DNA Damage in Nijmegen Breakage Syndrome Cells Leads to PARP Hyperactivation and Increased Oxidative Stress

Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the NBN gene, which codes for the protein nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage

Defizienz des DNA-Reparatur-Proteins Nibrin führt zu einer Erhöhung der spontanen nicht aber der strahlen-induzierten Mutationsrate in vivo

Einleitung: Nibrin (NBN) bildet zusammen mit MRE11 und RAD50 einen Komplex, der an der Reparatur von DNA-Doppelstrangbrüchen (DSB) und der Zellzyklusregulation beteiligt ist. Hypomorphe Mutationen von Komponenten des Komplexes führen beim Menschen zu Erkrankungen, die durch Radiosensitivität und eine stark erhöhte Tumorinzidenz des lymphoretikulären Systems charakterisiert sind. Mutationen des NBN- Gens resultieren im autosomal rezessiven Nijmegen Breakage Syndrom (NBS). Über 95% der NBS Patienten tragen dieselbe Mutation, eine 5bp-Deletion, die zu alternativer Translation und der Produktion eines aberranten Protein Fragments führt. Dieses Protein Fragment übernimmt partiell die Funktionen des Wildtyp-Nibrins. Null-Mutationen in Mäusen sind aufgrund erhöhter Apoptose im Blastozysten-Entwicklungsstadium embryonal letal. In der vorliegenden Arbeit haben wir den Zusammenhang von DNA- Schädigung, Mutationsrate und Mutationsspektrum in vivo und in vitro bei Mutationen von NBN untersucht. Zur in vivo Untersuchung wurden konditional null-mutante und humanisierte Mäuse mit der 5bp-Deletion verwendet. Diese Mäuse wurden mit einer transgenen Maus, die das bakterielle LacZ- Reporterplasmid trägt, gekreuzt. Es wurden sowohl homozygote als auch heterozygote Mäuse untersucht. Aus diesen Mäusen gewonnene Fibroblasten wurden verwendet, um den Zusammenhang von DNA- Schädigung, Mutationsrate und Mutationsspektrum in vitro zu untersuchen. Ergebnisse: Es zeigte sich, dass Mutationen von NBN zu erhöhten spontanen DNA-Schädigungen in vitro führen. In vivo konnte im Vergleich zu Kontroll-Mäusen eine 2.5-fache Erhöhung der basalen Mutationsrate in den lymphatischen Organen der humanisierten Maus gezeigt werden. Im Gegensatz zu den nach defekter DSB-Reparatur erwarteten komplexen Mutationen wiesen die untersuchten Zellen und Mäuse ein von Einzelbasenpaar-Veränderungen dominiertes Mutationsspektrum auf. Schlussfolgerung: Die Reparatur von spontanen Mutationen, möglicherweise entstanden durch fehlerhafte DNA-Replikation, spielt in Abwesenheit von Wildtyp-Nibrin eine große Rolle bei der Entstehung der erhöhten basalen Mutationsrate. Dies gilt auch für Zellen, die heterozygot für NBN- Nullmutationen sind. NBS Patienten-Zellen zeigen in vitro eine erhöhte Strahlensensitivität. Doch eine Erhöhung der Mutationsraten nach Bestrahlung ist in vivo nicht zu beobachten, was vermutlich auf eine intakte Apoptose in Abwesenheit von Wildtyp-Nibrin hindeutet.Introduction: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders, characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. Mutations of the NBN gene result in the autosomal recessive Nijmegen Breakage Syndrome (NBS). Over 95% of NBS patients carry the major NBN mutation, a 5bp deletion, which leads to alternative translation and the production of a truncated protein fragment. This protein fragment maintains partial nibrin function. Null mutations in mice are embryonically lethal due to massive apoptosis at the blastocyst stage. Little is actually known about the relationship between DNA damage, mutation frequency and mutation spectrum in the absence of wild type nibrin. We have utilised a conditional null mutant mouse and a humanized NBS mouse, which carries the 5bp deletion, to examine this relationship in vivo. Mice homozygous and heterozygous for nibrin mutations were examined. We have bred these mice with a transgenic mutagenesis mouse harbouring copies of the bacterial lacZ gene. Fibroblasts derived from the different mice were used to examine DNA damage, mutation frequency and spectrum in vitro. Results: We find that NBN mutation leads to increased DNA damage in vitro, even in the absence of an external genotoxic treatment. In vivo we find the basal mutation frequency measured in lymphatic tissue of the humanized NBS mice is approximately 2.5-fold higher than in control mice. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. Conclusion: We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, make a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin

Rapid NAD⁺ depletion in NBS patient fibroblasts after DNA damage.

<p>Relative levels of NAD⁺ in NBS-1LBI NBS patient fibroblasts (•) and LN9 control fibroblasts (▪) after DNA damage are shown. NAD⁺ levels in untreated cells were set at 100%.</p

Increased PARP activity in <i>Nbn^−/−</i> murine fibroblasts and NBS patient fibroblasts after DNA damage.

<p>Lysates from mouse (A) and human LN9 and GM166VA7 fibroblasts (B) with the given genotypes were harvested at the indicated timepoints (minutes) after a bleomycin treatment and probed on immunoblots with antibodies directed against poly(ADP-ribose) and ß-actin.</p

ROS levels in NBS patient fibroblasts after DNA damage are reduced by antioxidant scavengers but PARP remains hyperactivated.

<p>(A) FACS profiles of ROS measurements in NBS-1LBI patient cells 12 hours after treatment with bleomycin and in the presence or absence of the antioxidant TROLOX. Cells were stained with CM-H₂DCFDA for ROS detection. The data shown are from one of three experiments with essentially identical results. (B) Lysates from NBS-1LBI patient cells were harvested 15 minutes after DNA damage by bleomycin in the presence the PARP inhibitor KU-0058948 or the antioxidant TROLOX as indicated. Lysates were probed on immunoblots with antibodies directed against poly(ADP-ribose) and ß-actin.</p

High levels of ROS in <i>Nbn</i> null mutant murine fibroblasts and NBS patient cells after DNA damage.

<p>(A) FACS profiles of ROS measurements in murine cells with the indicated genotypes with or without treatment with bleomycin. Cells were stained with CM-H₂DCFDA 12 hours after treatment with bleomycin. Fluorescence intensity is proportional to ROS. The experiment was repeated six times and the same profiles were obtained. (B) Western-blot demonstration of conditional <i>Nbn</i> null mutation in murine fibroblasts. Lysates from <i>Nbn</i>^Ins-6/lox-6 fibroblasts with and without treatment with HTNC were probed on immunoblots with anti-nibrin and anti-actin antibodies. (C) Representative FACS profiles of ROS measurements in LN9 wild type and GM7166VA7 NBS patient fibroblasts with or without treatment with bleomycin. Cells were stained with CM-H₂DCFDA 12 hours after treatment with bleomycin. Fluorescence intensity is proportional to ROS. The experiment was repeated more than five times and essentially the same profiles were obtained.</p

ROS in <i>Nbn</i> null mutant murine fibroblasts and NBS patient cells after DNA damage.

<p>Relative levels of ROS after treatment with bleomycin are given for murine and human LN9, GM166VA7 and NBS-1LBI cells with the given genotypes and after the indicated treatments. ** p = 0.0095 in the Mann-Whitney U Test (two-tailed, n¹ = 4, n² = 6); * p = 0.017 in the Mann-Whitney U Test (two-tailed, n¹ = 3, n² = 12).</p