Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

SN1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O6-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks

Operationalization of a multidimensional sex/gender concept for quantitative environmental health research and implementation in the KORA study: Results of the collaborative research project INGER

BackgroundIn environmental health research, sex and gender are not yet adequately considered. There is a need to improve data collection in population-based environmental health studies by comprehensively surveying sex/gender-related aspects according to gender theoretical concepts. Thus, within the joint project INGER we developed a multidimensional sex/gender concept which we aimed to operationalize and to test the operationalization for feasibility.MethodsIn an iterative process, we created questionnaire modules which quantitatively captured the requirements of the INGER sex/gender concept. We deployed it in the KORA cohort (Cooperative Health Research in the Region of Augsburg, Germany) in 2019 and evaluated response and missing rates.ResultsThe individual sex/gender self-concept was surveyed via a two-step approach that asked for sex assigned at birth and the current sex/gender identity. Additionally, we used existing tools to query internalized sex/gender roles and externalized sex/gender expressions. Adapted to the KORA population, we asked for discrimination experiences and care and household activities contributing to explain structural sex/gender relations. Further intersectionality-related social categories (e.g., socio-economic position), lifestyle and psychosocial factors were covered through data available in KORA. We could not identify appropriate tools to assess the true biological sex, sexual orientation and ethnic/cultural identity, which have yet to be developed or improved. The response-rate was 71%, the evaluation of 3,743 questionnaires showed a low missing rate. Prevalence of marginalized groups regarding sex/gender identity and definable by experiences of discrimination was very low.ConclusionWe have shown how the multidimensional INGER sex/gender concept can be operationalized according to an European and North American understanding of sex/gender for use in quantitative research. The questionnaire modules proved feasible in an epidemiologic cohort study. Being a balancing act between theoretical concepts and its quantitative implementation our operationalization paves the way for an adequate consideration of sex/gender in environmental health research

B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death

Processing of O 6 - methylguanine in mammalian cells

DNA methylating compounds are widely used as anti-cancer chemotherapeutics. The pharmaceutical critical DNA lesion induced by these drugs is O6-methylguanine (O6MeG). O6MeG is highly mutagenic and genotoxic, by triggering apoptosis. Despite the potency of O6MeG to induce cell death, the mechanism of O6MeG induced toxicity is still poorly understood. Comparing the response of mouse fibroblasts wild-type (wt) and deficient for ataxia telangiectasia mutant protein (ATM), a kinase responsible for both the recognition and the signalling of DNA double-strand breaks (DSBs), it was shown that ATM deficient cells are more sensitive to the methylating agents N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), methyl methansulfonate (MMS) and the anti-cancer drug temozolomide, in both colony formation and apoptosis assays. This clearly shows that DSBs are involved in O6MeG toxicity. By inactivating the O6MeG repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) with the specific inhibitor O6-benzylguanine (O6BG), ATM wt and deficient cells became more sensitive to MNNG and MMS. The opposite effect was observed when over-expressing MGMT in ATM -/- cells. The results show that O6MeG is the critical DNA lesion causing death in ATM cells following MNNG treatment, and is partially responsible for the toxicity observed following MMS treatment. Furthermore, by inhibiting the ATM kinase activity with caffeine, it was shown that the resistance of wt cells to MNNG was due to the kinase activity of ATM, as wt cells underwent more apoptosis following methylating agent treatment in the presence of caffeine. Apoptosis and caspase-3 activation were late events, starting 48h after treatment. This lends support to the model where O6MeG lesions are converted into DSBs during replication. As ATM wt and deficient cells showed similar G2/M blockage and Chk1 activation following MNNG and MMS treatment, it was concluded that the protective effect of ATM is not due to cell cycle progression control. The hypersensitivity of ATM deficient cells was accompanied by their inability to activate the anti-apoptotic NFkB pathway. In a second part of this study, it was shown that the inflammatory cytokine IL-1 up-regulates the DNA repair gene apurinic endonuclease 2 (APEX2). Up-regulation of APEX2 occurred by transcriptional regulation as it was abrogated by actinomycin D. APEX2 mRNA accumulation was accompanied by increase in APEX2 protein level. IL-1 induced APEX2 expression as well as transfection of cells with APEX2 cDNA positively correlated with a decrease in apoptosis after treatment with genotoxic agents, particularly affecting cell death after H2O2. This indicates an involvement of APEX2 in the BER pathway in cells responding to IL-1.DNA-methylierende Agenzien werden verbreitet als anti-Krebs Therapeutika benutzt. Der durch diese Präparate verursachte DNA-Schaden ist die Bildung von O6–Methylguanin (O6MeG). Durch Auslösen von Apoptose ist O6MeG hoch mutagen und genotoxisch. Trotz der Fähigkeit von O6MeG den Zelltod zu induzieren, ist der Mechanismus der durch O6MeG erzeugten Toxizität kaum verstanden. Durch Vergleich von Maus wildtyp (wt) und für Ataxia Telangiectasia Mutant Protein (ATM), einer Kinase, die sowohl für das Erkennen als auch als Signalprotein von DNA-Doppelstrangbrüchen (DSBs) verantwortlich ist, defiziente Fibroblasten wurde gezeigt, daß ATM-defiziente Zellen sowohl in Colony Formation- als auch in Apoptose-Assays sensitiver gegen die methylierenden Agentien N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), Methyl methansulfonate (MMS) und das anti-Krebs Mittel Temozolomide sind. Dies zeigt klar, daß DSBs an der Toxizität von O6MeG beteiligt sind. Durch Inaktivierung des O6MeG-Reparaturenzyms O6–Methylguanin-DNA methyltransferase (MGMT) mit dem spezifischen Inhibitor O6-Benzylguanin wurden ATM wildtyp und defiziente Zellen sensitiver gegenüber MNNG und MMS. Der entgegengesetzte Effekt wurde beobachtet, wenn MGMT in ATM -/- Zellen überexprimiert wurde. Das Ergebnis zeigt, daß O6MeG der kritische DNA-Schaden ist, der den Tod von ATM-Zellen nach MNNG Behandlung bewirkt, und teilweise die Toxizität nach MMS Behandlung bewirkt. Weiterhin wurde durch Hemmung der ATM Kinase mit Koffein gezeigt, daß die Resistenz von wt Zellen auf der Kinaseaktivität von ATM beruht, da bei Behandlung mit methylierenden Agentien in Gegenwart von Koffein wt Zellen vermehrt Apoptose zeigten. Apoptose und Caspase-3 Aktivierung traten nach 48 h Behandlung auf. Dies unterstützt das Modell, in dem O6MeG-Schäden während der Replikation in DSBs umgewandelt werden. Da ATM wt und defiziente Zellen die gleiche G2/M Blockade und Chk1 Aktivierung nach MNNG und MMS Behandlung zeigten, kann geschlossen werden, daß der schützende Effekt von ATM nicht durch Kontrolle des Fortschritts des Zellzyklusses erfolgt. Die Hypersensivität von ATM defizienten Zellen wurde von der Unfähigkeit den anti-apoptotischen NFkB-Weg zu aktivieren begleitet. Im zweiten Teil der Arbeit wurde nachgewiesen, daß das inflammatorische Cytokin IL-1 das DNA-Reparaturgen für Apurinische Endonuclease 2 (APEX2) hoch reguliert. Die Hochregulation erfolgt auf Ebene der Transkription, da sie durch Actinomycin D aufgehoben werden konnte. Die Akkumulation von APEX2-mRNA ging einher mit einem Anstieg von APEX2-Protein. IL-1 induziert die APEX2-Expression, die Transfektion von Zellen mit APEX2-cDNA war positiv korreliert mit einer Abnahme der Apoptose nach Behandlung mit genotoxischen Agentien