Single-strand Scissions Research Articles

Biological activities of new anthracyclines containing fluorine, FAD 104 and its metabolites.

From various new anthracyclines containing fluorine in their sugar moieties, 7-O-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl)adriamycinone-14-hemi pimerate (FAD104) has been selected for clinical investigation, because of its excellent antitumor activity and solubility. In this paper, the mechanism whereby FAD104 exhibits good antitumor activity in vivo was studied through experiments in vitro in comparison with doxorubicin (DOX). FAD104 had weaker activity than DOX in DNA single and double strand scission in P388 cells and in binding to calf thymus DNA. FAD104 was taken up by P388 cells faster than DOX. The ester linkage in FAD104 was gradually hydrolyzed at neutral pH. FAD104 was metabolized to 7-O-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl)adriamycinone (FT-ADM) when incubated with mouse or human serum. In mouse serum the esterase activity was about 100 times higher than in human serum. The product of nonenzymatic and enzymatic esterolysis, FT-ADM, was rapidly taken up by P388 cells and accumulated, reaching at a 9.7-fold higher level than DOX. Thus FAD104 was less active than DOX in itself, but it showed much higher activity through conversion into FT-ADM, due to the action of esterase or to spontaneous and gradual hydrolysis at physiological pH. 7-O-(2,6-Dideoxy-2-fluoro-alpha-L-talopyranosyl)adriamycinol (FT-ADM-OH), another metabolite found in mouse serum had the least biological activity among the fluorine-containing anthracycline glycosides, but its activity was still higher than DOX.

Inductions of Ornithine Decarboxylase and Replicative DNA Synthesis but not DNA Single Strand Scission or Unscheduled DNA Synthesis in the Pyloric Mucosa of Rat Stomach by Catechol

The possible tumor‐promoting and genotoxic activities of catechol were examined. Administration of catechol by gastric intubation at doses of 10 to 90 mg/kg body weight to male F344 rats induced up to 19‐fold increase in ornithine decarboxylase activity with a maximum after 8 h and up to 8‐fold increase in replicative DNA synthesis with a maximum after 24 h in the pyloric mucosa of the stomach. These results suggest that catechol has tumor‐promoting activity in the pyloric mucosa of rat stomach. However, its administration at doses of 37.5 to 90 mg/kg body weight did not induce DNA single strand scission in the pyloric mucosa as determined by the alkaline elution method after 2 and 6 h or unscheduled DNA synthesis examined after 2 and 12 h.

Hydroxyl radical generation and DNA strand scission mediated by natural anticancer and synthetic quinones

Using ESR and spin-trapping techniques, it was found that synthetic 2-dimethylamino-3-chloro-1,4-naphthoquinone and the natural anticancer quinone daunomycin, when added to a system containing purified NADPH-cytochrome P-450 reductase, NADPH, ferric ions, and oxygen, (i) generated hydroxyl radicals and (ii) caused single-strand scission of super-coiled DNA of the plasmic pBR322. Since these two effects of the quinones were correlated to each other, we propose that potential anticancer quinones can be effectively screened by measuring their ability to form hydroxyl radicals in the above system.

Formation of endonuclease III-sensitive sites as a consequence of oxygen radical attack on DNA

Exposure of the plasmid pBR 322 to the aerobic xanthine oxidase reaction introduced single strand scissions and endonuclease III-sensitive sites. The latter may be residues of thymine glycol. Both forms of DNA damage were completely prevented by superoxide dismutase or catalase, whereas, bovine serum albumin was much less effective. Mannitol and benzoate, added as scavengers of HO·, and desferrioxamine or diethylene triamine pentaacetate, added to sequester Fe(III), also protected. There results inddicate a metal-catalyzed interaction of O 2 − with H 2O 2, which produces HO· which, in turn, causes DNA strand scission and oxidation of thymine residues III-sensitive sites than did plasmid from anaerobically-incubated cells, and a low molecular weight scavenger of O 2 − prevented the damage seen with the aerobic cells. Genetic defects in AP endonucleases rendered E. coli more susceptible to the dioxygen-dependent lethality of plumbagin, which mediates O 2 − production. Similarly, plasmid DNA, within the endonuclease-deficient cells, exhibited more strand scissions and endonuclease III-sensitive sites upon aerobic exposure to plumbagin than did endonuclease-sufficient cells, and a low molecular weight scavenger of O 2 − was protective. These results are consistent with the conclusions that strand scissions and formation of endonuclease III-sensitive sites are among the consequences of exposure of DNA to O 2 − plus H 2O 2 both in vitro and in vivo. to thymine glycol. Plasmid isolated from aerobically-incubated cells contained more strand scissions and endonuclease

Enhanced Sensitivity to Camptothecin in Ataxia-telangiectasia Cells and Its Relationship with the Expression of DNA Topoisomerase I

The antitumour drug camptothecin (CPT) can trap covalently bound topoisomerase I-DNA intermediates as complexes which conceal single-strand scissions. In an attempt to evaluate the cytotoxic potential of these lesions in human cells we have measured: (1) cell cycle delay and cell killing by CPT in primary and transformed fibroblasts, and in lymphoblastoid lines derived from normal, X-ray sensitive ataxia-telangiectasia (A-T) and xeroderma pigmentosum (XP) donors; (2) the properties of sublines obtained by high-dose selection in CPT: (3) levels of drug-induced DNA strand scission in intact cells; (4) the cellular availability of extractable topoisomerase I. The drug induced a marked cell cycle block in G2 phase, the magnitude of the block being closely related to cell kill. XP group A cells showed normal sensitivity to CPT, whereas A-T derived cells were consistently hypersensitive (3-5 fold) in a manner which could not be related to a primary deficiency in topoisomerase I activity, abnormal capacity for complex formation or anomalies in the intracellular generation of DNA strand breaks. A CPT-resistant A-T subline had reduced topoisomerase I activity but retained the characteristic of hypersensitivity to X-radiation. The subline lost resistance upon in vitro passage with evidence that resistance was initially an unstable feature of a subpopulation of cells. The findings have implications for the role of topoisomerase I in the in vitro phenotype of A-T cells, and the contribution made by topoisomerase I-dependent damage to the cytotoxic action of CPT.

The mechanism of action of quinocarmycin citrate (KW 2152) on mouse L1210 cells in vitro

The effects of the antitumor antibiotic, quinocarmycin citrate (KW 2152), on L1210 cells were studied in vitro. The cellular growth was completely inhibited at 10(-6) M KW 2152, and after 2 days no viable cell was seen. The incorporation of 3H-thymidine, 3H-uridine, or 3H-leucine into the acid-insoluble fraction was not affected at 10(-4) M for 1 h; however, when the cells were treated with 10(-6) M for 24 h, the radioactivity appearing in the acid-insoluble fraction was reduced to 20%, 30%, and 48%, respectively, of the control. The single strand scission of the DNA of L1210 cells was seen at 10(-7) M for 24 h, as revealed by an alkaline, sucrose density gradient. However, no damage to plasmid pBR322 was observed even at 10(-6) M KW 2152 for 24 h, as revealed by 0.8% agarose gel electrophoresis, indicating that some soluble factors of the cells might contribute to the damage to the DNA of L1210 cells. The processing of pre-rRNA of the cells was not inhibited at 10(-6) M of the drug for 24 h of incubation.

Photocleavage of DNA and photofootprinting of E. coli RNA polymerase bound to promoter DNA by azido-9-acridinylamines.

The long-wavelength ultraviolet (lambda approximately 420 nm) radiation induced reaction between 6-azido-2-methoxy-9-acridinylamines and supercoiled plasmid DNA results in single strand scissions and formation of covalent adducts (ratio approximately 1:10). By treating azidoacridine-photomodified DNA with piperidine at 90 degrees C, additional strand scissions are observed in a complex sequence dependent manner with an overall preference for T greater than or equal to G greater than C much greater than A. The resulting DNA fragments migrate as 5'-phosphates in polyacrylamide gels. Photofootprinting of the binding site of RNA-polymerase on promoter DNA is demonstrated with an azido-9-acridinylamino-octamethylene-9-aminoacridine. Similar experiments using 9-amino-6-azido-2-methoxyacridine indicate that this reagent recognizes changes in the DNA conformation induced by RNA polymerase binding, in relation to open complex formation.

Mechanism of ATP inhibition of mammalian type I DNA topoisomerase: DNA binding, cleavage, and rejoining are insensitive to ATP.

A general, unrefined mechanism of type I DNA topoisomerase action involves several steps including DNA binding, single-strand scission, strand passage resealing, and, possibly, readoption of an active enzyme conformation. None of these steps requires an energy cofactor; however, we have shown previously that several mammalian type I topoisomerases are, in fact, inhibited by ATP. In this study, we wanted to examine which steps in the gross topoisomerase mechanism were sensitive or insensitive to ATP. Nitrocellulose filter binding experiments showed that ATP did not interfere with the binding of DNA by the enzyme and that ATP binding by topoisomerase was 5-fold greater in the presence of DNA than in its absence. Agarose gel electrophoresis in the presence or absence of ethidium bromide indicated that resealing was unaffected by added ATP. The addition of the adenine nucleotide did not alter the pattern of camptothecin-stimulated cleavage of DNA, indicating that strand scission was not the point of inhibition. To test whether strand passage or the readoption of an active conformation was an inhibited step, we used a unique DNA topoisomer as substrate. The results argued against readoption of an active enzyme conformation as an ATP-sensitive process.

Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding

T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.

Enhanced bioreduction of 4-nitroquinoline 1-oxide by cultured ataxia telangiectasia cells.

When dermal fibroblast strains derived from ataxia telangiectasia (AT) and clinically normal donors were exposed to 4-nitroquinoline 1-oxide (4NQO) and their DNA subjected to velocity sedimentation analysis in alkaline sucrose gradients, the incidence of single-strand interruptions detected in the AT strains (AT2BE, AT3BI and AT4BI) was 1.4-1.8 times higher than that seen in the seven normal controls. Cellular uptake of exogenous radiolabelled 4NQO occurred at similar rates in AT and control cultures, arguing against increased influx of the chemical as the root cause of the elevated yield of strand breakage in the former cultures. However, sonicates of each AT strain contained an enhanced capacity to catalyze the reduction of 4NQO to the proximate carcinogen 4-hydroxyaminoquinoline 1-oxide; the differences in bioreductase activity between AT and normal cell sonicates correlated closely with those for the incidence of DNA strand openings in 4NQO-treated cultures. Our data further indicated that these single-strand scissions, seen under alkaline conditions, are not manifestations of intermediate reactions in the multistep excision repair process operative on 4NQO lesions because: (i) the interruptions were observed at comparable levels in AT2BE and AT3BI cells, the former purportedly deficient and the latter proficient in 4NQO adduct removal; and (ii) cells known to be defective in repairing all types of 4NQO lesions, namely, xeroderma pigmentosum complementation group A fibroblasts, accumulated breaks at normal rates during 4NQO treatment. Consequently, these breaks appear to represent a class of 4NQO lesions which are themselves alkali-labile and therefore become converted to single-strand interruptions in vitro during exposure of DNA to alkali before velocity sedimentation. We conclude that AT strains tend to sustain abnormally high amounts of DNA damage upon 4NQO exposure due to an elevated capacity to bioactivate the inert parent compound into a proximate carcinogen.

Crosslinking of DNA by dehydroretronecine, a metabolite of pyrrolizidine alkaloids.

Dehydroretronecine (DHR), a major metabolite of the pyrrolizidine alkaloids, has been shown to bind and crosslink pBR322 plasmid DNA and M13 viral DNA, as monitored by electron microscopy and agarose gel electrophoresis. The crosslinking was alkali-stable and varied with time, temperature, pH and reactant ratios. Although the binding of DHR to single-stranded M13 DNA was 3-fold higher than to double-stranded M13 DNA, only the double-stranded DNA was crosslinked. Intra- and inter-molecular crosslinking occurred with both linear and circular forms of double-stranded DNA. Electron microscopy showed that incubations with low DHR/DNA ratios yielded mostly DNA monomers and dimers while higher ratios produced larger aggregations, some of which were equivalent in size to dozens of individual DNA molecules. The average number of intermolecular crosslinks per molecule estimated by Poisson statistics was essentially the same using data from electron microscopy or DHR-binding. DHR generated single strand scissions equally well in both single- and double-stranded molecules.

Enhanced bleomycin-mediated damage of DNA opposite charged nicks. A model for bleomycin-directed double strand scission of DNA.

The anticancer drug, bleomycin, causes both single and double strand scission of duplex DNA in vitro, with double strand scission occurring in excess of that expected from the random accumulation of single strand nicks. The mechanism of the preferential double strand scission of DNA by bleomycin has been investigated through the synthesis of a series of double hairpin and linear oligonucleotides designed to contain a single nick-like structure at a defined site to serve as models of bleomycin-damaged duplex DNA. The 3' and/or 5' hydroxyls flanking the nick have been phosphorylated to model the increased negative charge at a bleomycin-generated nick. The ability of bleomycin to cleave the intact strand opposite the nick was then determined by autoradiography. The results demonstrate that phosphorylation at either the 3' or 5' hydroxyl, and especially when both sites are phosphorylated, strongly enhances selective cleavage by bleomycin of the opposite strand. These experiments indicate that bleomycin-mediated double strand scission is a form of self-potentiation in which the high affinity of bleomycin for the initially generated nicked sites leads to a greatly enhanced probability of scission of the strand opposite those sites.

Involvement of DNA polymerases in the repair of DNA damage by benzo[ a]pyrene in cultured Chinese hamster cells

Mechanisms for induction of single-strand scissions in DNA by S9-activated benzo[ a]pyrene (B[a]P) and their repair in cultured Chinese hamster V79 cells were investigated with inhibitors of DNA-repair synthesis using alkaline sucrose gradient sedimentation analysis. The marked induction of single-strand scissions in DNA was observed following 3 h treatment of V79 cells with 5 μg/ml of B[a]P. These DNA lesions were repaired to the control level within 4 h after removal of B[a]P. The simultaneous addition of inhibitors of DNA-repair synthesis, 1-β- D-arabinofuranosylcytosine (araC) plus hydroxyurea with B[a]P did not increase the formation of DNA single-strand scissions. When these inhibitors were added after removal of B[a]P, however, they significantly blocked the rejoining of DNA-strand scissions. On the other hand, when aphidicolin, a specific inhibitor of DNA polymerase α, was used instead of araC, a partial inhibition of the rejoining was observed, and further addition of 2′,3′-dideoxythymidine, an inhibitor of DNA polymerase β, augmented the inhibitory effect. These results indicate that B[a]P-induced single-strand scissions of DNA in V79 cells could be repaired mostly by excision repair which involved DNA polymerase α and a non-α polymerase, presumably polymerase β.

Light-induced modifications of DNA by gilvocarcin V and its aglycone

Gilvocarcins are antitumor agents that have been reported to damage DNA upon activation by visible light. This activation is dependent on interaction with DNA. Here it is shown that gilvocarcin V and its synthetic aglyclone analogue can both introduce single-strand scission into plasmid DNA. Light irradiation is required for the reaction. The binding of gilvocarcin V to plasmid DNA in the absence of light decreased the DNA linking number in a fashion similar to known intercalating agents such as ethidium bromide. The use of oligonucleotides as substrates for gilvocarcin V demonstrated that one of the steps of the reaction following binding of gilvocarcin V to DNA involves covalent modification at thymidine and to a lesser extent, cytosine residues.

Molecular evidence for cleavage of intradimer phosphodiester linkage as a novel step in excision repair of cyclobutyl pyrimidine photodimers in cultured human cells.

A re-analysis of the metabolic fate of ultraviolet light (u.v.)-induced cyclobutyl pyrimidine dimers in the DNA of dermal fibroblasts from patients with different genetic forms of xeroderma pigmentosum (XP), a rare cancer-prone skin disorder, has provided new insight into the mode of dimer repair in normal human cells. When DNA isolated from post-u.v. incubated cultures was subjected to enzymic photoreactivation (PR) to probe dimer authenticity, single-strand scissions were produced in the damaged DNA of incubated XP group A and D cells, but not in DNA from XP group C cells or normal controls. Since enzymic PR treatment ruptures only the cyclobutane ring, these results suggested that in dimer excision-defective XP group A and D strains, the intradimer phosphodiester bond may have been cleaved without site restoration. Such a cleavage event had not previously been detected; the possibility that this reaction may be an early step in the normal excision-repair process is supported by the observed release of free thymidine (dThd) and its monophosphate (TMP), but not of thymine, upon photochemical reversal of the dimer-containing excision fragments isolated from post-u.v. incubated normal cells. The combined number of dThd and TMP molecules released was equal to approximately equal to 80% of the number of dimers photoreversed; for such release to occur, the dimer must both be at one end of an excised fragment and contain an internal phosphodiester break. Taken together, these data lead us to propose a novel model for dimer repair in human cells in which hydrolysis of the intradimer phosphodiester linkage precedes the concerted action of a generalized 'bulky lesion-repair complex' involving conventional strand incision/lesion excision/repair resynthesis/strand ligation reactions.

DNA strand scission by D-glucosamine and its phosphates in plasmid pBR322.

DNA strand scission due to aminosugars and their derivatives in plasmid pBR322 was investigated through agarose gel electrophoresis. D-Glucosamine and its related aminosugars primarily caused single-strand scission of ccc-DNA to form oc-DNA in a time- and temperaturedependent manner. The degree of reaction was also related to the concentration of aminosugars. D-Glucosamine-6-phosphate was the most active among the compounds tested, and converted oc-DNA into linear-DNA and further fragmented DNA. The reaction was stimulated by Cu2+ and inhibited by catalase, superoxide dismutase, and some radical scavengers. The DNA strandscissoring activities of aminosugars were related to their nitroblue tetrazolium-reducing activities. These results support a hypothetic mechanism in which some free radicals, generated during autoxidation of aminosugars in aqueous solution, are involved in DNA strand scission.

In vitro maturation of circular bacteriophage P2 DNA. Purification of ter components and characterization of the reaction.

The protein components required for generation of cohesive ends in vitro from circular bacteriophage P2 DNA have been purified to near homogeneity. In the presence of ATP, the purified products of P2 genes M and P together with empty phage capsids (comprised primarily of the N protein) mediate site-specific cleavage of circular P2 DNA at the cohesive end site (cos). This terminase or ter system also utilizes circular DNAs of bacteriophages P4 and 186, introducing site-specific scissions at cos sites within these molecules. The ter reaction exhibits a peculiar requirement for a circular DNA substrate. Substrate activity is greatly reduced when circular P2, P4, or 186 DNAs are linearized by restriction endonuclease hydrolysis. Furthermore, multimeric P4 DNA molecule sites are also essentially inactive in the linear form but are active in the circular state. The dependence of ter action on a circular substrate is not due to inhibition of the system by linear DNA, nor does it appear to reflect a requirement for substrate superhelicity since circular P4 DNA containing single strand scissions is subject to terminase action. The terminase reaction is supported by ATP, dATP, or beta, gamma-imido ATP, but not by other ribonucleoside triphosphates ADP, alpha, beta-methylene ATP, or beta, gamma-methylene ATP. A DNA-dependent ATPase, which hydrolyzes ATP to AMP, copurifies with the P2 P protein and is inactivated with the same kinetics as P activity upon treatment with N-ethylmaleimide. The ATPase does not display specificity for P2 DNA in vitro.

Histochemical detection of DNA strand scissions in mammalian cells by [formula omitted] nick translation

A method to visualize in situ of single strand scissions of DNA in fixed mammalian cells has been developed. Using the nuclear nick translation with biotin-labeled dUTP followed by binding to avidin-biotin-peroxydase complex, the nuclei of HeLa cells which had been treated with a DNA-damaging antibiotic bleomycin were specifically stained, implicating that the histochemical detection of single strand scissions (nicks) of DNA in fixed cells was completed without destroying the morphology, and without using autoradiography.

Induction of single strand scission in .PHI.X174RFI DNA by D-isoglucosamine.

Induction of single strand scission in .PHI.X174RFI DNA by D-isoglucosamine.

Mechanism of in vitro inactivation of Lactobacillus phage PL-1 by D-glucosamine.

d-Glucosamine selectively inactivated free PL-1 phage under the conditions in which the growth of host cells, Lactobacillus casei ATCC 27092, was not affected. Both the N-acetylated and non-aminated forms little affected the phage infectivity. The rate of phage inactivation by D-glu-cosamine was proportional to the time and temperature of the incubation, and about 0.1 m d-glucosamine showed the maximum inactivation effect. The phage inactivation was stimulated by Cu2 +, and inhibited by catalase and several oxygen radical scavengers. The inactivated phage was identical with the intact one with respect to the electron microscopic appearance, the density and the ability of combining with host cells. The DNA in the virion was fragmented by D-glucosamine and the DNA fragmentation was also stimulated by Cu2 +. With plasmid pBR322, d-glucosamine caused single-strand scission of ccc-DNA to give oc- and in succession linear-DNA. The inactivation of PL-1 phage by d-glucosamine was considered to be due to single-st...