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Altered sensitivity to single-strand-specific reagents associated with the genomic vascular smooth muscle α-actin promoter during myofibroblast differentiation

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第20期275卷 15384-15391页

作者： Becker, NA Kelm, RJ Vrana, JA Getz, MJ Maher, LJ Mayo Clin & Mayo Fdn Dept Biochem & Mol Biol Rochester MN 55905 USA

Stimulation of quiescent AKR-2B mouse fibroblasts with transforming growth factor beta 1 results in uniform conversion to a myofibroblast-like phenotype as judged by a rapid accumulation of smooth muscle alpha-actin mRNA and protein. Because transcriptional regulation of the smooth muscle alpha-actin gene in these cells might be mediated by single-stranded DNA-binding proteins, we have examined the sensitivity of genomic DNA to chemical reagents with specificity for unpaired bases in a region of the promoter previously implicated in Pur alpha, Pur beta, and MSY1 binding in vitro (Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J, Biol. Chem. 274, 14238-14245), Our data reveal specific differences between purified DNA treated in vitro and nucleoprotein complexes treated in living cells. Although some differences were observed in quiescent cells, treatment with transforming growth factor beta 1 resulted in the development of additional sensitivity within 1 h. This enhancement was most pronounced in bases immediately upstream of an MCAT enhancer element-containing polypurine-polypyrimidine tract. A TATA-proximal element of similar base distribution showed no such hyperreactivities, These results suggest that activation of the endogenous smooth muscle alpha-actin gene during myofibroblast conversion is accompanied by specific structural changes in the promoter that are consistent with a decline in single-stranded DNA repressor protein binding.

关键词：肌动蛋白类/遗传学碱基序列细胞分化/药物作用 DNA 单链/药物作用增强子元件(遗传学) 表皮生长因子/药理学成纤维细胞/细胞学成纤维细胞/药物作用成纤维细胞/代谢胰岛素/药理学动力学分子序列数据肌平滑血管/代谢血小板源性生长因子/药理学启动区(遗传学) TATA盒转化生长因子β/药理学动物小鼠

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Antagonism of immunostimulatory CpG-oligodeoxynucleotides by 4-aminoquinolines and other weak bases: Mechanistic studies

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 1999年第3期291卷 1337-1347页

作者： Manzel, L Strekowski, L Ismail, FMD Smith, JC Macfarlane, DE Univ Iowa Dept Med Iowa City IA 52242 USA Vet Affairs Med Ctr Dept Med Iowa City IA 52242 USA

Oligodeoxynucleotides with unmethylated CpG motifs are immunostimulatory. Chloroquine and a number of structural analogs specifically and powerfully inhibit this effect at nanomolar concentrations. We explored the mechanism of this inhibition, with 4-aminoquinolines, quinacrine, 9-aminoacridines, and novel dibasic analogs, many of which are fluorescent. WEHI 231 murine B-lymphoma cells accumulated analogs up to a concentration several hundredfold higher than the medium. Uptake was rapid, nonsaturable, reversible, and partially inhibited by monensin, an agent that collapses pH gradients within cells. Uptake did not correlate highly with efficacy as inhibitors of CpG-oligodeoxynucleotide (ODN)-induced effects, suggesting that analogs act by a specific action. Confocal microscopy revealed analogs concentrating in large peripheral organelles. CpG-ODN is taken up by cells into acidifed, small, perinuclear vesicles. This uptake is thought to be necessary for immunostimulatory activity. Cellular uptake of fluorescent CpG-ODN was not inhibited by the analogs. The pH of intracellular CpG-ODN (6.4) was not affected by analogs at the concentration required for inhibition, but pH was increased by higher concentrations. UV spectroscopy revealed no binding of analogs to CpG-ODN. Nuclear Overhauser effect spectroscopy revealed that an analog bound to phosphatidylcholine vesicles, with the ring structure of the analog buried within the lipid and the side chain facing the aqueous environment. We conclude that the analogs do not inhibit the action of CpG-ODN by preventing the uptake or acidification of CpG-ODN. It seems more likely that the analogs inhibit the efficacy of CpG-ODN by a specific action within acidified vesicles, possibly at the interface of a phospholipid membrane.

关键词：佐剂免疫/拮抗剂和抑制剂佐剂免疫/药理学碱类/药理学氨基喹啉类/药理学 CpG岛 DNA 单链/药物作用氢离子浓度离子载体/药理学脂类/化学磁共振波谱学膜人工显微镜检查共焦莫能星/药理学寡核苷酸类/拮抗剂和抑制剂寡核苷酸类/代谢寡核苷酸类/药理学磷脂类/化学亚细胞部分/药物作用亚细胞部分/代谢肿瘤细胞培养的

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Identification and characterization of a bovine sperm protein that binds specifically to single-stranded telomeric deoxyribonucleic acid

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BIOLOGY OF REPRODUCTION 2000年第2期62卷 340-346页

作者： Kozik, A Bradbury, EM Zalensky, AO Univ Calif Davis Sch Med Dept Biol Chem Davis CA 95616 USA Univ Calif Los Alamos Natl Lab Los Alamos NM 87545 USA

Telomere DNA at the physical termini of chromosomes forms a single;stranded 3' overhang. In lower eukaryotes, e.g., ciliated protozoa, this DNA extension is capped by specific proteins that have been structurally and functionally characterized. Much less is known about single-stranded telomere DNA-binding proteins in vertebrates, Here we describe a new protein from bovine sperm designated bsSSTBP that specifically interacts with single-stranded (TTAGGG)(N) DNA. The bsSSTBP was extracted from nuclei by 0.6 M KCl. The native size of this protein, estimated by gel filtration, was 20-40 kDa. SOS-PACE of the UV cross-linked complex between bsSSTBP and telomere DNA indicated that several polypeptides are involved in complex formation. Bovine sSSTB had high specificity toward nucleotide sequence, since single nucleotide substitutions in the (TTAGGG), substrate suppressed binding. The minimal number of (TTAGGC) repeats required for binding of bsSSTBP was 3, and the protein recognized linear but not folded DNA structures. We propose that the bsSSTBP participates in telomere-telomere interactions and the telomere membrane localization observed in mature sperm. In mammals, somatic telomere-binding proteins are apparently substituted by sperm-specific ones that may lead to a structural reorganization of telomere domains to fulfill functions important during meiosis and fertilization.

关键词：细胞核/化学交联试剂/化学 DNA 单链/化学 DNA 单链/药物作用 DNA 单链/代谢电泳聚丙烯酰氨凝胶免疫酶技术分子量寡核苷酸探针蛋白质结合蛋白质类/化学蛋白质类/代谢精子/化学精子/代谢精子/超微结构端粒/代谢端粒/超微结构紫外线动物牛男(雄)性

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Selective photocleavage of DNA and RNA by anthraquinone derivatives: Targeting the single-strand region of hairpin structures

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PHOTOCHEMISTRY AND PHOTOBIOLOGY 1999年第6期70卷 847-852页

作者： Fernandez-Saiz, M Henderson, PT Wilson, WD Schuster, GB Georgia Inst Technol Sch Chem & Biochem Atlanta GA 30332 USA Georgia State Univ Dept Chem Atlanta GA 30303 USA

A tetracationic anthraquinone derivative (27AQS2) binds to hairpin DNA and RNA. Ultraviolet irradiation of the bound quinone causes cleavage in the loop region of both oligonucleotides and at guanines in the stem region of the DNA hairpin. The absence of observable strand cleavage at guanines in the RNA hairpin suggests that either aniline treatment does not cause cleavage at damaged guanines in RNA or that radical cation migration does not occur readily in RNA duplexes. The ability to target the single-stranded regions of DNA and RNA structures is an important property of this photonuclease.

关键词：蒽醌类/化学蒽醌类/代谢 DNA损伤 DNA 单链/化学 DNA 单链/药物作用 DNA 单链/代谢 DNA 单链/辐射效应嵌入剂/化学嵌入剂/代谢核酸构象光解光敏感药/化学光敏感药/代谢 RNA/化学 RNA/药物作用 RNA/代谢 RNA/辐射效应紫外线

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Base excision repair is efficient in cells lacking poly(ADP-ribose) polymerase 1

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NUCLEIC ACIDS RESEARCH 2000年第20期28卷 3887-3896页

作者： Vodenicharov, MD Sallmann, FR Satoh, MS Poirier, GG Univ Laval Med Res Ctr CHUQ Poly ADP Ribose Metab Grp Ste Foy PQ G1V 4G2 Canada Univ Laval Med Res Ctr CHUQ DNA Repair GrpHlth & Environm Unit Ste Foy PQ G1V 4G2 Canada Univ Laval Fac Med Ste Foy PQ G1V 4G2 Canada

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N-methyl-N'-nitro-N-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N-methyl-N'-nitro-N-nitrosoguanidine repaired their genomic DNA as efficiently as wildtype cells, Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to gamma -irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.

关键词：碱基错配/药物作用碱基错配/遗传学碱基错配/辐射效应细胞提取物细胞系无细胞系统 DNA损伤/药物作用 DNA损伤/遗传学 DNA损伤/辐射效应 DNA甲基化/药物作用 DNA修复/药物作用 DNA修复/遗传学 DNA修复/辐射效应 DNA 单链/生物合成 DNA 单链/药物作用 DNA 单链/遗传学 DNA 单链/辐射效应剂量效应关系辐射成纤维细胞基因缺失动力学甲硝基亚硝胍/药理学模型遗传学诱变剂/药理学 NAD/代谢质粒/药物作用质粒/遗传学质粒/辐射效应聚ADP核糖聚合酶类/缺乏聚ADP核糖聚合酶类/遗传学聚ADP核糖聚合酶类/生理学辐射耐受性 X线动物小鼠

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Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells

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INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 2000年第1期76卷 67-75页

作者： Dahm-Daphi, J Sass, C Alberti, W Univ Hamburg Dept Radiotherapy & Oncol D-20246 Hamburg Germany Inst Biophys & Radiobiol D-20246 Hamburg Germany

Purpose: Free OH radicals are considered to be the common mediator of DNA damage after ionizing radiation and oxidative stress. In particular, double-strand breaks (dsb) have a major impact on cell killing after irradiation, while the mechanism of cell killing is less clear for oxidative injury. The latter nor only affects DNA, but also equally other cell compartments, such as membranes and mitochondria, which may trigger cell death. This study intended to clarify the relationship between DNA damage induction, repair and cell inactivation for hydrogen peroxide and ionizing radiation. Materials and methods: Chinese hamster ovary (CHO) cells were treated with H2O2 in serum-free medium in combination with/without X-irradiation. DNA damage was measured using the alkaline unwinding method or neutral constant-field gel electrophoresis. Cell survival was recorded using the colony-formation assay. Results: Hydrogen peroxide induced a large number of single-strand breaks (ssb>36 000/cell) without impairing cell survival. This number reached a maximum (36 Gy-equiv. at 3 X 10(-4) mol/dm(3)) without further increase after higher concentrations. Repair kinetics of ssb were similar to those after irradiation. Dsb were found only after very high concentrations of H2O2 (>3 X 10(-2) mol/dm(3)), which is different from irradiation which generated ssb and dsb in the same dose range. A linear-quadratic increase of dsb was found with increasing concentrations of H2O2 suggesting a single or a pairwise action of OH radicals to form a dsb. After either irradiation or peroxide treatment cell killing was observed only after doses which also allowed dsb detection. The number of dsb calculated per lethal event was in the same range but slightly higher after irradiation (1.7-fold) than after H2O2 treatment. Conclusions: Cell killing after irradiation or hydrogen peroxide appears to be due to dsb, whereas cells withstand large numbers of single-strand lesions and other types of non-DNA damage oc

关键词： CHO细胞细胞存活/药物作用仓鼠亚科 DNA/药物作用 DNA/辐射效应 DNA损伤 DNA修复/遗传学 DNA 单链/药物作用 DNA 单链/辐射效应过氧化氢羟自由基/分析动力学辐射耐受性动物

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UVA-induced DNA single-strand cleavage by 1-hydroxypyrene and formation of covalent adducts between DNA and 1-hydroxypyrene

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CHEMICAL RESEARCH IN TOXICOLOGY 2000年第7期13卷 585-593页

作者： Dong, SM Hwang, HM Shi, XC Holloway, L Holloway, L Yu, HT Jackson State Univ Dept Chem Jackson MS 39217 USA Jackson State Univ Dept Biol Jackson MS 39217 USA

1-Hydroxypyrene (HOP), a metabolite found in the urine of humans and laboratory animals exposed to polycyclic aromatic hydrocarbons (PAHs), is known to be both acutely toxic and genotoxic. It has been widely used as a biomarker for studying PAH exposure. In this research, we have found that, upon UVA irradiation, HOP causes DNA single-strand cleavages and forms HOP-DNA covalent adducts. The UVA-induced cleavage of supercoiled plasmid Phi X174 DNA is dependent upon both HOP concentration and WA dosage. A longer irradiation time or higher HOP concentration induces more DNA cleavage. Results of the photocleavage experiments carried out in the presence of reactive oxygen species scavengers, histidine, sodium azide, mannitol, SOD, and desferal indicate that both the superoxide free radical and singlet oxygen are likely involved in causing DNA single-strand cleavage. The photocleavage is inhibited by the presence of an excited singlet-state quencher, KI, indicating that it is an excited-state reaction. Along with light-induced DNA cleavage, HOP also forms DNA covalent adducts while being degraded upon light irradiation. Light-induced degradation of 20 mu M HOP follows first-order reaction kinetics in a 10% methanolic buffer (10 mM phosphate) solution in the absence or presence of 40 mu M calf thymus DNA, with degradation half-lives of 20 or 15 min, respectively. The shorter degradation half-life in the presence of DNA is due to the formation of the HOP-DNA covalent adduct. The formation of the HOP-DNA covalent adduct is evidenced by comparing the UV-vis absorption and fluorescence emission spectra of the pure HOP with those of the HOP-DNA adduct. The covalent HOP-DNA adduct produced due to irradiation was purified by either extensive dialysis (3 x 500 mL buffer solutions), phenol and chloroform extraction followed by ethanol precipitation, or chloroform extraction alone. The isolated HOP-DNA adduct has an absorption peak at 353 nm, which is 8 nm red-shifted compared to th

关键词：色谱法高压液相 DNA/化学 DNA/辐射效应 DNA加合物/化学 DNA加合物/辐射效应 DNA 单链/化学 DNA 单链/药物作用 DNA 单链/辐射效应剂量效应关系药物剂量效应关系辐射诱变剂/化学诱变剂/药理学芘类/化学芘类/药理学芘类/辐射效应光谱法荧光胸腺/化学紫外线动物牛

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Tiaprofenic acid-photosensitized damage to nucleic acids:: A mechanistic study using complementary in vitro approaches

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PHOTOCHEMISTRY AND PHOTOBIOLOGY 2000年第5期71卷 499-505页

作者： Agapakis-Caussé, C Boscá, F Castell, JV Hernández, D Marín, ML Marrot, L Miranda, MA Univ Hosp La Fe Res Ctr E-46009 Valencia Spain LOreal Adv Res Life Sci Res Grp Genotox Aulnay Sous Bois France Univ Politecn Valencia UPV CSIC Inst Tecnol Quim Dept Quim E-46071 Valencia Spain

In order to determine whether or not tiaprofenic acid (TPA) could cause cellular DNA damage, human fibroblasts were irradiated in the presence of the drug and subsequently examined by means of the comet assay. This led to the observation that TPA actually sensitizes cellular DNA to the subsequent irradiation, When TPA was irradiated in the presence of supercoiled plasmid DNA, it produced large amounts of single-strand breaks (SSB);this is consistent with the effects observed on cellular genomic DNA by the comet assay. More importantly, low concentrations of TPA, unable to produce direct SSB, caused photo-oxidative damage to DNA as revealed by the use of excision-repair enzymes. The fact that TPA-irradiated DNA was a substrate of formamidopyrimidine glycosylase as well as endonuclease III revealed that both purine and pyrimidine bases were oxidized. This was further supported by the TPA-photosensitized oxidation of 2'-deoxyguanosine which led to a product mixture characteristic of mixed type-I/II mechanisms. Thymidine was less reactive under similar conditions, but it also decomposed to give a typical type-I product pattern. Accordingly, the TPA triplet was quenched by the two nucleosides with clearly different rate constants (10(8) vs 10(7) M-1 s(-1), respectively). As cellular RNA also contains oxidizable bases, it could be the target of similar processes, thus interfering with the biosynthesis of proteins by the cells. Extraction of total RNA from TPA-irradiated human fibroblasts, followed by gel electrophoresis and PCR analysis, confirmed this hypothesis. Finally, photosensitization experiments with Saccharomyces cerevisiae showed that, in spite of an efficient drug-yeast interaction leading to cytotoxicity, neither intergenic recombination nor gene conversion took place, Thus, while TPA-photosensitized damage to nucleic acids can result in genotoxicity, the risk of mutagenicity does not appear to be significant.

关键词：细胞培养的 DNA损伤 DNA 单链/药物作用 DNA 单链/辐射效应光敏感药/药理学丙酸盐类/药理学酿酒酵母菌/遗传学酿酒酵母菌/辐射效应紫外线人类

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铁与石棉体联合可导致φX174 RFI脱氧核糖核酸单链断裂

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国外医学（医学地理分册） 1995年第1期16卷 42-43页

作者：高永年

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N²-(1-carboxyethyl)deoxyguanosine, a nonenzymatic glycation adduct of DNA, induces single-strand breaks and increases mutation frequencies

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第2期264卷 544-549页

作者： Pischetsrieder, M Seidel, W Münch, G Schinzel, R Univ Wurzburg Theodor Boveri Inst Biozentrum D-97074 Wurzburg Germany Univ Erlangen Nurnberg Inst Pharm & Lebensmittelchem D-8520 Erlangen Germany

N-2-(l-Carboxyethyl)deoxyguanosine (CEdG) is a major nonenzymatic glycation product of DNA. The effect of CEdG modification, which was specifically prepared by incubation with dihydroxyacetone, on plasmid DNA topology was evaluated by gel electrophoresis. A time-dependent decrease of supercoiled plasmid-DNA was observed in parallel to the increase of CEdG adducts;the half-life time of the supercoiled plasmid-DNA was estimated to be approximately 16-18 h. CEdG-modified plasmid DNA showed a 25-fold reduced transformation efficiency. When modified DNA was used to transform Escherichia coli cells, a 6-fold increase in mutation frequency was determined by measuring loss of alpha-complementation. For the mutator strain BMH71-18mutS, an 8 fold increase in mutation frequency was observed. Although the exact mechanism of DNA damage is unclear, the occurrence of spontaneous depurination is likely. These findings suggest that a defined DNA glycation reaction can lead to DNA damage in vivo. (C) 1999 Academic Press.

关键词： DNA加合物/药理学 DNA损伤 DNA修复/遗传学 DNA 单链/化学 DNA 单链/药物作用 DNA 单链/代谢 DNA 超螺旋/化学 DNA 超螺旋/药物作用 DNA 超螺旋/代谢电泳琼脂凝胶大肠杆菌/遗传学大肠杆菌/代谢葡萄糖/化学半衰期突变氧化还原质粒/化学

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