP1 Nuclear Bodies.

Within the nucleus, sub-nuclear domains define territories where specific functions occur. Nuclear bodies (NBs) are dynamic structures that concentrate nuclear factors and that can be observed microscopically. Recently, NBs containing the p53 binding protein 1 (53BP1), a key component of the DNA damage response, were defined. Interestingly, 53BP1 NBs are visualized during G1 phase, in daughter cells, while DNA damage was generated in mother cells and not properly processed. Unlike most NBs involved in transcriptional processes, replication has proven to be key for 53BP1 NBs, with replication stress leading to the formation of these large chromatin domains in daughter cells. In this review, we expose the composition and organization of 53BP1 NBs and focus on recent findings regarding their regulation and dynamics. We then concentrate on the importance of the replication stress, examine the relation of 53BP1 NBs with DNA damage and discuss their dysfunction.

Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms.

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.

Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells.

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.

Genotoxicity of Cytolethal Distending Toxin (CDT) on Isogenic Human Colorectal Cell Lines: Potential Promoting Effects for Colorectal Carcinogenesis.

The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichia coli 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.

DNA damage in B and T lymphocytes of farmers during one pesticide spraying season.

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 (r (2) = 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.

RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex.

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-Gerold 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 GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-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.

Cell cycle modulation by Marek's disease virus: the tegument protein VP22 triggers S-phase arrest and DNA damage in proliferating cells.

Marek's disease is one of the most common viral diseases of poultry affecting chicken flocks worldwide. The disease is caused by an alphaherpesvirus, the Marek's disease virus (MDV), and is characterized by the rapid onset of multifocal aggressive T-cell lymphoma in the chicken host. Although several viral oncogenes have been identified, the detailed mechanisms underlying MDV-induced lymphomagenesis are still poorly understood. Many viruses modulate cell cycle progression to enhance their replication and persistence in the host cell, in the case of some oncogenic viruses ultimately leading to cellular transformation and oncogenesis. In the present study, we found that MDV, like other viruses, is able to subvert the cell cycle progression by triggering the proliferation of low proliferating chicken cells and a subsequent delay of the cell cycle progression into S-phase. We further identified the tegument protein VP22 (pUL49) as a major MDV-encoded cell cycle regulator, as its vector-driven overexpression in cells lead to a dramatic cell cycle arrest in S-phase. This striking functional feature of VP22 appears to depend on its ability to associate with histones in the nucleus. Finally, we established that VP22 expression triggers the induction of massive and severe DNA damages in cells, which might cause the observed intra S-phase arrest. Taken together, our results provide the first evidence for a hitherto unknown function of the VP22 tegument protein in herpesviral reprogramming of the cell cycle of the host cell and its potential implication in the generation of DNA damages.