scholarly journals Histone carbonylation in vivo and in vitro

2000 ◽  
Vol 351 (3) ◽  
pp. 769-777 ◽  
Author(s):  
Georg T. WONDRAK ◽  
Daniel CERVANTES-LAUREAN ◽  
Elaine L. JACOBSON ◽  
Myron K. JACOBSON
Keyword(s):  
Histone H1 ◽  
Nuclear Proteins ◽  
Carbonyl Groups ◽  
Cell Nuclei ◽  
Strand Breaks ◽  
Core Histones ◽  
Dna Strand ◽  
And Function

Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups into histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH4 (sodium borohydride). Histone H1 extracted from bovine thymus, liver and spleen was found to contain significantly elevated amounts of protein-bound carbonyl groups as compared with core histones. The carbonyl content of nuclear proteins of rat pheochromocytoma cells (PC12 cells) was not greatly increased following oxidative stress induced by H2O2, but was significantly increased following alkylating stress induced by N-methyl-N´-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing sugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei. Studies of the mechanism of histone H1 modification by ADP-ribose indicate that carbonylation involves formation of a stable acyclic ketoamine. Our results demonstrate preferential histone H1 carbonylation in vivo, with potentially important consequences for chromatin structure and function.

scholarly journals Recombinogenic Flap Ligation Pathway for Intrinsic Repair of Topoisomerase IB-Induced Double-Strand Breaks

2000 ◽  
Vol 20 (21) ◽  
pp. 8059-8068 ◽  
Author(s):  
Chonghui Cheng ◽  
Stewart Shuman
Keyword(s):  
High Efficiency ◽  
Strand Break ◽  
Strand Transfer ◽  
Strand Breaks ◽  
Genomic Deletions ◽  
Topoisomerase Ib ◽  
Dna Strand ◽  
Recombination Pathway

ABSTRACT Topoisomerase IB catalyzes recombinogenic DNA strand transfer reactions in vitro and in vivo. Here we characterize a new pathway of topoisomerase-mediated DNA ligation in vitro (flap ligation) in which vaccinia virus topoisomerase bound to a blunt-end DNA joins the covalently held strand to a 5′ resected end of a duplex DNA containing a 3′ tail. The joining reaction occurs with high efficiency when the sequence of the 3′ tail is complementary to that of the scissile strand immediately 5′ of the cleavage site. A 6-nucleotide segment of complementarity suffices for efficient flap ligation. Invasion of the flap into the duplex apparently occurs while topoisomerase remains bound to DNA, thereby implying a conformational flexibility of the topoisomerase clamp around the DNA target site. The 3′ flap acceptor DNA mimics a processed end in the double-strand-break-repair recombination pathway. Our findings suggest that topoisomerase-induced breaks may be rectified by flap ligation, with ensuing genomic deletions or translocations.

scholarly journals The STAGA Subunit ADA2b Is an Important Regulator of Human GCN5 Catalysis

2008 ◽  
Vol 29 (1) ◽  
pp. 266-280 ◽  
Author(s):  
Armin M. Gamper ◽  
Jaehoon Kim ◽  
Robert G. Roeder
Keyword(s):  
Saga Complex ◽  
Histone Tails ◽  
Functional Roles ◽  
Enzymatic Function ◽  
Core Histones ◽  
Human Proteins ◽  
Important Regulator ◽  
And Function

ABSTRACT Human STAGA is a multisubunit transcriptional coactivator containing the histone acetyltransferase GCN5L. Previous studies of the related yeast SAGA complex have shown that the yeast Gcn5, Ada2, and Ada3 components form a heterotrimer that is important for the enzymatic function of SAGA. Here, we report that ADA2a and ADA2b, two human homologues of yeast Ada2, each have the ability to form a heterotrimer with ADA3 and GCN5L but that only the ADA2b homologue is found in STAGA. By comparing the patterns of acetylation of several substrates, we found context-dependent requirements for ADA2b and ADA3 for the efficient acetylation of histone tails by GCN5. With human proteins, unlike yeast proteins, the acetylation of free core histones by GCN5 is unaffected by ADA2b or ADA3. In contrast, the acetylation of mononucleosomal substrates by GCN5 is enhanced by ADA2b, with no significant additional effect of ADA3, and the efficient acetylation of nucleosomal arrays (chromatin) by GCN5 requires both ADA2b and ADA3. Thus, ADA2b and ADA3 appear to act at two different levels of histone organization within chromatin to facilitate GCN5 function. Interestingly, although ADA2a forms a complex(es) with GCN5 and ADA3 both in vitro and in vivo, ADA2a-containing complexes are unable to acetylate nucleosomal H3. We have also shown the preferential recruitment of ADA2b, relative to ADA2a, to p53-dependent genes. This finding indicates that the previously demonstrated presence and function of GCN5 on these promoters reflect the action of STAGA and that the ADA2a and ADA2b paralogues have nonredundant functional roles.

Prevention of base excision repair by TRC102 (methoxyamine) potentiates the anti-tumor activity of pemetrexed in vitro and in vivo

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 13005-13005 ◽  
Author(s):  
L. Liu ◽  
A. Bulgar ◽  
J. Donze ◽  
B. J. Adams ◽  
C. P. Theuer ◽  
...  
Keyword(s):  
Excision Repair ◽  
Alkylating Agents ◽  
Phase 1 ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Ap Sites ◽  
Dna Strand ◽  
Anti Tumor Activity

13005 Background: TRC102 (methoxyamine) reverses resistance to alkylating agents by inhibiting base excision repair (BER; a mechanism of DNA repair), thereby increasing DNA strand breaks and potentiating the anti-tumor activity of alkylating agents without additional toxicity, Based on these data, TRC102 is currently being studied in combination with temozolomide in a phase 1 trial. We hypothesized that inhibition of BER by TRC102 would also increase DNA strand breaks and improve the anti-tumor activity of anti-metabolite chemotherapeutics, including pemetrexed, because these agents also produce AP sites that are recognized and repaired by BER. Methods: Pemetrexed- induced AP sites and BER inhibition was quantified using an apurinic/apyrimidinic (AP) site assay in vitro. Single and double DNA strand breaks were quantified by the Comet assay in vitro and anti-tumor activity was assessed in an in vivo xenograft study of subcutaneously implanted H460 human lung cancer cells. Results: Pemetrexed induced and TRC102 reduced the number of available AP sites in pemetrexed- treated H460 cells (by 60–80%), indicating successful inhibition of BER. TRC102 treatment increased DNA strand breaks in pemetrexed-treated H460 cells (2 fold increase versus treatment with pemetrexed alone). Premetrexed treatment alone and in combination with TRC 102 delayed tumor growth in vivo (tumor growth delay of 4.7 days in the 150 mg/m2 pemetrexed alone group, 5.7 days in the 150 mg/m2 pemetrexed + 2 mg/m2 TRC102 group and 6.9 days in the 150 mg/m2 pemetrexed + 4 mg/m2 TRC102 group); in vivo systemic toxicity was not increased. TRC102 alone had no effect in vitro or in vivo. Conclusions: TRC102 effectively inhibits BER in lung cancer cells treated with pemetrexed. Inhibition of DNA repair by TRC102 results in an increase in DNA strand breaks and improved anti-tumor activity versus treatment with pemetrexed alone. Given its preclinical efficacy and safety profile, study of TRC102 combined with pemetrexed in a phase 1 trial is warranted. No significant financial relationships to disclose.

Dual labeling of the cytoskeleton and DNA strand breaks in porcine embryos produced in vivo and in vitro

1998 ◽  
Vol 51 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Charles R. Long ◽  
John R. Dobrinsky ◽  
Wesley M. Garrett ◽  
Lawrence A. Johnson
Keyword(s):  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Dna Strand

scholarly journals Induction of CAF-1 Expression in Response to DNA Strand Breaks in Quiescent Human Cells

2006 ◽  
Vol 26 (5) ◽  
pp. 1839-1849 ◽  
Author(s):  
Arman Nabatiyan ◽  
Dávid Szüts ◽  
Torsten Krude
Keyword(s):  
Excision Repair ◽  
Dna Strand Breaks ◽  
Significant Loss ◽  
Human Cells ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Quiescent Cells ◽  
Dna Strand

ABSTRACT Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks.

scholarly journals Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats

Toxins ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 348 ◽  
Author(s):  
Leticia Díez-Quijada ◽  
Concepción Medrano-Padial ◽  
María Llana-Ruiz-Cabello ◽  
Giorgiana M. Cătunescu ◽  
Rosario Moyano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Fatty Degeneration ◽  
Dna Strand Breaks ◽  
Histopathological Study ◽  
Strand Breaks ◽  
Dna Strand

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

scholarly journals In vitro-in vivo correlations of pulmonary inflammogenicity and genotoxicity of MWCNT

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Emilio Di Ianni ◽  
Johanna Samulin Erdem ◽  
Peter Møller ◽  
Nicklas Mønster Sahlgren ◽  
Sarah Søs Poulsen ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Inflammatory Response ◽  
Dna Strand Breaks ◽  
Mass Spectrometry Analysis ◽  
Strand Breaks ◽  
Effective Doses ◽  
Dna Strand ◽  
Submerged Conditions

Abstract Background Multi-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (< 1, 1, 2, 3 or 4 standard deviation increases) for the increasing genotoxicity. Results Effective doses were shown to be 25 to 53%, and 21 to 57% of the doses administered to A549 and THP-1a, respectively. In submerged conditions (A549 and THP-1a cells), all NM induced dose-dependent IL-8 expression. NM-401 and NRCWE-006 caused the strongest pro-inflammatory response. In the ALI-exposed co-culture, only NM-401 caused increased IL-8 expression, and no DNA strand breaks were observed. Strong correlations were found between in vitro and in vivo inflammation when doses were normalized by surface area (also proxy for diameter and length). Significantly increased DNA damage was found for all MWCNT in THP-1a cells, and for short MWCNT in A549 cells. A concordance in genotoxicity of 83% was obtained between THP-1a cells and broncho-alveolar lavaged (BAL) cells. Conclusion This study shows correlations of pro-inflammatory potential in A549 and THP-1a cells with neutrophil influx in mice, and concordance in genotoxic response between THP-1a cells and BAL cells, for seven MWCNT.

Ascorbate potentiates DNA damage by 1-methyl-1-nitrosourea in vivo and generates DNA strand breaks in vitro

1987 ◽  
Vol 8 (11) ◽  
pp. 1657-1662 ◽  
Author(s):  
Paul V. Woolley ◽  
Shailendra Kumar ◽  
Peter Fitzgerald ◽  
Robert T. Simpson
Keyword(s):  
Dna Damage ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Dna Strand

scholarly journals DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro

PLoS ONE ◽  
2017 ◽  
Vol 12 (8) ◽  
pp. e0183684 ◽  
Author(s):  
Diane Penndorf ◽  
Vedrana Tadić ◽  
Otto W. Witte ◽  
Julian Grosskreutz ◽  
Alexandra Kretz
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Dna Strand ◽  
Lateral Sclerosis
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