DNA damage induced by catecholestrogens in the presence of copper (II): generation of reactive oxygen species and enhancement by NADH

Certain estrogen metabolites are involved in carcinogenesis and the development of resistance to methotrexate (MTX). In this study, we determined whether these well-established biological effects correlate with the relative efficiency of several estrogen metabolites to induce DNA strand breaks in the presence of copper, and investigated the potential enhancing effect of reduced nicotinamide adenine dinucleotide (NADH). DNA strand breaks induced by estradiol metabolites were measured by the conversion of supercoiled phage phiX-174 RF1 DNA to open circular and linear forms. The most active catecholestrogens were the 4-hydroxy derivatives, which produced about 2.5 times more DNA double strand breaks than the 2-hydroxy derivatives, while estradiol and 16alpha-hydroxyestrone were inactive. In addition, our results show that 4-hydroxyestradiol (4-OHE2) at physiological concentrations was capable of exhibiting DNA cleaving activity. The formation of these catecholestrogen-induced DNA strand breaks was associated with the utilization of oxygen and the generation of H2O2, because catalase inhibited the DNA cleaving activity of 4-OHE2. Interestingly, we also observed that NADH enhanced the induction of DNA strands breaks by 4-OHE2/Cu(II), probably by perpetuating the redox cycle between the quinone and the semiquinone forms of the catecholestrogen. In conclusion, this study demonstrated that the relative efficiency of 2-, and 4-hydroxyestrogen in carcinogenesis and for the enhancement of MTX resistance correlates with their relative capability to induce DNA strand breaks. In order to inhibit these estrogen-mediated biological effects, it may be important to develop different strategies to block the production of reactive oxygen species by the catecholestrogen-redox cycle.     

Synthesis and inhibitory activity of acyl-peptidyl-pyrrolidine derivatives toward post-proline cleaving enzyme; a study of subsite specificity.

Several pyrrolidine derivatives have been synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain. Almost all the compounds tested in this study inhibited the activity of both enzymes at low IC50 values (from nM to microM) but a specificity difference was observed with alkylacyl-peptidyl-pyrrolidine derivatives which strongly inhibited only the bacterial enzyme. The most effective inhibitors have a proline residue on their P2 sites and a substituted or unsubstituted phenoxybutyryl moiety on their P3 sites. Thus phenoxybutyryl-prolyl-pyrrolidine is the most effective partial structure of the inhibitors. The best inhibitors found were: 4-(4-benzylphenoxy)butyryl-prolyl-pyrrolidine for bacterial enzyme (IC50 1.4 nM) and 4-phenylbutyryl-thioprolyl-pyrrolidine for bovine brain enzyme (IC50 67 nM). In the passive avoidance test, using amnesic rats experimentally induced with scopolamine, the pyrrolidine derivatives which had potent inhibitory activity toward post-proline cleaving enzymes also showed strong anti-amnesic activities at doses of 1-5 mg/kg, i.p.     

Facile synthesis of a dimeric dipyrrole-polyamide and synergetic DNA-cleaving activity of its Cu(II) complex

Inspired by the potent DNA-cleaving activity of the Cu(II) complex of monopyrrole-polyamide dimer 1 (i.e., 1@Cu(2+)), we designed a new dimeric dipyrrole-polyamide analog 2 with the aim to optimize the catalytic activities of the metal complexes of this type of polypyrrole-polyamides. Compound 2 was prepared in 50% yield from the reaction of 1-methyl-4-[(1-methyl-4-nitro-1H-pyrrole-2-carbonyl)-amino]-1H-pyrrole-2-carboxylic acid with 2,2'-(ethane-1,2-diylbis(oxy))diethanamine, and fully characterized on the basis of NMR ((1)H and (13)C), MS (ESI and HR) and IR. Spectrophotometric titration, ESI-MS and conductivity measurements indicated that compound 2 formed a 1:1 complex with Cu(2+) ion (i.e., 2@Cu(2+)). Agarose gel electrophoresis studies indicated that 2@Cu(2+) was capable of efficiently converting pBR322 DNA into open circular and linear forms under physiological conditions, most probably via an oxidative mechanism. Its overall catalytic activity was estimated to be at least 30-fold higher than that of 1@Cu(2+). The fact that the cleaving activities of these Cu(II) complexes parallel, exactly, their binding affinities, raises the possibility that the cleaving activities of polypyrrole-polyamide derivatives of the type can be regulated by the binding affinities.     

Plant growth-regulating activities of batatasin III analogues

Nine bibenzyls and 10 stilbenes were synthesized as analoguesof batatasin III, a growth inhibitor isolated from dormant yambulbils, and examined for their plant growth-regulating activities.The bioassays used were the elongation of dark-grown intactrice coleoptiles, auxin-induced elongation of excised oat coleoptiles,and germination of rape and barnyard grass seeds. In the elongationof intact rice coleoptiles, 3,3'-dihydroxy-5-methoxy- (batatasinIII), 3,5-dimethoxy-3'-nitro-, 4'-bromo-3-nitro-, 3-amino-3'-chloro-,3-amino-4'-chloro-bibenzyls and 3-benzyloxy-4'-bromo-5-methoxy-,3-benzyloxy-3',4'-dichloro-5-methoxy-stilbenes were inhibitory,and 4'-bromo-3-nitrostilbene was promotive at a concentrationof 100 mg/liter. The results obtained by the other bioassayswere qualitatively consistent with these findings, although3-amino-4'- chlorobibenzyl and 4'-bromo-3-nitrostilbene werenot tested in all the bioassays. In the seed germination, which was rather tolerant to the testanalogues, batatasin III was inactive but 3-benzyloxy-4'-bromo-5-methoxy-and 3-benzyloxy-3',4'-dichloro- 5-methoxystilbenes were veryactive. Thus, if substituted properly, bibenzyls and stilbenes are activewithout hydroxyl and methoxyl group(s) as the functional group. ³ Present address: The National Institute for EnvironmentalStudies, Yatabc, Ibaraki 300-21, Japan. (Received November 19, 1975; )     

Apurinic endonuclease from Saccharomyces cerevisiae.

An endonuclease cleaving depurinated and alkylated double-stranded DNA has been purified 500-fold from Saccharomyces cerevisiae, strain MB 1052. The enzyme has an Mr of 31 000 +/- 2000, a sedimentation value of 3.2S and a diffusion coefficient of 9.5 X 10-7 cm2/s. The enzyme was active only at apurinic/apyridiminic sites, regardless of whether they were produced by heating the DNA at acidic pH or by alkylation with the ultimate carcinogen methyl methanesulphonate. Native DNA was not acted upon. U.v.-irradiated DNA and DNA treated with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene were cleaved to an extent related to the extent of apurinic/apyridiminic sites. Enzymic activity was not dependent upon Mg2+, but was stimulated approx. 3-fold by 4mM-Mg2+. The enzyme did not bind to DEAE-cellulose or CM-cellulose at KCl concentrations greater than 160 mM. The endonuclease was obtained free of exonuclease and 3-methyladenine-DNA glycosylase activity in five chromatographic steps.     

Synthesis and inhibitory activity of acyl-peptidyl-prolinalderivatives toward post-proline cleaving enzyme as nootropic agents

Several prolinal derivatives were synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain and their possible properties as nootropic agents. Almost all the compounds tested inhibited the activity of both enzymes at low IC50 values of the order of nM, but a specificity difference was observed with alkylacyl-prolinal derivatives which strongly inhibited only the bacterial enzyme. Prolyl-prolinal derivatives were the most effective inhibitors for both enzymes. In the passive avoidance test using amnesic rats experimentally induced with scopolamine, the prolinal derivatives that have potent inhibitory activity toward post-proline cleaving enzymes showed also strong anti-amnesic activities at dose of 10-1000 micrograms/kg, i.p. Some of the compounds showed a bell-shape dose dependency. These results suggest that the post-proline cleaving enzymes play an important role in the regulation of learning and memory consolidation in the brain and inhibitors of these enzymes are suggested as possible candidates for nootropic agents, particularly for an anti-amnesic drug.     

Interaction and cleavage of DNA with metal complexes of macrocyclic compound consisting of two 1, 10-phenanthroline.

We have synthesized two metal complexes of the macrocyclic compound (III, IV), with which either Cu2+ or Ru2+ is coordinated, consisting of two 1,10-phenanthroline molecules bridged by sulfur. Interaction of the metal complexes with DNA was examined by spectroscopic method and their binding constants were determined. The DNA cleaving activities of these complexes were also investigated.     

Inhibition of Calpains by Calmidazolium and Calpastatin

Abstract

Several prolinal derivatives were synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain and their possible properties as nootropic agents. Almost all the compounds tested inhibited the activity of both enzymes at low IC₅₀ values of the order of nM, but a specificity difference was observed with alkylacyl-prolinal derivatives which strongly inhibited only the bacterial enzyme. Prolyl-prolinal derivatives were the most effective inhibitors for both enzymes. In the passive avoidance test using amnesic rats experimentally induced with scopolamine, the prolinal derivatives that have potent inhibitory activity toward post-proline cleaving enzymes showed also strong anti-amnesic activities at doses of 10 ~ 1000μg/kg, i.p. Some of the compounds showed a bell-shape dose dependency. These results suggest that the post-proline cleaving enzymes play an important role in the regulation of learning and memory consolidation in the brain and inhibitors of these enzymes are suggested as possible candidates for nootropic agents, particularly for an anti-amnesic drug.     

DNA生物催化功能研究进展

近年来发现 ,不少结构特殊的DNA分子分别具有剪切RNA分子或DNA分子、T4聚核苷酸激酶样活性、DNA连接酶样活性以及催化卟啉金属离子化等多种生物催化功能 ,这些DNA分子被称为脱氧核酶或酶性DNA .它们在用作RNA和DNA工具酶、基因分析和诊断手段以及基因治疗药物等方面的潜力引人注目 .综述这些DNA分子的种类、结构特征、催化活性及应用现状和前景等方面的最新研究进展     

Photoinduced DNA cleavage by anthracene based hydroxamic acids

Two different series of naphthalene and anthracene based hydroxamic acids having amino acid derivatives were synthesized. Single strand DNA cleavage was achieved on irradiation of newly synthesized hydroxamic acids by UV light (≥350nm). Both reactive oxygen species (ROS) and generated radicals from hydroxamic acids were shown to be responsible for the DNA cleavage. Further, DNA cleaving ability of hydroxamic acids was found to be dependent on its concentration and on its structure.     

Fluorescent enkephalin derivatives with biological activity

Fluorescent dansylated derivatives of enkephalins were prepared, with the dansyl group either at the N- or C-terminal part of methionine-enkephalin (Met-E) or its peptidase-resistant analogue D-Ala₂-Met-E. An energy transfer, from the Tyr to the dansyl group, was found for all compounds and spectral properties were dependent upon the environment polarity. Met-E-(CH₂)₂ dansyl II and D-Ala₂-Met-E-(CH₂)₂ dansyl III exhibit biological activities on guinea-pig ileum and binding affinities on striatal membranes, similar to Met-E. III exhibits significant analgesic activity in mice after i.v. administration (about 35 % that of morphine). Fluorescent enkephalins potentially represent useful probes for the exploration of opiate receptors, studies of ligand-receptor interactions and evaluation of enkephalins cleaving peptidases in various tissues.     

The DNA cleavage induced by a chromium(V) complex and by chromate and glutathione is mediated by activated oxygen species

The number of strand breaks induced by the combination of chromate and glutathione (GSH) in PM2 DNA was effectively reduced upon addition of the hydroxyl radical scavengers dimethyl sulphoxide (DMSO), formate and benzoate. Administration of catalase also led to a depression of DNA degradation whereas superoxide dismutase (SOD) had very little influence. Essentially the same results were obtained in experiments employing a chromium(V) complex Na4(GSH)4Cr.8H20, which is an intermediate chromium species isolated from the reduction of chromate by glutathione. DNA cleavage was dependent on the presence of iron (FeCl3). When compared with the number of breaks produced by FeCl3 and GSH alone, chromate stimulated the generation of single-strand breaks. These findings suggest that hydroxyl radicals are one ultimate DNA cleaving agent in both reactions. A reaction scheme for the production of hydroxyl radicals is proposed.     

Pyrazolo-triazoles as light activable DNA cleaving agents

In view of the continuous interest in new DNA cleaving compounds, both for the development of new therapeutic agents and for the possible use as reagents in nucleic acids research, a few pyrazolo[3,4-d][1,2,3]triazole derivatives have been obtained and investigated for their antiproliferative activity and capability to cleave DNA, after light-activation. A possible in situ activation, i.e. in neoplastic tissues, of less cytotoxic derivatives, may lead to potential antitumor compounds endowed with high therapeutic indexes.     

DNA cleaving modes in minor groove of DNA helix by esperamicin and calicheamicin antitumor antibiotics.

This study examines and compares DNA cleavage modes by several esperamicin derivatives and calicheamicin. We found that the deoxyfucose-anthranilate moiety is a key factor to determine their DNA cutting modes. Probably, the bulky moiety hinders the abstraction of hydrogen atom from deoxyribose by the C-1 carbon radical of phenylene diradical. On the basis of the experimental results, detailed DNA cleaving modes in DNA minor groove by esperamicin and calicheamicin have been discussed.     

Sequence-specific base pair mimics are efficient topoisomerase IB inhibitors

Topoisomerase IB controls DNA topology by cleaving DNA transiently. This property is used by inhibitors, such as camptothecin, that stabilize, by inhibiting the religation step, the cleavage complex, in which the enzyme is covalently attached to the 3'-phosphate of the cleaved DNA strand. These drugs are used in clinics as antitumor agents. Because three-dimensional structural studies have shown that camptothecin derivatives act as base pair mimics and intercalate between two base pairs in the ternary DNA-topoisomerase-inhibitor complex, we hypothesized that base pairs mimics could act like campthotecin and inhibit the religation reaction after the formation of the topoisomerase I-DNA cleavage complex. We show here that three base pair mimics, nucleobases analogues of the aminophenyl-thiazole family, once targeted specifically to a DNA sequence were potent topoisomerase IB inhibitors. The targeting was achieved through covalent linkage to a sequence-specific DNA ligand, a triplex-forming oligonucleotide, and was necessary to position and keep the nucleobase analogue in the cleavage complex. In the absence of triplex formation, only a weak binding to the DNA and topoisomerase I-mediated DNA cleavage was observed. The three compounds were equally active once conjugated, implying that the intercalation of the nucleobase upon triplex formation is the essential feature for the inhibition activity.     

超氧化物歧化酶切割超螺旋DNA的活性

在体外系统中,发现超氧化物歧化酶(SOD)具有切割超螺旋DNA的活性. 猪血和牛血Cu/Zn-SOD以及烟草Mn-SOD都能将超螺旋DNA转变为非超螺旋结构的缺刻环状DNA,进一步产生线状DNA. 它们只作用于超螺旋DNA而不作用于线状DNA. 这个事实排除了SOD样品中污染核酸酶的可能性. 用H₂O₂、胍基抑制或蛋白酶降解的实验结果表明,这两种酶的活性中心处于酶蛋白的不同部位.     

Covalent modification and single-strand scission of DNA by a new antitumor antibiotic kapurimycin A3

Kapurimycin A3 is a new antitumor antibiotic isolated from a Streptomyces. It contains the anthrapyrone skeleton and a beta,gamma-unsaturated delta-keto carboxylic acid moiety in the structure. In vitro, kapurimycin causes single-strand cleavage of supercoiled pBR322 DNA. The diminished cytotoxicity and DNA cleaving activity for 13-decarboxykapurimycin A3 indicates that the beta, gamma-unsaturated delta-keto carboxylic acid moiety is important for the activity of kapurimycin. Kapurimycin A3 binds to calf thymus DNA at 4 degrees C, and the thermal treatment of this adduct results in release of a guanine covalently attached to C-16 of kapurimycin via one of its nitrogen atoms. Thus, the epoxide is the alkylating functional group of kapurimycin, and this is consistent with the lack of DNA cleaving and cytotoxic activities for 14,16-deoxy-14,16-dihydroxykapurimycin. These findings have revealed that DNA strand scission by kapurimycin is due to the alkylation of guanine by ring opening of the epoxide group of kapurimycin, depurination of modified guanine, and presumably subsequent hydrolysis of the phosphate ester backbone at the resultant apurinic sites.     

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24 h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10 h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.     

Nucleic acid synthesis in preimplantation mouse embryos

Summary Mouse embryos were collected at the 2-cell stage, cultured in vitro in the presence of³H deoxyuridine or uridine for 6 or 4 h and autoradiographed.Deoxyuridine is actively incorporated into the DNA of cleaving mouse embryos indicating the existence of thymidylate synthetase activity at least at the 4-cell stage and presumably already before this.RNAase treatment of embryos squashed on slides shows a weak but obvious incorporation of uridine into DNA of cleaving mouse embryos, from the 4-cell stage onwards; this incorporation is totally inhibited by hydroxyurea. The reduction of ribonucleotides to deoxyribonucleotides is a metabolic pathway already required for cleavage, as shown by hydroxyurea experiments.The second polar pody, known to incorporate thymidine, is unable to incorporate either deoxyuridine or uridine.