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

Low molecular weight cellulose ethers as aerosols for the the consolidation of cohesively weak paint layers

Due to Edvard Munch’s (1863-1944) unconventional painting technique, choice of materials, and the unstable climate conditions of his studios, where the paintings were stored during his lifetime, many of his paintings, now housed at MUNCH, have cohesively weak and loose paint layers. As a result, consolidation and re-adhesion of these fragile paint layers are the most frequently performed conservation treatments on his paintings. A selection of low molecular weight (lmw) hydroxypropyl methylcellulose ethers (HPMC), new to the field of conservation, have been evaluated in comparison to methylcellulose (MC) A4C and sturgeon glue regarding their suitability for the consolidation of cohesively weak paint layers. The mock-ups used for these investigations were of a similar composition (pigment, binding medium and pigment-binding medium ratio) and porosity to a paint sample from the painting “Beach Landscape with Trees and Boats” from 1905-06 by Edvard Munch. Viscosity and surface tension of aqueous solutions of the consolidants and their influence on the imbibition time and depth into porous paint layers were investigated. Fluorescence labelling was used to visualize the imbibition depth of an aqueous solution of the lmw HPMC E3 and MC A4C, applied as an aerosol. With this method it could be shown that the applied amount and the application method of the consolidant (with or without intermediate drying steps) can play a crucial role in the imbibition depth. To evaluate the consolidation effect of the tested polymers, the aerosols of their aqueous solutions were applied on the paint mock-ups in a reproducible and standardized way, using an automated two-axis-table. A customised abrasion test was developed to evaluate the comparative increase of the paint layer cohesion after consolidation. These preliminary investigations show lmw HPMC as promising alternatives to established consolidants. They allow for an ultrasonic nebulisation in higher concentrations and thus for the paint layer’s consolidation in a lower number of applications

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

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 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

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

Pitzer ion activities in mixed electrolytes for calibration of ion-selective electrodes used in clinical chemistry

Metrological comparability as well as reliability of ion activity results measured with ion-selective electrodes (ISE) was investigated within the framework of an interlaboratory comparison between eight partners from national metrology institutes and expert laboratories. Two electrolyte solutions containing the clinically most relevant ions sodium, potassium, magnesium, calcium and chloride having ion activities near the physiological range served as samples. The calibration of the measurement set-ups of the participants was carried out using gravimetrically prepared aqueous electrolyte solutions. The ion activities of these calibration standards were calculated by means of the semiempirical Pitzer model. The measurement uncertainty of the measurement results was calculated according to the guide to the expression of uncertainty in measurement, GUM. Based on a new scale for ion activities traceable to the SI system of units, comparability and reliability of ISE measurement results of clinically relevant ions is realised