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Cells are inevitably challenged by low-level/endogenous stresses that do not arrest DNA replication. Here, in human primary cells, we discovered and characterized a noncanonical cellular response that is specific to nonblocking replication stress. Although this response generates reactive oxygen species (ROS), it induces a program that prevents the accumulation of premutagenic 8-oxoguanine in an adaptive way. Indeed, replication stress-induced ROS (RIR) activate FOXO1-controlled detoxification genes such as SEPP1, catalase, GPX1, and SOD2. Primary cells tightly control the production of RIR: They are excluded from the nucleus and are produced by the cellular NADPH oxidases DUOX1/DUOX2, whose expression is controlled by NF-κB, which is activated by PARP1 upon replication stress. In parallel, inflammatory cytokine gene expression is induced through the NF-κB-PARP1 axis upon nonblocking replication stress. Increasing replication stress intensity accumulates DNA double-strand breaks and triggers the suppression of RIR by p53 and ATM. These data underline the fine-tuning of the cellular response to stress that protects genome stability maintenance, showing that primary cells adapt their responses to replication stress severity.

Several types of Quantum Dots (QDs) (CdS, CdSe and InP, as well as core-shell QDs such as type I InP-ZnS, quasi type-II CdSe-CdS and inverse type-I CdS-CdSe) were considered for generating α-aminoalkyl free radicals. The feasibility of the oxidation of the N-aryl amines and the generation of the desired radical was evidenced experimentally by quenching of the photoluminescence of the QDs and by testing a vinylation reaction using an alkenylsulfone radical trap. The QDs were tested in a radical [3+3]-annulation reaction giving access to tropane skeletons and that requires the completion of two consecutive catalytic cycles. Several QDs such as CdS core, CdSe core and inverted type I CdS-CdSe core-shell proved to be efficient photocatalysts for this reaction. Interestingly, the addition of a second shorter chain ligand to the QDs appeared to be essential to complete the second catalytic cycle and to obtain the desired bicyclic tropane derivatives. Finally, the scope of the [3+3]-annulation reaction was explored for the best performing QDs and isolated yields that compares well with classical iridium photocatalysis were obtained.

International audience; Exposure to solar UV is at the origin of numerous photodegradation pathways in biomolecules. Tryptophan is readily modified by UVB radiation into ring-opened and oxidized photoproducts. One of them, 6-formylindolo[3,2-b]carbazole (FICZ), has been extensively studied in the recent years because it very efficiently binds to AhR, a major factor in numerous biological processes, such as metabolism of xenobiotics. Unfortunately, little information is available on the actual yield of FICZ upon exposure to low and biologically relevant doses of UV radiation. In the present work, we used a sensitive and specific HPLC-tandem mass spectrometry assay to quantify a series of photoproducts induced by UVB and simulated sunlight (SSL) in solutions of tryptophan. FICZ represented only a minute amount of the photoproducts (0.02% and 0.03%, respectively). Experiments were repeated in culture medium where the yield of FICZ was also found to be very low, even when Trp was added. Last, no FICZ could be detected in cytosolic fractions of cultured cells exposed to SSL. Altogether, the present results show that FICZ is a very minor photoproduct and that it cannot be considered as the only endogenous photoproduct responsible for the induction of AhR-dependent responses in UV-irradiated cells. This article is protected by copyright. All rights reserved.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA [ADD1_IRSTEA]Systèmes aquatiques soumis à des pressions multiples; International audience; To improve the assessment of aquatic organism responses to environmental stressors, there is an interest in studying epigenetic marks in addition to other validated biomarkers. Indeed, the epigenetic marks may be influenced by the surrounding environment. Non-model invertebrates such as gammarids are sentinel organisms representative of the diversity of natural stream communities. Despite their ecologically relevance, the epigenetic responses have been to date poorly documented in these species. The present study explores the measurement of the global cytosine methylation level in the genome of the freshwater crustacean Gammarus fossarum. In a first step, natural variability of global cytosine methylation level (basal level) was assessed by studying the effect of sex, age and sampling site of organisms. Results showed a significant effect of age and sampling site. In a second step, effects of water temperature and food starvation were studied. For both factors, a hypermethylation was observed after 1 month of exposure. In a third step, gammarids were exposed to a range of environmentally relevant cadmium concentrations (0.05-5 µg/L) in order to assess the effect of a chemical stress. Whatever the cadmium concentration used, a significant hypomethylation was observed after 14 days followed by a trend for hypermethylation after 1 month of exposure. These results are the first ones dealing with the 5Cmethylation status in gammarids. The results constitute potential markers of environmental stresses in relevant sentinel species widely used in ecotoxicological studies.

International audience; Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermo-philic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a protein-bound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sul-fide, thus acting as a sulfur carrier. tRNA modification | thiolation | [Fe-S] cluster | thiouridine synthetase | U54-tRNA T he cellular translation machinery contains essential components such as tRNAs. To achieve their function, they feature a great variety of well-conserved posttranscriptional chemical modifications. Sulfur is present in several of these modified nucleosides: thiouridine and derivatives (s 4 U8, s 2 U34, and m 5 s 2 U54), 2-thioadenosine derivatives (ms 2 i 6 A37 and ms 2 t 6 A37), and 2-thiocytidine (s 2 C32). However, mechanisms of sulfur insertion into tRNAs are largely unknown, and the enzymes responsible for these reactions are incompletely characterized. Whereas redox conversion of a C-H to a C-S bond (synthesis of ms 2 i 6 A37 and ms 2 t 6 A37) depends on redox enzymes from the RadicalS -adenosyl-L-methionine iron-sulfur enzyme family, simple nonredox conversion of C = O to C = S group (synthesis of s 2 U34 and s 4 U8) is not expected to require such redox clusters. Intriguingly, we recently discovered that the ATP-dependent formation of s 2 C32 in some tRNAs is catalyzed by an iron-sulfur enzyme, TtcA (1). However, the role of its cluster has not been defined. In the same superfamily, TtuA enzymes catalyze the C2-thiolation of uridine 54 in the T loop of thermophilic tRNAs (Fig. 1A), allowing stabilization of tRNAs at high temperature in thermophilic microorganisms. Sequences analysis shows that they share conserved cysteines and ATP binding motif (Fig. S1). Here, we report a detailed biochemical and structural characterization of TtuA that shows the presence of a [4Fe-4S] cluster essential for activity. The crystal structures of Pyrococcus horikoshii TtuA (PhTtuA) show that the cluster, chelated by only three cysteines, is adjacent to the ATP binding site. The presence of electron density near the fourth iron, nonbonded to the protein, indicates that the cluster can bind an exogenous substrate. We propose that thiolation occurs via sulfur binding to the cluster and transfer to the tRNA substrate. The fact that the catalytic [4Fe-4S] cluster serves as a sulfur carrier during a nonredox thiolation reaction illustrates an unknown function in iron-sulfur enzymology. Results m 5 s 2 U but Not s 2 C Is Present in tRNAs from Thermotoga maritima. In the Thermotoga maritima genome, only one homolog of the ttuA gene was detected (2). It was earlier suggested that TtuA could perform thiolation of both C32 and m 5 U54 in this organism (2), because both s 2 C and m 5 s 2 U were detected in bulk tRNAs (3). However, our results show that, although 10.7 m 5 s 2 U modifications per 1,000 uridines were detected, both m 5 U and s 2 C were under the threshold of detection (below one modification per million normal nucleosides) (Fig. S2). T. maritima TtuA Binds an [Fe-S] Cluster. Recombinant T. maritima TtuA (TmTtuA) was purified as an apoprotein, apo-TmTtuA (Fig. 2A and Fig. S3A), then anaerobically treated with ferrous iron and L-cysteine in the presence of a cysteine desulfurase, and finally, purified (Fig. S3B) in the form of a homogeneous dimeric brownish protein, named holo-TmTtuA. Metal analysis (1.6 ± 0.1 Zn, 3.1 ± 0.2 Fe, and 2.6 ± 0.3 S per monomer) and UV-visible as well as EPR spectroscopy (Fig. 2) show that one TmTtuA monomer binds Significance Posttranscriptional modifications of tRNA are essential for trans-lational fidelity. More specifically, mechanisms of selective sul-furation of tRNAs are still largely unknown, and the enzymes responsible for these reactions are incompletely investigated. Therefore, characterizing such systems at the molecular level is greatly valuable to our understanding of a whole class of tRNA modification reactions. We study TtuA, a representative member of a tRNA modification enzyme superfamily, and show that it intriguingly catalyzes a nonredox sulfur insertion within tRNA using a catalytically essential [4Fe-4S] cluster. This report opens perspectives regarding functions of iron-sulfur proteins in biology as well as chemical reactions catalyzed by iron-sulfur clusters.

International audience; For some decades production of titanium dioxide nanoparticle (TiO$_2$-NP) has been increasing at a considerable rate; concerns as to the toxicity of these particles upon inhalation have been raised. Indeed, TiO$_2$-NPs have been shown to induce significant genotoxicity and to adversely affect both major DNA repair mechanisms: base excision repair (BER) and nucleotide excision repair (NER). The aims of the present study were to (i) compare the genotoxicity of TiO$_2$-NPs and their impact on DNA repair processes on A549 alveolar carcinoma and BEAS-2B normal bronchial lung cell lines and (ii) delve deeper into the mechanisms leading to these effects. To achieve these goals, TiO$_2$-NPs effects on cytotoxicity, genotoxicity, DNA repair activity and DNA repair gene expression were investigated in both cell lines upon exposure to 1–100 $\mu$g/mL of anatase/rutile, 21 nm TiO$_2$-NPs. Our results show that TiO$_2$-NPs induce comparable cytotoxic and genotoxic responses in BEAS-2B and A549 cells. Functional response to DNA damage is observed in both cell lines and consists of an overall downregulation in DNA repair processes. When evaluating the relative importance of the two DNA repair pathways, we observed a lower impact on BER compared with NER activities, suggesting that repair of oxidatively generated DNA damage is still triggered in these cells. This response becomes measureable at 4 h of exposure in BEAS-2B but only after 48 h of exposure in A549 cells. The delayed response in A549 cells is due to an initial overall and intense downregulation of the genes encoding DNA repair proteins. This overall downregulation correlates with increased methylation of DNA repair gene promoters and downregulation of NRF2 and BRCA1, which may thus be considered as upstream regulators. These results strengthen the evidence that TiO$_2$-NP induces indirect genotoxicity in lung cells, via modulation of DNA repair processes, and shed some light on the mechanisms behind this effect.

Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.

4-Hydroxy-2-nonenal (HNE), one of the main aldehydic compounds released during lipid peroxidation, has been proposed to react with DNA bases in cells. Several classes of DNA lesions involving addition of either HNE or its 2,3-epoxide (epox-HNE) have been identified. In the present work, HPLC associated with tandem mass spectrometry was used to determine the pattern of HNE-induced DNA lesions. First, adducts were quantified within isolated DNA treated with HNE under peroxidizing conditions. The 1,N2-propano-2'-deoxyguanosine adduct of HNE (HNE-dGuo) was found to be the major lesion under all conditions studied. 1,N6-Ethenoadenine and 1,N2-ethenoguanine together with their (1,2-dihydroxyheptyl)-substituted derivatives, which all arise from the reaction of epox-HNE with DNA, were produced in significantly lower yields, even in the presence of 20 mM H2O2. The pyrimidopurinone malondialdehyde-2'-deoxyguanosine adduct was also found to be produced, although in very low yield. Similar results were obtained in cultured human monocytes incubated with HNE, because the HNE-dGuo adduct represented more than 95% of the overall adducts to DNA. In addition, the former lesion was poorly repaired, in contrast to 1,N2-ethenoguanine and, to a lesser extent, 1,N6-ethenoadenine. Altogether, these results suggest than HNE-dGuo may represent the best biomarker of the genotoxic effects of HNE.

International audience; : DNA repair mechanisms are key components for the maintenance of the essential mitochondrial genome. Among them, base excision repair (BER) processes, dedicated in part to oxidative DNA damage, are individually well known in mitochondria. However, no large view of these systems in differential physiological conditions is available yet. Combining the use of pure mitochondrial fractions and a multiplexed oligonucleotide cleavage assay on a microarray, we demonstrated that a large range of glycosylase activities were present in Drosophila mitochondria. Most of them were quantitatively different from their nuclear counterpart. Moreover, these activities were modified during aging.

Despite active research and the development of target-specific therapies, resistance to standard cytotoxic drugs still represents a challenge in cancer treatment. Many conventional anticancer drugs such as alkylating agents, antimetabolites and topoisomerase inhibitors induce DNA lesions as part of their cytotoxic effect. An important factor that affects the cytotoxic effect of these drugs is the ability of tumour cells to sense a variety of DNA lesions and elicit a coordinated response including DNA repair processes. This global DNA damage response may lead to tolerance to the drug-induced DNA lesions or to enhanced repair preventing an ideal outcome for patients after chemotherapy. Because DNA Damage Response (DDR) and DNA repair mechanisms constitute a dynamic network of finely tuned pathways with coordinated back-up and redundancy, comprehensive functional assays that enable DNA repair activities to be measured are promising. In the present study, we used a specific multiplexed enzymatic DNA repair assay on biochip to simultaneously investigate several repair pathways: Base and Nucleotide Excision Repair, Inter-Strand Link Repair. We assessed the DNA repair signatures of a panel of 7 cancer cell lines, treated by 5 cytotoxic anticancer drugs. A rigorous strategy allowed us to present a new effective way of classifying the model cancer cell responses to the chemotherapeutic drugs: 3 specific classes of responses were identified according to up and down regulation of specific repair sub-pathways. It provided new indications on the mechanism of action of cytotoxic drugs and on the ability of cell lines to respond. A common hypothesis is that a poor DNA repair capacity is associated with higher cytotoxic effect of DNA-damaging drugs and that defective damage-related signalling pathways may lead to cell death. To determine the possible link between chemosensitivity and DNA repair, we investigated the association between the IC20 obtained for each drug and specific repair sub-pathways independently for each cell line. We identified associations between up-regulations of certain repair pathways and high IC20, as well as the inverted feature (low repair/low IC20). This information gives clues on the possible involvement of specific repair activities in chemoresistance. Our original approach is an interesting functional complement to molecular pharmacology strategies to understand the DDR and might prove highly effective as part of choosing the right treatment for optimized care of cancer patients. Finally, our functional and multiplexed approach could be integrated into systems biology approaches for more effective identification of cancer biomarkers. We thank the CEA's transversal program Technologies for Health for support. Citation Format: Sylvie Sauvaigo, Anne Forestier, Fanny Sarrazy, Sylvain Caillat, Yves Vandenbrouck. A multiplexed enzymatic repair assay on biochip reveals a functional DNA repair signature in cancer cell lines exposed to cytotoxic anticancer drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3419. doi:10.1158/1538-7445.AM2013-3419

International audience; The well established toxicity of cadmium and cadmium compounds results from their additive effects on several key cellular processes, including DNA repair. Mammalian cells have evolved several biochemical pathways to repair DNA lesions and maintain genomic integrity. By interfering with the homeostasis of redox metals and antioxidant systems, cadmium promotes the development of an intracellular environment that results in oxidative DNA damage which can be mutagenic if unrepaired. Small base lesions are recognised by specialized glycosylases and excised from the DNA molecule. The resulting abasic sites are incised, and the correct sequences restored by DNA polymerases using the opposite strands as template. Bulky lesions are recognised by a different set of proteins and excised from DNA as part of an oligonucleotide. As in base repair, the resulting gaps are filled by DNA polymerases using the opposite strands as template. Thus, these two repair pathways consist in excision of the lesion followed by DNA synthesis. In this study, we analysed in vitro the direct effects of cadmium exposure on the functionality of base and nucleotide DNA repair pathways. To this end, we used recently described dedicated microarrays that allow the parallel monitoring in cell extracts of the repair activities directed against several model base and/or nucleotide lesions. Both base and nucleotide excision/repair pathways are inhibited by CdCl₂, with different sensitivities. The inhibitory effects of cadmium affect mainly the recognition and excision stages of these processes. Furthermore, our data indicate that the repair activities directed against different damaged bases also exhibit distinct sensitivities, and the direct comparison of cadmium effects on the excision of uracile in different sequences even allows us to propose a hierarchy of cadmium sensibility within the glycosylases removing U from DNA. These results indicate that, in our experimental conditions, cadmium is a very potent DNA repair poison.

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

Background: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome associated with TP53 germline mutations and characterized by increased risk to multiple early-onset cancers. Studies in families from Southern and Southeastern Brazil have identified a germline founder mutation in the TP53 gene, the p.R337H, in high population prevalence (∼0.3%). Unlike the majority of the mutations in TP53, which are missense mutations located in the DNA-binding domain (DBD) of the protein, the TP53 p.R337H is located in the oligomerization domain (OD). Recently, we identified a new rare germline variant, the rs78378222 (A>C), in the 3′ UTR of the gene in 7/130 of the patients who tested negative for coding germline TP53 mutations. Based on the broad spectrum of p53 functions, which include metabolism and DNA damage response regulation, in the present study we aimed to characterize the functional impact of germline TP53 mutations identified in Brazilian families. Methods: Primary fibroblasts from p.R337H/WT (n = 2), p.R337H/p.R337H (n = 1), DBD mutations (n = 2), rs78378222[C] patients and from WT p53 controls (n = 2) were included. The DNA damage induction was performed using UVB (0.2W/m2) and ionizing irradiation (IR) (1 Gy). Results: First, we observed that all p53-mutated fibroblasts appeared to have lower p53 levels but higher levels of reactive oxygen species (ROS) and mitochondrial biomass compared to controls. Fibroblasts homozygous to p.R337H mutation presented more pronounced ROS production and, notably, higher expression of two antioxidant proteins, SOD2 and GPX1. On the other hand, the results of high-resolution respirometry showed higher oxygen consume in DBD-mutated fibroblasts. Second, using a specific multiplexed enzymatic DNA repair assay on biochip, we simultaneously investigated several DNA repair pathways. Overall, we observed a high DNA repair activation profile in p.R337H/p.R337H and poor DNA repair capacity in DBD-mutated fibroblasts. Interestingly, all p53-mutated fibroblasts showed higher repair activity of apurinic/pyrimidinic (AP) sites, a DNA lesion caused mainly by ROS attacks. Conclusions: Our data indicate important alterations in both metabolism and DNA damage response in normal cells (fibroblasts) from LFS patients, suggesting haploinsufficiency mechanism associated to these mutations. We were also able to demonstrate distinct phenotypes according to mutational status, which may explain, at least in part, the clinical variability in LFS. Citation Format: Gabriel Macedo, Sylvie Sauvaigo, Michele Silva Alves, Sylvain Caillat, Igor Araujo Vieira, Fernanda Timm, Cristina Brinckmann Oliveira Netto, Mauro Antonio Alves Castro, Ivi Bristot, Angela Fachel, Fabio Klamt, Patricia Ashton-Prolla. Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2015-804

Characterization of DNA-N-glycosylase activities in cell extract is a challenging problem and could represent a major concern for medical applications. Synthetic oligonucleotides which contain base lesions located on specific sites constitute suitable substrates for their study. An in vitro miniaturized assay was developed that allows the measurement of cleavage activities of DNA repair enzymes on a set of oligonucleotides (ODNs) that contained different lesions. The modified ODNs were indirectly hybridized onto probes chemically fixed at defined sites on a circular format within each well of a 96-well microtiter plate (Oligo Sorbent Array, OLISA). The lesions were selected among oxidative damage (8-oxo-7,8-dihydroguanine, formylamine), deaminated bases (uracil, hypoxanthine) and alkylated base (N(6)-etheno-adenine). Cleavage specificity was checked using different enzymes: Fapy-DNA-N-glycosylase, 3-methyladenine DNA glycosylase II, uracil-N-glycosylase, endonuclease V and endonuclease VIII. The extent of excision could be monitored simultaneously for the selected base damage. For this purpose, we used automated fluorescence imaging analysis of the residual ODNs that contained lesions and remained on the support after release of the cleaved ODNs recognized by the repair enzymes. The results indicated that this assay could advantageously replace the analysis of glycosylase activities by PAGE techniques. Finally we show that this in vitro repair assay represents an interesting tool for the determination of cellular repair activities.

The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a watersoluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkalilabile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

BCR-ABL expression in leukemic cells is associated with several types of DNA repair abnormalities, involving homologous recombination (HR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), mismatch repair (MMR) and base-excision repair (BER) pathways. These defects, combined with the genetic instability inherent to chronic myeloid leukemia (CML), are thought lead to ABL-kinase domain mutations which remain poorly repaired and selected by tyrosine kinase inhibitor (TKI) therapies, leading to drug resistance. Currently, there is no functional, practical, single step assay to determine if BCR-ABL expression in a given leukemic cell is predominantly associated with a specific DNA repair pathway. For this purpose, we have used a novel DNA repair test based on biopchips evaluating the functional capacity of cells to achieve DNA repair through the use of a functional excision/synthesis repair assay that establish DNA Repair Enzyme Signatures. This assay allows monitoring the repair of a set of DNA lesions handled by BER (8oxoG, alkylated bases, glycols and Abasic sites) and NER (photoproducts, psoralen and cisplatin adducts). For modelling CML, we have used the UT7 cell line expressing BCR-ABL and T315I-mutated BCR-ABL. Parental (P)-UT7 cells were used as controls. We have tested the effects of BCR-ABL tyrosine kinase activity on the DNA repair assays using two strategies: The first was the evaluation of the effects of the nuclear extracts of UT7 cells expressing either native (N) BCR-ABL or T315I-mutated BCR-ABL before and after incubation of the cells in the presence of Imatinib (IM) or Nilotinib whereas the second tested the effects of BCR-ABL in an inducible system without the use of TKI. As expected, UT7 cells with N-BCR-ABL were sensitive to the inhibitory effects of these drugs whereas UT7 cells with T315I-mutated BCR-ABL were totally resistant. Using this system, we have found that the inhibition of BCR-ABL by IM stimulated the DNA repair capacity of the cells and specially BER (8oxoG, alkylated bases and glycols) and NER (photoproducts and psoralen adducts) after 4 or 8 hours of treatment, as compared to baseline repair capacities ( p< 0.05). No effect was detected for the repair of Abasic sites and Cisplatin adducts. When the cells were treated with Nilotinib in the same conditions, we have observed also the stimulation of DNA repair capacities for all lesions except for the Abasic sites ( p< 0.125). The fact of treating P-UT-7 cells or UT7-T315I cells with IM or Nilotinib had no effect and led conversely, to the inhibition of DNA repair activity via Abasic and Cisplat adducts pathways (p< 0.05) or Pso (p < 0.125). To confirm directly the effect of the BCR-ABL activity in the repair modifications observed, we have used UT7 cell lines expressing N-BCR-ABL or T315I-mutated BCR-ABL under the control of a Doxycycline-inducible promoter. In this TET-OFF system, BCR-ABL expression is inhibited upon Doxycycline addition to the culture and BCR-ABL protein becomes undetectable by Western blots at day 8. In this system, the analysis of DNA repair capacity demonstrated a statistically significant stimulation of repair of Alkylated bases, Glycols, Psoralen adducts and photoproducts ( p < 0.05) upon inhibition of N-BCR-ABL or that of T315I-mutated BCR-ABL. Interestingly, the fact of reducing BCR-ABL T315I in the DOX-inducible system stimulated DNA repair capacity whereas in the non-inducible UT7 cells expressing T315I, IM did not have a stimulatory effect, whereas Nilotinib induced a slight stimulation of both NER and BER pathways after 8 hours of treatment. Overall, these results demonstrate that the novel microarrays that we describe can be efficiently detect the DNA repair ability of the leukemic cells and the reversal of these abnormalities on TKI therapies. The preliminay results suggest clearly a differential effect of IM as compared to Nilotinib in terms of the stimulation of DNA repair capacity. This new methodology can now be used to quantify the abnormality of the DNA repair pathways in CML cells at diagnosis, allowing the monitoring of the tyrosine kinase inhibitor therapies and potentially predicting the occurrence of ABL-kinase mutations according to the preferential involvement of NER or BER pathways. Disclosures Turhan: Bristol Myers Squibb, Novartis: Honoraria, Research Funding. Sauvaigo:LXRepair: Employment, Equity Ownership.

It is well established that mutations and combinations of polymorphisms in DNA Repair genes contribute to cancer occurrence. However the relationship between genotypes and cancer is not straightforward. As a matter of fact, individuals’ cancer susceptibility also depends on their genotoxic exposure history, age and lifestyle. In this context, functional DNA-repair tests better account for DNA Repair complexity and constitute powerful strategies to characterize effective individuals DNA Repair capacity. Currently available tools interrogate very limited aspects of DNA Repair mechanism and more informative assays are urgently needed. In humans Base Excision Repair (BER) involves several glycosylases that have the ability to recognize and cleave a set of small base lesions resulting from oxidation, alkylation, or deamination of normal bases, with the help of an AP endonuclease (APE1) that removes abasic sites. To precisely characterize the BER pathway and offer better chance to identify individuals with altered DNA repair capacities, we developed a comprehensive approach that quantifies simultaneously several BER repair enzymes in cell extracts. This multiplexed biochip version of the well-known oligonucleotide cleavage assay was implemented to characterize the DNA Repair Enzyme Signature of a cohort of 100 healthy individuals (50% males and 50% females, ranging in age from 18 to 70 years). All were non-smokers. Two 8 mL blood samples were taken the first day and another one one week apart. Whole cell extracts were prepared from the subjects Peripheral Blood Mononuclear Cells (PBMCs) and protein concentration was determined. Assay robustness was assessed and the inter- and intra-individual BER variability was also determined. We observed a large inter-individual variability in DNA repair capacities. We demonstrated that there was a gender-specific impact of aging on certain DNA Repair activities. Thanks to the multiplexed approach, using hierarchical clustering and principal component analysis, we gained new information on the different glycosylase activities co-regulations and assigned the samples to distinct subpopulation clusters characterized by their DNA Repair Signature. Here again, males and females exhibited different features. We demonstrated that our powerful assay is robust, can be implemented on a large scale and could be used for population studies to identify biomarkers of exposure and biomarkers of cancer susceptibility. We thank GRAVIT and CEA program Technologies for Health for financial support. Citation Format: Sylvie Sauvaigo, Lise Jacqueroux, Benoit Pitiot, Sylvain Caillat, Nicolas Gonnet, Jean-Luc Cracowski. DNA Repair Enzyme Signature of a cohort of 100 healthy individuals using peripheral blood mononuclear cells: In the search of cancer susceptibility biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 306. doi:10.1158/1538-7445.AM2014-306