Effects of acute versus chronic hypoxia on DNA damage responses and genomic instability.

Questions exist concerning the effects of acute versus chronic hypoxic conditions on DNA replication and genomic stability that may influence tumorigenesis. Severe hypoxia causes replication arrest independent of S-phase checkpoint, DNA damage response, or transformation status. Arrests occur during both the initiation and elongation phases of DNA replication, correlated with a rapid decrease in available deoxynucleotide triphosphates. With fluctuating oxygen tensions in tumors, arrested hypoxic cells may undergo rapid reperfusion and reoxygenation that leads to reoxygenation-induced DNA damage. In cells subjected to chronic hypoxia, we found that replicative restart was inhibited along with numerous replication factors, including MCM6 and RPA, the latter of which limits the hypoxia-induced DNA damage response. In contrast, in cells where replicative restart occurred, it was accompanied by extensive reoxygenation-induced DNA damage and compromised DNA repair. We found that cells reoxygenated after acute hypoxia underwent rapid p53-dependent apoptosis. Our findings suggest that cells lacking functional p53 are more susceptible to genomic instability and potentially tumorigenesis if they experience reoxygenation after acute exposure to hypoxia

Human AlkB Homologue 5 Is a Nuclear 2-Oxoglutarate Dependent Oxygenase and a Direct Target of Hypoxia-Inducible Factor 1α (HIF-1α)

Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia

Signalisation et réparation des cassures double-brin de l'ADN dans les gliomes : Modulation de la réponse aux traitements chimio-radiothérapeutiques.

6000 new cases of tumours of the nervous system are detected each year in France and their prognostic stay uncertain. This thesis aims to provide new insights in the molecular and cellular response of brain tumours to radio-chemotherapy. A DNA double-breaks repair depending on the MRE11 protein but independent of the phosphorylation of H2AX emerged from the study of artefacts of the immunofluorescence technique. The radiobiological characteristics of the 3 rodent glioma cell lines and 7 human glioma cell lines were analyzed. Functional impairments of the BRCA1 protein in response to radiation and/or cisplatin were observed in the majority of the models tested, raising the question of the role of this protein in the anti-glioma treatments and in gliomagenesis. We studied the effect of some protein kinases inhibitors on the quality of damage repair by the recombination or the DNA end-joining repair. The defect of repair results from the blockade of signaling pathways caused by these targeted treatments. The radiobiological characteristics of the neurofibromatosis of the type 1 (NF1), a genetic syndrome associated the tumors of the peripheral and central nervous system, were analyzed. NF1 appeared to be a syndrome with moderated radiosensitivity, associated with a weak deficiency of DNA end-joining repair but with a strong activity of MRE11.6000 nouveaux cas de tumeurs du système nerveux sont dépistés chaque année en France et leur pronostique reste incertaine. Nos travaux visent à éclaircir la réponse moléculaire et cellulaire de cette pathologie suite aux traitements radio-chimiothérapiques. Une nouvelle voie de réparation des cassures double-brin de l'ADN dépendant de la protéine MRE11 mais indépendante de la phosphorylation de H2AX a été mise en évidence. Les caractéristiques radiobiologiques des 3 modèles de gliomes de rongeurs et de 7 modèles de gliomes humaines ont été analysées. Des dysfonctionnements de la protéine BRCA1 en réponse aux radiations ou au cisplatine ont été observés dans la majorité de ces modèles testés, soulevant la question du rôle de cette protéine dans les traitements anti-gliomes ainsi que dans la gliomagenèse. Nous avons étudié l'effet de quelques drogues inhibitrices de protéine kinases sur la qualité de réparation par la recombinaison ou par la suture. Le défaut de réparation résulte du blocus de voies de signalisation causé par ces traitements ciblés. Les caractéristiques radiobiologiques d'un syndrome génétique associé aux tumeurs du système nerveux périphérique et central, la neurofibromatose de type 1 (NF1), ont été analysées. La NF1 a montré une radiosensibilité modérée, associée à une faible déficience de réparation de l'ADN par suture mais une forte activité de MRE11

Radiobiological features of the anti-cancer strategies involving synchrotron X-rays.

International audienceSynchrotrons are opening new paths in innovative anti-cancer radiotherapy strategies. Indeed, the fluence of X-rays induced by synchrotrons is so high (10(6) times higher than standard medical irradiators) that it enables the production of X-ray beams tunable in energy (monochromatic beams) and in size (micrometric beams). Monochromatic synchrotron X-ray beams theoretically permit photoactivate high-Z elements to be introduced in or close to tumours in order to increase the yield of damage by enhanced energy photoabsorption. This is notably the case of attempts with iodinated contrast agents used in tumour imaging (the computed tomography therapy approach) and with platinated agents used in chemotherapy (the PAT-Plat approach). Micrometric synchrotron X-ray beams theoretically permit very high radiation doses to accumulate in tumours by using arrays of parallel microplanar beams that spare the surrounding tissues (the microbeam radiation therapy approach). These anti-cancer applications of synchrotron radiation have been developed at the European Synchrotron Radiation Facility to be applied to glioma, one of the tumour tissues most refractory to standard treatments. In the present paper the molecular and cellular mechanisms involved in these three approaches are reviewed, in the context of recent advances in radiobiology. Furthermore, by considering the unavoidable biases, an attempt to propose a comparison of the different results obtained in preclinical trials dealing with rats bearing tumours is given

Exposure to acute hypoxia induces a transient DNA damage response which includes Chk1 and TLK1

Severe hypoxia has been demonstrated to induce a replication arrest which is associated with decreased levels of nucleotides. Chk1 is rapidly phosphorylated in response to severe hypoxia and in turn deactivates TLK1 through phosphorylation. Loss of Chk1 has been shown to sensitize cells to hypoxia/reoxygenation. After short (acute) exposure to hypoxia this is due to an increased rate of reoxygenation-induced replication restart and subsequent p53-dependent apoptosis. After longer (chronic) exposure to hypoxia S phase cells do not undergo reoxygenation-induced replication restart. Cells exposed to these levels of hypoxia are however sensitive to loss of Chk1. This suggests a new role for Chk1 in the cell cycle response to reoxygenation

Cadmium inhibits non-homologous end-joining and over-activates the MRE11-dependent repair pathway.

International audienceAlthough cadmium still represents a public health problem and despite the fact that it has been classified as an IARC Group-I carcinogen, the molecular and cellular mechanisms responsible for the toxicity and the carcinogenicity of cadmium compounds are poorly known. Since unrepaired DNA double-strand breaks (DSBs) are considered to be key-lesions in cell lethality, and because misrepaired DSBs are a source of genomic instability leading to cancer proneness, the activity of the major DSB-repair pathways, i.e. non-homologous end-joining (NHEJ) and recombination, has been evaluated in human endothelial cells exposed to cadmium chloride and cadmium diacetate. Exposure to cadmium results in the production of DSBs a few hours after incubation. These breaks trigger the phosphorylation of H2AX proteins, which was used as an indirect measure of DSB in this study. The presence of cadmium in cells decreases the repair rate of X-ray-induced DSBs, suggesting an impact of cadmium upon the reparability of DSBs. Such an interpretation was consolidated by the finding that the DNA-PK kinase activity, essential for NHEJ, is affected by the presence of cadmium. These results suggest that the toxicity of cadmium compounds may be explained by the propagation of persistent DSBs. In parallel, the presence of cadmium was also associated with an over-activation of the MRE11-dependent repair pathway that may favour genomic instability. Altogether, our data provide a first example of the impact of cadmium upon DSB repair and signalling

Consequences of the bleed-through phenomenon in immunofluorescence of proteins forming radiation-induced nuclear foci.

International audiencePURPOSE: By allowing the visualization of the proteins inside cells, the immunofluorescence technique has revolutionized our view of events that follow radiation response. Particularly, the formation of nuclear foci, their kinetic of appearance and disappearance, and the association-dissociation of protein partners are useful endpoints to better understand the effects of ionizing radiation. Recently, the technique based on the phosphorylation of the histone 2A family, member X (H2AX) has generated a plethora of reports concerning the interaction between the major proteins involved in DNA repair and stress signaling pathways. However, some unavoidable overlaps of excitation and emission wavelength spectra (the so-called bleed-through phenomenon) of the available fluorescent markers are still generating discrepancies and misinterpretations in the choreography of DNA damage response. Biases are particularly strong with the fluorescein isothiocyanate (FITC)-rhodamine couple, tetramethyl rhodamine iso-thiocyanate (TRITC), the most extensively used markers. METHOD AND RESULTS: Here, two representative examples of biased co-immunofluorescence with pH2AX proteins that form radiation-induced nuclear foci or not are presented. A brief review of literature points out differences in kinetic of appearance and association-dissociation of radiation-induced pH2AX and MRE11 foci. CONCLUSION: Through this report, we would like authors to consider more carefully protein co-localizations by performing systematically, before any co-immunofluorescence, immunofluorescence of each protein separately to avoid bleed-through artifacts

Lead contamination results in late and slowly repairable DNA double-strand breaks and impacts upon the ATM-dependent signaling pathways.

International audienceDespite a considerable amount of data, evaluation of the potential genotoxicity and cancer proneness of lead compounds remains unclear, probably due to the plethora of experimental procedures, biological endpoints and cellular models used. In parallel, the understanding in DNA damage formation, repair and signaling has considerably progressed all along these last years, notably for DNA double-strand breaks (DSBs). Here, were examined DNA damage formation and repair in human cells exposed to lead nitrate (Pb(NO(3))(2)) and their consequences upon the ATM-dependent stress signaling, cell cycle progression and cell death. As observed with anti-pH2AX immunofluorescence, exposure to Pb(NO(3))(2) results in formation of late DSBs, that would not originate from conversion of nucleotide damage but likely by a direct production of single-strand breaks. Lead contamination inhibits non-homologous end-joining repair process by preventing the DNA-PK kinase activity whereas the MRE11-dependent repair pathway is exacerbated. Lead contamination triggers successive synchronization of cells in G2/M phase in which the RAD51-dependent homologous recombination was found to be activated. Altogether, our findings support that lead contamination generates late unrepairable DSBs that impact upon the ATM-dependent stress signaling pathway by favoring propagation of errors. Such findings should help to consider more carefully the biological action of lead compounds in the frame of public and occupational exposures

DNA double-strand break repair defects in syndromes associated with acute radiation response: at least two different assays to predict intrinsic radiosensitivity?

International audiencePURPOSE: Human diseases associated with acute radiation responses are rare genetic disorders with common clinical and biological features including radiosensitivity, genomic instability, chromosomal aberrations, and frequently immunodeficiency. To determine what molecular assays are predictive of cellular radiosensitivity whatever the genes mutations, the existence of a quantitative correlation between cellular radiosensitivity and unrepaired DNA double-strand breaks (DSB) repair defects was examined in a collection of 40 human fibroblasts representing 8 different syndromes. MATERIALS AND METHODS: A number of techniques such as pulsed-field gel electrophoresis, plasmid assay and immunofluorescence with antibodies against MRE11, MDC1, 53BP1 and phosphorylated forms of H2AX, DNA-PK were applied systematically. RESULTS AND CONCLUSIONS: Survival fraction at 2 Gy was found to be inversely proportional to the amount of unrepaired DSB, whatever the genes mutations and the assay applied. However, no single assay discriminates the full range of human radiosensitivity. Particularly, nuclear foci formed by the phosphorylation of H2AX do not predict well moderate radiosensitivities. Our findings suggest the existence of an ATM-dependent interplay between the activation of DNA-PK and MRE11. A classification of diseases according their cellular radiosensitivity, their molecular response to radiation and the functional assays permitting their evaluation is proposed