The DNA polymerase λ is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway

New Topoisomerase I mutations are associated with resistance to camptothecin

<p>Abstract</p> <p>Background</p> <p>Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance.</p> <p>Methods</p> <p>We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of <it>TOP1 </it>mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing.</p> <p>Results</p> <p>In SN38 resistant HCT116 clones we identified three new <it>TOP1 </it>mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these <it>TOP1 </it>mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells.</p> <p>Conclusions</p> <p>These results indicate that the <it>TOP1 </it>mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.</p

The most likely time and place of introduction of BTV8 into belgian ruminants

Peer reviewe

The Necrotic Signal Induced by Mycophenolic Acid Overcomes Apoptosis-Resistance in Tumor Cells

The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA–mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL–overexpressing cells). All tested cells remained sensitive to MPA–mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells

Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains

Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzymelinked immunosorbent assays (ELISAs) to detect antibodies against different PEDV strains in pig serum. A total of 732 serum samples from North American or European pigs were tested. Samples included experimental samples from pigs infected with classical (G1a PEDV) or variant genogroup 1 PEDV (G1b PEDV), pandemic genogroup 2 PEDV (G2b PEDV) or noninfected controls. Field samples from herds with confirmed or unknown PEDV exposure were also used. Three indirect ELISAs based on G2b antigens (ELISAs 1, 2 and 3), a competitive ELISA based on the G2b antigen (ELISA 4) and a competitive ELISA based on the G1a antigen (ELISA 5) were compared. Overall, the tests had a moderate agreement (κ = 0.61). G1a PEDV infected pigs were earliest detected by ELISA 3, G1b PEDV infected pigs were earliest detected by ELISAs 4 and 5 and the performance of all tests was similar for the G2b PEDV group. ELISA 1 showed the overall lowest detection on experimentally and field derived samples. Diagnostic sensitivity and specificity with a 95% probability interval were estimated to be 68.2% (62.1 – 74.4%) and 97.5% (95.2 – 99.0%) for ELISA 1, 73.7% (71.5 – 79.6%) and 98.4% (96.6 – 99.5%) for ELISA 2, 86.2% (81.1 – 90.6%) and 91.6% (87.7 – 94.8%) for ELISA 3, 78.3% (72.8 – 83.5%) and 99.7% (98.2 – 100%) for ELISA 4, and 93.5% (90.3 – 96.0%) and 91.2% (83.8 – 97.9%) for ELISA 5. Differences in detection among assays seem to be more related to intrinsic factors of an assay than to the PEDV antigen used

[Alkylating agents].

International audienceWith the approval of mechlorethamine by the FDA in 1949 for the treatment of hematologic malignancies, alkylating agents are the oldest class of anticancer agents. Even though their clinical use is far beyond the use of new targeted therapies, they still occupy a major place in specific indications and sometimes represent the unique option for the treatment of refractory diseases. Here, we are reviewing the major classes of alkylating agents and their mechanism of action, with a particular emphasis for the new generations of alkylating agents. As for most of the chemotherapeutic agents used in the clinic, these compounds are derived from natural sources. With a complex but original mechanism of action, they represent new interesting alternatives for the clinicians, especially for tumors that are resistant to conventional DNA damaging agents. We also briefly describe the different strategies that have been or are currently developed to potentiate the use of classical alkylating agents, especially the inhibition of pathways that are involved in the repair of DNA lesions induced by these agents. In this line, the development of PARP inhibitors is a striking example of the recent regain of interest towards the "old" alkylating agents

L’inactivation de la réparation de l’ADN induit la formation de néoantigènes et dérégule la croissance tumorale

Comment onInactivation of DNA repair triggers neoantigen generation and impairs tumour growth. [Nature. 2017]International audienceno abstrac

De nouveaux rôles pour l’ADN topo-isomérase I

L’ADN topo-isomérase I eucaryote est une enzyme nucléaire dont le rôle essentiel est de supprimer les contraintes de torsion de l’ADN au cours des processus de réplication et de transcription. La découverte de son activité de protéine kinase, de sa grande similitude avec des enzymes de recombinaison de l’ADN, et de sa capacité de « reconnaître » une grande variété de lésions de l’ADN, a permis d’envisager de nouveaux rôles pour cette enzyme. Nous discuterons en particulier le rôle potentiel de la topo-isomérase I dans la signalisation des dommages de l’ADN aux systèmes de mort cellulaire

Topoisomerase I-mediated DNA damage.

International audienceTopoisomerase I is a ubiquitous and essential enzyme in multicellular organisms. It is involved in multiple DNA transactions including DNA replication, transcription, chromosome condensation and decondensation, and probably DNA recombination. Besides its activity of DNA relaxation necessary to eliminate torsional stresses associated with these processes, topoisomerase I may have other functions related to its interaction with other cellular proteins. Topoisomerase I is the target of the novel anticancer drugs, the camptothecins. Recently a broad range of physiological and environmentally-induced DNA modifications have also been shown to poison topoisomerases. This review summarizes the various factors that enhance or suppress top1 cleavage complexes and discusses the significance of such effects. We also review the different mechanisms that have been proposed for the repair of topoisomerase I-mediated DNA lesions