A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex

Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicin and its C-13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA-protein complex formation and double-strand DNA breaks in CCRF-CEM cells and KU-2 cells. In this study, we found that ICRF-193, a topo II catalytic inhibitor, antagonized both DNA-protein complex formation and double-strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF-193. Taken together, these findings indicate that the cell growth-inhibitory effects of amrubicin and amrubicinol are due to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topo II.     

Plasmid DNA strand breaks induced by laser irradiation of 193 nm wavelength

DNA of plasmid pBR322 irradiated with laser at a wavelength of 193 mm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid (in AB1157) is mainly determined by the number of directly formed laser-induced single-strand breaks, whereas the contribution of enzymatically produced single- and double-strand breaks is insignificant.     

Removal by human apurinic/apyrimidinic endonuclease 1 (Ape 1) and Escherichia coli exonuclease III of 3'-phosphoglycolates from DNA treated with neocarzinostatin, calicheamicin, and gamma-radiation

DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups. Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.     

DNA damage in folate deficiency

Folate deficiency significantly increases uracil content and chromosome breaks (as measured by micronucleated cells) in human leukocyte DNA. Folate supplementation reduces both the uracil content of DNA and the frequency of micronucleated cells, indicating that uracil misincorporation may play a causative role in folate deficiency-induced chromosome breaks. A calculation is presented to explain how the levels of uracil found in DNA could cause chromosome breaks. Based on this calculation, the frequency of uracil repair events that might result in double-strand DNA breaks increases by 1752-fold. These results are consistent with clinical and epidemiological evidence linking folate deficiency to DNA damage and cancer.     

In vitro repair of gaps in bacteriophage T7 DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.     

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.     

Hydroxyl radicals and DNA double-strand breaks in X-irradiated E. coli

We have used glycerol to study the relationship between hydroxyl radicals, one of the primary radiolytic products, and the production of DNA double-strand breaks in selected E. coli strains. Our results suggest that when bacteria are irradiated at doses up to about 120 Gray, hydroxyl radicals produce DNA lesions, but not double-strand breaks.     

Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells.     

Efficacy of novel antimicrobials against clinical isolates of opportunistic amebas

Using atomic force microscopy (AFM), we have investigated neutron-induced DNA double-strand breaks in plasmids in aqueous solution. AFM permits direct measurement of individual DNA molecules with an accuracy of a few nanometers. Furthermore, the analysis of the DNA fragment size distribution is non-parametric, whereas other methods are dependent on the model. Neutron irradiation of DNA results in the generation of many short fragments, an observation not made for damage induced by low-LET radiation. These data provide clear experimental evidence for the existence of clustered DNA double-strand breaks and demonstrate that short DNA fragments may be produced by such radiations in the absence of a nucleosomal DNA structure.     

Yeast DNA ligase IV mediates non-homologous DNA end joining

The discovery of homologues from the yeast Saccharomyces cerevisiae of the human Ku DNA-end-binding proteins (HDF1 and KU80) has established that this organism is capable of non-homologous double-strand end joining (NHEJ), a form of DNA double-strand break repair (DSBR) active in mammalian V(D)J recombination. Identification of the DNA ligase that mediates NHEJ in yeast will help elucidate the function of the four mammalian DNA ligases in DSBR, V(D)J recombination and other reactions. Here we show that S. cerevisiae has two typical DNA ligases, the known DNA ligase I homologue CDC9 and the previously unknown DNA ligase IV homologue DNL4. dnl4 mutants are deficient in precise and end-processed NHEJ. DNL4 and HDF1 are epistatic in this regard, with the mutation of each having equivalent effects. dnl4 mutants are complemented by overexpression of Dnl4 but not of Cdc9, and deficiency of Dnl4 alone does not impair either cell growth or the Cdc9-mediated responses to ionizing and ultraviolet radiation. Thus, S. cerevisiae has two distinct and separate ligation pathways.     

Contrast nephrotoxicity: predictive value of urinary enzyme markers in a rat model

Asbestos, proven to be carcinogenic in humans and animals, is reported to have no genotoxic effect. Asbestos workers have an increased risk of lung cancer, mesothelioma, and other tumours. Earlier findings showed that crocidolite can induce DNA strand breaks in cultured rat embryo cells as assessed by nick translation. We investigated DNA double-strand breaks in white blood cells (WBC) of ten workers occupationally exposed to asbestos. According to our results, obtained with neutral filter elution, individuals who had been exposed to asbestos fibres showed two to four times more DNA double-strand breaks (dsb) in white blood cells than ten non-exposed persons. The induced DNA fragments are of about 250 kb (compared to chromosomal DNA of Saccharomyces cerevisiae standard marker). Using additionally the chromosomal DNA protective method of agarose-plugs, DNA fragments in the range of 200 to 1000 kb have been found in the white blood cells of the same ten workers occupationally exposed to asbestos. In the white blood cells of non-exposed subjects no DNA fragments could be detected with this method. Compared to 51 non-exposed persons, elevated anti-ds DNA antibody concentrations were found in ten workers occupationally exposed to asbestos. The fact that workers occupationally exposed to asbestos have distinctly more double-strand breaks and anti-ds DNA antibodies could mean that an increased incidence of DNA-fragments may be an important indicator in the chronic effect of asbestos-associated carcinogenesis. Apparently, the chronic effects of asbestos observed here do not seem to be identical with that of previously reported acute in vitro effects.     

Interaction of Oct-1 and automodification domain of poly(ADP-ribose) synthetase

Ataxia telangiectasia (AT) cells display a profound sensitivity to ionizing radiation, exhibiting more frequent chromosomal breaks, increased micronuclei formation and abnormal DNA repair kinetics following exposure. Despite the recent cloning of the ATM gene there remains a need for a simple and rapid means of discriminating AT heterozygotes from normal individuals. Caffeine (1,3,7-trimethyl xanthine), known to inhibit the repair of double-strand DNA breaks following ionizing radiation, increases the frequency of radiation induced chromosomal breaks in normal cells. Here we report that caffeine potentiates the induction of chromosomal breaks in G2 arrested AT heterozygote and normal lymphoblastoid cells, but not in homozygous AT lymphoblastoid cells. This observation parallels the findings reported by others that caffeine fails to potentiate the effect of ionizing radiation in radiation-sensitive yeast strains and radiation sensitive CHO cells. It also suggests that caffeine may somehow mimic the effect of the ATM gene product in normal cells. We also report that caffeine is unlikely to be useful in helping to discriminate AT heterozygotes from normal individuals.     

Low levels of hydrogen peroxide and L-histidine induce DNA double-strand breakage and apoptosis

The results presented in this study demonstrate that L-histidine triggers a lethal response in U937 cells exposed to nontoxic, albeit growth-inhibitory, levels of H2O2. Treatment for 1 h with the cocktail H2O2/L-histidine promotes the formation of a low level of DNA double-strand breaks that are rapidly rejoined, and this process is followed by secondary DNA fragmentation at about 7 h of post-treatment incubation, at which time cells are still viable. The appearance of oligonucleosomal DNA fragments associated with the detection of morphological changes typical of apoptosis strongly suggests that a portion of the cells was undergoing an apoptotic process. The relative level of cells with fragmented chromatin never exceeded 15-20% throughout the 20 h post-treatment incubation. Treatment with high concentrations of H2O2 in the presence of L-histidine was found to trigger necrotic cell death. The results presented in this paper provide further experimental evidence in support of the notion that DNA double-strand breaks mediate the lethal effects of the cocktail H2O2/L-histidine and suggest that this type of DNA lesion can promote both apoptotic and necrotic cell death, depending on the concentration of the oxidant.     

Activation of nuclear factor kappa B inflammatory bowel disease

Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing double-strand DNA breaks. V(D)J recombination is a double-strand DNA breakage and rejoining process that relies on NHEJ for the joining steps. Here we show that the targeted disruption of both DNA ligase IV alleles in a human pre-B cell line renders the cells sensitive to ionizing radiation and ablates V(D)J recombination. This phenotype can only be reversed by complementation with DNA ligase IV but not by expression of either of the remaining two ligases, DNA ligase I or III. Hence, DNA ligase IV is the activity responsible for the ligation step in NHEJ and in V(D)J recombination.     

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.     

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.     

Spontaneous and photosensitiser-induced DNA single-strand breaks and formamidopyrimidine-DNA glycosylase sensitive sites at nucleotide resolutionin the nuclear and mitochondrial DNA of Saccharomyces cerevisiae

A system is described for mapping oxidative DNA damage (sites sensitive to formamidopyrimidine-DNA glycosylase and single-strand breaks) at nucleotide resolution in the nuclear and mitochondrial DNA of Saccharomyces cerevisiae. Our 3' end labelling method is sensitive and was first developed using the well-studied inducer of oxidative DNA damage, methylene blue (MB) plus light. We treated yeast DNA in vitro with this so as to maximise levels of damage for assay development. Unfortunately, MB does not remain in yeast cells and yeast DNA repair mutants sensitive to active oxygen species are not sensitive to this agent, thus for in vivo experiments we turned to a polycyclic aromatic, RO 19-8022 (RO). This resulted in oxidative DNA damage when light was applied to yeast cells in its presence. The spectra of enzyme-sensitive sites and single-strand breaks induced by MB in vitro or by RO plus light in vivo or in vitro were examined in two yeast reporter genes: the nuclear MFA2 and the mitochondrial OLI1. The experiments revealed that most of the enzyme-sensitive sites and single-strand breaks induced by MB or RO plus light are at the same positions in these sequences, and that these are guanines.