Selective alkylation of T–T mismatched DNA using vinyldiaminotriazine–acridine conjugate

Science.gov (United States)

Onizuka, Kazumitsu; Usami, Akira; Yamaoki, Yudai; Kobayashi, Tomohito; Hazemi, Madoka E; Chikuni, Tomoko; Sato, Norihiro; Sasaki, Kaname; Katahira, Masato

2018-01-01

Abstract The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T–T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)–acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T–T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T–T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT–acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1. PMID:29309639

Ada response - a strategy for repair of alkylated DNA in bacteria.

Science.gov (United States)

Mielecki, Damian; Grzesiuk, Elżbieta

2014-06-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N(3)-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N(1)-methyladenine (1meA) and N(3)-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O(6)-methylguanine (O(6) meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. © 2014 The Authors. FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

Ada response – a strategy for repair of alkylated DNA in bacteria

Science.gov (United States)

Mielecki, Damian; Grzesiuk, Elżbieta

2014-01-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. PMID:24810496

Chronic ethanol consumption inhibits repair of dimethylnitrosamine-induced DNA alkylation

International Nuclear Information System (INIS)

Mufti, S.I.; Salvagnini, M.; Lieber, C.S.; Garro, A.J.

1988-01-01

Chronic ethanol consumption causes a DNA repair deficiency. This was demonstrated in Sprague-Dawley rats injected with 14 C-labeled dimethylnitrosamine after being pair-fed isocaloric, ethanol, or carbohydrate control diets for 4 weeks. Hepatic DNA was isolated from rats killed at intervals over a 36 hour period after administration of the nitrosamine and concentrations of alkylated guanine derivatives were measured. While N7-methylguanine was lost at equivalent rates from the DNA of both diet groups, 06methylguanine, a promutagenic lesion, persisted at higher levels for longer periods of time in the DNA from the alcohol-fed animals

N-methylpurine DNA glycosylase inhibits p53-mediated cell cycle arrest and coordinates with p53 to determine sensitivity to alkylating agents.

Science.gov (United States)

Song, Shanshan; Xing, Guichun; Yuan, Lin; Wang, Jian; Wang, Shan; Yin, Yuxin; Tian, Chunyan; He, Fuchu; Zhang, Lingqiang

2012-08-01

Alkylating agents induce genome-wide base damage, which is repaired mainly by N-methylpurine DNA glycosylase (MPG). An elevated expression of MPG in certain types of tumor cells confers higher sensitivity to alkylation agents because MPG-induced apurinic/apyrimidic (AP) sites trigger more strand breaks. However, the determinant of drug sensitivity or insensitivity still remains unclear. Here, we report that the p53 status coordinates with MPG to play a pivotal role in such process. MPG expression is positive in breast, lung and colon cancers (38.7%, 43.4% and 25.3%, respectively) but negative in all adjacent normal tissues. MPG directly binds to the tumor suppressor p53 and represses p53 activity in unstressed cells. The overexpression of MPG reduced, whereas depletion of MPG increased, the expression levels of pro-arrest gene downstream of p53 including p21, 14-3-3σ and Gadd45 but not proapoptotic ones. The N-terminal region of MPG was specifically required for the interaction with the DNA binding domain of p53. Upon DNA alkylation stress, in p53 wild-type tumor cells, p53 dissociated from MPG and induced cell growth arrest. Then, AP sites were repaired efficiently, which led to insensitivity to alkylating agents. By contrast, in p53-mutated cells, the AP sites were repaired with low efficacy. To our knowledge, this is the first direct evidence to show that a DNA repair enzyme functions as a selective regulator of p53, and these findings provide new insights into the functional linkage between MPG and p53 in cancer therapy.

DNA minor groove targeted alkylating agents based on bisbenzimidazole carriers: synthesis, cytotoxicity and sequence-specificity of DNA alkylation.

Science.gov (United States)

Smaill, J B; Fan, J Y; Denny, W A

1998-12-01

A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucleotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, and 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with

Sequence-specific DNA alkylation by tandem Py-Im polyamide conjugates.

Science.gov (United States)

Taylor, Rhys Dylan; Kawamoto, Yusuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2014-09-01

Tandem N-methylpyrrole-N-methylimidazole (Py-Im) polyamides with good sequence-specific DNA-alkylating activities have been designed and synthesized. Three alkylating tandem Py-Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high-resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1, which contained a β-alanine linker, displayed the most-selective sequence-specific alkylation towards the target 10 bp DNA sequence. The tandem Py-Im polyamide conjugates displayed greater sequence-specific DNA alkylation than conventional hairpin Py-Im polyamide conjugates (4 and 5). For further research, the design of tandem Py-Im polyamide conjugates could play an important role in targeting specific gene sequences. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

International Nuclear Information System (INIS)

Hartley, J.A.; Forrow, S.M.; Souhami, R.L.

1990-01-01

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increase ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. The result differed with both the nitrogen mustard and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove

Balancing repair and tolerance of DNA damage caused by alkylating agents

OpenAIRE

Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D.

2012-01-01

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial ...

DNA modification by alkylating compounds

Energy Technology Data Exchange (ETDEWEB)

Kruglyakova, E.E.

1985-09-01

Results are given for research on the physico-chemical properties of alkylating compounds - nitroso alkyl ureas (NAU) which possess a broad spectrum of biological activity, such as mutagenic, carcinogenic, and anti-tumor action that is due to the alkylation and carbamoylation of DNA as well as other cellular components. Identified chemical products of NAU interaction with DNA and its components are cited. Structural conversions of a DNA macromolecule resulting from its chemical modification are examined. NAU are used to discuss possible biological consequences of DNA modification. 148 references.

Histone H2AX is a critical factor for cellular protection against DNA alkylating agents.

Science.gov (United States)

Meador, J A; Zhao, M; Su, Y; Narayan, G; Geard, C R; Balajee, A S

2008-09-25

Histone H2A variant H2AX is a dose-dependent suppressor of oncogenic chromosome translocations. H2AX participates in DNA double-strand break repair, but its role in other DNA repair pathways is not known. In this study, role of H2AX in cellular response to alkylation DNA damage was investigated. Cellular sensitivity to two monofunctional alkylating agents (methyl methane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)) was dependent on H2AX dosage, and H2AX null cells were more sensitive than heterozygous cells. In contrast to wild-type cells, H2AX-deficient cells displayed extensive apoptotic death due to a lack of cell-cycle arrest at G(2)/M phase. Lack of G(2)/M checkpoint in H2AX null cells correlated well with increased mitotic irregularities involving anaphase bridges and gross chromosomal instability. Observation of elevated poly(ADP) ribose polymerase 1 (PARP-1) cleavage suggests that MNNG-induced apoptosis occurs by PARP-1-dependent manner in H2AX-deficient cells. Consistent with this, increased activities of PARP and poly(ADP) ribose (PAR) polymer synthesis were detected in both H2AX heterozygous and null cells. Further, we demonstrate that the increased PAR synthesis and apoptotic death induced by MNNG in H2AX-deficient cells are due to impaired activation of mitogen-activated protein kinase pathway. Collectively, our novel study demonstrates that H2AX, similar to PARP-1, confers cellular protection against alkylation-induced DNA damage. Therefore, targeting either PARP-1 or histone H2AX may provide an effective way of maximizing the chemotherapeutic value of alkylating agents for cancer treatment.

DNA minor groove alkylating agents.

Science.gov (United States)

Denny, W A

2001-04-01

Recent work on a number of different classes of anticancer agents that alkylate DNA in the minor groove is reviewed. There has been much work with nitrogen mustards, where attachment of the mustard unit to carrier molecules can change the normal patterns of both regio- and sequence-selectivity, from reaction primarily at most guanine N7 sites in the major groove to a few adenine N3 sites at the 3'-end of poly(A/T) sequences in the minor groove. Carrier molecules discussed for mustards are intercalators, polypyrroles, polyimidazoles, bis(benzimidazoles), polybenzamides and anilinoquinolinium salts. In contrast, similar targeting of pyrrolizidine alkylators by a variety of carriers has little effect of their patterns of alkylation (at the 2-amino group of guanine). Recent work on the pyrrolobenzodiazepine and cyclopropaindolone classes of natural product minor groove binders is also reviewed.

Balancing repair and tolerance of DNA damage caused by alkylating agents.

Science.gov (United States)

Fu, Dragony; Calvo, Jennifer A; Samson, Leona D

2012-01-12

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity.

Bifunctional rhodium intercalator conjugates as mismatch-directing DNA alkylating agents.

Science.gov (United States)

Schatzschneider, Ulrich; Barton, Jacqueline K

2004-07-21

A conjugate of a DNA mismatch-specific rhodium intercalator, containing the bulky chrysenediimine ligand, and an aniline mustard has been prepared, and targeting of mismatches in DNA by this conjugate has been examined. The preferential alkylation of mismatched over fully matched DNA is found by a mobility shift assay at concentrations where untethered organic mustards show little reaction. The binding site of the Rh intercalator was determined by DNA photocleavage, and the position of covalent modification was established on the basis of the enhanced depurination associated with N-alkylation. The site-selective alkylation at mismatched DNA renders these conjugates useful tools for the covalent tagging of DNA base pair mismatches and new chemotherapeutic design.

Molecular design of sequence specific DNA alkylating agents.

Science.gov (United States)

Minoshima, Masafumi; Bando, Toshikazu; Shinohara, Ken-ichi; Sugiyama, Hiroshi

2009-01-01

Sequence-specific DNA alkylating agents have great interest for novel approach to cancer chemotherapy. We designed the conjugates between pyrrole (Py)-imidazole (Im) polyamides and DNA alkylating chlorambucil moiety possessing at different positions. The sequence-specific DNA alkylation by conjugates was investigated by using high-resolution denaturing polyacrylamide gel electrophoresis (PAGE). The results showed that polyamide chlorambucil conjugates alkylate DNA at flanking adenines in recognition sequences of Py-Im polyamides, however, the reactivities and alkylation sites were influenced by the positions of conjugation. In addition, we synthesized conjugate between Py-Im polyamide and another alkylating agent, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). DNA alkylation reactivies by both alkylating polyamides were almost comparable. In contrast, cytotoxicities against cell lines differed greatly. These comparative studies would promote development of appropriate sequence-specific DNA alkylating polyamides against specific cancer cells.

Concerted bis-alkylating reactivity of clerocidin towards unpaired cytosine residues in DNA

Science.gov (United States)

Richter, Sara N.; Menegazzo, Ileana; Fabris, Daniele; Palumbo, Manlio

2004-01-01

Clerocidin (CL) is a topoisomerase II poison, which cleaves DNA irreversibly at guanines (G) and reversibly at cytosines (C). Furthermore, the drug can induce enzyme-independent strand breaks at the G and C level. It has been previously shown that G-damage is induced by alkylation of the guanine N7, followed by spontaneous depurination and nucleic acid cleavage, whereas scission at C is obtained only after treatment with hot alkali, and no information is available to explain the nature of this damage. We present here a systematic study on the reactivity of CL towards C both in the DNA environment and in solution. Selected synthetic derivatives were employed to evaluate the role of each chemical group of the drug. The structure of CL–dC adduct was then characterized by tandem mass spectrometry and NMR: the adduct is a stable condensed ring system resulting from a concerted electrophilic attack of the adjacent carbonyl and epoxide groups of CL towards the exposed NH2 and N3, respectively. This reaction mechanism, shown here for the first time, is characterized by faster kinetic rates than alkylation at G, due to the fact that the rate-determining step, alkylation at the epoxide, is an intramolecular process, provided a Schiff base linking CL and C can rapidly form, whereas the corresponding reaction of G N7 is intermolecular. These results provide helpful hints to explain the reversible/irreversible nature of topoisomerase II mediated DNA damage produced by CL at C/G steps. PMID:15494453

Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak Inhibitors of the Human DNA Glycosylase OGG1

Directory of Open Access Journals (Sweden)

Tushar R. Mahajan

2015-09-01

Full Text Available The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER, one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.

Alkylation Induced DNA Repair and Mutagenesis in Escherichia coli.

Science.gov (United States)

1987-11-23

unrepaired 3-methyladenine in DNA 29 2.4.1 Cytotoxic effects of persisting m3A in DNA 30 2.4.2 Mutagenic bypass synthesis of depurinat ,d DNA 30 3 CONCLUDING...induced by a single exposure to the ca’rcinogen N- methyl-N- nitrosourea (MNU) due to activation of the malignant Ha-ras-i locus. Analysis of the induced...ing CO:A uolymerase I for repair synthesis . Since DNA polymerase I would be required to complete repair after the in~uial activity of TagII, we tested

Carboxymethyl chitin-glucan (CM-CG) protects human HepG2 and HeLa cells against oxidative DNA lesions and stimulates DNA repair of lesions induced by alkylating agents.

Science.gov (United States)

Slamenová, Darina; Kováciková, Ines; Horváthová, Eva; Wsólová, Ladislava; Navarová, Jana

2010-10-01

A large number of functional foods, including those that contain β-d-glucans, have been shown to prevent human DNA against genotoxic effects and associated development of cancer and other chronic diseases. In this paper, carboxymethyl chitin-glucan (CM-CG) isolated from Aspergillus niger was investigated from two standpoints: (1) DNA-protective effects against oxidative DNA damage induced by H(2)O(2) and alkylating DNA damage induced by MMS and MNNG, and (2) a potential effect on rejoining of MMS- and MNNG-induced single strand DNA breaks. The results obtained by the comet assay in human cells cultured in vitro showed that CM-CG reduced significantly the level of oxidative DNA lesions induced by H(2)O(2) but did not change the level of alkylating DNA lesions induced by MMS or MNNG. On the other side, the efficiency of DNA-rejoining of single strand DNA breaks induced by MMS and MNNG was significantly higher in HepG2 cells pre-treated with CM-CG. The antioxidative activity of carboxymethyl chitin-glucan was confirmed by the DPPH assay. Copyright © 2010 Elsevier Ltd. All rights reserved.

Base excision repair of chemotherapeutically-induced alkylated DNA damage predominantly causes contractions of expanded GAA repeats associated with Friedreich's ataxia.

Directory of Open Access Journals (Sweden)

Yanhao Lai

Full Text Available Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA, an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER. We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.

Synthesis and DNA cleavage activity of Bis-3-chloropiperidines as alkylating agents.

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Zuravka, Ivonne; Roesmann, Rolf; Sosic, Alice; Wende, Wolfgang; Pingoud, Alfred; Gatto, Barbara; Göttlich, Richard

2014-09-01

Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alkylation damage in DNA and RNA--repair mechanisms and medical significance

DEFF Research Database (Denmark)

Drabløs, Finn; Feyzi, Emadoldin; Aas, Per Arne

2004-01-01

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions...... are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase......, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents...

DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD).

Science.gov (United States)

Hašplová, Katarína; Hudecová, Alexandra; Magdolénová, Zuzana; Bjøras, Magnar; Gálová, Eliška; Miadoková, Eva; Dušinská, Mária

2012-01-05

3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Photo-triggered fluorescent theranostic prodrugs as DNA alkylating agents for mechlorethamine release and spatiotemporal monitoring.

Science.gov (United States)

Cao, Yanting; Pan, Rong; Xuan, Weimin; Wei, Yongyi; Liu, Kejian; Zhou, Jiahong; Wang, Wei

2015-06-28

We describe a new theranostic strategy for selective delivery and spatiotemporal monitoring of mechlorethamine, a DNA alkylating agent. A photo-responsive prodrug is designed and composed of a photolabile o-nitrophenylethyl group, a DNA alkylating mechlorethamine drug and a coumarin fluorophore. Masking of the "N" in mechlorethamine in a positively charged state in the prodrug renders it inactive, non-toxic, selective and non-fluorescent. Indeed, the stable prodrug shows negligible cytotoxicity towards normal cells with and without UV activation and is completely non-fluorescent. However, upon photo-irradiation, the active mechlorethamine is released and induces efficient DNA cross-links, accompanied by a strong fluorescence enhancement (152 fold). Furthermore, DNA cross-linking activity from the release can be transformed into anticancer activity observed in in vitro studies of tumor cells. Importantly, the drug release progress and the movement can be conveniently monitored by fluorescence spectroscopy. The mechanistic study proves that the DNA cross-linking activity is mainly due to the release of DNA alkylating mechlorethamine. Altogether, the studies show the power of the theranostic strategy for efficient therapy in cancer treatment.

Direct-acting DNA alkylating agents present in aqueous extracts of areca nut and its products.

Science.gov (United States)

Hu, Chiung-Wen; Chao, Mu-Rong

2012-11-19

Areca nut is a carcinogen to humans and has been strongly associated with oral premalignant and malignant diseases. Previous studies speculated the presence of unknown direct-acting mutagens present in aqueous extracts of areca nut. We hypothesized whether any direct-acting alkylating agents are present in areca nut and its commercial products. In this study, calf thymus DNA was treated with four different aqueous extracts obtained from unripe and ripe areca nuts or their commercial products, namely, pan masala (without tobacco) and gutkha (with tobacco). Three N-alkylated purines including N7-methylguanine (N7-MeG), N3-methyladenine (N3-MeA), and N7-ethylguanine (N7-EtG) were detected using sensitive and specific isotope-dilution liquid chromatography-tandem-mass spectrometry (LC-MS/MS) methods. The results showed that four types of aqueous extracts significantly induced the formation of N7-MeG and N3-MeA in a linear dose-response manner. Extracts from unripe areca nut exhibited higher methylating potency than those of ripe areca nut, while gutkha had higher methylating potency than pan masala. Meanwhile, gutkha made with areca nut and tobacco, was the only extract found to induce the formation of N7-EtG. Overall, this study first demonstrated that the presence of direct-acting alkylating agents in areca nut and its commercial products exist at a level that is able to cause significant DNA damage. Our findings may provide another mechanistic rationale for areca nut-mediated oral carcinogenesis and also highlight the importance and necessity of the identification of these direct-acting alkylating agents.

Aag-initiated base excision repair drives alkylation-induced retinal degeneration in mice.

Science.gov (United States)

Meira, Lisiane B; Moroski-Erkul, Catherine A; Green, Stephanie L; Calvo, Jennifer A; Bronson, Roderick T; Shah, Dharini; Samson, Leona D

2009-01-20

Vision loss affects >3 million Americans and many more people worldwide. Although predisposing genes have been identified their link to known environmental factors is unclear. In wild-type animals DNA alkylating agents induce photoreceptor apoptosis and severe retinal degeneration. Alkylation-induced retinal degeneration is totally suppressed in the absence of the DNA repair protein alkyladenine DNA glycosylase (Aag) in both differentiating and postmitotic retinas. Moreover, transgenic expression of Aag activity restores the alkylation sensitivity of photoreceptors in Aag null animals. Aag heterozygotes display an intermediate level of retinal degeneration, demonstrating haploinsufficiency and underscoring that Aag expression confers a dominant retinal degeneration phenotype.

PARP inhibitors protect against sex- and AAG-dependent alkylation-induced neural degeneration.

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Allocca, Mariacarmela; Corrigan, Joshua J; Fake, Kimberly R; Calvo, Jennifer A; Samson, Leona D

2017-09-15

Alkylating agents are commonly used to treat cancer. Although base excision repair (BER) is a major pathway for repairing DNA alkylation damage, under certain conditions, the initiation of BER produces toxic repair intermediates that damage healthy tissues. The initiation of BER by the alkyladenine DNA glycosylase (AAG, a.k.a. MPG) can mediate alkylation-induced cytotoxicity in specific cells in the retina and cerebellum of male mice. Cytotoxicity in both wild-type and Aag -transgenic ( AagTg ) mice is abrogated in the absence of Poly(ADP-ribose) polymerase-1 (PARP1). Here, we tested whether PARP inhibitors can also prevent alkylation-induced retinal and cerebellar degeneration in male and female WT and AagTg mice. Importantly, we found that WT mice display sex-dependent alkylation-induced retinal damage (but not cerebellar damage), with WT males being more sensitive than females. Accordingly, estradiol treatment protects males against alkylation-induced retinal degeneration. In AagTg male and female mice, the alkylation-induced tissue damage in both the retina and cerebellum is exacerbated and the sex difference in the retina is abolished. PARP inhibitors, much like Parp1 gene deletion, protect against alkylation-induced AAG-dependent neuronal degeneration in WT and AagTg mice, regardless of the gender, but their efficacy in preventing alkylation-induced neuronal degeneration depends on PARP inhibitor characteristics and doses. The recent surge in the use of PARP inhibitors in combination with cancer chemotherapeutic alkylating agents might represent a powerful tool for obtaining increased therapeutic efficacy while avoiding the collateral effects of alkylating agents in healthy tissues.

Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

Science.gov (United States)

Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

2015-10-15

Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Protein Recognition in Drug-Induced DNA Alkylation: When the Moonlight Protein GAPDH Meets S23906-1/DNA Minor Groove Adducts.

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Savreux-Lenglet, Gaëlle; Depauw, Sabine; David-Cordonnier, Marie-Hélène

2015-11-05

DNA alkylating drugs have been used in clinics for more than seventy years. The diversity of their mechanism of action (major/minor groove; mono-/bis-alkylation; intra-/inter-strand crosslinks; DNA stabilization/destabilization, etc.) has undoubtedly major consequences on the cellular response to treatment. The aim of this review is to highlight the variety of established protein recognition of DNA adducts to then particularly focus on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) function in DNA adduct interaction with illustration using original experiments performed with S23906-1/DNA adduct. The introduction of this review is a state of the art of protein/DNA adducts recognition, depending on the major or minor groove orientation of the DNA bonding as well as on the molecular consequences in terms of double-stranded DNA maintenance. It reviews the implication of proteins from both DNA repair, transcription, replication and chromatin maintenance in selective DNA adduct recognition. The main section of the manuscript is focusing on the implication of the moonlighting protein GAPDH in DNA adduct recognition with the model of the peculiar DNA minor groove alkylating and destabilizing drug S23906-1. The mechanism of action of S23906-1 alkylating drug and the large variety of GAPDH cellular functions are presented prior to focus on GAPDH direct binding to S23906-1 adducts.

DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis

OpenAIRE

Durbach, S I; Springer, B; Machowski, E E; North, R J; Papavinasasundaram, K G; Colston, M J; Böttger, E C; Mizrahi, V

2003-01-01

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N′-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is res...

Decreased stability of DNA in cells treated with alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Frankfurt, O.S. (Cedars Medical Center, Miami, FL (United States))

1990-12-01

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg{sup 2+}. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl{sub 2}. Thus, the presence of phosphates and MgCl{sub 2} during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S{sub 1} nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.

Atorvastatin Downregulates In Vitro Methyl Methanesulfonate and Cyclophosphamide Alkylation-Mediated Cellular and DNA Injuries

Directory of Open Access Journals (Sweden)

Carlos F. Araujo-Lima

2018-01-01

Full Text Available Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA reductase inhibitors, and this class of drugs has been studied as protective agents against DNA damages. Alkylating agents (AAs are able to induce alkylation in macromolecules, causing DNA damage, as DNA methylation. Our objective was to evaluate atorvastatin (AVA antimutagenic, cytoprotective, and antigenotoxic potentials against DNA lesions caused by AA. AVA chemopreventive ability was evaluated using antimutagenicity assays (Salmonella/microsome assay, cytotoxicity, cell cycle, and genotoxicity assays in HepG2 cells. The cells were cotreated with AVA and the AA methyl methanesulfonate (MMS or cyclophosphamide (CPA. Our datum showed that AVA reduces the alkylation-mediated DNA damage in different in vitro experimental models. Cytoprotection of AVA at low doses (0.1–1.0 μM was observed after 24 h of cotreatment with MMS or CPA at their LC50, causing an increase in HepG2 survival rates. After all, AVA at 10 μM and 25 μM had decreased effect in micronucleus formation in HepG2 cells and restored cell cycle alterations induced by MMS and CPA. This study supports the hypothesis that statins can be chemopreventive agents, acting as antimutagenic, antigenotoxic, and cytoprotective components, specifically against alkylating agents of DNA.

Design of novel antitumor DNA alkylating agents: the benzacronycine series.

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David-Cordonnier, Marie-Hélène; Laine, William; Gaslonde, Thomas; Michel, Sylvie; Tillequin, Francois; Koch, Michel; Léonce, Stéphane; Pierré, Alain; Bailly, Christian

2004-03-01

Acronycine, a natural alkaloid originally extracted from the bark of the Australian ash scrub Acronychia baueri, has shown a significant antitumor activity in animal models. Acronycine has been tested against human cancers in the early 1980s, but the clinical trials showed modest therapeutic effects and its development was rapidly discontinued. In order to optimize the antineoplastic effect, different benzoacronycine derivatives were synthesized. Among those, the di-acetate compound S23906-1 was recently identified as a promising anticancer drug candidate and a novel alkylating agent specifically reacting with the exocylic 2-NH2 group of guanines in DNA. The study of DNA bonding capacity of acronycine derivatives leads to the identification of the structural requirements for DNA alkylation. In nearly all cases, the potent alkylating agents, such as S23906-1, were found to be much more cytotoxic than the unreactive analogs such as acronycine itself or diol derivatives. Alkylation of DNA by the monoacetate derivative S28687-1, which is a highly reactive hydrolysis metabolite of S23906-1, occurs with a marked preference for the N2 position of guanine. Other bionucleophiles can react with S23906-1. The benzacronycine derivatives, which efficiently alkylate DNA, also covalently bind to the tripeptide glutathione (GSH) but not to the oxidized product glutathione disulfide. Here we review the reactivity of S23906-1 and some derivatives toward DNA and GSH. The structure-activity relationships in the benzacronycine series validate the reaction mechanism implicating DNA as the main molecular target. S23906-1 stands as the most promising lead of a medicinal chemistry program aimed at discovering novel antitumor drugs based on the acronycine skeleton.

SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

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Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

2013-01-01

Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for an organism's favorable response to alkylating agents. Furthermore, an individual's response to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity. PMID:22237395

Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea

International Nuclear Information System (INIS)

Swenson, D.H.; Lawley, P.D.

1978-01-01

The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp((Et) dT, was identified as a product from the reaction of DNA with N-ethyl-N-nitrosourea. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by this carcinogen, and by N-methyl-N-nitrosourea, dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. The results are related to steric factors, and the electrophilic character of each carcinogen. (author)

Synthesis and evaluation of sequence-specific DNA alkylating agents: effect of alkylation subunits.

Science.gov (United States)

Shimizu, Tatsuhiko; Sasaki, Shunta; Minoshima, Masafumi; Shinohara, Ken-ichi; Bando, Toshikazu; Sugiyama, Hiroshi

2006-01-01

We have demonstrated that hairpin pyrrole (Py)- imidazole (Im) polyamide-CBI conjugates selectively alkylate predetermined sequences. In this study, we investigated the effect of alkylation subunits, for example conjugates 1-4 with three types of DNA alkylating units, and Py-Im polyamides with indole linker. Conjugate 3 and 4 selectively alkylated the predetermined sequences as described previously, while conjugates 1 and 2 alkylate at mismatched sites.

Mechanism of the protective effects of long chain n-alkyl glucopyranosides against ultrasound-induced cytolysis of HL-60 cells

OpenAIRE

Cheng, Jason Y.; Riesz, Peter

2007-01-01

Recently it has been shown that long chain (C5 to C8) n-alkyl glucopyranosides completely inhibit ultrasound-induced cytolysis [1]. This protective effect has possible applications in HIFU (high intensity focused ultrasound) for tumor treatment, and in ultrasound assisted drug delivery and gene therapy. n-Alkyl glucopyranosides with hexyl (5mM), heptyl (3mM), octyl (2mM) n-alkyl chains protected 100% of HL-60 cells in-vitro from 1.057 MHz ultrasound induced cytolysis under a range of conditio...

SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

OpenAIRE

Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

2012-01-01

Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial fo...

Immunological detection of O6-methylguanine in alkylated DNA

International Nuclear Information System (INIS)

Briscoe, W.T.; Spizizen, J.; Tan, E.M.

1978-01-01

Antibodies to O 6 -methyldeoxyguanosine were produced in rabbits and utilized in a radioimmunoassay to detect this nucleoside at picomole levels. The specificity of the antibodies was demonstrated by the use of nucleoside analogues as inhibitors in the radioimmunoassay. The antibodies cross-reacted with O 6 -methylguanosine, O 6 -methylguanine, and O 6 -ethylguanosine. There was 10 4 to 10 6 times less sensitivity to inhibition by deoxyadenosine, deoxyguanosine, and guanosine than by O 6 -methyldeoxyguanosine. The radioimmunoassay also detected O 6 -methylguanine in DNA alkylated by agents known to produce O 6 -methylguanine, such as N'-methyl-N-nitrosourea. DNA alkylated with dimethyl sulfate, which does not produce O 6 -methylguanine in DNA, cross-reacted with the antibodies to a very limited extent. Such an assay system for modified nucleic acid components would be very useful in following the production, persistence, and repair of these lesions in a variety of cells and tissues treated with a broad spectrum of carcinogens and suspected carcinogens

A Theoretical Study of the Mechanism of the Alkylation of Guanine by N- Nitroso Compounds.

Science.gov (United States)

1992-01-01

these chemical agents alkylate DNA, but, as yet, the precise mechanism is unknown. What is known is that the result is a DNA-mutagen adduct with an alkyl ... nitrosoureas , Singer et. al. found that about 25% of the alkylation caused by MNU was on the DNA phospate backbone while, for ENU, phosphate...sites. 1.3 Mutagenicity of N-Nitroso Compounds In early experimental work with agents which alkylate DNA, comparisons of ultraviolet absorption

Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.

Science.gov (United States)

Dziegielewska, Barbara; Beerman, Terry A; Bianco, Piero R

2006-09-01

To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.

Complementarily addressed modification and cleavage of a single-stranded fragment of DNA with the aid of alkylating derivatives of oligonucleotides

International Nuclear Information System (INIS)

Brosalina, E.B.; Vlasov, V.V.; Kutyavin, I.V.; Mamaev, S.V.; Pletnev, A.G.; Podyminogin, M.A.

1986-01-01

The chemical modification of a 303-nucleotide single-stranded fragment of DNA by alkylating oligonucleotide derivatives bearing 4-[N-methyl-N-(2-chloroethyl)amino]benzyl groups in the 5'-terminal phosphate of the 3'-terminal ribose residue has been investigated. It has been shown that under the conditions of the formation of a complex with the DNA fragment both types of derivatives specifically alkylate nucleotides of the DNA fragments that are located directly adjacent to the sections complementary to the oligonucleotides bearing the reactive groups. Alkylation takes place with a high efficiency, and the DNA fragment can be cleaved specifically at the position of the alkylated nucleotides

Reduced DNA repair in mouse satellite DNA after treatment with methylmethanesulfonate, and N-methyl-N-nitrosourea.

Science.gov (United States)

Bodell, W J; Banerjee, M R

1976-01-01

We have measured DNA repair in mouse satellite and main band DNA as resolved by Ag+-Cs2SO4 centrifugation in response to treatment with the alkylating agents, methyl methanesulfonate, and N-methyl-N-nitrosourea. We find that there is a statistically significant lower incorporation of 3H-Tdr into the satellite DNA as compared to the main band at varying periods after treatment with the alkylating agents. This suggests a reduced repair activity in the satellite DNA. We have measured the extent of binding of 14C-methyl methanesulfonate to the satellite, and main band DNA, and no difference in binding was observed, indicating that the reduced repair activity of satellite DNA is not due to a difference in binding of alkylating agents. We believe that the reduced incorporation of 3H-Tdr into satellite DNA may be due to its location in the condensed chromatin fraction. PMID:184436

[The biochemical mechanisms of the action of N-alkyl-N-nitrosoureas. The possible reasons for drug resistance to these compounds].

Science.gov (United States)

Syrkin, A B; Gorbacheva, L B

1996-01-01

N-alkyl-N-nitrosoureas exhibit a wide spectrum of antitumor activity. They react as alkylating agents at nucleophilic sites in purine and pyrimidine moieties of DNA. The predominant site of this alkylation is N7 of guanine, which is followed by the site N3 of adenine and 06 of guanine. The formation and persistence of 0(6)-alkylguanine (0(6)-AG) may be of primary importance in cytotoxicity of the nitrosoureas. 0(6)-AG adducts of DNA of the tumor cells are repaired by protein 0(6)-alkylguanine-DNA transferase (0(6)-AGT) which transfers the alkyl group to internal cysteine residue being the acceptor protein for the alkyl group in an irreversible transfer reaction. 0(6)-AGT can protect the tumor cells against 0(6)-AG adducts by the way of inhibiting the formation of the DNA interstrand cross-links 0(6)-AGT plays an important role in the drug resistance because it repairs the DNA alkyl adducts at the 0(6) position of guanine. The 0(6)-AGT activity inversely correlates with the cytotoxic effect of the nitrosoureas. The agents like 0(6)-methylguanosine, 0(6)-methyl-2'-deoxyguanosine, and some 0(6)-benzylated guanine derivatives are effective inactivators of 0(6)-AGT, and thus can be used to enhance the cytotoxicity of N-nitrosoureas. The activation of 0(6)-AGT and other repairing enzymes such as alpha and beta DNA-polymerases as well as an increase in the level of reduced glutathione may be used in developing the resistance to the nitrosoureas.

DNA damage induced in mouse peritoneal exudate cells after in vivo administration of chemical and physical agents as determined by alkaline elution

International Nuclear Information System (INIS)

Nishi, Yoshisuke; Miyanaga, Kumiko; Sato, Sei-ichi; Inui, Naomichi

1990-01-01

The alkaline elution technique for detecting DNA strand breaks has been applied to the study of DNA damage in mouse peritoneal exudate cells resulting from the in vivo administration of chemical and physical agents. The direct methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea induced extensive breakage in samples taken 2 h after administration. The direct ethylating agents ethyl methanesulphonate and N-ethyl-N-nitrosourea also induced DNA strand breaks, but to a lesser extent than the methylating agents. The indirect methylating agent dimethylnitrosamine showed hardly any effect in this system. A weak but positive response was observed upon treatment with the anti-neoplastic alkylating agent procarbazine hydrochloride. The whole-body irradiation of mice with 60 Co γ-rays also induced DNA strand breaks. The elution profiles for γ-ray irradiation were different from those of alkylating agents, and indicate that alkylating agents produce many more secondary lesions leading to DNA strand breaks than γ-rays. N-methyl-N-nitrosourea produced slightly more DNA strand breaks in mutagen-sensitive mice, which are derived from the CD-1 strain, than in ICR mice. (Author)

DNA damage induced in mouse peritoneal exudate cells after in vivo administration of chemical and physical agents as determined by alkaline elution

Energy Technology Data Exchange (ETDEWEB)

Nishi, Yoshisuke (Japan Tobacco Inc., Yokohama (Japan). Central Research Inst.); Miyanaga, Kumiko; Sato, Sei-ichi (Japan Tobacco Inc., Hatano, Kanagawa (Japan). Toxicology Research Lab.); Inui, Naomichi (Japan Tobacco Inc., Yokohama, Kanagawa (Japan). Pharmaceutical Research Labs.)

1990-01-01

The alkaline elution technique for detecting DNA strand breaks has been applied to the study of DNA damage in mouse peritoneal exudate cells resulting from the in vivo administration of chemical and physical agents. The direct methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea induced extensive breakage in samples taken 2 h after administration. The direct ethylating agents ethyl methanesulphonate and N-ethyl-N-nitrosourea also induced DNA strand breaks, but to a lesser extent than the methylating agents. The indirect methylating agent dimethylnitrosamine showed hardly any effect in this system. A weak but positive response was observed upon treatment with the anti-neoplastic alkylating agent procarbazine hydrochloride. The whole-body irradiation of mice with {sup 60}Co {gamma}-rays also induced DNA strand breaks. The elution profiles for {gamma}-ray irradiation were different from those of alkylating agents, and indicate that alkylating agents produce many more secondary lesions leading to DNA strand breaks than {gamma}-rays. N-methyl-N-nitrosourea produced slightly more DNA strand breaks in mutagen-sensitive mice, which are derived from the CD-1 strain, than in ICR mice. (Author).

Mechanism of the protective effects of long chain n-alkyl glucopyranosides against ultrasound-induced cytolysis of HL-60 cells.

Science.gov (United States)

Cheng, Jason Y; Riesz, Peter

2007-07-01

Recently it has been shown that long chain (C5-C8) n-alkyl glucopyranosides completely inhibit ultrasound-induced cytolysis [J.Z. Sostaric, N. Miyoshi, P. Riesz, W.G. DeGraff, and J.B. Mitchell, Free Radical Biol. Med., 39 (2005) 1539]. This protective effect has possible applications in HIFU (high intensity focused ultrasound) for tumor treatment, and in ultrasound assisted drug delivery and gene therapy. n-Alkyl glucopyranosides with hexyl (5mM), heptyl (3mM), octyl (2mM) n-alkyl chains protected 100% of HL-60 cells in vitro from 1.057 MHz ultrasound-induced cytolysis under a range of conditions that resulted in 35-100% cytolysis in the absence of glucopyranosides. However the hydrophilic methyl-beta-d-glucopyranoside did not protect cells. The surface active n-alkyl glucopyranosides accumulate at the gas-liquid interface of cavitation bubbles. The OH radicals and H atoms formed in collapsing cavitation bubbles react by H-atom abstraction from either the n-alkyl chain or the glucose moiety of the n-alkyl glucopyranosides. Owing to the high concentration of the long chain surfactants at the gas-liquid interface of cavitation bubbles, the initially formed carbon radicals on the alkyl chains are transferred to the glucose moieties to yield radicals which react with oxygen leading to the formation of hydrogen peroxide. In this work, we find that the sonochemically produced hydrogen peroxide yields from oxygen-saturated solutions of long chain (hexyl, octyl) n-alkyl glucopyranosides at 614 kHz and 1.057 MHz ultrasound increase with increasing n-alkyl glucopyranoside concentration but are independent of concentration for methyl-beta-D-glucopyranoside. These results are consistent with the previously proposed mechanism of sonoprotection [J.Z. Sostaric, N. Miyoshi, P. Riesz, W.G. DeGraff, and J.B. Mitchell, Free Radical Biol. Med., 39 (2005) 1539]. This sequence of events prevents sonodynamic cell killing by initiation of lipid peroxidation chain reactions in cellular

L-β-N-methylamino-l-alanine (BMAA) nitrosation generates a cytotoxic DNA damaging alkylating agent: An unexplored mechanism for neurodegenerative disease.

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Potjewyd, G; Day, P J; Shangula, S; Margison, G P; Povey, A C

2017-03-01

L-β-N-methylamino-l-alanine (BMAA) is a non-proteinic amino acid, that is neurotoxic in vitro and in animals, and is implicated in the causation of amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS-PDC) on Guam. Given that natural amino acids can be N-nitrosated to form toxic alkylating agents and the structural similarity of BMAA to other amino acids, our hypothesis was that N-nitrosation of BMAA might result in a toxic alkylating agent, providing a novel mechanistic hypothesis for BMAA action. We have chemically nitrosated BMAA with sodium nitrite to produce nitrosated BMAA (N-BMAA) which was shown to react with the alkyl-trapping agent, 4-(p-nitrobenzyl)pyridine, cause DNA strand breaks in vitro and was toxic to the human neuroblastoma cell line SH-SY5Y under conditions in which BMAA itself was minimally toxic. Our results indicate that N-BMAA is an alkylating agent and toxin suggesting a plausible and previously unrecognised mechanism for the neurotoxic effects of BMAA. Copyright © 2017 Elsevier B.V. All rights reserved.

Cadmium inhibits repair of UV-, methyl methanesulfonate- and N-methyl-N-nitrosourea-induced DNA damage in Chinese hamster ovary cells

International Nuclear Information System (INIS)

Fatur, Tanja; Lah, Tamara T.; Filipic, Metka

2003-01-01

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl 2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl 2 , the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl 2 the MMS-induced DNA strand breaks accumulated during the first 2 h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl 2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair

Comparison of alkylating agent and radiation carcinogenesis: some aspects of the possible involvement of effects on DNA

International Nuclear Information System (INIS)

Lawley, P.D.

1976-01-01

A series of alkylating agents was classified in terms of increasing relative ability to react at O-atom sites in DNA, MMS 6 -alkylguanines are directly miscoding bases. This series of agents was also used in a study of comparative carcinogenicity with respect to induction of thymic lymphoma in mice, a system in which x-irradiation yields tumors. A positive correlation between ability of agents to alkylate O-6 of guanine in DNA of thymus and carcinogenic potency was found. Although MMS was not active in this system, it was noted that it can induce tumors in other systems. The relationship between repair of alkylation and radiation induced damage in DNA was briefly discussed. The methylating agents induce single-strand breaks in DNA, and the principal repair system appears to fall into the category of short repair as denoted by Regan and Setlow (1974). These single-strand breaks may result from spontaneous hydrolytic depurinations of 3- and 7-methylpurines, or from enzymatic depurinations, e.g., of 3-alkyladenine and O 6 -alkylguanine. Aralkylating agents, which are also carcinogens, can evoke an alternative repair response of the uvr type

Sequence-specific DNA alkylation targeting for Kras codon 13 mutation by pyrrole-imidazole polyamide seco-CBI conjugates.

Science.gov (United States)

Taylor, Rhys Dylan; Asamitsu, Sefan; Takenaka, Tomohiro; Yamamoto, Makoto; Hashiya, Kaori; Kawamoto, Yusuke; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

2014-01-27

Hairpin N-methylpyrrole-N-methylimidazole polyamide seco-CBI conjugates 2-6 were designed for synthesis by Fmoc solid-phase synthesis, and their DNA-alkylating activities against the Kras codon 13 mutation were compared by high-resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5'-ACGTCACCA-3' (site 2), including minor 1 bp-mismatch alkylation against wild type 5'-ACGCCACCA-3' (site 3). Conjugate 6, which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5'-ACGTCACCA-3' (site 2). The hairpin polyamide seco-CBI conjugate 5 thus alkylates according to Dervan's pairing rule with the pairing recognition which β/β pair targets T-A and A-T pairs. SPR and a computer-minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATM regulates 3-methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents.

Science.gov (United States)

Agnihotri, Sameer; Burrell, Kelly; Buczkowicz, Pawel; Remke, Marc; Golbourn, Brian; Chornenkyy, Yevgen; Gajadhar, Aaron; Fernandez, Nestor A; Clarke, Ian D; Barszczyk, Mark S; Pajovic, Sanja; Ternamian, Christian; Head, Renee; Sabha, Nesrin; Sobol, Robert W; Taylor, Michael D; Rutka, James T; Jones, Chris; Dirks, Peter B; Zadeh, Gelareh; Hawkins, Cynthia

2014-10-01

Alkylating agents are a first-line therapy for the treatment of several aggressive cancers, including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed, increasing therapeutic response while minimizing toxicity. Using an siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular, the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM), were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. Inhibition of ATM and MPG-mediated BER cooperate to sensitize tumor cells to alkylating agents, impairing tumor growth in vitro and in vivo with no toxicity to normal cells, providing an ideal therapeutic window. ©2014 American Association for Cancer Research.

Mutagenesis by alkylating agents: coding properties for DNA polymerase of poly (dC) template containing 3-methylcytosine

Energy Technology Data Exchange (ETDEWEB)

Boiteux, S.; Laval, J. (Institut Gustave-Roussy, 94 - Villejuif (France))

After treatment of poly(dC) by the simple alkylating agent (/sup 3/H)dimethylsulfate, 90 percent of the radioactivity cochromatographed with 3-methylcytosine and 10 percent with 5-methylcytosine which is the normally occurring methylated base. In order to study the influence of 3-methylcytosine on DNA replication, untreated and MDS-treated poly(dC) were used as templates for E. coli DNA polymerase I. The alkylation of poly(dC) inhibits DNA chain elongation, and does not induce any mispairing under high fidelity conditions. The alteration of DNA polymerase I fidelity by manganese ions allows some replication of 3-methylcytosine which mispairs with either dAMP or dTMP. Our results suggest that 3-methylcytosine could be responsible, at least partially, for killing and the mutagenesis observed after cell treatment by alkylating agents.

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

Science.gov (United States)

Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

2015-07-09

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase.

Science.gov (United States)

Shiraishi, A; Sakumi, K; Sekiguchi, M

2000-10-01

O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

Mechanisms of action of quinone-containing alkylating agents: DNA alkylation by aziridinylquinones.

Science.gov (United States)

Hargreaves, R H; Hartley, J A; Butler, J

2000-11-01

Aziridinyl quinones can be activated by cellular reductases eg. DT-diaphorase and cytochrome P450 reductase to form highly reactive DNA alkylating agents. The mechanisms by which this activation and alkylation take place are many and varied. Using clinically relevant and experimental agents this review will describe many of these mechanisms. The agents discussed are Mitomycin C, EO9 and analogues, diaziridinylbenzoquinones and the pyrrolo[1, 2-alpha]benzimidazolequinones.

DNA damage-inducible transcripts in mammalian cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

1988-01-01

Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKβ inhibition

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Anfernee Kai-Wing Tse

2017-04-01

Full Text Available Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKKβ inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKKβ inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKKβ inhibitors with nitrosoureas can be potentially exploited for melanoma therapy.

Antimitotic antitumor agents: synthesis, structure-activity relationships, and biological characterization of N-aryl-N'-(2-chloroethyl)ureas as new selective alkylating agents.

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Mounetou, E; Legault, J; Lacroix, J; C-Gaudreault, R

2001-03-01

A series of N-aryl-N'-(2-chloroethyl)ureas (CEUs) and derivatives were synthesized and evaluated for antiproliferative activity against a wide panel of tumor cell lines. Systematic structure--activity relationship (SAR) studies indicated that: (i) a branched alkyl chain or a halogen at the 4-position of the phenyl ring or a fluorenyl/indanyl group, (ii) an exocyclic urea function, and (iii) a N'-2-chloroethyl moiety were required to ensure significant cytotoxicity. Biological experiments, such as immunofluorescence microscopy, confirmed that these promising compounds alter the cytoskeleton by inducing microtubule depolymerization via selective alkylation of beta-tubulin. Subsequent evaluations demonstrated that potent CEUs were weak alkylators, were non-DNA-damaging agents, and did not interact with the thiol function of either glutathione or glutathione reductase. Therefore, CEUs are part of a new class of antimitotic agents. Finally, among the series of CEUs evaluated, compounds 12, 15, 16, and 27 were selected for further in vivo trials.

Synthesis and evaluation of novel caged DNA alkylating agents bearing 3,4-epoxypiperidine structure.

Science.gov (United States)

Kawada, Yuji; Kodama, Tetsuya; Miyashita, Kazuyuki; Imanishi, Takeshi; Obika, Satoshi

2012-07-14

Previously, we reported that the 3,4-epoxypiperidine structure, whose design was based on the active site of DNA alkylating antitumor antibiotics, azinomycins A and B, possesses prominent DNA cleavage activity. In this report, novel caged DNA alkylating agents, which were designed to be activated by UV irradiation, were synthesized by the introduction of four photo-labile protecting groups to a 3,4-epoxypiperidine derivative. The DNA cleavage activity and cytotoxicity of the caged DNA alkylating agents were examined under UV irradiation. Four caged DNA alkylating agents showed various degrees of bioactivity depending on the photosensitivity of the protecting groups.

Repair of Alkylation Damage in Eukaryotic Chromatin Depends on Searching Ability of Alkyladenine DNA Glycosylase.

Science.gov (United States)

Zhang, Yaru; O'Brien, Patrick J

2015-11-20

Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway by excising alkylated and deaminated purine lesions. In vitro biochemical experiments demonstrate that AAG uses facilitated diffusion to efficiently search DNA to find rare sites of damage and suggest that electrostatic interactions are critical to the searching process. However, it remains an open question whether DNA searching limits the rate of DNA repair in vivo. We constructed AAG mutants with altered searching ability and measured their ability to protect yeast from alkylation damage in order to address this question. Each of the conserved arginine and lysine residues that are near the DNA binding interface were mutated, and the functional impacts were evaluated using kinetic and thermodynamic analysis. These mutations do not perturb catalysis of N-glycosidic bond cleavage, but they decrease the ability to capture rare lesion sites. Nonspecific and specific DNA binding properties are closely correlated, suggesting that the electrostatic interactions observed in the specific recognition complex are similarly important for DNA searching complexes. The ability of the mutant proteins to complement repair-deficient yeast cells is positively correlated with the ability of the proteins to search DNA in vitro, suggesting that cellular resistance to DNA alkylation is governed by the ability to find and efficiently capture cytotoxic lesions. It appears that chromosomal access is not restricted and toxic sites of alkylation damage are readily accessible to a searching protein.

Nearest neighbor affects G:C to A:T transitions induced by alkylating agents.

OpenAIRE

Glickman, B W; Horsfall, M J; Gordon, A J; Burns, P A

1987-01-01

The influence of local DNA sequence on the distribution of G:C to A:T transitions induced in the lacI gene of E. coli by a series of alkylating agents has been analyzed. In the case of nitrosoguanidine, two nitrosoureas and a nitrosamine, a strong preference for mutation at sites proceeded 5' by a purine base was noted. This preference was observed with both methyl and ethyl donors where the predicted common ultimate alkylating species is the alkyl diazonium ion. In contrast, this preference ...

Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKβ inhibition.

Science.gov (United States)

Tse, Anfernee Kai-Wing; Chen, Ying-Jie; Fu, Xiu-Qiong; Su, Tao; Li, Ting; Guo, Hui; Zhu, Pei-Li; Kwan, Hiu-Yee; Cheng, Brian Chi-Yan; Cao, Hui-Hui; Lee, Sally Kin-Wah; Fong, Wang-Fun; Yu, Zhi-Ling

2017-04-01

Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKKβ inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKKβ inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKKβ inhibitors with nitrosoureas can be potentially exploited for melanoma therapy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Sequence-selective single-molecule alkylation with a pyrrole-imidazole polyamide visualized in a DNA nanoscaffold.

Science.gov (United States)

Yoshidome, Tomofumi; Endo, Masayuki; Kashiwazaki, Gengo; Hidaka, Kumi; Bando, Toshikazu; Sugiyama, Hiroshi

2012-03-14

We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole-imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (∼200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named "five-well DNA frame" carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA.

CC-1065 and the duocarmycins: unraveling the keys to a new class of naturally derived DNA alkylating agents.

Science.gov (United States)

Boger, D L; Johnson, D S

1995-01-01

Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065, duocarmycin A, and duocarmycin SA are reviewed. Recent studies conducted with synthetic agents containing deep-seated structural changes and the unnatural enantiomers of the natural products and related analogs have defined the structural basis for the sequence-selective alkylation of duplex DNA and fundamental relationships between chemical structure, functional reactivity, and biological properties. The agents undergo a reversible, stereoelectronically controlled adenine-N3 addition to the least substituted carbon of the activated cyclopropane within selected AT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agents within the narrower, deeper AT-rich minor groove and the steric accessibility to the alkylation site that accompanies deep AT-rich minor groove penetration control the sequence-selective DNA alkylation reaction and stabilize the resulting adduct. For the agents that possess sufficient reactivity to alkylate DNA, a direct relationship between chemical or functional stability and biological potency has been defined. Images Fig. 1 Fig. 2 PMID:7731958

Inducible Alkylation of DNA by a Quinone Methide-Peptide Nucleic Acid Conjugate†

Science.gov (United States)

Liu, Yang; Rokita, Steven E.

2012-01-01

The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alkylation of non-complementary sequences is only possible when a template strand is present to co-localize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self adducts formed between the PNA and its attached QM remained active and reversible over more than eight days in aqueous solution prior to reaction with a chosen target added subsequently. PMID:22243337

Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells

International Nuclear Information System (INIS)

Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin

2016-01-01

Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O 6 -methylguanine–DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1 h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24 h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair. - Highlights: • Nitrogen mustard-induced MGMT-DNA cross-linking was detected in a living cell. • Concentration- and time-dependent manners of MGMT-DNA cross-linking were revealed. • Proteolysis played an important role in protein (MGMT)-DNA cross-linking repair. • DVC1 acts as a proteolytic enzyme in cross-linking repair in a p

The role of the HCR system in the repair of lethal lesions of Bacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

International Nuclear Information System (INIS)

Vizdalova, M.; Janovska, E.; Zhestyanikov, V.D.

1980-01-01

The role of the HCR system in the repair of prelethal lesions induced by UV light, γ radiation and alkylating agents was studied in the Bacillus subtilis SPP1 phage, its heat sensitive mutants (N3, N73 nad ts 1 ) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher in hcr + cells than in hcr cells, the differences being more striking in intact phages than in their transfecting DNA's. Repair inhibitors reduce survival in hcr + cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growing hcr + cells but has no effect on its survival in competent hcr + cells; acriflavin and ethidium bromide decrease the survival of the UV-irradiated SPP1 phage in both exponentially growing and competent hcr + cells to the level of survival observed in hcr cells; moreover, ethidium bromide lowers the number of infective centres in hcr + cells of the UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of the UV-irradiated phages or their DNA in hcr cells. The repair mechanism under study also effectively repairs lesions induced by polyfunctional alkylating agents in the transfecting DNA's of B. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in the phages and their DNA by ethyl methanesulphonate or γ radiation. (author)

Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3'-5')thymidine.

Science.gov (United States)

Swenson, D H; Lawley, P D

1978-01-01

1. The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates. PMID:208508

O6-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C

International Nuclear Information System (INIS)

Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

2006-01-01

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O 6 -methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC 5 values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N 7 guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism

O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

Science.gov (United States)

Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

2006-03-01

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

DNA-Directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles.

Science.gov (United States)

Atwell, G J; Fan, J Y; Tan, K; Denny, W A

1998-11-19

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine dicarboxylates, coupling of these with the desired carrier, and reduction to give the required bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators, followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5'-NCCN, 5'-NGCN and 5'-NCGN sequences in the PCR stopping assay ( indicates block sites). The data suggest that these targeted compounds, like the known thioimidazole bis(carbamate) carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic (IC50s >1 microM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic than the corresponding minor-groove-targeted ones. This was true also for the more reactive thioimidazole bis(carbamates) (IC50s 0.8 and 11 microM, respectively), but both were more active than the analogous "untargeted" carmethizole (IC50 20

Parp1 protects against Aag-dependent alkylation-induced nephrotoxicity in a sex-dependent manner.

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Calvo, Jennifer A; Allocca, Mariacarmela; Fake, Kimberly R; Muthupalani, Sureshkumar; Corrigan, Joshua J; Bronson, Roderick T; Samson, Leona D

2016-07-19

Nephrotoxicity is a common toxic side-effect of chemotherapeutic alkylating agents. Although the base excision repair (BER) pathway is essential in repairing DNA alkylation damage, under certain conditions the initiation of BER produces toxic repair intermediates that damage healthy tissues. We have shown that the alkyladenine DNA glycosylase, Aag (a.k.a. Mpg), an enzyme that initiates BER, mediates alkylation-induced whole-animal lethality and cytotoxicity in the pancreas, spleen, retina, and cerebellum, but not in the kidney. Cytotoxicity in both wild-type and Aag-transgenic mice (AagTg) was abrogated in the absence of Poly(ADP-ribose) polymerase-1 (Parp1). Here we report that Parp1-deficient mice expressing increased Aag (AagTg/Parp1-/-) develop sex-dependent kidney failure upon exposure to the alkylating agent, methyl methanesulfonate (MMS), and suffer increased whole-animal lethality compared to AagTg and wild-type mice. Macroscopic, histological, electron microscopic and immunohistochemical analyses revealed morphological kidney damage including dilated tubules, proteinaceous casts, vacuolation, collapse of the glomerular tuft, and deterioration of podocyte structure. Moreover, mice exhibited clinical signs of kidney disease indicating functional damage, including elevated blood nitrogen urea and creatinine, hypoproteinemia and proteinuria. Pharmacological Parp inhibition in AagTg mice also resulted in sensitivity to MMS-induced nephrotoxicity. These findings provide in vivo evidence that Parp1 modulates Aag-dependent MMS-induced nephrotoxicity in a sex-dependent manner and highlight the critical roles that Aag-initiated BER and Parp1 may play in determining the side-effects of chemotherapeutic alkylating agents.

Quantitative estimation of the extent of alkylation of DNA following treatment of mammalian cells with non-radioactive alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Snyder, R.D. (Univ. of Tennessee, Oak Ridge); Regan, J.D.

1981-01-01

Alkaline sucrose sedimentation has been used to quantitate phosphotriester formation following treatment of human cells with the monofunctional alkylating agents methyl and ethyl methanesulfonate. These persistent alkaline-labile lesions are not repaired during short-term culture conditions and thus serve as a useful and precise index of the total alkylation of the DNA.Estimates of alkylation by this procedure compare favorably with direct estimates by use of labeled alkylating agents.

VP-16 and alkylating agents activate a common metabolic pathway for suppression of DNA replication

International Nuclear Information System (INIS)

Das, S.K.; Berger, N.A.

1986-01-01

The cytotoxic effects of etoposide (VP-16) are mediated by topoisomerase II production of protein crosslinked DNA strand breaks. Previous studies have shown that alkylating agent induced DNA damage results in expansion of dTTP pools and reduction of dCTP pools and DNA replication. Studies were conducted with V79 cells to determine whether the metabolic consequences of VP-16 treatment were similar to those induced by alkylating agents. Treatment with 0.5μM VP-16 prolonged the doubling time of V79 cells from 12 to 18 hrs and caused cell volume to increase from 1.1 to 1.6 x 10 -12 l. 2mM caffeine completely blocked the volume increase and substantially prevented the prolongation of doubling time. 5μM VP-16 reduced the rate of [ 3 H]TdR incorporation by 70%, whereas in the presence of 2mM caffeine, VP-16 caused only a 10% decrease in the rate of [ 3 H]TdR incorporation. 4 hr treatment with 5.0μM VP-16 increased dTTP levels from 65 +/- 10 pmol/10 6 cells to 80 +/- 13 pmol/10 6 cells and caused dCTP level to decline from 113 +/- 23 pmol/10 6 cells to 92 +/- 17 pmol/10 6 cells. These results indicate that the metabolic consequences of VP-16 treatment are similar to alkylating agent treatment and that an increase in dTTP pools with a subsequent effect on ribonucleotide reductase may be a final common pathway by which many cytotoxic agents suppress DNA synthesis

MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents.

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Kaina, Bernd; Christmann, Markus; Naumann, Steffen; Roos, Wynand P

2007-08-01

O(6)-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against alkylating agents that generate, among other lesions, O(6)-alkylguanine in DNA (collectively termed O(6)-alkylating agents [O(6)AA]). The defense is highly important, since O(6)AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O(6)AA induced DNA damage is subject to repair, which is executed by MGMT, AlkB homologous proteins (ABH) and base excision repair (BER). Although this review focuses on MGMT, the mechanism of repair by ABH and BER will also be discussed. Experimental systems, in which MGMT has been modulated, revealed that O(6)-methylguanine (O(6)MeG) and O(6)-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O(6)MeG-induced clastogenicity and cell death require MutS alpha-dependent mismatch repair (MMR), whereas O(6)-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O(6)MeG to provoke cytotoxicity. In MGMT depleted cells, O(6)MeG induces apoptosis almost exclusively, barely any necrosis, which is presumably due to the remarkable ability of secondarily formed DNA double-strand breaks (DSBs) to trigger apoptosis via ATM/ATR, Chk1, Chk2, p53 and p73. Depending on the cellular background, O(6)MeG activates both the death receptor and the mitochondrial apoptotic pathway. The inter-individual expression of MGMT in human lymphocytes is highly variable. Given the key role of MGMT in cellular defense, determination of MGMT activity could be useful for assessing a patient's drug sensitivity. MGMT is expressed at highly variable amounts in human tumors. In gliomas, a correlation was found between MGMT activity, MGMT promoter methylation and response to O(6)AA. Although the human MGMT gene is inducible by glucocorticoids and genotoxins such as radiation and

Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.

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Cai, Shanbao; Xu, Yi; Cooper, Ryan J; Ferkowicz, Michael J; Hartwell, Jennifer R; Pollok, Karen E; Kelley, Mark R

2005-04-15

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

Immunological detection and quantification of DNA components structurally modified by alkylating carcinogens, mutagens and chemotherapeutic agents

International Nuclear Information System (INIS)

Rajewsky, M.F.

1983-01-01

The detection and quantification of defined reaction products of chemical mutagens and carcinogens (and of many cancer chemotherapeutic agents) with DNA require highly sensitive analytical techniques. The exceptional capability of immunoglobulins to recognize subtle alterations of molecular structure (especially when monoclonal antibodies are used to maximize specificity), outstanding sensitivity of immunoanalysis by high-affinity antibodies, and the fact that radioactively-labelled agents are not required suggest the utility of a radioimmunoassay to recognize and quantitate alkylated DNA products. We have recently developed a set of high-affinity monoclonal antibodies (secreted by mouse x mouse as well as by rat x rat hybridomas; antibody affinity constants, 10 9 to > 10 10 lmol) specifically directed against several DNA alkylation products with possible relevance in relation to both mutagenesis and malignant transformation of mammalian cells. These alkylation products include 0 6 -N-butyldeoxyguanosine, and 0 4 -ethyldeoxythymidine. When used in a radioimmunassay, an antibody specific for 0 6 -ethyldeoxyguanosine, for example, will detect this product at an 0 6 -ethyldeoxyguanosine/deoxyguanosine molar ratio of approx. 3 x 10 -7 in a hydrolysate of 100 ug of DNA. The limit of detection can be lowered further if the respective alkyldeoxynucleosides are separated by HPLC from the DNA hydrolysate prior to the RIA. The anti-alkyldeoxynucleoside monoclonal antibodies can also be used to visualize, by immunostaining and fluorescence microscopy combined with electronic image intensification, specific alkylation products in the nuclear DNA of individual cells, and to localize structurally modified bases in double-stranded DNA molecules by transmission electron microscopy

[Alkylating agents].

Science.gov (United States)

Pourquier, Philippe

2011-11-01

With the approval of mechlorethamine by the FDA in 1949 for the treatment of hematologic malignancies, alkylating agents are the oldest class of anticancer agents. Even though their clinical use is far beyond the use of new targeted therapies, they still occupy a major place in specific indications and sometimes represent the unique option for the treatment of refractory diseases. Here, we are reviewing the major classes of alkylating agents and their mechanism of action, with a particular emphasis for the new generations of alkylating agents. As for most of the chemotherapeutic agents used in the clinic, these compounds are derived from natural sources. With a complex but original mechanism of action, they represent new interesting alternatives for the clinicians, especially for tumors that are resistant to conventional DNA damaging agents. We also briefly describe the different strategies that have been or are currently developed to potentiate the use of classical alkylating agents, especially the inhibition of pathways that are involved in the repair of DNA lesions induced by these agents. In this line, the development of PARP inhibitors is a striking example of the recent regain of interest towards the "old" alkylating agents.

DNA unwinding by ASCC3 helicase is coupled to ALKBH3 dependent DNA alkylation repair and cancer cell proliferation

Science.gov (United States)

Dango, Sebastian; Mosammaparast, Nima; Sowa, Mathew E.; Xiong, Li-Jun; Wu, Feizhen; Park, Keyjung; Rubin, Mark; Gygi, Steve; Harper, J. Wade; Shi, Yang

2011-01-01

Summary Demethylation by the AlkB dioxygenases represents an important mechanism for repair of N-alkylated nucleotides. However, little is known about their functions in mammalian cells. We report the purification of the ALKBH3 complex and demonstrate its association with the Activating Signal Co-integrator Complex (ASCC). ALKBH3 is overexpressed in various cancers, and both ALKBH3 and ASCC are important for alkylation damage resistance in these tumor cell lines. ASCC3, the largest subunit of ASCC, encodes a 3′-5′ DNA helicase, whose activity is crucial for the generation of single-stranded DNA upon which ALKBH3 preferentially functions for dealkylation. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 leads to increased 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. Our data provide a molecular mechanism by which ALKBH3 collaborates with ASCC to maintain genomic integrity in a cell type specific manner. PMID:22055184

The Fanconi anemia pathway sensitizes to DNA alkylating agents by inducing JNK-p53-dependent mitochondrial apoptosis in breast cancer cells.

Science.gov (United States)

Zhao, Lin; Li, Yanlin; He, Miao; Song, Zhiguo; Lin, Shu; Yu, Zhaojin; Bai, Xuefeng; Wang, Enhua; Wei, Minjie

2014-07-01

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to DNA alkylating agents and greatly influences drug response in cancer treatment. However, the molecular mechanisms underlying the FA/BRCA pathway reversed resistance have received limited attention. In the present study, we investigated the effect of Fanconi anemia complementation group F protein (FANCF), a critical factor of the FA/BRCA pathway, on cancer cell apoptosis induced by DNA alkylating agents such as mitomycin c (MMC). We found that FANCF shRNA potentiated MMC-induced cytotoxicity and apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. At a mechanistic level, FANCF shRNA downregulated the anti-apoptotic protein Bcl-2 and upregulated the pro-apoptotic protein Bax, accompanied by release of cyt-c and smac into the cytosol in MMC-treated cells. Furthermore, activation of caspase-3 and -9, other than caspase-8, cleavage of poly(ADP ribose) polymerase (PARP), and a decrease of mitochondrial membrane potential (MMP) indicated that involvement of the mitochondrial apoptotic pathway in FANCF silencing of MMC-treated breast cancer cells. A decrease in IAP family proteins XIAP and survivin were also observed following FANCF silencing in MMC-treated breast cancer cells. Notably, FANCF shRNA was able to increase p53 levels through activation of the JNK pathway in MMC-treated breast cancer cells. Furthermore, p53 inhibition using pifithrin-α abolished the induction of caspase-3 and PARP by FANCF shRNA and MMC, indicating that MMC-induced apoptosis is substantially enhanced by FANCF shRNA via p53-dependent mechanisms. To our knowledge, we provide new evidence for the potential application of FANCF as a chemosensitizer in breast cancer therapy.

Effects of inhibitors of DNA repair on the frequencies of chromosomal aberrations induced by x-rays or alkylating agents in cultured human lymphocytes

International Nuclear Information System (INIS)

Kihlman, B.A.; Andersson, H.C.

1986-01-01

In the first part of this presentation the authors give examples of the synergistic enhancements that are obtained with various inhibitor combinations in G/sub 2/. The second part of the presentation deals with the effects of two agents, also well known for their capacity to potentiate the frequency of chromosomal aberrations induced by physical and chemical agents, but with a different mechanism of action. These agents are caffeine and 3-aminobenzamide (3AB). Caffeine has for decades been used as an inhibitor of DNA repair although its mechanism of action has not been fully understood. 3AB has more recently come into focus as an efficient inhibitor of the synthesis of poly-(ADP-ribose), a substance believed to be of importance in connection with the repair of certain types of DNA damage. The results presented do not quite fit in with the general idea about the mode of action of these agents. All experiments were carried out with whole-blood cultures of human lymphocytes. When inhibitors were used as post-treatments, chromosomal aberrations were induced by X-rays or by the alkylating agents thiotepa (TT) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). X-rays were generated by a Siemens Stabilipan 200 apparatus, at a dose rate of 0.5 Gy/min. The tube (TR 200f) was operated at 180 kV, 10 mA and the radiation filtered through 4 mm Al

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila.

Science.gov (United States)

Vogel, E W; Dusenbery, R L; Smith, P D

1985-04-01

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in

Determination of alkylation of bacterial DNA as a rapid test for toxicological evaluation of alkylating xenobiotic agents

Energy Technology Data Exchange (ETDEWEB)

Botzenhart, K.; Waldner-Sander, S.; Schweinsberg, F.

1986-05-01

Alkylated purine bases from hydrolized DNA can be separated by HPLC and quantified with a fluorescence detector. We applied this method to bacterial DNA. 7-methylguanine was detected after treatment of Serratia marcescens with iodoacetamide, dimethyl sulfate and with polluted air.

N-Alkylation Using Sodium Triacetoxyborohydride with Carboxylic Acids as Alkyl Sources.

Science.gov (United States)

Tamura, Satoru; Sato, Keigo; Kawano, Tomikazu

2018-01-01

A versatile N-alkylation was performed using sodium triacetoxyborohydride and carboxylic acid as an alkyl source. The combination of these reagents furnished products different from those given previously by a similar reaction. Moreover, the mild conditions of our method allowed some functional groups to remain through the reaction, whereas they would react and be converted into other moieties in the similar reductive N-alkylation reported previously. Herein, we provide a new procedure for the preparation of various compounds containing nitrogen atoms.

Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents.

Science.gov (United States)

Hickman, M J; Samson, L D

1999-09-14

All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O(6)-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O(6)-methylguanine (O(6)MeG) and O(6)-chloroethylguanine (O(6)CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSalpha branch of the DNA mismatch repair pathway (but not the MutSbeta branch) is absolutely required for signaling the initiation of apoptosis in response to O(6)MeGs and is partially required for signaling apoptosis in response to O(6)CEGs. Further, O(6)MeG lesions signal the stabilization of the p53 tumor suppressor, and such signaling is also MutSalpha-dependent. Despite this, MutSalpha-dependent apoptosis can be executed in a p53-independent manner. DNA mismatch repair status did not influence the response of cells to other inducers of p53 and apoptosis. Thus, it appears that mismatch repair status, rather than p53 status, is a strong indicator of the susceptibility of cells to alkylation-induced apoptosis. This experimental system will allow dissection of the signal transduction events that couple a specific type of DNA base lesion with the final outcome of apoptotic cell death.

Toward hypoxia-selective DNA-alkylating agents built by grafting nitrogen mustards onto the bioreductively activated, hypoxia-selective DNA-oxidizing agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine).

Science.gov (United States)

Johnson, Kevin M; Parsons, Zachary D; Barnes, Charles L; Gates, Kent S

2014-08-15

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a heterocyclic di-N-oxide that undergoes enzymatic deoxygenation selectively in the oxygen-poor (hypoxic) cells found in solid tumors to generate a mono-N-oxide metabolite. This work explored the idea that the electronic changes resulting from the metabolic deoxygenation of tirapazamine analogues might be exploited to activate a DNA-alkylating species selectively in hypoxic tissue. Toward this end, tirapazamine analogues bearing nitrogen mustard units were prepared. In the case of the tirapazamine analogue 18a bearing a nitrogen mustard unit at the 6-position, it was found that removal of the 4-oxide from the parent di-N-oxide to generate the mono-N-oxide analogue 17a did indeed cause a substantial increase in reactivity of the mustard unit, as measured by hydrolysis rates and DNA-alkylation yields. Hammett sigma values were measured to quantitatively assess the magnitude of the electronic changes induced by metabolic deoxygenation of the 3-amino-1,2,4-benzotriazine 1,4-dioxide heterocycle. The results provide evidence that the 1,2,4-benzotiazine 1,4-dioxide unit can serve as an oxygen-sensing prodrug platform for the selective unmasking of bioactive agents in hypoxic cells.

Oxidants and not alkylating agents induce rapid mtDNA loss and mitochondrial dysfunction

Science.gov (United States)

Furda, Amy M.; Marrangoni, Adele M.; Lokshin, Anna; Van Houten, Bennett

2013-01-01

Mitochondrial DNA (mtDNA) is essential for proper mitochondrial function and encodes 22 tRNAs, 2 rRNAs and 13 polypeptides that make up subunits of complex I, III, IV, in the electron transport chain and complex V, the ATP synthase. Although mitochondrial dysfunction has been implicated in processes such as premature aging, neurodegeneration, and cancer, it has not been shown whether persistent mtDNA damage causes a loss of oxidative phosphorylation. We addressed this question by treating mouse embryonic fibroblasts with either hydrogen peroxide (H2O2) or the alkylating agent methyl methanesulfonate (MMS) and measuring several endpoints, including mtDNA damage and repair rates using QPCR, levels of mitochondrial- and nuclear-encoded proteins using antibody analysis, and a pharmacologic profile of mitochondria using the Seahorse Extracellular Flux Analyzer. We show that a 60 min treatment with H2O2 causes persistent mtDNA lesions, mtDNA loss, decreased levels of a nuclear-encoded mitochondrial subunit, a loss of ATP-linked oxidative phosphorylation and a loss of total reserve capacity. Conversely, a 60 min treatment with 2 mM MMS causes persistent mtDNA lesions but no mtDNA loss, no decrease in levels of a nuclear-encoded mitochondrial subunit, and no mitochondrial dysfunction. These results suggest that persistent mtDNA damage is not sufficient to cause mitochondrial dysfunction. PMID:22766155

40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for the...

Alkylation induced cerebellar degeneration dependent on Aag and Parp1 does not occur via previously established cell death mechanisms.

Directory of Open Access Journals (Sweden)

Carrie M Margulies

Full Text Available Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg. Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose polymerase 1 (Parp1. Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD+ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.

Alkylation induced cerebellar degeneration dependent on Aag and Parp1 does not occur via previously established cell death mechanisms.

Science.gov (United States)

Margulies, Carrie M; Chaim, Isaac Alexander; Mazumder, Aprotim; Criscione, June; Samson, Leona D

2017-01-01

Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER) pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg). Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS) undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose) polymerase 1 (Parp1). Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN) sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD+ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.

Nature of chromosome gaps induced by alkylating agents and γ-rays as revealed by caffeine treatment

International Nuclear Information System (INIS)

Dimitrov, B.

1981-01-01

In the cells of primary roots of Crepis capillaris, post-treatment with caffeine increased the frequency of gaps and chromosomal aberrations induced by the alkylating agents ethyleneimine and N-nitroso-N-methylurethane and γ-rays. The increase in the frequency of gaps was considerably lower than that observed in chromosomal aberrations, this being more strongly expressed in the case fo the alkylating agents. The potentiating effect of caffeine on the γ-ray-induced chromosomal gaps was a little higher in S as compared in G 2 . These results lead to the conclusion that the alkylating agents and the γ-rays might induce 2 types of chromosomal gap. (orig.)

Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.

Science.gov (United States)

Jansen, M; Bardenheuer, W; Sorg, U R; Seeber, S; Flasshove, M; Moritz, T

2001-07-01

Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

Role of gene 59 of bacteriophage T4 in repair of uv-irradiated and alkylated DNA in vivo

International Nuclear Information System (INIS)

Wu, R.; Wu, J.L.; Yeh, Y.C.

1975-01-01

Nonsense mutants in gene 59 (amC5, am HL628) were used to study the role of this gene in the repair of uv-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to uv irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after uv irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to uv irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that uv repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B(su - ), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of uv lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Hammond, R.A.; Miller, M.R.; McClung, J.K.

1990-01-01

The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

N-Alkylation by Hydrogen Autotransfer Reactions.

Science.gov (United States)

Ma, Xiantao; Su, Chenliang; Xu, Qing

2016-06-01

Owing to the importance of amine/amide derivatives in all fields of chemistry, and also the green and environmentally benign features of using alcohols as alkylating reagents, the relatively high atom economic dehydrative N-alkylation reactions of amines/amides with alcohols through hydrogen autotransfer processes have received much attention and have developed rapidly in recent decades. Various efficient homogeneous and heterogeneous transition metal catalysts, nano materials, electrochemical methods, biomimetic methods, asymmetric N-alkylation reactions, aerobic oxidative methods, and even certain transition metal-free, catalyst-free, or autocatalyzed methods, have also been developed in recent years. With a brief introduction to the background and developments in this area of research, this chapter focuses mainly on recent progress and technical and conceptual advances contributing to the development of this research in the last decade. In addition to mainstream research on homogeneous and heterogeneous transition metal-catalyzed reactions, possible mechanistic routes for hydrogen transfer and alcohol activation, which are key processes in N-alkylation reactions but seldom discussed in the past, the recent reports on computational mechanistic studies of the N-alkylation reactions, and the newly emerged N-alkylation methods based on novel alcohol activation protocols such as air-promoted reactions and transition metal-free methods, are also reviewed in this chapter. Problems and bottlenecks that remained to be solved in the field, and promising new research that deserves greater future attention and effort, are also reviewed and discussed.

Nearest neighbor affects G:C to A:T transitions induced by alkylating agents.

Science.gov (United States)

Glickman, B W; Horsfall, M J; Gordon, A J; Burns, P A

1987-01-01

The influence of local DNA sequence on the distribution of G:C to A:T transitions induced in the lacI gene of E. coli by a series of alkylating agents has been analyzed. In the case of nitrosoguanidine, two nitrosoureas and a nitrosamine, a strong preference for mutation at sites proceeded 5' by a purine base was noted. This preference was observed with both methyl and ethyl donors where the predicted common ultimate alkylating species is the alkyl diazonium ion. In contrast, this preference was not seen following treatment with ethylmethanesulfonate. The observed preference for 5'PuG-3' site over 5'-PyG-3' sites corresponds well with alterations observed in the Ha-ras oncogene recovered after treatment with NMU. This indicates that the mutations recovered in the oncogenes are likely the direct consequence of the alkylation treatment and that the local sequence effects seen in E. coli also appear to occur in mammalian cells. PMID:3329097

Nearest neighbor affects G:C to A:T transitions induced by alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Glickman, B.W.; Horsfall, M.J.; Gordon, A.J.E.; Burns, P.A.

1987-12-01

The influence of local DNA sequence on the distribution of G:C to A:T transitions induced in the lacI gene of E. coli by a series of alkylating agents has been analyzed. In the case of nitrosoguanidine, two nitrosoureas and a nitrosamine, a strong preference for mutation at sites proceeded 5' by a purine base was noted. This preferences was observed with both methyl and ethyl donors where the predicted common ultimate alkylating species in the alkyl diazonium ion. In contrast, this preferences was not seen following treatment with ethylmethanesulfonate. The observed preference for 5'PuG-3' site over 5'-PyG-3' sites corresponds well with alterations observed in the Ha-ras oncogene recovered after treatment with NMU. This indicates that the mutations recovered in the oncogenes are likely the direct consequence of the alkylation treatment and that the local sequence effects seen in E. coli also appear to occur in mammalian cells.

Regioselective 1-N-Alkylation and Rearrangement of Adenosine Derivatives.

Science.gov (United States)

Oslovsky, Vladimir E; Drenichev, Mikhail S; Mikhailov, Sergey N

2015-01-01

Several methods for the preparation of some N(6)-substituted adenosines based on selective 1-N-alkylation with subsequent Dimroth rearrangement were developed. The proposed methods seem to be effective for the preparation of natural N(6)-isopentenyl- and N(6)-benzyladenosines, which are known to possess pronounced biological activities. Direct 1-N-alkylation of 2',3',5'-tri-O-acetyladenosine and 3',5'-di-O-acetyl-2'-deoxyadenosine with alkyl halides in N,N-dimethylformamide (DMF) in the presence of BaCO3 and KI gave 1-N-substituted derivatives with quantitative yields, whereas 1-N-alkylation of adenosine was accompanied by significant O-alkylation. Moreover, the reaction of trimethylsilyl derivatives of N(6)-acetyl-2',3',5'-tri-O-acetyladenosine and N(6)-acetyl-3',5'-di-O-acetyl-2'-deoxyadenosine with alkyl halides leads to the formation of the stable 1-N-substituted adenosines. Dimroth rearrangement of 1-N-substituted adenosines in aqueous ammonia yields pure N(6)-substituted adenosines.

DFT investigations of phosphotriesters hydrolysis in aqueous solution: a model for DNA single strand scission induced by N-nitrosoureas.

Science.gov (United States)

Liu, Tingting; Zhao, Lijiao; Zhong, Rugang

2013-02-01

DNA phosphotriester adducts are common alkylation products of DNA phosphodiester moiety induced by N-nitrosoureas. The 2-hydroxyethyl phosphotriester was reported to hydrolyze more rapidly than other alkyl phosphotriesters both in neutral and in alkaline conditions, which can cause DNA single strand scission. In this work, DFT calculations have been employed to map out the four lowest activation free-energy profiles for neutral and alkaline hydrolysis of triethyl phosphate (TEP) and diethyl 2-hydroxyethyl phosphate (DEHEP). All the hydrolysis pathways were illuminated to be stepwise involving an acyclic or cyclic phosphorane intermediate for TEP or DEHEP, respectively. The rate-limiting step for all the hydrolysis reactions was found to be the formation of phosphorane intermediate, with the exception of DEHEP hydrolysis in alkaline conditions that the decomposition process turned out to be the rate-limiting step, owing to the extraordinary low formation barrier of cyclic phosphorane intermediate catalyzed by hydroxide. The rate-limiting barriers obtained for the four reactions are all consistent with the available experimental information concerning the corresponding hydrolysis reactions of phosphotriesters. Our calculations performed on the phosphate triesters hydrolysis predict that the lower formation barriers of cyclic phosphorane intermediates compared to its acyclic counter-part should be the dominant factor governing the hydrolysis rate enhancement of DEHEP relative to TEP both in neutral and in alkaline conditions.

Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase.

Science.gov (United States)

Paik, Johanna; Duncan, Tod; Lindahl, Tomas; Sedgwick, Barbara

2005-11-15

One of the major cytotoxic lesions generated by alkylating agents is DNA 3-alkyladenine, which can be excised by 3-alkyladenine DNA glycosylase (AAG). Inhibition of AAG may therefore result in increased cellular sensitivity to chemotherapeutic alkylating agents. To investigate this possibility, we have examined the role of AAG in protecting human tumor cells against such agents. Plasmids that express small interfering RNAs targeted to two different regions of AAG mRNA were transfected into HeLa cervical carcinoma cells and A2780-SCA ovarian carcinoma cells. Stable derivatives of both cell types with low AAG protein levels were sensitized to alkylating agents. Two HeLa cell lines with AAG protein levels reduced by at least 80% to 90% displayed a 5- to 10-fold increase in sensitivity to methyl methanesulfonate, N-methyl-N-nitrosourea, and the chemotherapeutic drugs temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea. These cells showed no increase in sensitivity to UV light or ionizing radiation. After treatment with methyl methanesulfonate, AAG knockdown HeLa cells were delayed in S phase but accumulated in G2-M. Our data support the hypothesis that ablation of AAG activity in human tumor cells may provide a useful strategy to enhance the efficacy of current chemotherapeutic regimens that include alkylating agents.

Bifunctional Rhodium Intercalator Conjugates as Mismatch-Directing DNA Alkylating Agents

OpenAIRE

Schatzschneider, Ulrich; Barton, Jacqueline K.

2004-01-01

A conjugate of a DNA mismatch-specific rhodium intercalator, containing the bulky chrysenediimine ligand, and an aniline mustard has been prepared, and targeting of mismatches in DNA by this conjugate has been examined. The preferential alkylation of mismatched over fully matched DNA is found by a mobility shift assay at concentrations where untethered organic mustards show little reaction. The binding site of the Rh intercalator was determined by DNA photocleavage, and the position of covale...

Iminium ion chemistry of mitosene DNA alkylating agents. Enriched 13C NMR and isolation studies.

Science.gov (United States)

Ouyang, A; Skibo, E B

2000-05-16

Described herein is a study of the reductive alkylation chemistry of mitosene antitumor agents. We employed a 13C-enriched electrophilic center to probe the fate of the iminium ion resulting from reductive activation. The 13C-labeled center permitted the identification of complex products resulting from alkylation reactions. In the case of DNA reductive alkylation, the type and number of alkylation sites were readily assessed by 13C NMR. Although there has been much excellent work done in the area of mitosene chemistry and biochemistry, the present study provides a number of new findings: (1) The major fate of the iminium ion is head-to-tail polymerization, even in dilute solutions. (2) Dithionite reductive activation results in the formation of mitosene sulfite esters as well as the previously observed sulfonate adducts. (3) The mitosene iminium ion alkylates the adenosine 6-amino group as well as the guanosine 2-amino group. The identification of the latter adduct was greatly facilitated by the 13C-label at the electrophilic center. (4) The mitosene iminium ion alkylates DNA at both nitrogen and oxygen centers without any apparent base selectivity. The complexity of mitosene reductive alkylation of DNA will require continued adduct isolation studies.

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids.

Science.gov (United States)

Lawley, P D; Brookes, P

1968-09-01

. Effects of the sulphur mustards on nucleic acid synthesis in sensitive and resistant strains were studied. DNA synthesis was inhibited in both strains at low doses in a dose-dependent manner, but RNA and protein synthesis were not affected in this way. 9. DNA synthesis in E. coli B(s-1) was permanently inhibited by low doses of mustards. In the resistant strains 15T(-) and B/r a characteristic recovery in DNA synthesis was observed after a dose-dependent time-lag. This effect could be shown at low doses in the region of the mean lethal dose. 10. Cellular DNA was isotopically prelabelled and the effect of mustards on stability of DNA was investigated. With resistant strains a dose-dependent release of DNA nucleotide material into acid-soluble form was found; this was much more extensive with the difunctional mustard (about 400 nucleotides released per DNA alkylation) than with the monofunctional mustard (about 10 nucleotides per alkylation). With the sensitive strain no dose-dependent release was found, though the DNA was less stable independent of cellular alkylation. 11. The results are discussed in terms of the concepts that alkylation of cellular DNA induces lesions which interfere with DNA replication, but which can be enzymically ;repaired'. The possible nature of these lesions is discussed in terms of the known reactions of the alkylating agents with DNA.

DNA alkylation and tumor induction in regenerating rat liver after cell cycle-related continuous N-nitrosodimethylamine infusion

Energy Technology Data Exchange (ETDEWEB)

Rabes, H.M.; Kerler, R.; Wilhelm, R.

1983-01-01

Synchronized regenerating rat liver after partial hepatectomy was used to study cell cycle-related DNA base alkylation and liver carcinogenesis. A continuous iv infusion of (/sup 14/C)N-nitrosodimethylamine (DMN) at a dose of 0.5 mg/kg/hour was given to inbred male Wistar Af/Han rats over a period of 8 hours either during the G1 phase, hydroxyurea-synchronized DNA synthesis, or the G2+M-phase of regenerating liver or to untreated rats (G0-phase liver--carcinogen dose, 1.5 mg/kg/hour). Two hours after the end of the infusion, the amount of 7-methylguanine was highest in the G0-phase liver, with a decrease in the G1 phase, the S-phase, and the G2+M-phase. After continuous DMN exposure, the O/sub 6/-methylguanine:7-methylguanine ratio was lower in the S-phase and G2+M-phase livers than in the G0-phase and G1-phase livers, indicating an increased O/sub 6/-methylguanine repair during DNA synthesis and the G2+M-phase. Liver tumors in rats treated by continuous DMN infusion either during the G0 phase or the S-phase developed only after carcinogen exposure during DNA synthesis.

Possible targets for the aneugenic activity of alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Pellerano, P. [IST-National Institute for Research on Cancer, Genova (Italy); Abbondandolo, A. [Univ. of Genova (Italy); Bonatti, S.; Simili, M. [CNR Institute of Mutagenesis and Differentiation, Pisa (Italy)

1993-12-31

Alkylating agents have been of invaluable help in mutation research for half a century. In all tested organisms, they have proved able to induce a large variety of genetic effects, including aneuploidy. Credible molecular models exist to explain the ability of alkylating agents to induce gene mutation and to act as initiators in carcinogenesis as a consequence of DNA alkylation at specific sites. On the contrary, neither the mechanism of aneuploidy induction nor the relevant cellular targets are known.

Alkylation of N-substituted 2-phenylacetamides

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SLOBODAN D. PETROVIC

2004-10-01

Full Text Available Various N-substituted phenylacetamides were alkylated using different alkylating agents under neutral and basic conditions. Reactions were performed at different reaction temperatures and in various solvents. Also, a number of various catalysts were used including phase-transfer catalysts. Reactions were followed using GC or GC-MS technique and the presence as well as the yields of the alkylation products were established. Generally, the best yield and high selectivity in the studied reactions were achieved under basic conditions where in the certain cases some products, mostly N-product, were obtained solely in quantitative yields.

Effect O6-guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations.

Science.gov (United States)

Kara, Mahmut; Drsata, Tomas; Lankas, Filip; Zacharias, Martin

2015-01-01

Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O(6)-alkylguanin-DNA-Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O(6)-alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O(6)-methyl-guanine (O(6)-MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O(6)-MeG:C or O(6)-MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O(6)-MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O(6)-MeG:C or O(6)-MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes. © 2014 Wiley Periodicals, Inc.

Mechanisms of chemoresistance to alkylating agents in malignant glioma.

Science.gov (United States)

Sarkaria, Jann N; Kitange, Gaspar J; James, C David; Plummer, Ruth; Calvert, Hilary; Weller, Michael; Wick, Wolfgang

2008-05-15

Intrinsic or acquired chemoresistance to alkylating agents is a major cause of treatment failure in patients with malignant brain tumors. Alkylating agents, the mainstay of treatment for brain tumors, damage the DNA and induce apoptosis, but the cytotoxic activity of these agents is dependent on DNA repair pathways. For example, O6-methylguanine DNA adducts can cause double-strand breaks, but this is dependent on a functional mismatch repair pathway. Thus, tumor cell lines deficient in mismatch repair are resistant to alkylating agents. Perhaps the most important mechanism of resistance to alkylating agents is the DNA repair enzyme O6-methylguanine methyltransferase, which can eliminate the cytotoxic O6-methylguanine DNA adduct before it causes harm. Another mechanism of resistance to alkylating agents is the base excision repair (BER) pathway. Consequently, efforts are ongoing to develop effective inhibitors of BER. Poly(ADP-ribose)polymerase plays a pivotal role in BER and is an important therapeutic target. Developing effective strategies to overcome chemoresistance requires the identification of reliable preclinical models that recapitulate human disease and which can be used to facilitate drug development. This article describes the diverse mechanisms of chemoresistance operating in malignant glioma and efforts to develop reliable preclinical models and novel pharmacologic approaches to overcome resistance to alkylating agents.

Dynamic adsorption properties of n-alkyl glucopyranosides determine their ability to inhibit cytolysis mediated by acoustic cavitation

OpenAIRE

Sostaric, Joe Z.; Miyoshi, Norio; Cheng, Jason Y.; Riesz, Peter

2008-01-01

Suspensions of human leukemia (HL-60) cells readily undergo cytolysis when exposed ultrasound above the acoustic cavitation threshold. However, n-alkyl glucopyranosides (hexyl-,heptyl- and octyl-) completely inhibit ultrasound-induced (1057 kHz) cytolysis (Sostaric, et al., Free Radic. Biol. Med. 2005, 39, 1539–1548). The efficacy of protection from ultrasound-induced cytolysis was determined by the n-alkyl chain length of the glucopyranosides, indicating that protection efficacy depended on ...

Identification and Characterization of uvrA, a DNA Repair Gene of Deinococcus radiodurans

Science.gov (United States)

1996-01-01

alkylating agents , such as methyl-N-nitro~N~nitrosoguanidine(MNNG), N-methyl-N~ nitrosourea (MNU), and to a lesser extent methyl methanesulfonate (MMS...6,4) Photoproduct 17 c. Thymine Glycols and Cross-links 17 3. Ionizing Radiation Damage " 17 4. Chemical Damage 20 a. Alkylating Agents .20 b. Cross...Examples of base damage induced by ionizing radiation 19 6. Nucleotide centers in DNA that are most reactive to alkylating agents 21 7. Schematic

Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.

Science.gov (United States)

Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R

2000-04-14

There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos.

NARCIS (Netherlands)

Feitsma, H.; Akay, A.; Cuppen, E.

2008-01-01

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in

Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA - in vivo, in vitro, and in silico studies.

Directory of Open Access Journals (Sweden)

Damian Mielecki

Full Text Available Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB, characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB, PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems

Pseudomonas putida AlkA and AlkB Proteins Comprise Different Defense Systems for the Repair of Alkylation Damage to DNA – In Vivo, In Vitro, and In Silico Studies

Science.gov (United States)

Mielecki, Damian; Saumaa, Signe; Wrzesiński, Michał; Maciejewska, Agnieszka M.; Żuchniewicz, Karolina; Sikora, Anna; Piwowarski, Jan; Nieminuszczy, Jadwiga; Kivisaar, Maia; Grzesiuk, Elżbieta

2013-01-01

Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against

Dynamic adsorption properties of n-alkyl glucopyranosides determine their ability to inhibit cytolysis mediated by acoustic cavitation.

Science.gov (United States)

Sostaric, Joe Z; Miyoshi, Norio; Cheng, Jason Y; Riesz, Peter

2008-10-09

Suspensions of human leukemia (HL-60) cells readily undergo cytolysis when exposed to ultrasound above the acoustic cavitation threshold. However, n-alkyl glucopyranosides (hexyl, heptyl, and octyl) completely inhibit ultrasound-induced (1057 kHz) cytolysis (Sostaric, et al. Free Radical Biol. Med. 2005, 39, 1539-1548). The efficacy of protection from ultrasound-induced cytolysis was determined by the n-alkyl chain length of the glucopyranosides, indicating that protection efficacy depended on adsorption of n-alkyl glucopyranosides to the gas/solution interface of cavitation bubbles and/or the lipid membrane of cells. The current study tests the hypothesis that "sonoprotection" (i.e., protection of cells from ultrasound-induced cytolysis) in vitro depends on the adsorption of glucopyranosides at the gas/solution interface of cavitation bubbles. To test this hypothesis, the effect of ultrasound frequency (from 42 kHz to 1 MHz) on the ability of a homologous series of n-alkyl glucopyranosides to protect cells from ultrasound-induced cytolysis was investigated. It is expected that ultrasound frequency will affect sonoprotection ability since the nature of the cavitation bubble field will change. This will affect the relative importance of the possible mechanisms for ultrasound-induced cytolysis. Additionally, ultrasound frequency will affect the lifetime and rate of change of the surface area of cavitation bubbles, hence the dynamically controlled adsorption of glucopyranosides to their surface. The data support the hypothesis that sonoprotection efficiency depends on the ability of glucopyranosides to adsorb at the gas/solution interface of cavitation bubbles.

Mechanochemical N-alkylation of imides

Directory of Open Access Journals (Sweden)

Anamarija Briš

2017-08-01

Full Text Available The mechanochemical N-alkylation of imide derivatives was studied. Reactions under solvent-free conditions in a ball mill gave good yields and could be put in place of the classical solution conditions. The method is general and can be applied to various imides and alkyl halides. Phthalimides prepared under ball milling conditions were used in a mechanochemical Gabriel synthesis of amines by their reaction with 1,2-diaminoethane.

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

Science.gov (United States)

Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

2014-01-01

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

Anticancer activity of botanical alkyl hydroquinones attributed to topoisomerase II poisoning

International Nuclear Information System (INIS)

Huang, C.-P.; Fang, W.-H.; Lin, L.-I.; Chiou, Robin Y.; Kan, L.-S.; Chi, N.-H.; Chen, Y.-R.; Lin, T.-Y.; Lin, S.-B.

2008-01-01

Cytotoxic alkyl hydroquinone compounds have been isolated from many plants. We previously isolated 3 structurally similar cytotoxic alkyl hydroquinone compounds from the sap of the lacquer tree Rhus succedanea L. belonging to the sumac family, which have a long history of medicinal use in Asia. Each has an unsaturated alkyl chain attached to the 2-position of a hydroquinone ring. One of these isolates, 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)], being the most cytotoxic, was chosen for studying the anticancer mechanism of these compounds. We found that HQ17(3) was a topoisomerase (Topo) II poison. It irreversibly inhibited Topo IIα activity through the accumulation of Topo II-DNA cleavable complexes. A cell-based assay showed that HQ17(3) inhibited the growth of leukemia HL-60 cells with an EC 50 of 0.9 μM, inhibited the topoisomerase-II-deficient cells HL-60/MX2 with an EC 50 of 9.6 μM, and exerted no effect on peripheral blood mononuclear cells at concentrations up to 50 μM. These results suggest that Topo II is the cellular drug target. In HL-60 cells, HQ17(3) promptly inhibited DNA synthesis, induced chromosomal breakage, and led to cell death with an EC 50 about one-tenth that of hydroquinone. Pretreatment of the cells with N-acetylcysteine could not attenuate the cytotoxicity and DNA damage induced by HQ17(3). However, N-acetylcysteine did significantly reduce the cytotoxicity of hydroquinone. In F344 rats, intraperitoneal injection of HQ17(3) for 28 days induced no clinical signs of toxicity. These results indicated that HQ17(3) is a potential anticancer agent, and its structural features could be a model for anticancer drug design

Antibody Drug Conjugates Differentiate Uptake and DNA Alkylation of Pyrrolobenzodiazepines in Tumors from Organs of Xenograft Mice.

Science.gov (United States)

Ma, Yong; Khojasteh, S Cyrus; Hop, Cornelis E C A; Erickson, Hans K; Polson, Andrew; Pillow, Thomas H; Yu, Shang-Fan; Wang, Hong; Dragovich, Peter S; Zhang, Donglu

2016-12-01

Pyrrolobenzodiazepine (PBD)-dimer is a DNA minor groove alkylator, and its CD22 THIOMAB antibody drug conjugate (ADC) demonstrated, through a disulfide linker, an efficacy in tumor reduction for more than 7 weeks with minimal body weight loss in xenograft mice after a single 0.5-1 mg/kg i.v. dose. The DNA alkylation was investigated here in tumors and healthy organs of mice to understand the sustained efficacy and tolerability. The experimental procedures included the collection of tumors and organ tissues of xenograft mice treated with the ADC followed by DNA isolation/hydrolysis/quantitation and payload recovery from reversible DNA alkylation. PBD-dimer formed a considerable amount of adducts with tissue DNA, representing approximately 98% (at 24 hours), and 99% (at 96 hours) of the total PBD-dimer in tumors, and 78-89% in liver and lung tissues, suggesting highly efficient covalent binding of the released PBD-dimer to tissue DNA. The amount of PBD-DNA adducts in tumor tissues was approximately 24-fold (at 24 hours) and 70-fold (at 96 hours) greater than the corresponding amount of adducts in liver and lung tissues. In addition, the DNA alkylation levels increased 3-fold to 4-fold from 24 to 96 hours in tumors [41/10 6 base pairs (bp) at 96 hours] but remained at the same level (1/10 6 bp) in livers and lungs. These results support the typical target-mediated cumulative uptake of ADC into tumors and payload release that offers an explanation for its sustained antitumor efficacy. In addition, the low level of DNA alkylation in normal tissues is consistent with the tolerability observed in mice. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Alkyltransferase-like proteins: brokers dealing with alkylated DNA bases.

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Schärer, Orlando D

2012-07-13

A new pathway for the repair of DNA alkylation damage is described in this issue of Molecular Cell (Latypov et al., 2012). Alkyltransferase-like enzymes mark O(6)-alkylguanine lesions and, depending on adduct size, channel them into global genome or transcription-coupled nucleotide excision repair pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Manganese-catalyzed Dehydrogenative Alkylation or α-Olefination of Alkyl-N-Heteroaromatics by Alcohols.

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Kempe, Rhett; Zhang, Guoying; Irrgang, Torsten; Dietel, Thomas; Kallmeier, Fabian

2018-05-02

Catalysis involving earth-abundant transition metals is an option to help save our rare noble metal resources and is especially interesting if novel reactivity or selectivity patterns are observed. We report here on a novel reaction: the dehydrogenative alkylation or α-olefination of alkyl-N-heteroaromatics by alcohols. Manganese complexes developed in our laboratory catalyze the reaction efficiently. Fe and Co complexes stabilized by such ligands are essentially inactive. Hydrogen is liberated during the reaction and bromo or iodo functional groups and olefins can be tolerated. A variety of alkyl-N-heteroaromatics can be functionalized, and benzyl and aliphatic alcohols undergo the reaction. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, December 15, 1982-July 15, 1983

International Nuclear Information System (INIS)

Lee, W.R.

1983-01-01

The molecular changes in DNA of mutations induced at the well-defined locus alcohol dehydrogenase (Adh) in Drosophila melanogaster were compared between null mutants induced by x-rays, the alkylating agent N-ethyl-N-nitrosourea (ENU) and decay of tritium incorporated into specific sites of DNA

Oncometabolite D-2-Hydroxyglutarate Inhibits ALKBH DNA Repair Enzymes and Sensitizes IDH Mutant Cells to Alkylating Agents.

Science.gov (United States)

Wang, Pu; Wu, Jing; Ma, Shenghong; Zhang, Lei; Yao, Jun; Hoadley, Katherine A; Wilkerson, Matthew D; Perou, Charles M; Guan, Kun-Liang; Ye, Dan; Xiong, Yue

2015-12-22

Chemotherapy of a combination of DNA alkylating agents, procarbazine and lomustine (CCNU), and a microtubule poison, vincristine, offers a significant benefit to a subset of glioma patients. The benefit of this regimen, known as PCV, was recently linked to IDH mutation that occurs frequently in glioma and produces D-2-hydroxyglutarate (D-2-HG), a competitive inhibitor of α-ketoglutarate (α-KG). We report here that D-2-HG inhibits the α-KG-dependent alkB homolog (ALKBH) DNA repair enzymes. Cells expressing mutant IDH display reduced repair kinetics, accumulate more DNA damages, and are sensitized to alkylating agents. The observed sensitization to alkylating agents requires the catalytic activity of mutant IDH to produce D-2-HG and can be reversed by the deletion of mutant IDH allele or overexpression of ALKBH2 or AKLBH3. Our results suggest that impairment of DNA repair may contribute to tumorigenesis driven by IDH mutations and that alkylating agents may merit exploration for treating IDH-mutated cancer patients. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

N-(2-chloroethyl)-N-nitrosoureas covalently bound to nonionic and monocationic lexitropsin dipeptides. Synthesis, DNA affinity binding characteristics, and reactions with 32P-end-labeled DNA

International Nuclear Information System (INIS)

Church, K.M.; Wurdeman, R.L.; Zhang, Yi; Chen, Faxian; Gold, B.

1990-01-01

The synthesis and characterization of a series of compounds that contain an N-alkyl-N-nitrosourea functionality linked to DNA minor groove binding bi- and tripeptides (lexitropsins or information-reading peptides) based on methylpyrrole-2-carboxamide subunits are described. The lexitropsins (lex) synthesized have either a 3-(dimethylamino)propyl or propyl substituent on the carboxyl terminus. The preferred DNA affinity binding sequences of these compounds were footprinted in 32 P-end-labeled restriction fragments with methidiumpropyl-EDTA·Fe(II), and in common with other structural analogues, e.g., distamycin and netropsin, these nitrosoureas recognize A-T-rich runs. The affinity binding of the compound with the dimethylamino terminus, which is ionized at near-neutral pH, appeared stronger than that observed for the neutral dipeptide. The sequence specificity for DNA alkylation by (2-chloroethyl)nitrosourea-lex dipeptides (Cl-ENU-lex), with neutral and charged carboxyl termini, using 32 P-end-labeled restriction fragments, was determined by the conversion of the adducted sites into single-strand breaks by sequential heating at neutral pH and exposure to base. The DNA cleavage sites were visualized by polyacrylamide gel electrophoresis and autoradiography. Linking the Cl-ENU moiety to minor groove binders is a viable strategy to qualitatively and quantitatively control the delivery and release of the ultimate DNA alkylating agent in a sequence-dependent fashion

Effect O6-Guanine Alkylation on DNA Flexibility Studied by Comparative Molecular Dynamics Simulations

Czech Academy of Sciences Publication Activity Database

Kara, M.; Dršata, Tomáš; Lankaš, Filip; Zacharias, M.

2015-01-01

Roč. 103, č. 1 (2015), s. 23-32 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : DNA damage * DNA alkylation * DNA repair * molecular simulation * molecular dynamics simulation Subject RIV: BO - Biophysics Impact factor: 2.248, year: 2015

Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin. Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves As An Atypical DNA Intercalator

Science.gov (United States)

Fekry, Mostafa I.; Szekely, Jozsef; Dutta, Sanjay; Breydo, Leonid; Zang, Hong; Gates, Kent S.

2012-01-01

Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product leinamycin. Previous studies suggested that alkylation of duplex DNA by activated leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because leinamycin does not contain a classical noncovalent DNA-binding motif such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by leinamycin provided evidence that activated leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 103 M−1. Activated leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated leinamycin with palindromic duplexes containing 5'-CG and 5'-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of leinamycin with respect to target guanine residues. The data allows construction of a model for the leinamycin-DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA. PMID:21954957

Profiling the nucleobase and structure selectivity of anticancer drugs and other DNA alkylating agents by RNA sequencing.

Science.gov (United States)

Gillingham, Dennis; Sauter, Basilius

2018-05-06

Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically the chemical reactivity of DNA alkylators has been determined in vitro with short oligonucleotides. Here we use next generation RNA sequencing to report on the chemoselectivity of alkylating agents. We develop the method with the well-known clinically used DNA modifiying drugs streptozotocin and temozolomide, and then apply the technique to profile RNA modification with uncharacterized alkylation reactions such as with powerful electrophiles like trimethylsilyldiazomethane. The multiplexed and massively parallel format of NGS offers analyses of chemical reactivity in nucleic acids to be accomplished in less time with greater statistical power. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Visible light- and radiation-induced alkylation of pyridine ring with alkanoic acid

International Nuclear Information System (INIS)

Sugimori, Akira; Yamada, Tetsuo

1986-01-01

Quinoline and 4-methylquinoline are efficiently alkylated with alkanoic acid in the presence of iron(III) sulfate upon visible light-irradiation. Iron(III) sulfate not only accelerates the photoreaction but also increases the yield of alkylation. Gamma-irradiation also brings about the alkylation. In the photo- and radiation-induced alkylation with alkanoic acid, alkyl radicals play important roles. (author)

Persistence of DNA adducts, hypermutation and acquisition of cellular resistance to alkylating agents in glioblastoma.

Science.gov (United States)

Head, R J; Fay, M F; Cosgrove, L; Y C Fung, K; Rundle-Thiele, D; Martin, J H

2017-12-02

Glioblastoma is a lethal form of brain tumour usually treated by surgical resection followed by radiotherapy and an alkylating chemotherapeutic agent. Key to the success of this multimodal approach is maintaining apoptotic sensitivity of tumour cells to the alkylating agent. This initial treatment likely establishes conditions contributing to development of drug resistance as alkylating agents form the O 6 -methylguanine adduct. This activates the mismatch repair (MMR) process inducing apoptosis and mutagenesis. This review describes key juxtaposed drivers in the balance between alkylation induced mutagenesis and apoptosis. Mutations in MMR genes are the probable drivers for alkylation based drug resistance. Critical to this interaction are the dose-response and temporal interactions between adduct formation and MMR mutations. The precision in dose interval, dose-responses and temporal relationships dictate a role for alkylating agents in either promoting experimental tumour formation or inducing tumour cell death with chemotherapy. Importantly, this resultant loss of chemotherapeutic selective pressure provides opportunity to explore novel therapeutics and appropriate combinations to minimise alkylation based drug resistance and tumour relapse.

Nanostructured poly(benzimidazole membranes by N-alkylation

Directory of Open Access Journals (Sweden)

J. Weber

2014-01-01

Full Text Available Modification of poly(benzimidazole (PBI by N-alkylation leads to polymers capable of undergoing microphase separation. Polymers with different amounts of C18 alkyl chains have been prepared. The polymers were analyzed by spectroscopy, thermal analysis, electron microscopy and X-ray scattering. The impact of the amount of alkyl chains on the observed microphase separation was analyzed. Membranes prepared from the polymers do show microphase separation, as evidenced by scattering experiments. While no clear morphology could be derived for the domains in the native state, evidence for the formation of lamellar morphologies upon doping with phosphoric acid is provided. Finally, the proton conductivity of alkyl-modified PBI is compared with that of pure PBI, showing that the introduction of alkyl side chains does not result in significant conductivity changes.

Suppression of alkylating agent induced cell transformation and gastric ulceration by low-dose alkylating agent pretreatment

International Nuclear Information System (INIS)

Onodera, Akira; Kawai, Yuichi; Kashimura, Asako; Ogita, Fumiya; Tsutsumi, Yasuo; Itoh, Norio

2013-01-01

Highlights: •Low-dose MNNG pretreatment suppresses high-dose MNNG induced in vitro transformation. •Gastric ulcers induced by high-dose MNNG decreased after low-dose MNNG pretreatment. •Efficacy of low-dose MNNG related to resistance of mutation and oxidative stress. -- Abstract: Exposure to mild stress by chemicals and radiation causes DNA damage and leads to acquired stress resistance. Although the linear no-threshold (LNT) model of safety assessment assumes risk from any dose, evidence from radiological research demonstrates a conflicting hormetic phenomenon known as the hormesis effect. However, the mechanisms underlying radiation hormesis have not yet been clarified, and little is known about the effects of low doses of chemical carcinogens. We analyzed the efficacy of pretreatment with low doses of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) on the subsequent induction of cell transformation and gastric ulceration by high-dose MNNG. We used an in vitro Balb/3T3 A31-1-1 cell transformation test and monitored the formation of gastric ulcers in 5-week-old male ICR mice that were administered MNNG in drinking water. The treatment concentrations of MNNG were determined by the cell survival rate and past reports. For low-dose in vitro and in vivo experiments, MNNG was used at 0.028 μM, and 2.8 μg/mL, respectively. The frequency of cell transformation induced by 10 μm MNNG was decreased by low-dose MNNG pretreatment to levels similar to that of spontaneous transformation. In addition, reactive oxygen species (ROS) and mutation frequencies induced by 10 μm MNNG were decreased by low-dose MNNG pretreatment. Importantly, low-dose MNNG pretreatment had no effect on cell proliferation. In vivo studies showed that the number of gastric ulcers induced by 1 mg/mL MNNG decreased after low-dose MNNG pretreatment. These data indicate that low-dose pretreatment with carcinogens may play a beneficial role in the prevention of chemical toxicity

Suppression of alkylating agent induced cell transformation and gastric ulceration by low-dose alkylating agent pretreatment

Energy Technology Data Exchange (ETDEWEB)

Onodera, Akira, E-mail: onodera@pharm.kobegakuin.ac.jp [Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pharmaceutical Sciences, Kobegakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586 (Japan); Kawai, Yuichi [Department of Pharmaceutical Sciences, Kobegakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586 (Japan); Kashimura, Asako; Ogita, Fumiya; Tsutsumi, Yasuo; Itoh, Norio [Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871 (Japan)

2013-06-14

Highlights: •Low-dose MNNG pretreatment suppresses high-dose MNNG induced in vitro transformation. •Gastric ulcers induced by high-dose MNNG decreased after low-dose MNNG pretreatment. •Efficacy of low-dose MNNG related to resistance of mutation and oxidative stress. -- Abstract: Exposure to mild stress by chemicals and radiation causes DNA damage and leads to acquired stress resistance. Although the linear no-threshold (LNT) model of safety assessment assumes risk from any dose, evidence from radiological research demonstrates a conflicting hormetic phenomenon known as the hormesis effect. However, the mechanisms underlying radiation hormesis have not yet been clarified, and little is known about the effects of low doses of chemical carcinogens. We analyzed the efficacy of pretreatment with low doses of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) on the subsequent induction of cell transformation and gastric ulceration by high-dose MNNG. We used an in vitro Balb/3T3 A31-1-1 cell transformation test and monitored the formation of gastric ulcers in 5-week-old male ICR mice that were administered MNNG in drinking water. The treatment concentrations of MNNG were determined by the cell survival rate and past reports. For low-dose in vitro and in vivo experiments, MNNG was used at 0.028 μM, and 2.8 μg/mL, respectively. The frequency of cell transformation induced by 10 μm MNNG was decreased by low-dose MNNG pretreatment to levels similar to that of spontaneous transformation. In addition, reactive oxygen species (ROS) and mutation frequencies induced by 10 μm MNNG were decreased by low-dose MNNG pretreatment. Importantly, low-dose MNNG pretreatment had no effect on cell proliferation. In vivo studies showed that the number of gastric ulcers induced by 1 mg/mL MNNG decreased after low-dose MNNG pretreatment. These data indicate that low-dose pretreatment with carcinogens may play a beneficial role in the prevention of chemical toxicity

Structural Characterization of N-Alkylated Twisted Amides: Consequences for Amide Bond Resonance and N-C Cleavage.

Science.gov (United States)

Hu, Feng; Lalancette, Roger; Szostak, Michal

2016-04-11

Herein, we describe the first structural characterization of N-alkylated twisted amides prepared directly by N-alkylation of the corresponding non-planar lactams. This study provides the first experimental evidence that N-alkylation results in a dramatic increase of non-planarity around the amide N-C(O) bond. Moreover, we report a rare example of a molecular wire supported by the same amide C=O-Ag bonds. Reactivity studies demonstrate rapid nucleophilic addition to the N-C(O) moiety of N-alkylated amides, indicating the lack of n(N) to π*(C=O) conjugation. Most crucially, we demonstrate that N-alkylation activates the otherwise unreactive amide bond towards σ N-C cleavage by switchable coordination. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tripodal (N-alkylated) CMP(O) and malonamide ligands: synthesis, extraction of metal ions, and potentiometric studies

International Nuclear Information System (INIS)

Janczewski, D.; Reinhoudt, D.N.; Verboom, W.; Malinowska, E.; Pietrzak, M.; Hill, C.; Allignol, C.

2007-01-01

Tripodal ligands build on the C-pivot (9b-e, 13b-d, and 17a-d) and tri-alkyl-benzene platforms (10a,b, 11, 12, 14a,b, and 18a,b) bearing (N-alkylated) carbamoyl-methyl-phosphine oxide (CMPO), carbamoyl-methyl-phosphonate (CMP), and malonamide moieties were synthesized. Extraction studies with Am 3+ and Eu 3+ show that in general there is a positive influence of the N-alkyl substituents in C-pivot CMP(O) ligands on the D(distribution) coefficients. The tri-alkyl-benzene CMPO ligands 10a,b, 11, and 12 have considerably larger D coefficients than the corresponding C-pivot analogues 9a-e, although hardly having any selectivity, while N-alkylation gives rise to smaller D coefficients. Although less effective the extraction behavior of the C-pivot CMP analogues 13b-d shows more or less the same trend as the corresponding CMPO ligands 9b-e upon substitution of the carboxamide N-atom with different alkyl chains. The different malonamide ligands 17a-d and 18a,b are bad extractants, while N-alkylation makes them even worse. Potentiometric studies of CMP(O) and malonamide ligands in polymeric membranes on Pb 2+ , Cu 2+ , Ca 2+ , Mg 2+ , Na + , and K + salts revealed that N-alkyl substituents increase the stability constants of ion-ionophore complexes compared to unsubstituted ligands. In polymeric membrane electrodes the ligands induce a selectivity pattern that differs significantly from the so-called Hofmeister series, giving the highest selectivity coefficients for UO 2 2+ among all examined cations (Pb 2+ , Cu 2+ , Ca 2+ , Mg 2+ , Na + , K + ). (authors)

UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

International Nuclear Information System (INIS)

Arimoto-Kobayashi, Sakae; Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko

2010-01-01

We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in φX174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

Energy Technology Data Exchange (ETDEWEB)

Arimoto-Kobayashi, Sakae [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan); Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan)

2010-09-10

We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in {phi}X174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses

International Nuclear Information System (INIS)

Hu, Xiaolan; Yu, Yingnian; Li, Qian; Wu, Danxiao; Tan, Zhengning; Wang, Cheng; Wang, Jvping; Wu, Meiping

2011-01-01

Highlights: → MNNG-induced appearance of DUT-N in the extracellular fluid has cellular specificity. → MNNG alters the subcellular distribution of DUT-N in human cells in different ways. → DUT-N may be a potential biomarker to assess the risk of alkylating agents exposure. -- Abstract: Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25 μM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.

A powerful selection assay for mixture libraries of DNA alkylating agents.

Science.gov (United States)

Ham, Young-Wan; Boger, Dale L

2004-08-04

A simple and powerful selection assay that permits the separation (rpHPLC), quantitation (ELSD), and identification (ESI-MS) of thermally released adenine adducts derived from duocarmycin analogues is detailed that can establish the most effective DNA alkylating agents in synthetic combinatorial mixtures.

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of mouse spermatogenic cells to O6-alkylating agents.

Science.gov (United States)

Thompson, M J; Abdul-Rahman, S; Baker, T G; Rafferty, J A; Margison, G P; Bibby, M C

2000-07-01

The O(6)-alkylguanine-DNA alkyltransferase inactivator O(6)-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea to study the role of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O(6)-alkylating agents. Exposure of the mice to 1, 3-bis(2-chloroethyl)-1-nitrosourea or O(6)-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O(6)-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O(6)-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O(6)-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1, 3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O(6)-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.

DNA Repair Modulates The Vulnerability of The Developing Brain to Alkylating Agents

Science.gov (United States)

Kisby, G.E.; Olivas, A.; Park, T.; Churchwell, M.; Doerge, D.; Samson, L. D.; Gerson, S.L.; Turker, M.S.

2009-01-01

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag-/-) or O6-methylguanine methyltransferase (Mgmt-/-), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt-/- neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag-/- neurons were for the most part significantly less sensitive than wild type or Mgmt-/- neurons to MAM and HN2. Aag-/- neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt-/- mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM treated Aag-/- or MGMT overexpressing (MgmtTg+) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in MgmtTg+ mice treated with HN2. Collectively, these in vitro

Complex responses to alkylating agents

International Nuclear Information System (INIS)

Samson, L.D.

2003-01-01

Using Affymetrix oligonucleotide GeneChip analysis, we previously found that, upon exposure to the simple alkylating agent methylmethane sulfonate, the transcript levels for about one third of the Saccharomyces cerevisiae genome (∼2,000 transcripts) are induced or repressed during the first hour or two after exposure. In order to determine whether the responsiveness of these genes has any relevance to the protection of cells against alkylating agents we have undertaken several follow-up studies. First, we explored the specificity of this global transcriptional response to MMS by measuring the global response of S. cerevisiae to a broad range of agents that are known to induce DNA damage. We found that each agent produced a very different mRNA transcript profile, even though the exposure doses produced similar levels of toxicity. We also found that the selection of genes that respond to MMS is highly dependent upon what cell cycle phase the cells are in at the time of exposure. Computational clustering analysis of the dataset derived from a large number of exposures identified several promoter motifs that are likely to control some of the regulons that comprise this large set of genes that are responsive to DNA damaging agents. However, it should be noted that these agents damage cellular components other than DNA, and that the responsiveness of each gene need not be in response to DNA damage per se. We have also begun to study the response of other organisms to alkylating agents, and these include E. coli, cultured mouse and human cells, and mice. Finally, we have developed a high throughput phenotypic screening method to interrogate the role of all non-essential S. cerevisiae genes (about 4,800) in protecting S. cerevisiae against the deleterious effects of alkylating agents; we have termed this analysis 'genomic phenotyping'. This study has uncovered a plethora of new pathways that play a role in the recovery of eukaryotic cells after exposure to toxic

Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

Energy Technology Data Exchange (ETDEWEB)

Wang Zheng; Wang Guliang; Yang Jun; Guo Lei; Yu Yingnian

2003-07-25

Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-{beta}, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 {mu}M) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor {alpha} (TNF{alpha}) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.

A Short Review on the Synthetic Strategies of Duocarmycin Analogs that are Powerful DNA Alkylating Agents.

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Patil, Pravin C; Satam, Vijay; Lee, Moses

2015-01-01

The duocarmycins and CC-1065 are members of a class of DNA minor groove, AT-sequence selective, and adenine-N3 alkylating agents, isolated from Streptomyces sp. that exhibit extremely potent cytotoxicity against the growth of cancer cells grown in culture. Initial synthesis and structural modification of the cyclopropa[c] pyrrolo[3,2-e]indole (CPI) DNA-alkylating motif as well as the indole non-covalent binding region in the 1980s have led to several compounds that entered clinical trials as potential anticancer drugs. However, due to significant systemic toxicity none of the analogs have passed clinical evaluation. As a result, the intensity in the design, synthesis, and development of novel analogs of the duocarmycins has continued. Accordingly, in this review, which covers a period from the 1990s through the present time, the design and synthesis of duocarmycin SA are described along with the synthesis of novel and highly cytotoxic analogs that lack the chiral center. Examples of achiral analogs of duocarmycin SA described in this review include seco-DUMSA (39 and 40), seco-amino-CBI-TMI (13, Centanamycin), and seco-hydroxy-CBI-TMI (14). In addition, another novel class of biologically active duocarmycin SA analogs that contained the seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) DNA alkylating submit was also designed and synthesized. The synthesis of seco-iso-CFI-TMI (10, Tafuramycin A) and seco-CFQ-TMI (11, Tafuramycin B) is included in this review.

HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress.

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Khanra, Kalyani; Chakraborty, Anindita; Bhattacharyya, Nandan

2015-01-01

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta (polβ Δ208-304) specific for ovarian cancer. Pol β Δ208-304 has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. Polβ Δ208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H2O2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol β Δ208-304 transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

Science.gov (United States)

Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

Bis(trialkylsilyl) peroxides as alkylating agents in the copper-catalyzed selective mono-N-alkylation of primary amides.

Science.gov (United States)

Sakamoto, Ryu; Sakurai, Shunya; Maruoka, Keiji

2017-06-13

The copper-catalyzed selective mono-N-alkylation of primary amides with bis(trialkylsilyl) peroxides as alkylating agents was reported. The results of a mechanistic study suggest that this reaction should proceed via a free radical process that includes the generation of alkyl radicals from bis(trialkylsilyl) peroxides.

Tripodal (N-alkylated) CMP(O) and malonamide ligands: synthesis, extraction of metal ions, and potentiometric studies

Energy Technology Data Exchange (ETDEWEB)

Janczewski, D.; Reinhoudt, D.N.; Verboom, W. [Twente Univ., Lab. of Supramolecular Chemistry and Technology, Mesa Research Institute for Nanotechnology, Enschede (Netherlands); Malinowska, E.; Pietrzak, M. [Warsaw Univ. of Technology, Dept. of Analytical Chemistry, Faculty of Chemistry (Poland); Hill, C.; Allignol, C. [CEA Valrho, 30 - Marcoule (France)

2007-01-15

Tripodal ligands build on the C-pivot (9b-e, 13b-d, and 17a-d) and tri-alkyl-benzene platforms (10a,b, 11, 12, 14a,b, and 18a,b) bearing (N-alkylated) carbamoyl-methyl-phosphine oxide (CMPO), carbamoyl-methyl-phosphonate (CMP), and malonamide moieties were synthesized. Extraction studies with Am{sup 3+} and Eu{sup 3+} show that in general there is a positive influence of the N-alkyl substituents in C-pivot CMP(O) ligands on the D(distribution) coefficients. The tri-alkyl-benzene CMPO ligands 10a,b, 11, and 12 have considerably larger D coefficients than the corresponding C-pivot analogues 9a-e, although hardly having any selectivity, while N-alkylation gives rise to smaller D coefficients. Although less effective the extraction behavior of the C-pivot CMP analogues 13b-d shows more or less the same trend as the corresponding CMPO ligands 9b-e upon substitution of the carboxamide N-atom with different alkyl chains. The different malonamide ligands 17a-d and 18a,b are bad extractants, while N-alkylation makes them even worse. Potentiometric studies of CMP(O) and malonamide ligands in polymeric membranes on Pb{sup 2+}, Cu{sup 2+}, Ca{sup 2+}, Mg{sup 2+}, Na{sup +}, and K{sup +} salts revealed that N-alkyl substituents increase the stability constants of ion-ionophore complexes compared to unsubstituted ligands. In polymeric membrane electrodes the ligands induce a selectivity pattern that differs significantly from the so-called Hofmeister series, giving the highest selectivity coefficients for UO{sub 2}{sup 2+} among all examined cations (Pb{sup 2+}, Cu{sup 2+}, Ca{sup 2+}, Mg{sup 2+}, Na{sup +}, K{sup +}). (authors)

Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.

Science.gov (United States)

Guo, Jun; Cui, Qiu; Jiang, WeiHao; Liu, Cheng; Li, DingFeng; Zeng, Yanjun

2013-08-01

The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

Photogeneration and reactivity of naphthoquinone methides as purine selective DNA alkylating agents.

Science.gov (United States)

Verga, Daniela; Nadai, Matteo; Doria, Filippo; Percivalle, Claudia; Di Antonio, Marco; Palumbo, Manlio; Richter, Sara N; Freccero, Mauro

2010-10-20

A one-step protecting-group-free synthesis of both 6-hydroxy-naphthalene-2-carbaldehyde and the bifunctional binaphthalenyl derivative afforded 6-hydroxymethylnaphthalen-2-ol, 6-methylaminomethyl-naphthalen-2-ol, [(2-hydroxy-3-naphthyl)methyl]trimethyl ammonium iodide, and a small library of bifunctional binol analogues in good yields. Irradiation of naphthol quaternary ammonium salt and binol-derivatives (X = OH, NHR, NMe(3)(+), OCOCH(3), and L-proline) at 310 and 360 nm resulted in the photogeneration of the 2,6-naphthoquinone-6-methide (NQM) and binol quinone methide analogues (BQMs) by a water-mediated excited-state proton transfer (ESPT). The hydration, the mono- and bis-alkylation reactions of morpholine and 2-ethanethiol, as N and S prototype nucleophiles, by the transient NQM (λ(max) 310, 330 nm) and BQMs (λ(max) 360 nm) were investigated in water by product distribution analysis and laser flash photolysis (LFP). Both the photogeneration and the reactivity of NQM and BQMs exhibited striking differences. BQMs were at least 2 orders of magnitude more reactive than NQM, and they were generated much more efficiently from a greater variety of photoprecursors including the hydroxymethyl, quaternary ammonium salt and several binol-amino acids. On the contrary, the only efficient precursor of NQM was the quaternary ammonium salt. All water-soluble BQM precursors were further investigated for their ability to alkylate and cross-link plasmid DNA and oligonucleotides by gel electrophoresis: the BQMs were more efficient than the isomeric o-BQM (binol quinone methide analogue of 2,3-naphthoquinone-3-methide). Sequence analysis by gel electrophoresis, HPLC, and MS showed that the alkylation occurred at purines, with a preference for guanine. In particular, a BQM was able to alkylate N7 of guanines resulting in depurination at the oligonucleotide level, and ribose loss at the nucleotide level. The photoreactivity of BQM precursors translated into photocytotoxic and

Quantitative assessment of the dose-response of alkylating agents in DNA repair proficient and deficient ames tester strains.

Science.gov (United States)

Tang, Leilei; Guérard, Melanie; Zeller, Andreas

2014-01-01

Mutagenic and clastogenic effects of some DNA damaging agents such as methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) have been demonstrated to exhibit a nonlinear or even "thresholded" dose-response in vitro and in vivo. DNA repair seems to be mainly responsible for these thresholds. To this end, we assessed several mutagenic alkylators in the Ames test with four different strains of Salmonella typhimurium: the alkyl transferases proficient strain TA1535 (Ogt+/Ada+), as well as the alkyl transferases deficient strains YG7100 (Ogt+/Ada-), YG7104 (Ogt-/Ada+) and YG7108 (Ogt-/Ada-). The known genotoxins EMS, MMS, temozolomide (TMZ), ethylnitrosourea (ENU) and methylnitrosourea (MNU) were tested in as many as 22 concentration levels. Dose-response curves were statistically fitted by the PROAST benchmark dose model and the Lutz-Lutz "hockeystick" model. These dose-response curves suggest efficient DNA-repair for lesions inflicted by all agents in strain TA1535. In the absence of Ogt, Ada is predominantly repairing methylations but not ethylations. It is concluded that the capacity of alkyl-transferases to successfully repair DNA lesions up to certain dose levels contributes to genotoxicity thresholds. Copyright © 2013 Wiley Periodicals, Inc.

Abnormal sensitivity of skin fibroblasts from familial polyposis patients to DNA alkylating agents

International Nuclear Information System (INIS)

Barfknecht, T.R.; Little, J.B.

1982-01-01

Fibroblast cell strains derived from different patients all afflicted with genetic predisposing to the development of intestinal polyposis and cancer were tested for their sensitivity to the lethal effects of the DNA alkylating agents methylmethanesulfonate (MMS), ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, and 4-nitroquinoline 1-oxide. The genetic syndromes studied were: (a) adenomatosis of the colon and rectum only, an autosomal dominant trait; (b) Turcot's syndrome, a rare autosomal recessive polyposis syndrome also characterized by central nervous system tumors; and (c) Gardner's syndrome, an autosomal dominant syndrome which, in addition to intestinal polyposis, is also clinically characterized by osteomas and soft tissue tumors. Fibroblasts from a patient with Turcot's syndrome were hypersensitive to MMS, having a D0 value of 0.24 mM (p less than 0.01) versus the normal average D0 of 0.36 mM and a D10 value of 0.95 mM (p less than 0.01) compared with the normal average value of 1.3 mM. Fibroblasts from the Gardner's syndrome proband were moderately sensitive to MMS, ethyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine due to significant differences of D10 values of 0.60 mM (p less than 0.01), 15 mM (p less than 0.01), and 4.8 microM (p less than 0.025), respectively, versus the normal average values of 1.3 mM, 28 mM, and 9.4 microM. Fibroblasts from the clinically affected Gardner's syndrome daughter of the proband were significantly more sensitive to MMS treatment, D0 of 0.22 mM (p less than 0.01) versus the normal average D0 of 0.36 mM and a D10 of 0.97 mM (p less than 0.01) versus the normal average. This differential sensitivity to the several DNA alkylating agents suggests that different mechanisms of hypersensitivity to these chemicals may be associated with fibroblasts from the various forms of familial polyposis

Sorbate-nitrite interactions: acetonitrile oxide as an alkylating agent.

Science.gov (United States)

Pérez-Prior, M Teresa; Gómez-Bombarelli, Rafael; González-Pérez, Marina; Manso, José A; García-Santos, M Pilar; Calle, Emilio; Casado, Julio

2009-07-01

Because chemical species with DNA-damaging and mutagenic activity are formed in sorbate-nitrite mixtures and because sorbic acid sometimes coexists with nitrite occurring naturally or incorporated as a food additive, the study of sorbate-nitrite interactions is important. Here, the alkylating potential of the products resulting from such interactions was investigated. Drawn were the following conclusions: (i) Acetonitrile oxide (ACNO) is the compound responsible for the alkylating capacity of sorbate-nitrite mixtures; (ii) ACNO alkylates 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases, forming an adduct (AD; epsilon = 1.4 x 10(4) M(-1) cm(-1); lambda = 519 nm); (iii) the NBP alkylation reaction complies with the rate equation, r = d[AD]/dt = k(alk)(ACNO)[ACNO][NBP]-k(hyd)(AD)[AD], k(alk)(ACNO) being the NBP alkylation rate constant for ACNO and k(hyd)(AD) the rate constant for the adduct hydrolysis reaction; (iv) the small fraction of ACNO forming the adduct with NBP, as well as the small magnitude of the quotient (k(alk) (ACNO)/k(hyd)(ACNO)) as compared with those reported for other alkylating agents, such as some lactones and N-alkyl-N-nitrosoureas, reveals the ACNO effective alkylating capacity to be less significant; (v) the low value of the NBP-ACNO adduct life (defined as the total amount of adduct present along the progression of the NBP alkylation per unit of alkylating agent concentration) points to the high instability of this adduct; and (vi) the obtained results are in accordance with the low carcinogenicity of ACNO.

Synthesis of N-methyl-N-nitrosourea linked to a methidium chloride analogue and its reactions with 32P-end-labeled DNA

International Nuclear Information System (INIS)

Konakahara, T.; Wurdeman, R.L.; Gold, B.

1988-01-01

The synthesis and characterization of an N-methyl-N-nitrosourea (MNU) analogue that is covalently linked to methidium nucleus (9) is described. At 37/degrees/C in pH 8.0 buffer 9 hydrolyzes via pseudo-first-order kinetics, with a calculated t/sub 1/2/ = 77 min. By use of polyacrylamide sequencing gels the formation of piperidine-labile N 7 -methylguanine adducts from the reaction of 9 and MNU with 5'- 32 P-end-labeled DNA restriction fragments is reported. DNA methylation by 9 in 10 mM Tris buffer is enhanced with increasing ionic strength (50-200 mM NaCl), which contrasts to the inhibition of MNU-induced cleavage with increasing salt. In addition, 9 methylates all G sites equally, while MNU shows a clear preference for d(G)/sub n/ (n ≥ 3) runs and an asymmetrical methylation pattern within these G-rich regions. The results are discussed in terms of the delivery of the MNU moiety to the DNA target by a non-sequence-specific intercalation process and the subsequent hydrolytic generation of a nondiffusible alkylating intermediate

Synthesis of no-carrier-added radiobrominated n-alkylated analogues of spiperone

International Nuclear Information System (INIS)

Moerlein, S.M.; Laufer, P.; Stoecklin, G.

1985-01-01

The synthesis of a series of p-bromo-3-N-alkyl spiperone analogues is described. N-alkylation was achieved via reaction of the potassium salt of the spiperone lactam ring with alkyl iodide; subsequent reactions with elemental bromine gave the p-brominated isomers. Optimization studies using no-carrier-added (n.c.a.) 77 Br - indicated that radio-bromination of N-alkyl spiperone analogues occurs with higher yields and in shorter reaction times when dichloramine-T (DCT) is used rather than H 2 0 2 /acetic acid as an oxidant. The production of the title compounds in high effective specific activity with radiochemical yields of 20-30 % using n.c.a. 77 Br - and DCT is reported. (author)

Sequence selectivity of azinomycin B in DNA alkylation and cross-linking: a QM/MM study.

Science.gov (United States)

Senthilnathan, Dhurairajan; Kalaiselvan, Anbarasan; Venuvanalingam, Ponnambalam

2013-01-01

Azinomycin B--a well-known antitumor drug--forms cross-links with DNA through alkylation of purine bases and blocks tumor cell growth. This reaction has been modeled using the ONIOM (B3LYP/6-31+g(d):UFF) method to understand the mechanism and sequence selectivity. ONIOM results have been checked for reliability by comparing them with full quantum mechanics calculations for selected paths. Calculations reveal that, among the purine bases, guanine is more reactive and is alkylated by aziridine ring through the C10 position, followed by alkylation of the epoxide ring through the C21 position of Azinomycin B. While the mono alkylation is controlled kinetically, bis-alkylation is controlled thermodynamically. Solvent effects were included using polarized-continuum-model calculations and no significant change from gas phase results was observed.

The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents.

Science.gov (United States)

Rasmussen, L J; Rasmussen, M; Lützen, A; Bisgaard, H C; Singh, K K

2000-05-25

DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.

Probing Conformational Changes in Human DNA Topoisomerase IIα by Pulsed Alkylation Mass Spectrometry*

Science.gov (United States)

Chen, Yu-tsung; Collins, Tammy R. L.; Guan, Ziqiang; Chen, Vincent B.; Hsieh, Tao-Shih

2012-01-01

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5′-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193. PMID:22679013

Mono- and Di-Alkylation Processes of DNA Bases by Nitrogen Mustard Mechlorethamine.

Science.gov (United States)

Larrañaga, Olatz; de Cózar, Abel; Cossío, Fernando P

2017-12-06

The reactivity of nitrogen mustard mechlorethamine (mec) with purine bases towards formation of mono- (G-mec and A-mec) and dialkylated (AA-mec, GG-mec and AG-mec) adducts has been studied using density functional theory (DFT). To gain a complete overview of DNA-alkylation processes, direct chloride substitution and formation through activated aziridinium species were considered as possible reaction paths for adduct formation. Our results confirm that DNA alkylation by mec occurs via aziridine intermediates instead of direct substitution. Consideration of explicit water molecules in conjunction with polarizable continuum model (PCM) was shown as an adequate computational method for a proper representation of the system. Moreover, Runge-Kutta numerical kinetic simulations including the possible bisadducts have been performed. These simulations predicted a product ratio of 83:17 of GG-mec and AG-mec diadducts, respectively. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Orientational diffusion of n-alkyl cyanides

International Nuclear Information System (INIS)

Zhu Xiang; Farrer, Richard A; Zhong Qin; Fourkas, John T

2005-01-01

Ultrafast optical Kerr effect spectroscopy has been used to study the temperature-dependent orientational dynamics of a series of nitriles with n-alkyl chains ranging from one to 11 carbons in length. In all cases the orientational diffusion is found to be described by a single-exponential decay. Analysis of the orientational correlation times using the Debye-Stokes-Einstein equation suggests that the molecules adopt extended configurations and reorient as rigid rods. The liquids with shorter alkyl chains undergo an apparent ordering transition as they are cooled

Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents.

Science.gov (United States)

Esteller, M; Garcia-Foncillas, J; Andion, E; Goodman, S N; Hidalgo, O F; Vanaclocha, V; Baylin, S B; Herman, J G

2000-11-09

The DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents. MGMT activity is controlled by a promoter; methylation of the promoter silences the gene in cancer, and the cells no longer produce MGMT. We examined gliomas to determine whether methylation of the MGMT promoter is related to the responsiveness of the tumor to alkylating agents. We analyzed the MGMT promoter in tumor DNA by a methylation-specific polymerase-chain-reaction assay. The gliomas were obtained from patients who had been treated with carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, or BCNU). The molecular data were correlated with the clinical outcome. The MGMT promoter was methylated in gliomas from 19 of 47 patients (40 percent). This finding was associated with regression of the tumor and prolonged overall and disease-free survival. It was an independent and stronger prognostic factor than age, stage, tumor grade, or performance status. Methylation of the MGMT promoter in gliomas is a useful predictor of the responsiveness of the tumors to alkylating agents.

Synthesis and Biological Evaluation of N-Alkyl-3-(alkylamino-pyrazine-2-carboxamides

Directory of Open Access Journals (Sweden)

Lucia Semelkova

2015-05-01

Full Text Available A series of N-alkyl-3-(alkylaminopyrazine-2-carboxamides and their N-alkyl-3-chloropyrazine-2-carboxamide precursors were prepared. All compounds were characterized by analytical methods and tested for antimicrobial and antiviral activity. The antimycobacterial MIC values against Mycobacterium tuberculosis H37Rv of the most effective compounds, 3-(hexylamino-, 3-(heptylamino- and 3-(octylamino-N-methyl-pyrazine-2-carboxamides 14‒16, was 25 μg/mL. The compounds inhibited photosystem 2 photosynthetic electron transport (PET in spinach chloroplasts. This activity was strongly connected with the lipophilicity of the compounds. For effective PET inhibition longer alkyl chains in the 3-(alkylamino substituent in the N-alkyl-3-(alkylaminopyrazine-2-carboxamide molecule were more favourable than two shorter alkyl chains.

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

Science.gov (United States)

Klapacz, Joanna; Pottenger, Lynn H.; Engelward, Bevin P.; Heinen, Christopher D.; Johnson, George E.; Clewell, Rebecca A.; Carmichael, Paul L.; Adeleye, Yeyejide; Andersen, Melvin E.

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. PMID:27036068

Damage-induced DNA repair processes in Escherichia coli cells

International Nuclear Information System (INIS)

Slezarikova, V.

1986-01-01

The existing knowledge is summed up of the response of Escherichia coli cells to DNA damage due to various factors including ultraviolet radiation. So far, three inducible mechanisms caused by DNA damage are known, viz., SOS induction, adaptation and thermal shock induction. Greatest attention is devoted to SOS induction. Its mechanism is described and the importance of the lexA recA proteins is shown. In addition, direct or indirect role is played by other proteins, such as the ssb protein binding the single-strand DNA sections. The results are reported of a study of induced repair processes in Escherichia coli cells repeatedly irradiated with UV radiation. A model of induction by repeated cell irradiation discovered a new role of induced proteins, i.e., the elimination of alkali-labile points in the daughter DNA synthetized on a damaged model. The nature of the alkali-labile points has so far been unclear. In the adaptation process, regulation proteins are synthetized whose production is induced by the presence of alkylation agents. In the thermal shock induction, new proteins synthetize in cells, whose function has not yet been clarified. (E.S.)

Direct, Regioselective N-Alkylation of 1,3-Azoles.

Science.gov (United States)

Chen, Shuai; Graceffa, Russell F; Boezio, Alessandro A

2016-01-04

Regioselective N-alkylation of 1,3-azoles is a valuable transformation. Organomagnesium reagents were discovered to be competent bases to affect regioselective alkylation of various 1,3-azoles. Counterintuitively, substitution selectively occurred at the more sterically hindered nitrogen atom. Numerous examples are provided, on varying 1,3-azole scaffolds, with yields ranging from 25 to 95%.

An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents.

Directory of Open Access Journals (Sweden)

Ryan W Benson

Full Text Available DinB (DNA Pol IV is a translesion (TLS DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N(2-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ, a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site "aromatic triad", namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS. Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.

Mutational specificity of alkylating agents and the influence of DNA repair

Energy Technology Data Exchange (ETDEWEB)

Horsfall, M.J.; Gordon, A.J.; Burns, P.A.; Zielenska, M.; van der Vliet, G.M.; Glickman, B.W. (York Univ., Toronto, Ontario (Canada))

1990-01-01

Alkylating treatments predominantly induce G:C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G:C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence. 199 references.

Alkylation sensitivity screens reveal a conserved cross-species functionome

Science.gov (United States)

Svilar, David; Dyavaiah, Madhu; Brown, Ashley R.; Tang, Jiang-bo; Li, Jianfeng; McDonald, Peter R.; Shun, Tong Ying; Braganza, Andrea; Wang, Xiao-hong; Maniar, Salony; St Croix, Claudette M.; Lazo, John S.; Pollack, Ian F.; Begley, Thomas J.; Sobol, Robert W.

2013-01-01

To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases UNG and MYH as well as MPG to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in E. coli and S. cerevisiae. The conserved biological processes across all three species composes an Alkylation Functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance. PMID:23038810

Chemotherapy-induced pulmonary hypertension: role of alkylating agents.

Science.gov (United States)

Ranchoux, Benoît; Günther, Sven; Quarck, Rozenn; Chaumais, Marie-Camille; Dorfmüller, Peter; Antigny, Fabrice; Dumas, Sébastien J; Raymond, Nicolas; Lau, Edmund; Savale, Laurent; Jaïs, Xavier; Sitbon, Olivier; Simonneau, Gérald; Stenmark, Kurt; Cohen-Kaminsky, Sylvia; Humbert, Marc; Montani, David; Perros, Frédéric

2015-02-01

Pulmonary veno-occlusive disease (PVOD) is an uncommon form of pulmonary hypertension (PH) characterized by progressive obstruction of small pulmonary veins and a dismal prognosis. Limited case series have reported a possible association between different chemotherapeutic agents and PVOD. We evaluated the relationship between chemotherapeutic agents and PVOD. Cases of chemotherapy-induced PVOD from the French PH network and literature were reviewed. Consequences of chemotherapy exposure on the pulmonary vasculature and hemodynamics were investigated in three different animal models (mouse, rat, and rabbit). Thirty-seven cases of chemotherapy-associated PVOD were identified in the French PH network and systematic literature analysis. Exposure to alkylating agents was observed in 83.8% of cases, mostly represented by cyclophosphamide (43.2%). In three different animal models, cyclophosphamide was able to induce PH on the basis of hemodynamic, morphological, and biological parameters. In these models, histopathological assessment confirmed significant pulmonary venous involvement highly suggestive of PVOD. Together, clinical data and animal models demonstrated a plausible cause-effect relationship between alkylating agents and PVOD. Clinicians should be aware of this uncommon, but severe, pulmonary vascular complication of alkylating agents. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

Science.gov (United States)

Klapacz, Joanna; Pottenger, Lynn H; Engelward, Bevin P; Heinen, Christopher D; Johnson, George E; Clewell, Rebecca A; Carmichael, Paul L; Adeleye, Yeyejide; Andersen, Melvin E

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

NARCIS (Netherlands)

M. Molinete; W. Vermeulen (Wim); A. Bürkle; J. Mé nissier-de Murcia; J.H. Küpper; J.H.J. Hoeijmakers (Jan); G. de Murcia

1993-01-01

textabstractThe zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during

DNA repair in B. subtilis: an inducible dimer-specific W-reactivation system

International Nuclear Information System (INIS)

Fields, P.I.; Yasbin, R.E.

1982-01-01

The W-reactivation system of Bacillus subtilis can repair pyrimidine dimers in bacteriophage DNA. This inducible repair system can be activated by treatment of the bacteria with uv, alkylating agents, cross-linking agents and gamma irradiation. However, bacteriophage treated with agents other than those that cause pyrimidine dimers to be produced was not repaired by this unique form of W-reactivation. In contrast, the W-reactivation system of Escherichia coli can repair a variety of damages placed in the bacteriophage DNA

Thermodynamic Interactions between Polystyrene and Long-Chain Poly(n-Alkyl Acrylates) Derived from Plant Oils.

Science.gov (United States)

Wang, Shu; Robertson, Megan L

2015-06-10

Vegetable oils and their fatty acids are promising sources for the derivation of polymers. Long-chain poly(n-alkyl acrylates) and poly(n-alkyl methacrylates) are readily derived from fatty acids through conversion of the carboxylic acid end-group to an acrylate or methacrylate group. The resulting polymers contain long alkyl side-chains with around 10-22 carbon atoms. Regardless of the monomer source, the presence of alkyl side-chains in poly(n-alkyl acrylates) and poly(n-alkyl methacrylates) provides a convenient mechanism for tuning their physical properties. The development of structured multicomponent materials, including block copolymers and blends, containing poly(n-alkyl acrylates) and poly(n-alkyl methacrylates) requires knowledge of the thermodynamic interactions governing their self-assembly, typically described by the Flory-Huggins interaction parameter χ. We have investigated the χ parameter between polystyrene and long-chain poly(n-alkyl acrylate) homopolymers and copolymers: specifically we have included poly(stearyl acrylate), poly(lauryl acrylate), and their random copolymers. Lauryl and stearyl acrylate were chosen as model alkyl acrylates derived from vegetable oils and have alkyl side-chain lengths of 12 and 18 carbon atoms, respectively. Polystyrene is included in this study as a model petroleum-sourced polymer, which has wide applicability in commercially relevant multicomponent polymeric materials. Two independent methods were employed to measure the χ parameter: cloud point measurements on binary blends and characterization of the order-disorder transition of triblock copolymers, which were in relatively good agreement with one another. The χ parameter was found to be independent of the alkyl side-chain length (n) for large values of n (i.e., n > 10). This behavior is in stark contrast to the n-dependence of the χ parameter predicted from solubility parameter theory. Our study complements prior work investigating the interactions between

Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKK? inhibition

OpenAIRE

Tse, Anfernee Kai-Wing; Chen, Ying-Jie; Fu, Xiu-Qiong; Su, Tao; Li, Ting; Guo, Hui; Zhu, Pei-Li; Kwan, Hiu-Yee; Cheng, Brian Chi-Yan; Cao, Hui-Hui; Lee, Sally Kin-Wah; Fong, Wang-Fun; Yu, Zhi-Ling

2017-01-01

Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Eleva...

Effect of 3-aminobenzamide on the rate of ligation during repair of alkylated DNA in human fibroblasts

International Nuclear Information System (INIS)

Morgan, W.F.; Cleaver, J.E.

1983-01-01

3-Aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase, produced rapid reversible changes in single-strand break frequencies in DNA from primary human fibroblasts damaged by alkylating agents, but it did not cause such changes in the DNA of cells damaged by ultraviolet light. The increase in single-strand peak frequencies was not due to an accumulation of blocked repair sites, such as occurs with DNA polymerase inhibitors, but to a delay in the rejoining of induced breaks. 3-Aminobenzamide increases the net break frequency that results from a dynamic balance between excision and ligation. This balance appears to be regulated at the ligation step by adenosine diphosphate ribosylation, which is rapidly altered by addition or removal of 3-aminobenzamide. The rapidity with which strand break frequencies change in the presence of 3-aminobenzamide implies that individual strand breaks resulting from excision at any time after exposure have a lifetime of no more than about 30 min in the cell

Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link".

Science.gov (United States)

Oh, H K; Teo, A K; Ali, R B; Lim, A; Ayi, T C; Yarosh, D B; Li, B F

1996-09-24

Human O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA by transferring alkyl (R-) adducts from O6-alkylguanine (6RG) in DNA to its own cysteine residue at codon 145 (formation of R-MGMT). We show here that R-MGMT in cell extracts, which is sensitive to protease V8 cleavage at the glutamic acid residues at codons 30 (E30) and 172 (E172), can be specifically immunoprecipitated with an MGMT monoclonal antibody, Mab.3C7. This Mab recognizes an epitope of human MGMT including the lysine 107 (K107) which is within the most basic region that is highly conserved among mammalian MGMTs. Surprisingly, the K107L mutant protein is repair-deficient and readily cleaved by protease V8 similar to R-MGMT. We propose that R-MGMT adopted an altered conformation which exposed the Mab.3C7 epitope and rendered that protein sensitive to protease V8 attack. This proposal could be explained by the disruption of a structural "salt-link" within the molecule based on the available structural and biochemical data. The specific binding of Mab.3C7 to R-MGMT has been compared with the protease V8 method in the detection of R-MGMT in extracts of cells treated with low dosages of methyliodide (SN2) and O6-benzylguanine. Their identical behaviors in producing protease V8 sensitive R-MGMT and Mab.3C7 immunoprecipitates suggest that probably methyl iodide (an ineffective agent in producing 6RG in DNA) can directly alkylate the active site of cellular MGMT similar to O6-benzylguanine. The effectiveness of MeI in producing R-MGMT, i.e., inactivation of cellular MGMT, indicates that this agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic O6-alkylguanine residues in DNA in vivo.

A novel acetylation cycle of transcription co-activator Yes-associated protein that is downstream of Hippo pathway is triggered in response to SN2 alkylating agents.

Science.gov (United States)

Hata, Shoji; Hirayama, Jun; Kajiho, Hiroaki; Nakagawa, Kentaro; Hata, Yutaka; Katada, Toshiaki; Furutani-Seiki, Makoto; Nishina, Hiroshi

2012-06-22

Yes-associated protein (YAP) is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes. Although cytoplasmic retention of YAP is known to be mediated by Hippo pathway-dependent phosphorylation, post-translational modifications that regulate YAP in the nucleus remain unclear. Here we report the discovery of a novel cycle of acetylation/deacetylation of nuclear YAP induced in response to S(N)2 alkylating agents. We show that after treatment of cells with the S(N)2 alkylating agent methyl methanesulfonate, YAP phosphorylation mediated by the Hippo pathway is markedly reduced, leading to nuclear translocation of YAP and its acetylation. This YAP acetylation occurs on specific and highly conserved C-terminal lysine residues and is mediated by the nuclear acetyltransferases CBP (CREB binding protein) and p300. Conversely, the nuclear deacetylase SIRT1 is responsible for YAP deacetylation. Intriguingly, we found that YAP acetylation is induced specifically by S(N)2 alkylating agents and not by other DNA-damaging stimuli. These results identify a novel YAP acetylation cycle that occurs in the nucleus downstream of the Hippo pathway. Intriguingly, our findings also indicate that YAP acetylation is involved in responses to a specific type of DNA damage.

DNA adduct profiling of in vitro colonic meat digests to map red vs. white meat genotoxicity.

Science.gov (United States)

Hemeryck, Lieselot Y; Rombouts, Caroline; De Paepe, Ellen; Vanhaecke, Lynn

2018-05-01

The consumption of red meat has been linked to an increased colorectal cancer (CRC) risk. One of the major hypotheses states that heme iron (present in red meat) stimulates the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs). By means of DNA adductomics, chemically induced DNA adduct formation can be mapped in relation to e.g. dietary exposures. In this study, this state-of-the-art methodology was used to investigate alkylation and (lipid per)oxidation induced DNA adduct formation in in vitro red vs. white meat digests. In doing so, 90 alkylation and (lipid per)oxidation induced DNA adduct types could be (tentatively) identified. Overall, 12 NOC- and/or LPO-related DNA adduct types, i.e. dimethyl-T (or ethyl-T), hydroxymethyl-T, tetramethyl-T, methylguanine (MeG), guanidinohydantoin, hydroxybutyl-C, hydroxymethylhydantoin, malondialdehyde-x3-C, O 6 -carboxymethylguanine, hydroxyethyl-T, carboxyethyl-T and 3,N 4 -etheno-C were singled out as potential heme-rich meat digestion markers. The retrieval of these DNA adduct markers is in support of the heme, NOC and LPO hypotheses, suggesting that DNA adduct formation may indeed contribute to red meat related CRC risk. Copyright © 2018 Elsevier Ltd. All rights reserved.

CORRELATION ANALYSIS OF IR, 1 H- AND 13 C-NMR SPECTRAL DATA OF N-ALKYL AND N-CYCLOALKYL CYANOACETAMIDES

Directory of Open Access Journals (Sweden)

Aleksandar D. Marinković

2011-09-01

Full Text Available Linear free energy relationships (LFER were applied to the IR, 1H- and 13C--NMR spectral data in N-alkyl and N-cycloalkyl cyanoacetamides. N-alkyl and N-cycloalkyl cyanocetamides were synthesized from corresponding amine and ethyl cyanoacetate. A number of substituents were employed for alkyl substitution, and fairly good correlations were obtained, using simple Hammett equation. In N-alkyl and N-cycloalkyl cyanoacetamides substituent cause SCS of N-H hydrogen primarily by steric interaction, polar subtituent effect influences SCS shift of C=O carbon, while steric effect of N-alkyl substituent causes IR stretching frequencies of N-H, C=O and CN group. The conformations of investigated compounds have been studied by the use of semiempirical PM6 method, and together with LFER analysis, give a better insight into the influence of such a structure on the transmission of electronic substituent effects. Negative ρ values for several correlations (reverse substituent effect were found.

Repairability during G1 of the inductor leisure of exchanges in the sister chromatid induced by alkylating agents in DNA substituted and no substituted with BUDR, in cells of the salivary gland of mouse In vivo

International Nuclear Information System (INIS)

Gonzalez B, F.

2004-01-01

In this work you determines the repair of the lesions inductoras of Sister chromatid exchange (ICHs) generated in the cells of the salivary gland of mouse, for the treatment with the N-Methyl-N-Nitrosourea (MNU), the N-Ethyl-N-Nitrosourea (ENU), the Methyl methanesulfonate (MMS) and the Ethyl methanesulfonate (EMS) in early and slow G1 of the first one and the second cellular division, that is to say before and after the cells incorporate 5-bromine-2 -Desoxyuridine (BrdU) in the DNA. Groups witness non treaties were included with mutagen. The cells of the salivary gland repaired the generated lesions partially by the MNU, the MMS and the EMS in the 1st division, and only the lesions induced by the ENU and MMS were repaired partially in the 2nd division. The ENU generates injure that they were not repaired in the 1st division and those taken place by the EMS were little repaired in the 2nd division. The methylating agents generated but ICHs that the ethylating. One observes that the BrdU makes to the molecule of the DNA but susceptible to the damage generated by the alkylating agents that induce the formation of the ICHs. This susceptibility was incremented around 150% for the treatment with the MNU, the ENU and the MMS, on the other hand for the EMS it was 3 times minor. It is proposed that the one electronegative atom of this analog of the timine would to work as a nucleophyllic center with which the electrophyllic compounds react. (Author)

Cu(I)-Catalyzed Enantioselective Friedel-Crafts Alkylation of Indoles with 2-Aryl-N-sulfonylaziridines as Alkylating Agents.

Science.gov (United States)

Ge, Chen; Liu, Ren-Rong; Gao, Jian-Rong; Jia, Yi-Xia

2016-07-01

A highly enantioselective Friedel-Crafts alkylation of indoles with N-sulfonylaziridines as alkylating agents has been developed by utilizing the complex of Cu(CH3CN)4BF4/(S)-Segphos as a catalyst. A range of optically active tryptamine derivatives are obtained in good to excellent yields and enantioselectivities (up to >99% ee) via a kinetic resolution process.

Alkylsilyl Peroxides as Alkylating Agents in the Copper-Catalyzed Selective Mono-N-Alkylation of Primary Amides and Arylamines.

Science.gov (United States)

Sakamoto, Ryu; Sakurai, Shunya; Maruoka, Keiji

2017-07-06

The copper-catalyzed selective mono-N-alkylation of primary amides or arylamines using alkylsilyl peroxides as alkylating agents is reported. The reaction proceeds under mild reaction conditions and exhibits a broad substrate scope with respect to the alkylsilyl peroxides, as well as to the primary amides and arylamines. Mechanistic studies suggest that the present reaction should proceed through a free-radical process that includes alkyl radicals generated from the alkylsilyl peroxides. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amphetamine in rat brain after intraperitoneal injection of N-alkylated analogues.

Science.gov (United States)

Nazarali, A J; Baker, G B; Coutts, R T; Pasutto, F M

1983-01-01

Three N-alkylated analogues of amphetamine were administered intraperitoneally to male Sprague-Dawley rats and whole brain levels of amphetamine (AM) and the N-alkyl analogue were determined one hour after injection of the N-alkylated compounds. The drugs administered were the N-2-cyanoethyl-(I) (fenproporex), the N-3-chloropropyl-(II) (mefenorex) and the N-n-propyl-(III) derivatives of AM: the first two of these are used clinically as anorexiants, and the latter has been used extensively to study aspects of metabolism of AM-like compounds. Analysis of AM, I, II and III was performed using electron-capture gas chromatography with a capillary column after reaction of compounds with pentafluorobenzoyl chloride under aqueous conditions. In a second comparative study, equimolar doses (0.05 mMole/kg) of I or AM were administered intraperitoneally to the rats and brain levels determined after one hour. Results indicate extensive N-dealkylation occurs for compounds I, II and III in the rat.

The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine triggers apoptosis through p53-dependent and -independent pathways

International Nuclear Information System (INIS)

Kim, W.-J.; Beardsley, Dillon I.; Adamson, Aaron W.; Brown, Kevin D.

2005-01-01

One of the cellular responses to DNA damaging events is the activation of programmed cell death, also known as apoptosis. Apoptosis is an important process in limiting tumorigenesis by eliminating cells with damaged DNA. This view is reinforced by the finding that many genes with pro-apoptotic function are absent or altered in cancer cells. The tumor suppressor p53 performs a significant role in apoptotic signaling by controlling expression of a host of genes that have pro-apoptotic or pro-survival function. The S N 1 DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers apoptosis and the upregulation/phosphorylation of p53; however, the mechanism(s) governing MNNG-induced cell death remain unresolved. We observed that the human lymphoblastoid cell line WTK-1, which expresses mutant p53, shows far less sensitivity to the cytotoxic effects of MNNG than the closely related, p53-normal line TK-6. Exposure to 15 μM MNNG (LD50 at 24 h in TK-6) leads to a kinetically slower rate of apoptotic onset in WTK-1 cells compared to TK-6 as judged by viability assays and approaches that directly examine apoptotic onset. Similar results were obtained using an unrelated human lymphoblastoid line B310 expressing reduced levels of p53 due to E6 oncoprotein expression, indicating that MNNG activates both p53-dependent and -independent apoptotic mechanisms and that these two mechanisms are discernable by the rates which they trigger apoptotic onset. We document, during time points corresponding to peak apoptotic response in TK6, WTK-1, B310, and B310-E6, that these cell lines show marked decreases in mitochondrial transmembrane potential and increases in cytochrome c within the cytosolic fraction of MNNG-treated cells. Consistent with these events, we observed that both caspase-9 and -3 are activated in our panel of lymphoblastoid cells after MNNG exposure. We also found, using both broad spectrum and specific inhibitors, that blocking caspase activity in TK-6 and

UV-induced influence of N-nitrosoamines on melting parameters of DNA in vitro

International Nuclear Information System (INIS)

Yamshanov, V.A.

1979-01-01

The results of studies have shown the UV-induced decrease of melting temperatures of the DNA of E. coli and chick erythrocytes under the influence of simple N-nitrosoamines (NDMA, NDEA, NDPA). Either UV or nitrosoamines separately failed to effect the DNA or their action was insignificant. It is suggested that this effect may be partly due to the action of UV on DNA

UV-induced influence of N-nitrosoamines on melting parameters of DNA in vitro

Energy Technology Data Exchange (ETDEWEB)

Yamshanov, V A [Nauchno-Issledovatel' skij Inst. Onkologii, Leningrad (USSR)

1979-07-01

The results of studies have shown the UV-induced decrease of melting temperatures of the DNA of E. coli and chick erythrocytes under the influence of simple N-nitrosoamines (NDMA, NDEA, NDPA). Either UV or nitrosoamines separately failed to effect the DNA or their action was insignificant. It is suggested that this effect may be partly due to the action of UV on DNA.

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

International Nuclear Information System (INIS)

Okui, T.; Endoh, D.; Arai, S.; Isogai, E.; Hayashi, M.

1996-01-01

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

A yeast mutant specifically sensitive to bifunctional alkylation

International Nuclear Information System (INIS)

Ruhland, A.; Kircher, M.; Wilborn, F.; Brendel, M.

1981-01-01

A mutation that specifically confers sensitivity to bi- and tri-functional alkylating agents is presented. No or little cross-sensitivity to radiation or monofunctional agents could be detected. Sensitivity does not seem to be due to preferential alkylation of mutant DNA as parent and mutant strain exhibit the same amount of DNA alkylation and the same pattern of DNA lesions including interstrand crosslinks. The mutation is due to a defect in a nuclear gene which has been designated SNM1 (sensitive to nitrogen mustard); it may control an important step in the repair of DNA interstrand crosslinks (orig.(AJ)

Time-resolved fluorescence sensing of N-acetyl amino acids, nucleobases, nucleotides and DNA by the luminescent Tb (III) - 8-alkyl-2-oxo-2H-chromene-3-carbaldehyde probe

Energy Technology Data Exchange (ETDEWEB)

Azab, Hassan A. [Chemistry Department, Faculty of Science, Suez Canal University, 41522 Ismailia (Egypt); Khairy, Gasser M., E-mail: gasser_chemist@yahoo.com [Chemistry Department, Faculty of Science and Arts, Aljouf University, P.O. Box # 2014, Skaka 41421 (Saudi Arabia); Chemistry Department, Faculty of Science, Suez Canal University, 41522 Ismailia (Egypt); Abd El-Ghany, N.; Ahmed, Marwa A. [Chemistry Department, Faculty of Science, Suez Canal University, El-Arish (Egypt)

2016-08-15

A time-resolved (gated) luminescence-based method for the detection of some of N-acetyl amino acids, nucleobases, nucleotides, and DNA using terbium- 8-alkyl-2-oxo-2H-chromene-3-carbaldehyde (AOCC) complex in 1:2 metal: ligand ratio in microtiterplate format has been evolved. The linear range for determination of the selected biomolecules is 0.1–1.0 µM. The detection limit was in the range of 0.0371–0.106 µM. The thermodynamic parameters, and binding constants (K) of N-acetyl amino acids, nucleobases, nucleotides with Tb (III) –(AOCC) {sub 2} complex were calculated. Positive and negative values of entropy (ΔS) and enthalpy (ΔH) changes for Tb (III) –(AOCC){sub 2}– N-acetyl amino acids, nucleobases or nucleotides ternary complexes were evaluated. Selectivity of Tb (III) -complex towards different biomolecules has been studied using ratiometric methods of analysis by comparison of biomolecules binding affinities for Tb (III) -complex. Interaction of Tb (III) complex with DNA has been studied.

Time-resolved fluorescence sensing of N-acetyl amino acids, nucleobases, nucleotides and DNA by the luminescent Tb (III) - 8-alkyl-2-oxo-2H-chromene-3-carbaldehyde probe

International Nuclear Information System (INIS)

Azab, Hassan A.; Khairy, Gasser M.; Abd El-Ghany, N.; Ahmed, Marwa A.

2016-01-01

A time-resolved (gated) luminescence-based method for the detection of some of N-acetyl amino acids, nucleobases, nucleotides, and DNA using terbium- 8-alkyl-2-oxo-2H-chromene-3-carbaldehyde (AOCC) complex in 1:2 metal: ligand ratio in microtiterplate format has been evolved. The linear range for determination of the selected biomolecules is 0.1–1.0 µM. The detection limit was in the range of 0.0371–0.106 µM. The thermodynamic parameters, and binding constants (K) of N-acetyl amino acids, nucleobases, nucleotides with Tb (III) –(AOCC) 2 complex were calculated. Positive and negative values of entropy (ΔS) and enthalpy (ΔH) changes for Tb (III) –(AOCC) 2 – N-acetyl amino acids, nucleobases or nucleotides ternary complexes were evaluated. Selectivity of Tb (III) -complex towards different biomolecules has been studied using ratiometric methods of analysis by comparison of biomolecules binding affinities for Tb (III) -complex. Interaction of Tb (III) complex with DNA has been studied.

Synthesis and characterization of DNA minor groove binding alkylating agents.

Science.gov (United States)

Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K; Mascara, Gerard P; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W; Bobola, Michael S; Silber, John R; Gold, Barry

2013-01-18

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine

International Nuclear Information System (INIS)

Ward, E.J.; Stewart, B.W.

1987-01-01

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine. Either hepatic DNA was prelabeled with [ 3 H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length. The data suggest that structural lesions in heterochromatin, which may be a consequence of incomplete repair, are preferentially degraded by endogenous nuclease(s)

Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Morohoshi, F.; Munakata, N.

1985-03-01

Three mutant strains exhibiting hyper-sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine, but not to methyl methanesulfonate, were selected by a replica method from mutagenized spores of Bacillus subtilis. All three were totally deficient in the adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine with regard to both lethality and mutagenesis. The activity to destroy O/sup 6/-methylguanine residues in the methylated DNA was not elevated in the mutant cells by the pretreatment with sublethal concentrations of N-methyl-N-nitro-N-nitrosoguanidine. This deficiency corresponded to the persistance of O/sup 6/-methylguanine residues in the DNA of both control and pretreated mutant cells challenged with the drug. The lethal and mutagenic sensitivity of the mutant strains were observed only for methyl- or ethyl-nitroso compounds that are thought to be active as inducers and are also active in O-alkylation. Except for the insensitivity to methyl methanesulfonate, the phenotypes of these mutants look very similar to those of ada mutants isolated previously in Escherichia coli.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

Energy Technology Data Exchange (ETDEWEB)

Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Kakadiya, Rajesh B.; Su, Tsann-Long [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China); Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

2014-08-01

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

Science.gov (United States)

Raj, Nitya; Klimstra, David S; Horvat, Natally; Zhang, Liying; Chou, Joanne F; Capanu, Marinela; Basturk, Olca; Do, Richard Kinh Gian; Allen, Peter J; Reidy-Lagunes, Diane

2017-07-01

Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

Role of complex formation in the photosensitized degradation of DNA induced by N'-formylkynurenine

International Nuclear Information System (INIS)

Walrant, P.; Santus, R.; Charlier, M.

1976-01-01

N'-Formylkynurenine derivatives efficiently bind to DNA or polynucleotides. Homopolynucleotides and DNA displayed marked differences in the binding process. Association constants were derived which indicated that the oxidized indole ring is more strongly bound to DNA than the unoxidized one. Irradiation of such complexes with wavelengths greater than 320 nm induced pyrimidine dimer formation as well as DNA chain breaks. Complex formation is shown to play an important role in these photosensitized reactions. The photodynamic action of N-formylkynurenine on DNA constituents was negligible at neutral pH but guanine and xanthine derivatives were sensitizable at higher pH. Thymine dimer splitting can occur in aggregated frozen aqueous solutions of N'-formylkynurenine and thymine dimer but this photosensitized splitting was negligible in liquid solutions at room temperature. (author)

ALKBH7 drives a tissue and sex-specific necrotic cell death response following alkylation-induced damage

Science.gov (United States)

Jordan, Jennifer J; Chhim, Sophea; Margulies, Carrie M; Allocca, Mariacarmela; Bronson, Roderick T; Klungland, Arne; Samson, Leona D; Fu, Dragony

2017-01-01

Regulated necrosis has emerged as a major cell death mechanism in response to different forms of physiological and pharmacological stress. The AlkB homolog 7 (ALKBH7) protein is required for regulated cellular necrosis in response to chemotherapeutic alkylating agents but its role within a whole organism is unknown. Here, we show that ALKBH7 modulates alkylation-induced cellular death through a tissue and sex-specific mechanism. At the whole-animal level, we find that ALKBH7 deficiency confers increased resistance to MMS-induced toxicity in male but not female mice. Moreover, ALKBH7-deficient mice exhibit protection against alkylation-mediated cytotoxicity in retinal photoreceptor and cerebellar granule cells, two cell types that undergo necrotic death through the initiation of the base excision repair pathway and hyperactivation of the PARP1/ARTD1 enzyme. Notably, the protection against alkylation-induced cerebellar degeneration is specific to ALKBH7-deficient male but not female mice. Our results uncover an in vivo role for ALKBH7 in mediating a sexually dimorphic tissue response to alkylation damage that could influence individual responses to chemotherapies based upon alkylating agents. PMID:28726787

Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents.

Science.gov (United States)

Beranek, D T

1990-07-01

Alkylating agents, because of their ability to react directly with DNA either in vitro or in vivo, or following metabolic activation as in the case of the dialkylnitrosamines, have been used extensively in studying the mechanisms of mutagenicity and carcinogenicity. Their occurrence is widespread in the environment and human exposure from natural and pollutant sources is universal. Since most of these chemicals show varying degrees of both carcinogenicity and mutagenicity, and exhibit compound-specific binding patterns, they provide an excellent model for studying molecular dosimetry. Molecular dosimetry defines dose as the number of adducts bound per macromolecule and relates the binding of these adducts to the human mutagenic or carcinogenic response. This review complies DNA alkylation data for both methylating and ethylating agents in a variety of systems and discusses the role these alkylation products plays in molecular mutagenesis.

Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O(6)-methylguanine DNA-methyltransferase in other organisms.

Science.gov (United States)

Kleibl, Karol

2002-09-01

Alkylating agents are environmental genotoxic agents with mutagenic and carcinogenic potential, however, their properties are also exploited in the treatment of malignant diseases. O(6)-Methylguanine is an important adduct formed by methylating agents that, if not repaired, can lead to mutations and death. Its repair is carried out by O(6)-methylguanine DNA-methyltransferase (MTase) in an unique reaction in which methyl groups are transferred to the cysteine acceptor site of the protein itself. Exposure of Escherichia coli cells to sublethal concentrations of methylating agents triggers the expression of a set of genes, which allows the cells to tolerate DNA lesions, and this kind of inducible repair is called the adaptive response. The MTase of E. coli, encoded by the ada gene was the first MTase to be discovered and one of best characterised. Its repair and regulatory mechanisms are understood in considerable detail and this bacterial protein played a key role in identification of its counterparts in other living organisms. This review summarises the nature of alkylation damage in DNA and our current knowledge about the adaptive response in E. coli. I also include a brief mention of MTases from other organisms with the emphasis on the human MTase, which could play a crucial role in both cancer prevention and cancer treatment.

DNA-adducts in fish exposed to alkylating carcinogens

International Nuclear Information System (INIS)

Giam, C.S.; Holliday, T.L.; Williams, J.L.; Bahnson, A.; Weller, R.; Hinton, D.E.

1988-01-01

There are limited studies on DNA-adduct formation following exposure of fish or fish cells to carcinogens. It will be essential to determine if procarcinogens and carcinogens form the same DNA-adducts in different liver cells and how these compare to those reported in mammalian livers. They are also interested in the influence of different alkylating agents on the type and quantity of DNA-adduct formation and repair in fish. While eggs or small fish are ideal for routine screening, large fish such as trout (Salmo gairdneri) is needed initially for the development of analytical procedures for the isolation, quantitation and identification of various adducts. Trout (Salmo gairdneri) weighing approximately 250 grams were acclimatized at 13 degree C before being given i.p. injection of diethylnitrosoamine (DEN). The exposure period varied, though most animals were sacrificed after 24 hours. Their livers were excised and DNA was isolated mainly according the procedure of Croy et al. The neutral thermal hydrolysate and the acid hydrolysate were analyzed by HPLC-Fluorescent detector for 7-ethylguanine and O 6 -ethylguanine, respectively. O 6 -ethylguanine was detected, 7-ethylguanine was not detected. Attempts are being made to improve the detection of the latter compound. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used to establish nanogram quantities of the ethylated bases. Laser desorption FT-IC-MS is particularly useful for characterizing thermally-labile and involatile nucleosides or nucleotides. Excretion of DEN was rapid and high. Exposure of trout (and other fish) to various ethylating agents will be discussed

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

International Nuclear Information System (INIS)

Wolff, Sheldon; Afzal, Veena; Wiencke, J.K.; Olivieri, G.; Michaeli, A.

1988-01-01

The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive reponse that follows exposure to low doses of alkylating agents. (author)

Building blocks for ionic liquids: Vapor pressures and vaporization enthalpies of 1-(n-alkyl)-imidazoles

International Nuclear Information System (INIS)

Emel'yanenko, Vladimir N.; Portnova, Svetlana V.; Verevkin, Sergey P.; Skrzypczak, Andrzej; Schubert, Thomas

2011-01-01

Highlights: → We measured vapor pressures of the 1-(n-alkyl)-imidazoles by transpiration method. → Variations on the alkyl chain length n were C 3 , C 5 -C 7 , and C 9 -C 10 . → Enthalpies of vaporization were derived from (p, T) dependencies. → Enthalpies of vaporization at 298.15 K were linear dependent on the chain length. - Abstract: Vapor pressures of the linear 1-(n-alkyl)-imidazoles with the alkyl chain C 3 , C 5 -C 7 , and C 9 -C 10 have been measured by the transpiration method. The molar enthalpies of vaporization Δ l g H m of these compounds were derived from the temperature dependencies of vapor pressures. A linear correlation of enthalpies of vaporization Δ l g H m (298.15 K) of the 1-(n-alkyl)-imidazoles with the chain length has been found.

Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents

OpenAIRE

Hickman, Mark J.; Samson, Leona D.

1999-01-01

All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O6-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O6-methylguanine (O6MeG) and O6-chloroethylguanine (O6CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSα branch of the DNA mismatch repair pathway (but not the MutSβ branch) is absolutely required for signaling the ...

Targeting the plasma membrane of neoplastic cells through alkylation: a novel approach to cancer chemotherapy.

Science.gov (United States)

Trendowski, Matthew; Fondy, Thomas P

2015-08-01

Although DNA-directed alkylating agents and related compounds have been a mainstay in chemotherapeutic protocols due to their ability to readily interfere with the rapid mitotic progression of malignant cells, their clinical utility is limited by DNA repair mechanisms and immunosuppression. However, the same destructive nature of alkylation can be reciprocated at the cell surface using novel plasma membrane alkylating agents. Plasma membrane alkylating agents have elicited long term survival in mammalian models challenged with carcinomas, sarcomas, and leukemias. Further, a specialized group of plasma membrane alkylating agents known as tetra-O-acetate haloacetamido carbohydrate analogs (Tet-OAHCs) potentiates a substantial leukocyte influx at the administration and primary tumor site, indicative of a potent immune response. The effects of plasma membrane alkylating agents may be further potentiated through the use of another novel class of chemotherapeutic agents, known as dihydroxyacetone phosphate (DHAP) inhibitors, since many cancer types are known to rely on the DHAP pathway for lipid synthesis. Despite these compelling data, preliminary clinical trials for plasma membrane-directed agents have yet to be considered. Therefore, this review is intended for academics and clinicians to postulate a novel approach of chemotherapy; altering critical malignant cell signaling at the plasma membrane surface through alkylation, thereby inducing irreversible changes to functions needed for cell survival.

Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo

International Nuclear Information System (INIS)

Satoh, M.S.; Huh, N.; Rajewsky, M.F.; Kuroki, T.

1988-01-01

DNA repair is essential for maintaining the integrity of the genetic material, and a number of DNA repair mechanisms have been fairly well characterized for the nuclear DNA of eukaryotic cells as well as prokaryotes. However, little is know about DNA repair in mitochondria. Using highly sensitive immunoanalytical methods to detect specific DNA alkylation products, the authors found active removal of O 6 -ethyl-2'-deoxyguanosine (O 6 -EtdGuo) from rat liver mitochondrial DNA after pulse-exposure to N-ethyl-N-nitrosourea in vivo. In the kidney, O 6 -EtdGuo was removed from mitochondrial DNA with moderate efficiency, but nearly no removal was observed from the DNA of brain mitochondria. Among the rat tissues examined, the kinetics of O 6 -EtdGuo elimination from mitochondrial DNA was very similar to the kinetics of removal from nuclear DNA. O 4 -Ethyl-2'-deoxythymidine, another premutagenic DNA ethylation product, was stable in both mitochondrial and nuclear DNA of rat liver

Read-across of ready biodegradability based on the substrate specificity of N-alkyl polypropylene polyamine-degrading microorganisms

NARCIS (Netherlands)

Geerts, R.; Ginkel, van C.G.; Plugge, C.M.

2017-01-01

The biodegradation of N-alkyl polypropylene polyamines (NAPPs) was studied using pure and mixed cultures to enable read-across of ready biodegradability test results. Two Pseudomonas spp. were isolated from activated sludge with N-oleyl alkyl propylene diamine and N-coco alkyl dipropylene triamine,

Cellular response to alkylating agent MNNG is impaired in STAT1-deficients cells.

Science.gov (United States)

Ah-Koon, Laurent; Lesage, Denis; Lemadre, Elodie; Souissi, Inès; Fagard, Remi; Varin-Blank, Nadine; Fabre, Emmanuelle E; Schischmanoff, Olivier

2016-10-01

The SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1, an anti-proliferative and pro-apoptotic transcription factor is known to potentiate p53 and to affect DNA-damage cellular response. We studied whether STAT1 may modulate cell fate following activation of the mismatch repair system upon exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Using STAT1-proficient or -deficient cell lines, we found that STAT1 is required for: (i) reduction in the extent of DNA lesions, (ii) rapid phosphorylation of T68-CHK2 and of S15-p53, (iii) progression through the G2 /M checkpoint and (iv) long-term survival following treatment with MNNG. Presence of STAT1 is critical for the formation of a p53-DNA complex comprising: STAT1, c-Abl and MLH1 following exposure to MNNG. Importantly, presence of STAT1 allows recruitment of c-Abl to p53-DNA complex and links c-Abl tyrosine kinase activity to MNNG-toxicity. Thus, our data highlight the important modulatory role of STAT1 in the signalling pathway activated by the mismatch repair system. This ability of STAT1 to favour resistance to MNNG indicates the targeting of STAT1 pathway as a therapeutic option for enhancing the efficacy of SN1 alkylating agent-based chemotherapy. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model.

Science.gov (United States)

Kelner, Michael J; McMorris, Trevor C; Rojas, Rafael J; Estes, Leita A; Suthipinijtham, Pharnuk

2008-12-01

Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

Light of DNA-alkylating agents in castration-resistant prostate cancer cells: a novel mixed EGFR/DNA targeting combi-molecule.

Science.gov (United States)

Liang, Guan-Can; Zheng, Hao-Feng; Chen, Yan-Xiong; Li, Teng-Cheng; Liu, Wei; Fang, You-Qiang

2017-01-01

The mechanism underlying the therapeutic effects of combi-molecule JDF12 on prostate cancer (PCa) DU145 cells remains still unclear. This study aimed to investigate the proteomic profile after JDF12 treatment in DU145 cells by comparing with that in Iressa treated cells and untreated cells. MTT was used to evaluate drug cytotoxicity, DAPI staining was done to assess apoptosis of cells, and flow cytometry was used to analyze cell cycle. iTRAQ and qPCR were employed to obtain the proteomic profiles of JDF12 treated, Iressa treated, and untreated DU145 cells, and validate the expression of selected differentially expressed proteins, respectively. JDF12 could significantly inhibit the proliferation and increase the apoptosis of DU145 cells when compared with Iressa or blank group. In total, 5071 proteins were obtained, out of which, 42, including 21 up-regulated and 21 down-regulated proteins, were differentially expressed in JDF12 group when compared with Iressa and blank groups. The up-regulated proteins were mainly involved in DNA damage/repair and energy metabolism; while the down-regulated proteins were mainly associated with cell apoptosis. qPCR confirmed the expression of several biologically important proteins in DU145 cells after JDF12 treatment. The molecular mechanisms of DNA alkylating agents on PCa therapy that with the assistant of EGFR-blocker were revealed on proteomic level, which may increase the possible applications of DNA alkylating agents and JDF12 on PCa therapy.

Masked N-Heterocyclic Carbene-Catalyzed Alkylation of Phenols with Organic Carbonates.

Science.gov (United States)

Lui, Matthew Y; Yuen, Alexander K L; Masters, Anthony F; Maschmeyer, Thomas

2016-09-08

An easily prepared masked N-heterocyclic carbene, 1,3-dimethylimidazolium-2-carboxylate (DMI-CO2 ), was investigated as a "green" and inexpensive organocatalyst for the alkylation of phenols. The process made use of various low-toxicity and renewable alkylating agents, such as dimethyl- and diethyl carbonate, in a focused microwave reactor. DMI-CO2 was found to be a very active catalyst and excellent yields of a range of aryl alkyl ethers were obtained under relatively benign conditions. The observed difference in the conversion behavior of phenol methylation, in the presence of either the carbene or 1,8-diazabicycloundec-7-ene (DBU) catalyst, was rationalized on the basis of mechanistic investigations. The primary mode of action for the N-heterocyclic carbene is nucleophilic catalysis. Activation of the dialkyl carbonate electrophile results in concomitant evolution of an organo-soluble alkoxide, which deprotonates the phenolic starting material. In contrast, DBU is initially protonated by the phenol and thus consumed. Subsequent regeneration and participation in nucleophilic catalysis only becomes significant after some phenolate alkylation occurs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Study on the Alkylation Reactions of N(7)-Unsubstituted 1,3-Diazaoxindoles.

Science.gov (United States)

Kókai, Eszter; Halász, Judit; Dancsó, András; Nagy, József; Simig, Gyula; Volk, Balázs

2017-05-19

The chemistry of the 5,7-dihydro-6 H -pyrrolo[2,3- d ]pyrimidin-6-one (1,3-diazaoxindole) compound family, possessing a drug-like scaffold, is unexplored. In this study, the alkylation reactions of N (7)-unsubstituted 5-isopropyl-1,3-diazaoxindoles bearing various substituents at the C (2) position have been investigated. The starting compounds were synthesized from the C (5)-unsubstituted parent compounds by condensation with acetone and subsequent catalytic reduction of the 5-isopropylidene moiety. Alkylation of the thus obtained 5-isopropyl derivatives with methyl iodide or benzyl bromide in the presence of a large excess of sodium hydroxide led to 5,7-disubstituted derivatives. Use of butyllithium as the base rendered alkylation in the C (5) position possible with reasonable selectivity, without affecting the N (7) atom. During the study on the alkylation reactions, some interesting by-products were also isolated and characterized.

N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA

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Narayan Satya

2004-01-01

Full Text Available Abstract Background Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG is able to induce senescence in the HCT-116 colon cancer cell line. Results Cells treated with lower concentrations of MNNG (0–25 microM for 50 h showed a dose-dependent increase in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50–100 microM showed a senescence-like G0/G1 phase arrest which was confirmed by increased expression of β-galactosidase, a senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of p53 protein, but the senescence-like G0/G1 phase arrest was dissociated with p53 protein levels, since the p53 protein levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-related proteins such as p21, p27, cyclin B1, Cdc2, c-Myc and max. The expression levels of these proteins were increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals with cellular senescence in MNNG-treated cells. We found that protein and m

Effect of green juice and their bioactive compounds on genotoxicity induced by alkylating agents in mice.

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Fagundes, Gabriela Elibio; Damiani, Adriani Paganini; Borges, Gabriela Daminelli; Teixeira, Karina Oliveira; Jesus, Maiellen Martins; Daumann, Francine; Ramlov, Fernanda; Carvalho, Tiago; Leffa, Daniela Dimer; Rohr, Paula; Moraes De Andrade, Vanessa

2017-01-01

Kale juice (Brassica oleracea L. var. acephala D.C.) is a reliable source of dietary carotenoids and typically contains the highest concentrations of lutein (LT) and beta-carotene (BC) among green leafy vegetables. As a result of their antioxidant properties, dietary carotenoids are postulated to decrease the risk of disease occurrence, particularly certain cancers. The present study aimed to (1) examine the genotoxic and antigenotoxic activity of natural and commercially available juices derived from Brassica oleracea and (2) assess influence of LT or BC against DNA damage induced by alkylating agents such as methyl methanesulfonate (MS) or cyclophosphamide (CP) in vivo in mice. Male Swiss mice were divided into groups of 6 animals, which were treated with water, natural, or commercial Brassica oleraceae juices (kale), LT, BC, MMS, or CP. After treatment, DNA damage was determined in peripheral blood lymphocytes using the comet assay. Results demonstrated that none of the Brassica oleraceae juices or carotenoids produced genotoxic effects. In all examined cell types, kale juices or carotenoids inhibited DNA damage induced by MMS or CP administered either pre- or posttreatment by 50 and 20%, respectively. Under our experimental conditions, kale leaf juices alone exerted no marked genotoxic or clastogenic effects. However, a significant decrease in DNA damage induced by MMS or CP was noted. This effect was most pronounced in groups that received juices, rather than carotenoids, suggesting that the synergy among constituents present in the food matrix may be more beneficial than the action of single compounds. Data suggest that the antigenotoxic properties of kale juices may be of therapeutic importance.

N- and C-alkylation of seven-membered iminosugars generates potent glucocerebrosidase inhibitors and F508del-CFTR correctors.

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Désiré, J; Mondon, M; Fontelle, N; Nakagawa, S; Hirokami, Y; Adachi, I; Iwaki, R; Fleet, G W J; Alonzi, D S; Twigg, G; Butters, T D; Bertrand, J; Cendret, V; Becq, F; Norez, C; Marrot, J; Kato, A; Blériot, Y

2014-11-28

The glycosidase inhibitory properties of synthetic C-alkyl and N-alkyl six-membered iminosugars have been extensively studied leading to therapeutic candidates. The related seven-membered iminocyclitols have been less examined despite the report of promising structures. Using an in house ring enlargement/C-alkylation as well as cross-metathesis methodologies as the key steps, we have undertaken the synthesis and biological evaluation of a library of fourteen 2C- and eight N-alkyl tetrahydroxylated azepanes starting from an easily available glucopyranose-derived azidolactol. Four, six, nine and twelve carbon atom alkyl chains have been introduced. The study of two distinct D-gluco and L-ido stereochemistries for the tetrol pattern as well as R and S configurations for the C-2 carbon bearing the C-alkyl chain is reported. We observed that C-alkylation of the L-ido tetrahydroxylated azepane converts it from an α-L-fucosidase to a β-glucosidase and β-galactosidase inhibitor while N-alkylation of the D-gluco iminosugar significantly improves its inhibition profile leading to potent β-glucosidase, β-galactosidase, α-L-rhamnosidase and β-glucuronidase inhibitors whatever the stereochemistry of the alkyl chain. Interestingly, the N-alkyl chain length usually parallels the azepane inhibitor potency as exemplified by the identification of a potent glucocerebrosidase inhibitor (Ki 1 μM) bearing a twelve carbon atom chain. Additionally, several C-alkyl azepanes demonstrated promising F508del-CFTR correction unlike the parent tetrahydroxyazepanes. None of the C-alkyl and N-alkyl azepanes did inhibit ER α-glucosidases I or II.

Deuterium isotope effect on metabolism of N-nitrosodimethylamine in vivo in rat

International Nuclear Information System (INIS)

Swann, P.F.; Mace, R.; Angeles, R.M.; Keefer, L.K.

1983-01-01

The maximal rates of metabolic oxidation of N-nitrosodimethylamine (NDMA) and N-nitrosodimethylamine-d6 (NDMA-d6) in vivo (VH and VD, respectively) have been measured by following 14CO2 exhalation in rats after intraperitoneal injection of the two 14C-labelled carcinogens at high doses (20 or 40 mg/kg). Complete deuteration of NDMA reduced only slightly the maximal rate of metabolism when the two substrates were administered separately (VH/VD approximately 1.2). However, much larger (approximately 4-fold) deuterium isotope effects were observed when mixtures of NDMA with NDMA-d6 were injected. These results are tentatively interpreted as evidence that C-H bond cleavage is not a rate limiting feature of overall metabolism, but that the complex between NDMA and the principal enzyme(s) metabolizing it in vivo freely equilibrates with unbound substrate. Single, large, intraperitoneal doses of NDMA and NDMA-d6 produced a similar alkylation of rat liver DNA and also of kidney DNA. However, a small oral dose (54 micrograms/kg) of NDMA-d6 produced 1/3 less alkylation of liver DNA and 3 times as much alkylation of kidney DNA as did an equimolar dose of NDMA. The reduction in alkylation of liver DNA correlates well with, and possibly explains, the decreased ability of NDMA-d6 to induce liver tumors in rats. The associated increase in the alkylation of kidney DNA suggests that this change is due to a decrease in the amount of nitrosamine removed from the portal blood on the first pass through the liver

Aryl sulfonate based anticancer alkylating agents.

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Sheikh, Hamdullah Khadim; Arshad, Tanzila; Kanwal, Ghazala

2018-05-01

This research work revolves around synthesis of antineoplastic alkylating sulfonate esters with dual alkylating sites for crosslinking of the DNA strands. These molecules were evaluated as potential antineoplastic cross linking alkylating agents by reaction with the nucleoside of Guanine DNA nucleobase at both ends of the synthesized molecule. Synthesis of the alkylating molecules and the crosslinking with the guanosine nucleoside was monitored by MALDITOF mass spectroscopy. The synthesized molecule's crosslinking or adduct forming rate with the nucleoside was compared with that of 1,4 butane disulfonate (busulfan), in form of time taken for the appearance of [M+H] + . It was found that aryl sulfonate leaving group was causing higher rate of nucleophilic attack by the Lewis basic site of the nucleobase. Furthermore, the rate was also found to be a function of electron withdrawing or donating nature of the substituent on the aryl ring. Compound with strong electron withdrawing substituent on the para position of the ring reacted fastest. Hence, new alkylating agents were synthesized with optimized or desired reactivity.

Photosensitive interaction of RSU 1069 with DNA

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Edwards, D.I.; Knox, R.J.; Skolimowski, I.M.; Zahoor, A.; Knight, R.C.

1984-08-01

RSU 1069 is a 2-nitroimidazole radiosensitizer with an aziridine-containing side chain. In light (360 nm) the absorbance maximum of the nitro group at 325 nm disappears, which is accompanied by expulsion of the nitro group as the nitrite ion. This photosensitive effect was used to determine separately the damage of DNA induced by the reduced nitro group and the alkylating property of the aziridine. The aziridine-induced DNA damage is maximized in the dark when the nitro group is either absent (electrolytically reduced prior to the addition of DNA) or non functional (unreduced). In the light, damage is reduced. Typical DNA damage includes helix disruption leading to single strand breaks and the release of thymidine. Alkaline filter elution studies show evidence only for strand breakage and none for cross-linking indicating the drug is capable of mono-functional alkylation only.

Photosensitive interaction of RSU 1069 with DNA

International Nuclear Information System (INIS)

Edwards, D.I.; Knox, R.J.; Skolimowski, I.M.; Zahoor, A.; Knight, R.C.

1984-01-01

RSU 1069 is a 2-nitroimidazole radiosensitizer with an aziridine-containing side chain. In light (360 nm) the absorbance maximum of the nitro group at 325 nm disappears, which is accompanied by expulsion of the nitro group as the nitrite ion. This photosensitive effect was used to determine separately the damage of DNA induced by the reduced nitro group and the alkylating property of the aziridine. The aziridine-induced DNA damage is maximized in the dark when the nitro group is either absent (electrolytically reduced prior to the addition of DNA) or non functional (unreduced). In the light, damage is reduced. Typical DNA damage includes helix disruption leading to single strand breaks and the release of thymidine. Alkaline filter elution studies show evidence only for strand breakage and none for cross-linking indicating the drug is capable of mono-functional alkylation only

DNA repair in mutagen-injured higher plants

International Nuclear Information System (INIS)

Veleminsky, J.; Gichner, T.

1978-01-01

Data are summarized proving the occurrence of photoreactivation of UV-induced pyrimidine dimers in cells of Nicotiana tabucum, Gingko and carrot, the excision of dimers in cells of Nicotiana tabacum, Gingko and carrot, the excision of dimers in protoplasts of carrot and in embryos of Lathyrus sativus, and the repair of DNA single-strand breaks induced in carrot protoplasts and barley embryonic cells by ionizing radiation. In irradiated barley embryos the unscheduled DNA synthesis and higher accessibility of induced primers to DNA polymerase I of E. coli were observed preferentially in G 1 cells with diffused chromatin. These reactions were inhibited by caffeine and EDTA. Unscheduled DNA synthesis was also observed in synchronized irradiated root cuttings of Vicia faba and in barley embryos treated with 4-nitroquinoline oxide, the latter being inhibited by caffeine and hydroxyurea. Repair synthesis was also established in barley embryos treated with mutagenic N-methyl-N-nitrosourea under conditions that postponed the onset of germination after the treatment. The same conditions enhanced the repair of DNA single-strand breaks induced by this mutagen and several other monofunctional alkylating compounds. From tissues of barley and of Phaseolus multiflorus, endonucleases for apurinic sites were isolated and characterized. Some of them are located in chromatin, others in chloroplasts. The relation between DNA repair and genetic effects of mutagens in higher plants is also discussed. (Auth.)

Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents

International Nuclear Information System (INIS)

Hoy, C.A.; Thompson, L.H.; Mooney, C.L.; Salazar, E.P.

1985-01-01

DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity. Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Although the initial number of cross-links was similar for the three cell lines, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes

Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

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Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

2013-01-01

Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1

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Matthew J. Randall

2013-01-01

Full Text Available Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal. Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1, a critical enzyme involved in regulation of thioredoxin (Trx-mediated redox signaling, by alkylation at its selenocysteine (Sec residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases

Quinone methides tethered to naphthalene diimides as selective G-quadruplex alkylating agents.

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Di Antonio, Marco; Doria, Filippo; Richter, Sara N; Bertipaglia, Carolina; Mella, Mariella; Sissi, Claudia; Palumbo, Manlio; Freccero, Mauro

2009-09-16

We have developed novel G-quadruplex (G-4) ligand/alkylating hybrid structures, tethering the naphthalene diimide moiety to quaternary ammonium salts of Mannich bases, as quinone-methide precursors, activatable by mild thermal digestion (40 degrees C). The bis-substituted naphthalene diimides were efficiently synthesized, and their reactivity as activatable bis-alkylating agents was investigated in the presence of thiols and amines in aqueous buffered solutions. The electrophilic intermediate, quinone-methide, involved in the alkylation process was trapped, in the presence of ethyl vinyl ether, in a hetero Diels-Alder [4 + 2] cycloaddition reaction, yielding a substituted 2-ethoxychroman. The DNA recognition and alkylation properties of these new derivatives were investigated by gel electrophoresis, circular dichroism, and enzymatic assays. The alkylation process occurred preferentially on the G-4 structure in comparison to other DNA conformations. By dissecting reversible recognition and alkylation events, we found that the reversible process is a prerequisite to DNA alkylation, which in turn reinforces the G-quadruplex structural rearrangement.

Mechanisms of resistance to alkylating agents

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Damia, G.; D‘Incalci, M.

1998-01-01

Alkylating agents are the most widely used anticancer drugs whose main target is the DNA, although how exactly the DNA lesions cause cell death is still not clear. The emergence of resistance to this class of drugs as well as to other antitumor agents is one of the major causes of failure of cancer treatment. This paper reviews some of the best characterized mechanisms of resistance to alkylating agents. Pre- and post-target mechanisms are recognized, the former able to limit the formation of...

Development of laser-induced fluorescence detection to assay DNA damage

International Nuclear Information System (INIS)

Sharma, M.; Freund, H.G.

1991-01-01

A precolumn derivation method has been developed for high performance liquid chromatographic (HPLC) analysis of DNA damage using fluorescence detection. The modified nucleotide, having excised enzymatically from the exposed DNA, is enriched from the normal nucleotides and labeled with a fluorescent reagent. The labeling procedure involves phosphoramidation of the nucleotide with ethylenediamine (EDA) followed by conjugation of the free amino end of the phosphoramidate with 5-dimethylaminonaphthalene 1-sulfonyl chloride, commonly known as Dansyl chloride. The dansylated nucleotide can be analyzed with a sub-picomole limit of detection (LOD) by conventional HPLC using a conventional fluorescence detector. By combining microbore HPLC with laser-induced fluorescence (LIF) detection, the authors present the development of an analytical system that has sub-femtomole LOD for real-time analysis of the dansylated nucleotide. In this paper the application of the developed system in fluorescence postlabeling assay of a small alkyl-modified nucleotide (5-methyl CMP) in calf-thymus DNA is discussed

Copper(II)/amine synergistically catalyzed enantioselective alkylation of cyclic N-acyl hemiaminals with aldehydes.

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Sun, Shutao; Mao, Ying; Lou, Hongxiang; Liu, Lei

2015-07-07

The first catalytic asymmetric alkylation of N-acyl quinoliniums with aldehydes has been described. A copper/amine synergistic catalytic system has been developed, allowing the addition of functionalized aldehydes to a wide range of electronically varied N-acyl quinoliniums in good yields with excellent enantiocontrol. The synergistic catalytic system was also effective for N-acyl dihydroisoquinoliniums and β-caboliniums, demonstrating the general applicability of the protocol in the enantioselective alkylation of diverse cyclic N-acyl hemiaminals.

Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents.

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Christmann, M; Kaina, B

2000-11-17

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O(6)-methylguanine in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSalpha complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation. The results demonstrate that O(6)-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O(6)-methylguanine-generating mutagens results in an increase of MutSalpha and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.

Neurotoxicity induced by alkyl nitrites: Impairment in learning/memory and motor coordination.

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Cha, Hye Jin; Kim, Yun Ji; Jeon, Seo Young; Kim, Young-Hoon; Shin, Jisoon; Yun, Jaesuk; Han, Kyoungmoon; Park, Hye-Kyung; Kim, Hyung Soo

2016-04-21

Although alkyl nitrites are used as recreational drugs, there is only little research data regarding their effects on the central nervous system including their neurotoxicity. This study investigated the neurotoxicity of three representative alkyl nitrites (isobutyl nitrite, isoamyl nitrite, and butyl nitrite), and whether it affected learning/memory function and motor coordination in rodents. Morris water maze test was performed in mice after administrating the mice with varying doses of the substances in two different injection schedules of memory acquisition and memory retention. A rota-rod test was then performed in rats. All tested alkyl nitrites lowered the rodents' capacity for learning and memory, as assessed by both the acquisition and retention tests. The results of the rota-rod test showed that isobutyl nitrite in particular impaired motor coordination in chronically treated rats. The mice chronically injected with isoamyl nitrite also showed impaired function, while butyl nitrite had no significant effect. The results of the water maze test suggest that alkyl nitrites may impair learning and memory. Additionally, isoamyl nitrite affected the rodents' motor coordination ability. Collectively, our findings suggest that alkyl nitrites may induce neurotoxicity, especially on the aspect of learning and memory function. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Ionic liquid containing hydroxamate and N-alkyl sulfamate ions

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Friesen, Cody A.; Wolfe, Derek; Johnson, Paul Bryan

2016-03-15

Embodiments of the invention are related to ionic liquids and more specifically to ionic liquids used in electrochemical metal-air cells in which the ionic liquid includes a cation and an anion selected from hydroxamate and/or N-alkyl sulfamate anions.

N-fluoroalkylated and N-alkylated analogues of the dopaminergic D-2 receptor antagonist raclopride

International Nuclear Information System (INIS)

Lannoye, G.S.; Moerlein, S.M.; Parkinson, D.; Welch, M.J.

1990-01-01

A series of raclopride [(S)-2-[(3,5-dichloro-6-methoxy-2- hydroxybenzamido)methyl]-1-ethylpyrrolidine] derivatives bearing pyrrolidino N-fluoroalkyl or -alkyl substituents were synthesized and evaluated as potential dopaminergic receptor-based positron tomography radiopharmaceuticals. Radiosynthetic procedures for producing the corresponding N-[18F]fluoroalkylated analogues of raclopride from 18F- (beta+, t1/2 = 110 min) in high specific activity were also developed. In vitro binding assays using competitive displacement of [3H]spiperone from primate caudate tissue indicated that the N-alkylated analogues of raclopride had Ki values of 5-40 nM, whereas the corresponding values for analogous N-fluoroalkylated derivatives ranged from 90-160 nM. The relatively low D-2 binding affinity of these fluorinated salicylamides was corroborated by in vivo tissue biodistribution results in rodents. On the basis of structure-binding correlations, the impact of intramolecular hydrogen bonding, ligand basicity, and steric bulk on the affinity of the benzamides for D-2 receptor binding are discussed. Strategies are presented for the development of alternative fluorinated salicylamides that are both receptor active and metabolically stable

Comparative analysis of oxidative synthesis of N-alkyl, N,N-dialkyl and N-cykloalkyl-O-isobutyl thioncarbamate

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Sovrlić Milica Ž.

2011-01-01

Full Text Available A optimized synthesis of N-alkyl, N,N-dialkyl- and N-cycloalkyl-O-isobutyl thioncarbamates by aminolysis of sodium isobutylxanthogenic acid (NaiBXAc and primary, secondary and cycloalkyl amines was developed at laboratory scale and applied at semi-industrial level. Studies on dependence of N-n-propyl-O-isopropylthiocarbamate yield and purity with respect to reaction parameters: reaction time and molar ratio of n-propylamine and NaiBXAc, were performed. In such way, optimal reaction conditions for synthesis of N-alkyl, N,N-dialkyl- and N-cycloalkyl-O-isobutyl thioncarbamates, by aminolysis of NaiBXAc, were established. Also, comparative results of thioncarbamates synthesis starting from potassium isobutyl xanthate (KiBX and corresponding amines in presence of different oxidants: hydrogen peroxide, sodium hypochlorite and new oxidative agent potassium peroxodisulfate were evaluated. Synthesized compounds have been fully characterized by FTIR, 1H and 13C NMR and MS data, elemental analysis and purity have been determined by gas chromatographic method (GC. According to our knowledge, ten synthesized thioncarbamates are for the first time characterized. Synthesized compounds could be used as selective reagents for flotation of copper and zinc ores. The presented methods offer several benefits, namely, high product yields and purity, simple operation, mild reaction conditions without use of hazardous organic solvents, while some of them could be implemented on industrial scale production.

N,N'-(Hexane-1,6-diylbis(4-methyl-N-(oxiran-2-ylmethylbenzenesulfonamide: Synthesis via cyclodextrin mediated N-alkylation in aqueous solution and further Prilezhaev epoxidation

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Julian Fischer

2013-12-01

Full Text Available N-alkylation of N,N'-(hexane-1,6-diylbis(4-methylbenzenesulfonamide with allyl bromide and subsequent Prilezhaev reaction with m-chloroperbenzoic acid to give N,N'-(hexane-1,6-diylbis(4-methyl-N-(oxiran-2-ylmethylbenzenesulfonamide is described. This twofold alkylation was performed in aqueous solution, whereby α-, and randomly methylated β-cyclodextrin were used as adequate phase transfer catalysts and the cyclodextrin–guest complexes were characterized by 1H NMR and 2D NMR ROESY spectroscopy. Finally, the curing properties of the diepoxide with lysine-based α-amino-ε-caprolactam were analyzed by rheological measurements.

TRANSPORT PROPERTIES FOR 1-ETHYL-3-METHYLIMIDAZOLIUM n-ALKYL SULFATES: POSSIBLE EVIDENCE OF GROTTHUSS MECHANISM

International Nuclear Information System (INIS)

García-Garabal, S.; Vila, J.; Rilo, E.; Domínguez-Pérez, M.; Segade, L.; Tojo, E.; Verdía, P.; Varela, L.M.; Cabeza, O.

2017-01-01

The objective of this work was to study the effect of the temperature and the lengthening of the linear alkyl chain of the anion in the transport physical properties of the pure ionic liquids 1-ethyl-3-methyl imidazolium n-alkyl sulphate (being n = 0, 1, 2, 4, 6 and 8). Density, viscosity and electrical conductivities were measured at atmospheric pressure in a wide temperature range. In the bibliography, data existed for these magnitudes for all ionic liquids studied but none of these had information about the electrical conductivity of 1-ethyl-3-methyl imidazolium n-alkyl sulfate whith n = 0, 4, 6 and 8. The experimental results show clearly 1-ethyl-3-methyl imidazolium hydrogen sulphate cannot be considered part of the 1-ethyl-3-methyl imidazolium n-alkyl sulphate family because of its hydrogen bonding ability. Results of density and viscosity behave as expected. However, in the case of the electrical conductivity due to the lack of alkyl chain in the hydrogen sulfate we expected to get extreme values but in practise, we obtained intermediate values between 1-ethyl-3-methyl imidazolium butyl sulphate and 1-ethyl-3-methyl imidazolium hexyl sulphate. This suggests that a Grotthus mechanism exists as result of a protonic current in addition to ionic conductivity, being Waldeńs plot consistent with this idea.

Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts

International Nuclear Information System (INIS)

Shaham, M.; Adler, B.; Ganguly, S.; Chaganti, R.S.K.

1987-01-01

Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). The authors report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental of Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 μgml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEB/sup r/) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEB/sup r/ colonies independently derived from transfection of human DNA and one of three DEB/sup r/ colonies independently derived from transfection of Chinese hamster DNA. The results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA

Alkylating Agent-Induced NRF2 Blocks Endoplasmic Reticulum Stress-Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis.

Science.gov (United States)

Zanotto-Filho, Alfeu; Masamsetti, V Pragathi; Loranc, Eva; Tonapi, Sonal S; Gorthi, Aparna; Bernard, Xavier; Gonçalves, Rosângela Mayer; Moreira, José C F; Chen, Yidong; Bishop, Alexander J R

2016-12-01

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR. ©2016 American Association for Cancer Research.

Alkylating agent induced NRF2 blocks endoplasmic reticulum stress-mediated apoptosis via control of glutathione pools and protein thiol homeostasis

Science.gov (United States)

Zanotto-Filho, Alfeu; Masamsetti, V. Pragathi; Loranc, Eva; Tonapi, Sonal S.; Gorthi, Aparna; Bernard, Xavier; Gonçalves, Rosângela Mayer; Moreira, José C. F.; Chen, Yidong; Bishop, Alexander J. R.

2016-01-01

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival; an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine (BSO) caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine (NAC) or glutathione-ethyl-ester (GSH-E), decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 targets overexpression and poor survival. In KEAP1 mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. PMID:27638861

Chemical carcinogenesis in the nervous system. Preferential accumulation of O6-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-N-nitrosourea.

Science.gov (United States)

Margison, G P; Kleihues, P

1975-01-01

The alkylation of purine bases in DNA of several rat tissues was determined during weekly injections (10 mg/kg) of N-[3H]methyl-N-nitrosourea, a dose schedule known to selectively induce tumours of the nervous system. Each group of animals was killed 1 week after the final injection, and the DNA hydrolysates were analysed by chromatography on Sephadex G-10. After five weekly applications, O6-methylguanine had accumulated in brain DNA to an extent which greatly exceeded that in kidney, spleen and intestine. In the liver, the final O6-methylguanine concentration was less than 1% of that in brain. Between the first and the fifth injection, the O6-methylguanine/7-methylguanine ratio in cerebral DNA increased from 0.28 to 0.68. In addition, 3-methylguanine was found to accumulate in brain DNA whereas in the other organs no significant quantities of this base were detectable. The results are compatible with the hypothesis that O6-alkylation of guanine in DNA plays a major role in the induction of tumours by N-methyl-N-nitrosourea and related carcinogens. The kinetics of the increase of O6-methylguanine in cerebral DNA suggest that there is no major cell fraction in the brain which is capable of excising chemically methylated bases from DNA. This repair deficiency could be a determining factor in the selective induction of nervous-system tumours by N-methyl-N-nitrosourea and other neuro-oncogenic compounds. PMID:1200992

Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts

International Nuclear Information System (INIS)

McCormick, J.J.; Maher, V.M.

1985-01-01

A comparison of the cytotoxicity and mutagenicity of a series of carcinogens in normal diploid human fibroblasts and in cells deficient in one or more DNA repair processes has provided insight into the specific DNA adduct(s) responsible for these biological effects. The carcinogens tested include ultraviolet radiation; reactive derivatives of structurally related aromatic amides; metabolites of benzo(a)pyrene; the simple alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea; and aflatoxin B 1 dichloride, a model for the reactive 2,3-epoxide of aflatoxin B 1 . Exponentially growing cells were exposed to agents and assayed for mutations and cell killing. Cells deficient in repair of particular DNA adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. Many of the carcinogens were compared for their mutagenic and/or cytotoxic effect, not only as a function of dose administered, but also as a function of the initial number of adducts or photoproducts induced in DNA and the number remaining at critical times posttreatment. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and frequency of mutations induced. Comparative studies of the frequency of UV-induced transformation of normal and repair-deficient cells showed this also to be true for transformation

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

Science.gov (United States)

O'Hanlon, Karen A; Margison, Geoffrey P; Hatch, Amy; Fitzpatrick, David A; Owens, Rebecca A; Doyle, Sean; Jones, Gary W

2012-09-01

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O(6)-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system.

Globular, Sponge-like to Layer-like Morphological Transition in 1-n-Alkyl-3-methylimidazolium Octylsulfate Ionic Liquid Homologous Series.

Science.gov (United States)

Kapoor, Utkarsh; Shah, Jindal K

2018-01-11

Segregation of polar and nonpolar domains in ionic liquids for which either the cation or anion is responsible for inducing nonpolar domains is well understood. On the other hand, information regarding the nanoscale heterogeneities originating due to the presence of nonpolar content on both the ions is rudimentary at this point. The present contribution is aimed at addressing this question and focuses on a molecular dynamics simulation study to probe nanoscale structural and aggregation features of the 1-n-alkyl-3-methylimidazolium [C n mim] octylsulfate [C 8 SO 4 ] ionic liquid homologous series (n = 2, 4, 6, 8, 10, and 12). The objective of this work is to determine the effect of increasing alkyl chain length in the cation on nonpolar domain formation, especially when the alkyl chain lengths from both the ions participate in defining such domains. The results indicate that all the ionic liquids form nonpolar domains, morphology of which gradually changes from globular, sponge-like to layer-like structure with increase in the cationic alkyl chain length. The length of the nonpolar domains calculated from the total structure factor for [C 10 mim][C 8 SO 4 ] is considerably higher than that reported for other imidazolium-based ionic liquid containing smaller anions. The structure factor for [C 12 mim][C 8 SO 4 ] ionic liquid contains multiple intermediate peaks separating the charge alternation peak and pre-peak, which points to nonpolar domains of varying lengths, an observation that remains to be validated. Analysis of the heterogeneous order parameters and orientational correlation functions of the alkyl chains further suggests an increase in the spatial heterogeneity and long-range order along the homologous series. The origin of rich diversity of structures obtained by introducing nonpolar content on both the ions is discussed.

Development of the adverse outcome pathway "alkylation of DNA in male premeiotic germ cells leading to heritable mutations" using the OECD's users' handbook supplement.

Science.gov (United States)

Yauk, Carole L; Lambert, Iain B; Meek, M E Bette; Douglas, George R; Marchetti, Francesco

2015-12-01

The Organisation for Economic Cooperation and Development's (OECD) Adverse Outcome Pathway (AOP) programme aims to develop a knowledgebase of all known pathways of toxicity that lead to adverse effects in humans and ecosystems. A Users' Handbook was recently released to provide supplementary guidance on AOP development. This article describes one AOP-alkylation of DNA in male premeiotic germ cells leading to heritable mutations. This outcome is an important regulatory endpoint. The AOP describes the biological plausibility and empirical evidence supporting that compounds capable of alkylating DNA cause germ cell mutations and subsequent mutations in the offspring of exposed males. Alkyl adducts are subject to DNA repair; however, at high doses the repair machinery becomes saturated. Lack of repair leads to replication of alkylated DNA and ensuing mutations in male premeiotic germ cells. Mutations that do not impair spermatogenesis persist and eventually are present in mature sperm. Thus, the mutations are transmitted to the offspring. Although there are some gaps in empirical support and evidence for essentiality of the key events for certain aspects of this AOP, the overall AOP is generally accepted as dogma and applies broadly to any species that produces sperm. The AOP was developed and used in an iterative process to test and refine the Users' Handbook, and is one of the first publicly available AOPs. It is our hope that this AOP will be leveraged to develop other AOPs in this field to advance method development, computational models to predict germ cell effects, and integrated testing strategies. © 2015 Her Majesty the Queen in Right of Canada.

Design and synthesis of BODIPY sensitizers with long alkyl chains tethered to N-carbazole and their application for dye sensitized solar cells

Energy Technology Data Exchange (ETDEWEB)

Cheema, Hammad [Polymer and Color Chemistry Program, North Carolina State University, Raleigh, NC, 27695 (United States); Younts, Robert; Gautam, Bhoj; Gundogdu, Kenan [Physics Department, North Carolina State University, Raleigh, NC, 27695 (United States); El-Shafei, Ahmed, E-mail: Ahmed_El-Shafei@ncsu.edu [Polymer and Color Chemistry Program, North Carolina State University, Raleigh, NC, 27695 (United States)

2016-12-01

In this study, three boron dipyrromethenes (BODIPY) dyes with extended conjugation and electron donating carbazole groups with different alkyl chain lengths tethered to N-carbazole were synthesized and characterized for dye-sensitized solar cells. The goal was to study the effect of different alkyl chain lengths on dye aggregation at TiO{sub 2} surface. The proposed molecular strategy resulted in BODIPY dyes which showed interesting electronic absorption and fluorescence properties. It was observed that intramolecular energy transfer decreases with the increase in alkyl chain length possibly due to induced changes in molecular geometry caused by long alkyl chains. Additionally, interface analysis by impedance spectroscopy in comparison to N719 sensitized TiO{sub 2} solar cell showed significant charge transport related losses (Nyquist plot) most likely due to impedance resulted from aggregated BODIPY dye on TiO{sub 2} surface. Femtosecond transient absorption studies showed the loss of excited electrons by recombination with oxidized ground state of the sensitizers. - Highlights: • BODIPY dyes with carbazole electron donating groups are characterized. • Photophysics is discussed based on transient and steady state spectroscopy results. • Impedance spectroscopy found huge charge transport related losses on TiO{sub 2.}.

Design and synthesis of BODIPY sensitizers with long alkyl chains tethered to N-carbazole and their application for dye sensitized solar cells

International Nuclear Information System (INIS)

Cheema, Hammad; Younts, Robert; Gautam, Bhoj; Gundogdu, Kenan; El-Shafei, Ahmed

2016-01-01

In this study, three boron dipyrromethenes (BODIPY) dyes with extended conjugation and electron donating carbazole groups with different alkyl chain lengths tethered to N-carbazole were synthesized and characterized for dye-sensitized solar cells. The goal was to study the effect of different alkyl chain lengths on dye aggregation at TiO_2 surface. The proposed molecular strategy resulted in BODIPY dyes which showed interesting electronic absorption and fluorescence properties. It was observed that intramolecular energy transfer decreases with the increase in alkyl chain length possibly due to induced changes in molecular geometry caused by long alkyl chains. Additionally, interface analysis by impedance spectroscopy in comparison to N719 sensitized TiO_2 solar cell showed significant charge transport related losses (Nyquist plot) most likely due to impedance resulted from aggregated BODIPY dye on TiO_2 surface. Femtosecond transient absorption studies showed the loss of excited electrons by recombination with oxidized ground state of the sensitizers. - Highlights: • BODIPY dyes with carbazole electron donating groups are characterized. • Photophysics is discussed based on transient and steady state spectroscopy results. • Impedance spectroscopy found huge charge transport related losses on TiO_2_.

Synthesis and Antiangiogenic Activity of N-Alkylated Levamisole Derivatives

DEFF Research Database (Denmark)

Hansen, Anders N.; Bendiksen, Christine D.; Sylvest, Lene

2012-01-01

profile, was recently shown to be an inhibitor of angiogenesis in vitro and exhibited tumor growth inhibition in mice. Here we describe the synthesis and in vitro evaluation of a series of N-alkylated analogues of levamisole with the aim of characterizing structure-activity relationships with regard...

Exploring the Role of Genetic Modifiers in DNA Repair and Breast Cancer

Science.gov (United States)

2013-09-01

organismal sensitivity to the alkylating agent N-methyl-N- nitrosourea . Can- cer Res. 63: 7047–7050. Goytisolo, F. A., E. Samper, J. Martin-Caballero, P...this study refers to this distinction. DNA- alkylating agents (methyl methanesulfonate [MMS], ethylmethanesulfonate [EMS], melphalan, etc.) are of...particular interest at low doses, as this class of genotoxic agents encompasses a number of natural and industrial environmental carcinogens (2). Alkylating

7-N-Acetylcysteine-pyrrole conjugate-A potent DNA reactive metabolite of pyrrolizidine alkaloids.

Science.gov (United States)

He, Xiaobo; Ma, Liang; Xia, Qingsu; Fu, Peter P

2016-10-01

Plants containing pyrrolizidine alkaloids (PAs) are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs) that are capable of alkylating cellular DNA and proteins, form (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP) and DHP. Reactions of 7-NAC-DHP with 2'-deoxyguanosine (dG), 2'-deoxyadenosine (dA), and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation. Copyright © 2016. Published by Elsevier B.V.

7-N-Acetylcysteine-pyrrole conjugate—A potent DNA reactive metabolite of pyrrolizidine alkaloids

Directory of Open Access Journals (Sweden)

Xiaobo He

2016-10-01

Full Text Available Plants containing pyrrolizidine alkaloids (PAs are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs that are capable of alkylating cellular DNA and proteins, form (±-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP and DHP. Reactions of 7-NAC-DHP with 2′-deoxyguanosine (dG, 2′-deoxyadenosine (dA, and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation.

Neighbor-directed histidine N(τ) alkylation. A route to imidazolium-containing phosphopeptide macrocycles

Energy Technology Data Exchange (ETDEWEB)

Qian, Wen-Jian [National Cancer Inst., Frederick, MD (United States); Park, Jung-Eun [National Cancer Inst., Bethesda, MD (United States); Grant, Robert [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Lai, Christopher C. [National Cancer Inst., Frederick, MD (United States); Kelley, James A. [National Cancer Inst., Frederick, MD (United States); Yaffe, Michael B. [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Lee, Kyung S. [National Cancer Inst., Bethesda, MD (United States); Burke, Terrence R. [National Cancer Inst., Frederick, MD (United States)

2015-07-07

Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. These cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Furthermore, neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.

Design rules for carbazole derivatized n-alkyl methacrylate polymeric memristors

Czech Academy of Sciences Publication Activity Database

McFarlane, T. M.; Zdyrko, B.; Bandera, Y.; Worley, D.; Klep, O.; Jurča, M.; Tonkin, C.; Foulger, S. H.; Vilčáková, J.; Sáha, P.; Pfleger, Jiří

2018-01-01

Roč. 6, č. 10 (2018), s. 2533-2545 ISSN 2050-7526 Institutional support: RVO:61389013 Keywords : n-alkyl methacrylate polymers * charge transport * composites Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 5.256, year: 2016

Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2-or 4-pyrenyl-functionalized O2 '-alkylated RNA monomers

DEFF Research Database (Denmark)

Karmakar, Saswata; Madsen, Andreas Stahl; Guenther, Dale C.

2014-01-01

'-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA...

Determination of rate constants of N-alkylation of primary amines by 1H NMR spectroscopy.

Science.gov (United States)

Li, Chenghong

2013-09-05

Macromolecules containing N-diazeniumdiolates of secondary amines are proposed scaffolds for controlled nitrogen oxide (NO) release medical applications. Preparation of these compounds often involves converting primary amine groups to secondary amine groups through N-alkylation. However, N-alkylation results in not only secondary amines but tertiary amines as well. Only N-diazeniumdiolates of secondary amines are suitable for controlled NO release; therefore, the yield of secondary amines is crucial to the total NO load of the carrier. In this paper, (1)H NMR spectroscopy was used to estimate the rate constants for formation of secondary amine (k1) and tertiary amine (k2) for alkylation reagents such as propylene oxide (PO), methyl acrylate (MA), and acrylonitrile (ACN). At room temperature, the ratio of k2/k1 for the three reactions was found to be around 0.50, 0.026, and 0.0072.

Regulation of DNA Alkylation Damage Repair: Lessons and Therapeutic Opportunities.

Science.gov (United States)

Soll, Jennifer M; Sobol, Robert W; Mosammaparast, Nima

2017-03-01

Alkylation chemotherapy is one of the most widely used systemic therapies for cancer. While somewhat effective, clinical responses and toxicities of these agents are highly variable. A major contributing factor for this variability is the numerous distinct lesions that are created upon alkylation damage. These adducts activate multiple repair pathways. There is mounting evidence that the individual pathways function cooperatively, suggesting that coordinated regulation of alkylation repair is critical to prevent toxicity. Furthermore, some alkylating agents produce adducts that overlap with newly discovered methylation marks, making it difficult to distinguish between bona fide damaged bases and so-called 'epigenetic' adducts. Here, we discuss new efforts aimed at deciphering the mechanisms that regulate these repair pathways, emphasizing their implications for cancer chemotherapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis and Performance of a Biomimetic Indicator for Alkylating Agents.

Science.gov (United States)

Provencher, Philip A; Love, Jennifer A

2015-10-02

4-(4-Nitrobenzyl)pyridine (NBP) is a colorimetric indicator compound for many types of carcinogenic alkylating agents. Because of the similar reactivity of NBP and guanine in DNA, NBP serves as a DNA model. NBP assays are used in the toxicological screening of pharmaceutical compounds, detection of chemical warfare agents, environmental hygiene technology, preliminary toxicology tests, mutagenicity of medicinal compounds, and other chemical analyses. Nevertheless, the use of NBP as a DNA model suffers from the compound's low water solubility, its lack of reactive oxygen sites, and dissimilar steric encumbrance compared to DNA. We report herein the design and synthesis of NBP derivatives that address some of these issues. These derivatives have been tested in solution and found to be superior in the colorimetric assay of the alkylating anticancer drug cyclophosphamide. The derivatives have also been integrated into a polymeric silica material which changes color upon the exposure to dangerous alkylating agents, such as iodomethane vapor, without the need for an exogenous base. This material modernizes the NBP assay from a time-consuming laboratory analysis to a real-time solid state sensor, which requires neither solvent nor additional reagents and can detect both gas- and solution-phase alkylating agents.

Selective N-alkylation of amines using nitriles under hydrogenation conditions: facile synthesis of secondary and tertiary amines.

Science.gov (United States)

Ikawa, Takashi; Fujita, Yuki; Mizusaki, Tomoteru; Betsuin, Sae; Takamatsu, Haruki; Maegawa, Tomohiro; Monguchi, Yasunari; Sajiki, Hironao

2012-01-14

Nitriles were found to be highly effective alkylating reagents for the selective N-alkylation of amines under catalytic hydrogenation conditions. For the aromatic primary amines, the corresponding secondary amines were selectively obtained under Pd/C-catalyzed hydrogenation conditions. Although the use of electron poor aromatic amines or bulky nitriles showed a lower reactivity toward the reductive alkylation, the addition of NH(4)OAc enhanced the reactivity to give secondary aromatic amines in good to excellent yields. Under the same reaction conditions, aromatic nitro compounds instead of the aromatic primary amines could be directly transformed into secondary amines via a domino reaction involving the one-pot hydrogenation of the nitro group and the reductive alkylation of the amines. While aliphatic amines were effectively converted to the corresponding tertiary amines under Pd/C-catalyzed conditions, Rh/C was a highly effective catalyst for the N-monoalkylation of aliphatic primary amines without over-alkylation to the tertiary amines. Furthermore, the combination of the Rh/C-catalyzed N-monoalkylation of the aliphatic primary amines and additional Pd/C-catalyzed alkylation of the resulting secondary aliphatic amines could selectively prepare aliphatic tertiary amines possessing three different alkyl groups. According to the mechanistic studies, it seems reasonable to conclude that nitriles were reduced to aldimines before the nucleophilic attack of the amine during the first step of the reaction.

Formation of enamines by alkylation of imines

NARCIS (Netherlands)

Heiszwolf, G.J.; Kloosterziel, H.

1966-01-01

cf. CA 64, 12473c. With ice-cooling, 1 equiv. alkylating agent was added to one equiv. of the imine in 1M soln. in a solvent in the presence of NaH to give both N- and C-alkylated products. The following summarizes the date (imine, solvent, alkylating agent, % unreacted imine, % N-alkylated product,

Noncanonical regulation of alkylation damage resistance by the OTUD4 deubiquitinase.

Science.gov (United States)

Zhao, Yu; Majid, Mona C; Soll, Jennifer M; Brickner, Joshua R; Dango, Sebastian; Mosammaparast, Nima

2015-06-12

Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors. © 2015 The Authors.

Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1☆☆☆

Science.gov (United States)

Randall, Matthew J.; Spiess, Page C.; Hristova, Milena; Hondal, Robert J.; van der Vliet, Albert

2013-01-01

Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated1 kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK

N- versus O-alkylation: utilizing NMR methods to establish reliable primary structure determinations for drug discovery.

Science.gov (United States)

LaPlante, Steven R; Bilodeau, François; Aubry, Norman; Gillard, James R; O'Meara, Jeff; Coulombe, René

2013-08-15

A classic synthetic issue that remains unresolved is the reaction that involves the control of N- versus O-alkylation of ambident anions. This common chemical transformation is important for medicinal chemists, who require predictable and reliable protocols for the rapid synthesis of inhibitors. The uncertainty of whether the product(s) are N- and/or O-alkylated is common and can be costly if undetermined. Herein, we report an NMR-based strategy that focuses on distinguishing inhibitors and intermediates that are N- or O-alkylated. The NMR strategy involves three independent and complementary methods. However, any combination of two of the methods can be reliable if the third were compromised due to resonance overlap or other issues. The timely nature of these methods (HSQC/HMQC, HMBC. ROESY, and (13)C shift predictions) allows for contemporaneous determination of regioselective alkylation as needed during the optimization of synthetic routes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation.

Science.gov (United States)

Ayala-García, Víctor M; Valenzuela-García, Luz I; Setlow, Peter; Pedraza-Reyes, Mario

2016-12-15

Aag from Bacillus subtilis has been implicated in in vitro removal of hypoxanthine and alkylated bases from DNA. The regulation of expression of aag in B. subtilis and the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptional aag-lacZ fusion expressed low levels of β-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably, aag-lacZ expression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in a sigG-deficient background, suggesting a forespore-specific mechanism of aag transcription. Two additional findings supported this suggestion: (i) expression of an aag-yfp fusion was observed in the forespore, and (ii) in vivo mapping of the aag transcription start site revealed the existence of upstream regulatory sequences possessing homology to σ G -dependent promoters. In comparison with the wild-type strain, disruption of aag significantly reduced survival of sporulating B. subtilis cells following nitrous acid or methyl methanesulfonate treatments, and the Rif r mutation frequency was significantly increased in an aag strain. These results suggest that Aag protects the genome of developing B. subtilis sporangia from the cytotoxic and genotoxic effects of base deamination and alkylation. In this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles

Energy Technology Data Exchange (ETDEWEB)

Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of); Shahverdi, Ahmad Reza [Department of Pharmaceutical Biotechnology and Biotechnology Research Centre, Faculty of Pharmacy, TUMS, Tehran (Iran, Islamic Republic of); Ahmadi, Abbas [Department of Histology and Embryology, Faculty of Veterinary Medicine, Urmia University, Urmia (Iran, Islamic Republic of); Baeeri, Maryam; Mohammadirad, Azadeh [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of); Abdollahi, Mohammad, E-mail: mohammad.abdollahi@utoronto.ca [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of)

2013-02-01

Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential. Highlights: ► Cisplatin (CIS) affects spermatozoa as a male reproductive toxicant. ► Effect of Nano-Se on CIS-induced spermatotoxicity was investigated. ► CIS-exposure induces oxidative sperm DNA damage

Read-across of ready biodegradability based on the substrate specificity of N-alkyl polypropylene polyamine-degrading microorganisms.

Science.gov (United States)

Geerts, R; van Ginkel, C G; Plugge, C M

2017-04-01

The biodegradation of N-alkyl polypropylene polyamines (NAPPs) was studied using pure and mixed cultures to enable read-across of ready biodegradability test results. Two Pseudomonas spp. were isolated from activated sludge with N-oleyl alkyl propylene diamine and N-coco alkyl dipropylene triamine, respectively. Both strains utilized all NAPPs tested as the sole source of carbon, nitrogen and energy for growth. Mineralization of NAPPs was independent of the alkyl chain length and the size of the polyamine moiety. NAPPs degraded in closed bottle tests (CBTs) using both river water and activated sludge. However, ready biodegradability of NAPPs with alkyl chain lengths of 16-18 carbon atoms and polyamine moieties with three and four nitrogen atoms could not be demonstrated. Biodegradation in the CBT was hampered by their limited bioavailability, making assessment of the true ready biodegradability of these highly adsorptive surfactants impossible. All NAPPs are therefore classified as readily biodegradable through read-across. Read-across is justified by the broad substrate specificity of NAPP-degrading microorganisms, their omnipresence and the mineralization of NAPPs.

Development of novel alkylating drugs as anticancer agents.

Science.gov (United States)

Izbicka, Elzbieta; Tolcher, Anthony W

2004-06-01

Although conventional alkylating drugs have proven efficacy in the treatment of malignancies, the agents themselves are not selective. Therefore, non-specific alkylation of cellular nucleophilic targets may contribute to many of the observed toxic effects. Novel approaches to drug discovery have resulted in candidate agents that are focused on 'soft alkylation'--alkylators with greater target selectivity. This review highlights the discovery of small molecule drugs that bind to DNA with higher selectivity, act in a unique hypoxic tumor environment, or covalently bind specific protein targets overexpressed in cancer, such as topoisomerase II, glutathione transferase pi1, beta-tubulin and histone deacetylase.

Synthesis of triated N1'-alkyl derivatives of the delta opioid receptor ligand naltrindole

International Nuclear Information System (INIS)

Lever, J.R.; Johnson, S.M.

1997-01-01

Tritiated N1'-methyl and N1'-ethyl analogues of naltrindole (NTI) have been synthesized for evaluation as radioligands for studies of delta opioid receptors. The two N1'-alkyl-5',7'-dibromoNTI precursors for radiolabeling were prepared by base-promoted alkylation of 2,4-dibromophenylhydrazine with either iodomethane or iodoethane followed by condensation with naltrexone using the Fischer indole synthesis. Catalytic debromotritiation followed by HPLC purification afforded [ 3 H]MeNTI (17.3 Ci/mmol) and [ 3 H]EtNTI (22.5 Ci/mmol) with high chemical and radiochemical purities (≥ 99.8%). (author)

A biochemical defect in the repair of alkylated DNA in cells from an immunodeficient patient (46BR)

International Nuclear Information System (INIS)

Teo, I.A.; Broughton, B.C.; Day, R.S.; James, M.R.; Karran, P.; Mayne, L.V.; Lehmann, A.R.

1983-01-01

The fibroblast cell strain 46BR, derived from an immunodeficient individual, is hypersensitive to the lethal effects of a variety of DNA-damaging agents, this effect being particularly marked for monofunctional methylating agents. After U.V. irradiation 46BR cells show normal unscheduled DNA synthesis, daughter strand repair, and recovery of DNA and RNA synthesis. The inhibition of DNA replicative synthesis by U.V. is slightly less than that of normal cells. After gamma-irradiation the rejoining of strand breaks is normal as are the kinetics of replicative DNA synthesis. Following treatment with dimethylsulphate, replicative DNA synthesis is affected in a similar way to normal cells, unscheduled DNA synthesis may be increased relative to normal cells, but more strand breaks persist in 46BR than in normal cells. In addition 46BR cells are hypersensitive to the toxic effects of 3-aminobenzamide, an inhibitor of ADP-ribosyl transferase. This enzyme is involved in the ligation step of repair of alkylation damage. A hypothesis is presented suggesting that 46BR may be defective in DNA ligase I

Synthesis and evaluation of N,N-di-alkyl-2-methoxyacetamides for the separation of U(VI) and Pu(IV) from nitric acid medium

Energy Technology Data Exchange (ETDEWEB)

Kumaresan, R.; Prathibha, T.; Selvan, B. Robert; Venkatesan, K.A.; Antony, M.P. [Indira Gandhi Centre for Atomic Research, Kalpakkam (India). Fuel Chemistry Div.

2017-07-01

The homologs of N,N-di-alkyl-2-methoxyacetamides (DAMeOA) having three different alkyl chains varying from hexyl to decyl (C{sub 6}, C{sub 8} and C{sub 10}) were synthesized and characterized by NMR and IR spectral analyses. Extraction behavior of U(VI) and Pu(IV) from nitric acid medium in a solution of 0.5 M of DAMeOA in n-dodecane (n-DD) was studied and the results were compared with those obtained using N,N-di-hexyloctanamide (DHOA) in n-dodecane. The effect of various parameters on the distribution ratio of U(VI) and Pu(IV) in DAMeOA was studied. The extraction of nitric acid increased with decrease in chain length of alkyl group attached to amidic nitrogen atom of DAMeOA and the conditional nitric acid extraction constant was determined. The extraction of nitric acid in DAMeOA/n-DD resulted in the formation of third phase in organic phase and the third phase occurred early with DAMeOA having smaller alkyl chain length. In contrast to this, the distribution ratio (D) of U(VI) and Pu(IV) in DAMeOA/n-DD increased with increase in the concentration of nitric acid and with increase in the chain length of alkyl group attached to amidic nitrogen atom of DAMeOA. The stoichiometry of the metal - solvate was determined from the slope of extraction data. Quantitative recovery of uranium and plutonium from the loaded organic phase was achieved using dilute nitric acid.

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus

Science.gov (United States)

O’Hanlon, Karen A.; Margison, Geoffrey P.; Hatch, Amy; Fitzpatrick, David A.; Owens, Rebecca A.; Doyle, Sean; Jones, Gary W.

2012-01-01

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O6-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O6-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system. PMID:22669901

Kinetic investigation of the oxidation of N-alkyl anilines by ...

Indian Academy of Sciences (India)

Home; Journals; Journal of Chemical Sciences; Volume 112; Issue 6. Kinetic investigation of the oxidation of N-alkyl anilines by peroxomonophosphoric acid in anionic surfactant sodium lauryl sulphate. G P Panigrahi Jagannath Panda. Physical and Theoretical Volume 112 Issue 6 December 2000 pp 615-622 ...

Mono-alkylation of cyanoimide at a molybdenum(IV) diphosphinic center by alkyl halides: synthesis, cathodically induced isomerization and theoretical studies

International Nuclear Information System (INIS)

Alegria, Elisabete C.B.A.; Guedes da Silva, M. Fátima C.; Kuznetsov, Maxim L.; Martins, Luísa M.D.R.S.; Pombeiro, Armando J.L.

2016-01-01

Treatment of trans-[Mo(NCN) 2 (dppe) 2 ] with alkyl halides (RX) affords the alkylated cyanoimido-complexes trans-[Mo(NCN)(NCNR)(dppe) 2 ]X [R = Me, X = I (1); R = Et, X = I (2); R = Pr, X = I (3); R = i Pr, X = I (4); R = CH 2 Ph, X = Br (5); R = CH 2 C 6 H 4 NO 2 -4, X = Br (6)], while its reaction with the trimethyloxonium salt [Me 3 O][BF 4 ] affords trans-[Mo(NCN)(NCNMe)(dppe) 2 ][BF 4 ] (7). The reactions are accelerated by microwave irradiation. Complexes 1-7 were fully characterized by elemental analyses, IR and NMR spectroscopies, FAB-MS spectrometry, cyclic voltammetry and controlled potential electrolysis. The electrophilic addition to the exo-N atom of one of the cyanoimide ligands was confirmed by single crystal X-ray crystal analysis of 1. In aprotic medium and at a Pt electrode, compounds 1-7 undergo, apart from two consecutive single-electron reversible oxidations, also two successive single-electron reductions at different potentials, involving a cathodically induced trans-to-cis isomerization, following a double square ECEC-type mechanism which was studied in detail by digital simulation of the cyclic voltammograms. Quantum-chemical calculations indicate that the oxidations and reductions are mainly metal centered (although the latter with some involvement of the cyanoimide moieties), and that the reduction leads to a decrease of the relative stability of the trans isomer vs. the cis one.

Alcohols as alkylating agents in heteroarene C-H functionalization

Science.gov (United States)

Jin, Jian; MacMillan, David W. C.

2015-09-01

Redox processes and radical intermediates are found in many biochemical processes, including deoxyribonucleotide synthesis and oxidative DNA damage. One of the core principles underlying DNA biosynthesis is the radical-mediated elimination of H2O to deoxygenate ribonucleotides, an example of `spin-centre shift', during which an alcohol C-O bond is cleaved, resulting in a carbon-centred radical intermediate. Although spin-centre shift is a well-understood biochemical process, it is underused by the synthetic organic chemistry community. We wondered whether it would be possible to take advantage of this naturally occurring process to accomplish mild, non-traditional alkylation reactions using alcohols as radical precursors. Because conventional radical-based alkylation methods require the use of stoichiometric oxidants, increased temperatures or peroxides, a mild protocol using simple and abundant alkylating agents would have considerable use in the synthesis of diversely functionalized pharmacophores. Here we describe the development of a dual catalytic alkylation of heteroarenes, using alcohols as mild alkylating reagents. This method represents the first, to our knowledge, broadly applicable use of unactivated alcohols as latent alkylating reagents, achieved via the successful merger of photoredox and hydrogen atom transfer catalysis. The value of this multi-catalytic protocol has been demonstrated through the late-stage functionalization of the medicinal agents, fasudil and milrinone.

Alcohols as alkylating agents in heteroarene C-H functionalization.

Science.gov (United States)

Jin, Jian; MacMillan, David W C

2015-09-03

Redox processes and radical intermediates are found in many biochemical processes, including deoxyribonucleotide synthesis and oxidative DNA damage. One of the core principles underlying DNA biosynthesis is the radical-mediated elimination of H2O to deoxygenate ribonucleotides, an example of 'spin-centre shift', during which an alcohol C-O bond is cleaved, resulting in a carbon-centred radical intermediate. Although spin-centre shift is a well-understood biochemical process, it is underused by the synthetic organic chemistry community. We wondered whether it would be possible to take advantage of this naturally occurring process to accomplish mild, non-traditional alkylation reactions using alcohols as radical precursors. Because conventional radical-based alkylation methods require the use of stoichiometric oxidants, increased temperatures or peroxides, a mild protocol using simple and abundant alkylating agents would have considerable use in the synthesis of diversely functionalized pharmacophores. Here we describe the development of a dual catalytic alkylation of heteroarenes, using alcohols as mild alkylating reagents. This method represents the first, to our knowledge, broadly applicable use of unactivated alcohols as latent alkylating reagents, achieved via the successful merger of photoredox and hydrogen atom transfer catalysis. The value of this multi-catalytic protocol has been demonstrated through the late-stage functionalization of the medicinal agents, fasudil and milrinone.

Thermal phase behaviour of N-alkyl-N-methylpyrrolidinium and piperidinium bis(trifluoromethanesulfonyl)imide salts

International Nuclear Information System (INIS)

Henderson, W A; Jr, V G Young; Pearson, W; Passerini, S; Long, H C De; Trulove, P C

2006-01-01

The phases, ion crystal packing and thermal properties of the N-alkyl-N-methylpyrrolidinium and piperidinium bis(trifluoromethanesulfonyl)imide (PYR 1R TFSI and PIP 1R TFSI (subscript R = 1 for methyl and 2 for ethyl), respectively) salts are compared using powder and single-crystal x-ray diffraction (XRD) and differential scanning calorimetry (DSC). The crystal structure of PIP 12 TFSI has been determined at 123 K. The salt crystallizes in the triclinic space group P1-bar with Z 8. Structural data are also reported for PYR 11 TFSI at 153 K and PIP 12 TFSI at 223 K. PIP 11 TFSI has identical ion crystal packing to the analogous pyrrolidinium salt PYR 11 TFSI. Since increasing the cation alkyl chain length to propyl or butyl (R = 3 or 4) reduces the melting point of the salts below room temperature, this study may provide valuable insight into why these pyrrolidinium and piperidinium salts form room-temperature ionic liquids

Hybrid ligand-alkylating agents targeting telomeric G-quadruplex structures.

Science.gov (United States)

Doria, Filippo; Nadai, Matteo; Folini, Marco; Di Antonio, Marco; Germani, Luca; Percivalle, Claudia; Sissi, Claudia; Zaffaroni, Nadia; Alcaro, Stefano; Artese, Anna; Richter, Sara N; Freccero, Mauro

2012-04-14

The synthesis, physico-chemical properties and biological effects of a new class of naphthalene diimides (NDIs) capable of reversibly binding telomeric DNA and alkylate it through an electrophilic quinone methide moiety (QM), are reported. FRET and circular dichroism assays showed a marked stabilization and selectivity towards telomeric G4 DNA folded in a hybrid topology. NDI-QMs' alkylating properties revealed a good reactivity on single nucleosides and selectivity towards telomeric G4. A selected NDI was able to significantly impair the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression. These findings points to our hybrid ligand-alkylating NDIs as possible tools for the development of novel targeted anticancer therapies. This journal is © The Royal Society of Chemistry 2012

Pd@[nBu₄][Br] as a Simple Catalytic System for N-Alkylation Reactions with Alcohols.

Science.gov (United States)

Cacciuttolo, Bastien; Pascu, Oana; Aymonier, Cyril; Pucheault, Mathieu

2016-08-10

Palladium nanoparticles, simply and briefly generated in commercial and cheap onium salts using supercritical carbon dioxide, have been found to be an effective catalytic system for additive free N-alkylation reaction using alcohols via cascade oxidation/condensation/reduction steps.

Slx4 becomes phosphorylated after DNA damage in a Mec1/Tel1-dependent manner and is required for repair of DNA alkylation damage

Science.gov (United States)

Flott, Sonja; Rouse, John

2005-01-01

Members of the RecQ family of DNA helicases, mutated in several syndromes associated with cancer predisposition, are key regulators of genome stability. The Saccharomyces cerevisiae SLX4 gene is required for cell viability in the absence of Sgs1, the only yeast RecQ helicase. SLX4 encodes one subunit of the heterodimeric Slx1–Slx4 endonuclease, although its cellular function is not clear. Slx1–Slx4 was reported to preferentially cleave replication fork-like structures in vitro, and cells lacking SLX4 are hypersensitive to DNA alkylation damage. Here we report that Slx4 becomes phosphorylated in cells exposed to a wide range of genotoxins. Even though it has been proposed that the role of Slx4 is restricted to S-phase, Slx4 phosphorylation is observed in cells arrested in G1 or G2 phases of the cell cycle, but not during an unperturbed cell cycle. Slx4 phosphorylation is completely abolished in cells lacking the Mec1 and Tel1 protein kinases, critical regulators of genome stability, but is barely affected in the absence of both Rad53 and Chk1 kinases. Finally we show that, whereas both Slx1 and Slx4 are dispensable for activation of cell-cycle checkpoints, Slx4, but not Slx1, is required for repair of DNA alkylation damage in both aynchronously growing cells and in G2-phase-arrested cells. These results reveal Slx4 as a new target of the Mec1/Tel1 kinases, with a crucial role in DNA repair that is not restricted to the processing of stalled replisomes. PMID:15975089

Structural aspects of DNA in its replication and repair

International Nuclear Information System (INIS)

Mitra, S.; Pal, B.C.; Foote, R.S.; Bates, R.C.; Bhattacharyya, A.; Snow, E.T.; Wobbe, C.R.; Morse, C.C.; Snyder, C.E.

1984-01-01

The research objective of this laboratory is to investigate the structure of DNA, the mechanism of DNA replication and its regulation, and the mechanism and role of repair of the altered DNA in the expression of heritable changes. This research has two broad aims, namely investigation of (a) the regulation of DNA replication in mammals, using parvovirus DNA as a model system and (b) the role of DNA repair in mutagenesis and carcinogenesis induced by simple alkylating mutagens

Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus

International Nuclear Information System (INIS)

Schmitt, J.; Schlehofer, J.R.; Mergener, K.; Gissmann, L.; zur Hausen, H.

1989-01-01

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur

Effects of Photo-chemically Activated Alkylating Agents of the FR900482 Family on Chromatin

OpenAIRE

Subramanian, Vidya; Ducept, Pascal; Williams, Robert M.; Luger, Karolin

2007-01-01

Bioreductive alkylating agents are an important class of clinical antitumor antibiotics that cross-link and mono-alkylate DNA. Here we use a synthetic photochemically activated derivative of FR400482 to investigate the molecular mechanism of this class of drugs in a biologically relevant context. We find that the organization of DNA into nucleosomes effectively protects it against drug-mediated cross-linking, while permitting mono-alkylation. This modification has the potential to form covale...

The formation of quasi-alicyclic rings in alkyl-aromatic compounds

Science.gov (United States)

Straka, Pavel; Buryan, Petr; Bičáková, Olga

2018-02-01

The alkyl side chains of n-alkyl phenols, n-alkyl benzenes and n-alkyl naphthalenes are cyclised, as demonstrated by GC measurements, FTIR spectroscopy and molecular mechanics calculations. Cyclisation occurs due to the intramolecular interaction between an aromatic ring (-δ) and a hydrogen of the terminal methyl group (+δ) of an alkyl chain. In fact, conventional molecules are not aliphatic-aromatic, but quasi-alicyclic-aromatic. With the aromatic molecules formed with a quasi-alicyclic ring, the effect of van der Waals attractive forces increases not only intramolecularly but also intermolecularly. This effect is strong in molecules with propyl and higher alkyl substituents. The increase of intermolecular van der Waals attractive forces results in bi-linearity in the GC retention time of the compounds in question, observed in the dependence of the logarithm of the relative retention time on the number of carbons in a molecule in both polar and nonpolar stationary phases with both capillary and packed columns. The role of van der Waals forces has been demonstrated using the potential energies of covalent and noncovalent interactions for 2-n-alkyl phenols, n-alkyl benzenes and 1-n-alkyl- and 2-n-alkyl naphthalenes.

Influence of some DNA-alkylating drugs on thermal stability, acid and osmotic resistance of the membrane of whole human erythrocytes and their ghosts.

Science.gov (United States)

Ivanov, I T; Gadjeva, V

2000-09-01

Human erythrocytes and their resealed ghosts were alkylated under identical conditions using three groups of alkylating antitumor agents: mustards, triazenes and chloroethyl nitrosoureas. Osmotic fragility, acid resistance and thermal stability of membranes were changed only in alkylated ghosts in proportion to the concentration of the alkylating agent. All the alkylating agents decreased acid resistance in ghosts. The clinically used drugs sarcolysine, dacarbazine and lomustine all decreased osmotic fragility and thermal stability of ghost membranes depending on their lipophilicity. DM-COOH did not decrease osmotic fragility and thermal stability of ghost membranes, while NEM increased thermal stability of membranes. The preliminary but not subsequent treatment of ghosts with DM-COOH fully abolished the alkylation-induced thermal labilization of ghost membrane proteins while NEM had a partial effect only. The present study gives direct evidence that alkylating agents, having a high therapeutic activity against malignant growth, bind covalently to proteins of cellular membranes.

Synthesis and enzyme inhibitory studies of some new N-alkylated/aralkylated N-(4-ethoxyphenyl)-2,3-drobenzo-(1,4)-dioxin-6-sulfonamides

International Nuclear Information System (INIS)

Abbasi, M.A.; Islam, M.; Rehman, A.U.; Siddiqui, S.Z.

2016-01-01

The research endeavor was aimed to synthesize N-alkyl/aralkylated-N-(4-ethoxyphenyl)-2,3-dihydrobenzo-(1,4)-dioxine-6 sulfonamides and to evaluate their enzyme inhibitory potential. The target molecules were synthesized in two steps. The first step involved the reaction of 4-ethoxyaniline (1) with N-2,3-dihydrobenzo(1,4)-dioxin-6-sulfonyl chloride (2) under dynamic pH control maintained by 10% aqueous Na/sub 2/CO/sub 3/ to yield N-(4-ethoxyphenyl)-2,3-dihydrobenzo-(1,4)-dioxine-6-sulfonamide (3). In second step parent compound 3 was reacted with various alkyl/aralkyl halides (4a-l) in N,N'-dimethylformamide and catalytic amount of lithium hydride to accomplish some new N-alkyl/aralkylated-N-(4-ethoxyphenyl)-2,3-dihydrobenzo-(1,4)-dioxine-6 sulfonamides (5a-l). Probable structures of the synthesized compounds were characterized by contemporary spectral techniques i.e. IR, 1H-NMR and EIMS and were finally evaluated for enzyme inhibitory potential against a-glucosidase and urease. The synthesized compounds exhibited moderate to weak therapeutic potential throughout the series. (author)

Glutathione Depletion Induced by c-Myc Downregulation Triggers Apoptosis on Treatment with Alkylating Agents1

Science.gov (United States)

Biroccio, Annamaria; Benassi, Barbara; Fiorentino, Francesco; Zupi, Gabriella

2004-01-01

Abstract Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by l-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis. PMID:15153331

Amino acid nitrosation products as alkylating agents.

Science.gov (United States)

García-Santos, M del P; Calle, E; Casado, J

2001-08-08

Nitrosation reactions of alpha-, beta-, and gamma-amino acids whose reaction products can act as alkylating agents of DNA were investigated. To approach in vivo conditions for the two-step mechanism (nitrosation and alkylation), nitrosation reactions were carried out in aqueous acid conditions (mimicking the conditions of the stomach lumen) while the alkylating potential of the nitrosation products was investigated at neutral pH, as in the stomach lining cells into which such products can diffuse. These conclusions were drawn: (i) The alkylating species resulting from the nitrosation of amino acids with an -NH(2) group are the corresponding lactones; (ii) the sequence of alkylating power is: alpha-lactones > beta-lactones > gamma-lactones, coming respectively from the nitrosation of alpha-, beta-, and gamma-amino acids; and (iii) the results obtained may be useful in predicting the mutagenic effectiveness of the nitrosation products of amino acids.

[Methaemoglobinaemia induced by ingestion of alkyl nitrite, "poppers"].

Science.gov (United States)

Kragsfeldt, Celina Thelberg; Nissen, Christoffer B; Brandt, Frans

2016-08-22

We present a case report of an 18-year-old male who was referred to the emergency department with evidence of methaemoglobinaemia. He presented with classic symptoms with peripheral cyanosis and hypoxia. Arterial blood gas showed a methaemoglobin level of 36%. This was caused by ingestion of alkyl nitrate, a widespread party drug called "poppers". When inhaled it causes euphoria, reduced pain and relaxation of the anal sphincter, but oral use may induce life-threatening methaemoglobinaemia. The treatment of choice is the antidote methylene blue. After treatment the patient regained full recovery and was discharged on the following day. We discuss classic symptoms, diagnosis and treatment of intoxication with methylene blue.

O6-Methylguanine-DNA methyltransferase status in neuroendocrine tumours: prognostic relevance and association with response to alkylating agents.

Science.gov (United States)

Walter, T; van Brakel, B; Vercherat, C; Hervieu, V; Forestier, J; Chayvialle, J-A; Molin, Y; Lombard-Bohas, C; Joly, M-O; Scoazec, J-Y

2015-02-03

O(6)-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins. A total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or streptozotocin), were retrospectively studied. In each case, we used methyl-specific PCR (MS-PCR) and pyrosequencing for evaluation of promoter methylation and immunohistochemistry for evaluation of protein status. MGMT promoter methylation was detected in 12 out of 99 (12%) interpretable cases by MS-PCR and in 24 out of 99 (24%) by pyrosequencing. O(6)-Methylguanine-DNA methyltransferase loss of expression was observed in 29 out of 89 (33%) interpretable cases. Status of MGMT was not correlated with overall survival (OS) from diagnosis. Progression-free survival and OS from first alkylant use (temozolomide, dacarbazine and streptozotocin) were higher in patients with MGMT protein loss (respectively, 20.2 vs 7.6 months, Palkylant-based chemotherapy in NETs.

Effect of alkyl-N-phenylcarbamates on photochemical activity of spinach chloroplasts

International Nuclear Information System (INIS)

Sersen, F.; Kralova, K.; Macho, V.

1999-01-01

This study is aimed to investigate the effect of alkyl-N-phenylcarbamates on photosynthetic electron transport in spinach chloroplasts, to determine site of action in the photosynthetic apparatus of spinach chloroplasts and to find correlations between their structure and biological activity. (authors)

DNA Polymerase α (swi7) and the Flap Endonuclease Fen1 (rad2) Act Together in the S-Phase Alkylation Damage Response in S. pombe

Science.gov (United States)

Koulintchenko, Milana; Vengrova, Sonya; Eydmann, Trevor; Arumugam, Prakash; Dalgaard, Jacob Z.

2012-01-01

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase. PMID:23071723

Determination of reaction rate constants for alkylation of 4-(p-nitrobenzyl) pyridine by different alkylating agents.

Science.gov (United States)

Walles, S A

1980-02-01

The rate constants have been determined for the reaction between some different alkylating agents and 4-(p-nitrobenzyl) pyridine (NBP) in methanol. These constants have been compared with those for alkylation of aniline in water. All the constants were lower in methanol than in water but in different degrees. The rate constants of the different alkylating agents have been calculated at a nucleophilic strength n=2. The genetic risk defined as the degree of alkylation of a nucleophile (n=2) is equivalent to the rate constant kn=2 and the target dose. The dependence of the genetic risk on the rate constant (kn=2) is discussed.

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

Energy Technology Data Exchange (ETDEWEB)

Shi, Rongxin; Mullins, Elwood A.; Shen, Xing; #8208; Xing; Lay, Kori T.; Yuen, Philip K.; David, Sheila S.; Rokas, Antonis; Eichman, Brandt F. (UCD); (Vanderbilt)

2017-10-20

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

Alcohols as alkylating agents in heteroarene C–H functionalization

Science.gov (United States)

Jin, Jian; MacMillan, David W. C.

2015-01-01

Redox processes and radical intermediates are found in many biochemical processes, including deoxyribonucleotide synthesis and oxidative DNA damage1. One of the core principles that underlies DNA biosynthesis is the radical-mediated elimnation of H2O to deoxygenate ribonucleotides, an example of ‘spin-center shift’ (SCS)2, during which an alcohol C–O bond is cleaved, resulting in a carbon-centered radical intermediate. While SCS is a well-understood biochemical process, it is underutilized by the synthetic organic chemistry community. We wondered whether it would be possible to take advantage of this naturally occurring process to accomplish mild, non-traditional alkylations using alcohols as radical precursors. Considering traditional radical-based alkylation methods require the use of stoichiometric oxidants, elevated temperatures, or peroxides3–7, the development of a mild protocol using simple and abundant alkylating agents would have significant utility in the synthesis of diversely functionalized pharmacophores. In this manuscript, we describe the successful execution of this idea via the development of a dual catalytic alkylation of heteroarenes using alcohols as mild alkylating reagents. This method represents the first broadly applicable use of unactivated alcohols as latent alkylating reagents, achieved via the successful merger of photoredox and hydrogen atom transfer (HAT) catalysis. The utility of this multi-catalytic protocol has been demonstrated through the late-stage functionalization of the medicinal agents, fasudil and milrinone. PMID:26308895

Exacerbation of N-nitrosodiethylamine Induced Hepatotoxicity and DNA Damage in Mice Exposed to Chronic Unpredictable Stress

Directory of Open Access Journals (Sweden)

Nayeem Bilal

2017-06-01

Full Text Available Psychological stress contributes to increased susceptibility to a number of diseases including cancer. The present study was designed to assess the effect of chronic unpredictable stress on N-nitrosodiethylamine induced liver toxicity in terms of in vivo antioxidant status and DNA damage in Swiss albino mice. The animals used in this study were randomized into different groups based on the treatment with N-nitrosodiethylamine or chronic unpredictable stress alone and post-stress administration of N-nitrosodiethylamine. The mice were sacrificed after 12 weeks of treatment, and the status of major enzymatic and non-enzymatic antioxidants, liver function markers, lipid peroxidation and the extent of DNA damage were determined in circulation and liver tissues of all the groups. The N-nitrosodiethylamine treated group showed significantly compromised levels of the antioxidant enzymes, lipid peroxidation, and the liver function markers with enhanced DNA damage as compared to chronic unpredictable stress or control groups. A similar but less typical pattern observed in the chronic unpredictable stress treated mice. All the measured biochemical parameters were significantly altered in the group treated with the combination of chronic unpredictable stress and N-nitrosodiethylamine when compared to controls, or chronic unpredictable stress alone and/or N-nitrosodiethylamine alone treated groups. Thus, exposure to continuous, unpredictable stress conditions even in general life may significantly enhance the hepatotoxic potential of N-nitrosodiethylamine through an increase in the oxidative stress and DNA damage.

Phase behavior of polystyrene-block-poly(n-alkyl methacrylate) copolymers investigated by SANS, SAXS, and temperature-dependent FTIR spectroscopy

International Nuclear Information System (INIS)

Ryu, Du Yeol; Lee, Dong Hyun; Kim, Hye Jeong; Kim, Jin Kon; Jung, Y. M.; Kim, S. B.

2005-01-01

The phase behavior of polystyrene-block -poly(n-alkyl methacrylate) (PS-PnAMA) copolymer were investigated by Small-Angle Neutron Scattering (SANS), Small-Angle X-ray Scattering (SAXS), and temperature-dependent Fourier Transform Infrared (FTIR) spectroscopy. Also, the effect of hydrostatic pressure on the transition temperatures was studied by using SANS with pressure controller. Phase behavior was changed significantly with the change of alkyl number (n). For n = 2∼4, only Lower Disordered-to-Order Tansition (LDOT) was observed, whereas the Ordered-to-Disorder (ODT) was found for n =1 and n =6. Finally, a closed-loop phase behavior was found for n =5. Using incompressible random phase approximation, the segmental interactions (χ) between PS and PnAMA for all n values were obtained. The standard expression of χ = a + b/T (where T is the absolute temperature) was valid only for n =1 and n =6. But, this relationship was not valid any more for n = 2∼4. For n =5, a more complex behavior of χ upon temperature was observed. We investigated, by using temperature-dependent FTIR, the mechanism why as closed loop phase behavior was observed for n =5. Interestingly, the conformation of C-C-O stretching band of the PnPMA chain (n=5) (and thus the directional enthapic gain) was different in the two disordered states, and, therefore, the driving force to induce the disordered state at lower temperatures was different from that at higher temperatures

Effects of derivatization reagents consisting of n-alkyl chloroformate/n-alcohol combinations in LC-ESI-MS/MS analysis of zwitterionic antiepileptic drugs.

Science.gov (United States)

Kostić, Nađa; Dotsikas, Yannis; Malenović, Anđelija; Medenica, Mirjana

2013-11-15

In the current study, three antiepileptic drugs with zwitterionic properties, namely vigabatrin, pregabalin and gabapentin, were chosen as model analytes to undergo derivatization by applying various n-alkyl chloroformate/n-alcohol combinations, followed by LC-ESI-MS/MS analysis. The employment of 16 combinations per drug using methyl, ethyl, propyl or butyl chloroformate coupled with methanol, ethanol, propanol or butanol, greatly affected a series of parameters of the derivatives, such as retention time on C8 column, signal expressed via areas, limit of detection values, as well as the yields of the main and side reactions. Practically, even slight modification of n-alkyl group of either chloroformate or alcohol resulted in significant changes in the chromatographic and mass spectrometric behavior of the novel derivative. It was clearly demonstrated that all the estimated parameters were highly correlated with the length of n-alkyl groups of the involved chloroformate and alcohol. The most significant influence was monitored in peak area values, indicating that the length of the n-alkyl chain plays an important role in electrospray ionization efficiency. For this parameter, increasing the n-alkyl chain from methyl to butyl led to increment up to 2089%, 508.7% and 1075% for area values of derivatized vigabatrin, pregabalin and gabapentin, respectively. These changes affected also the corresponding values of limits of detection, with the estimated improvements up to 1553%, 397.7% and 875.0% for the aforementioned derivatized drugs, respectively. Besides the obvious utilization of these conclusions in the development of bioanalytical methods for these analytes with the current protocol, this study offers valuable data which can be useful in more general approaches, giving insights into the effects of this derivatization reaction and its performances. Copyright © 2013 Elsevier B.V. All rights reserved.

Microtubule disruption induced in vivo by alkylation of beta-tubulin by 1-aryl-3-(2-chloroethyl)ureas, a novel class of soft alkylating agents.

Science.gov (United States)

Legault, J; Gaulin, J F; Mounetou, E; Bolduc, S; Lacroix, J; Poyet, P; Gaudreault, R C

2000-02-15

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-isopropyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G2 transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3-2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC50 of 21.3+/-1.1 microM as compared with an IC50 of 11.6+/-0.7 microM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive beta-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-14C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional beta-tubulin band, indicating that CEU could covalently bind to the beta-tubulin. The 4-tBCEU-binding site on beta-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional beta-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site. To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the betaIII-tubulin, an isoform where Cys239

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

International Nuclear Information System (INIS)

Stefanovsky, V.Yu.; Dimitrov, S.I.; Angelov, D.; Pashev, I.G.

1989-01-01

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA

Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids: implications for multicellular resistance to alkylating agents.

Science.gov (United States)

Francia, Giulio; Green, Shane K; Bocci, Guido; Man, Shan; Emmenegger, Urban; Ebos, John M L; Weinerman, Adina; Shaked, Yuval; Kerbel, Robert S

2005-10-01

Similar to other anticancer agents, intrinsic or acquired resistance to DNA-damaging chemotherapeutics is a major obstacle for cancer therapy. Current strategies aimed at overcoming this problem are mostly based on the premise that tumor cells acquire heritable genetic mutations that contribute to drug resistance. Here, we present evidence for an epigenetic, tumor cell adhesion-mediated, and reversible form of drug resistance that is associated with a reduction of DNA mismatch repair proteins PMS2 and/or MLH1 as well as other members of this DNA repair process. Growth of human breast cancer, human melanoma, and murine EMT-6 breast cancer cell lines as multicellular spheroids in vitro, which is associated with increased resistance to many chemotherapeutic drugs, including alkylating agents, is shown to lead to a reproducible down-regulation of PMS2, MLH1, or, in some cases, both as well as MHS6, MSH3, and MSH2. The observed down-regulation is in part reversible by treatment of tumor spheroids with the DNA-demethylating agent, 5-azacytidine. Thus, treatment of EMT-6 mouse mammary carcinoma spheroids with 5-azacytidine resulted in reduced and/or disrupted cell-cell adhesion, which in turn sensitized tumor spheroids to cisplatin-mediated killing in vitro. Our results suggest that antiadhesive agents might sensitize tumor spheroids to alkylating agents in part by reversing or preventing reduced DNA mismatch repair activity and that the chemosensitization properties of 5-azacytidine may conceivably reflect its role as a potential antiadhesive agent as well as reversal agent for MLH1 gene silencing in human tumors.

Current approaches to improve the anticancer chemotherapy with alkylating agents: state of the problem in world and Ukraine.

Directory of Open Access Journals (Sweden)

Iatsyshyna A. P.

2012-01-01

Full Text Available Alkylating agents are frequently used in many established anticancer chemotherapies. They alkylate the genomic DNA at various sites. Alkylation of the guanine at the O6-position is cytotoxic, it has the strongest mutagenic potential, as well as can cause the tumor development. Alkyl groups at the O6-position of guanine are removed by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT. The effectiveness of alkylating chemotherapy is limited by MGMT in cancer cells and adverse toxic side effects in normal cells. Different approaches consisting in the modulation of the MGMT expression and activity are under development now to improve the cancer chemotherapy. They include two main directions, in particular, the increase in chemosensitivity of cancer cells to alkylating drugs and the protection of normal cells from the toxic side effects of chemotherapy. This review is focused on current attempts to improve the alkylating chemotherapy of malignant tumours worldwide and state of the issue in Ukraine

Tribology of monolayer films: comparison between n-alkanethiols on gold and n-alkyl trichlorosilanes on silicon.

Science.gov (United States)

Booth, Brandon D; Vilt, Steven G; McCabe, Clare; Jennings, G Kane

2009-09-01

This Article presents a quantitative comparison of the frictional performance for monolayers derived from n-alkanethiolates on gold and n-alkyl trichlorosilanes on silicon. Monolayers were characterized by pin-on-disk tribometry, contact angle analysis, ellipsometry, and electrochemical impedance spectroscopy (EIS). Pin-on-disk microtribometry provided frictional analysis at applied normal loads from 10 to 1000 mN at a speed of 0.1 mm/s. At low loads (10 mN), methyl-terminated n-alkanethiolate self-assembled monolayers (SAMs) exhibited a 3-fold improvement in coefficient of friction over SAMs with hydroxyl- or carboxylic-acid-terminated surfaces. For monolayers prepared from both n-alkanethiols on gold and n-alkyl trichlorosilanes on silicon, a critical chain length of at least eight carbons is required for beneficial tribological performance at an applied load of 9.8 mN. Evidence for disruption of chemisorbed alkanethiolate SAMs with chain lengths n tribology wear tracks. The direct comparison between the tribological stability of alkanethiolate and silane monolayers shows that monolayers prepared from n-octadecyl dimethylchlorosilane and n-octadecyl trichlorosilane withstood normal loads at least 30 times larger than those that damaged octadecanethiolate SAMs. Collectively, our results show that the tribological properties of monolayer films are dependent on their internal stabilities, which are influenced by cohesive chain interactions (van der Waals) and the adsorbate-substrate bond.

The synthesis and biological evaluation of new DNA-directed alkylating agents, phenyl N-mustard-4-anilinoquinoline conjugates containing a urea linker.

Science.gov (United States)

Marvania, Bhavin; Kakadiya, Rajesh; Christian, Wilson; Chen, Tai-Lin; Wu, Ming-Hsi; Suman, Sharda; Tala, Kiran; Lee, Te-Chang; Shah, Anamik; Su, Tsann-Long

2014-08-18

We synthesized a series of phenyl N-mustard-4-anilinoquinoline conjugates to study their antitumorigenic effects. These agents were prepared by the condensation of 4-[N,N-bis(2-chloroethyl)amino]phenyl isocyanate with 6-amino-4-methylamino or 4-anilinoquinolines. The structure-activity relationship (SAR) studies revealed that the C2-methylquinoline derivatives (18a-o) were generally more cytotoxic than the C2-phenylquinoline conjugates (23a-d) in inhibiting the cell growth of various human tumor cell lines in vitro. However, the methylamino or aniline substituents at C4 of quinoline did not influence the cytotoxic effects. The title conjugates were capable of inducing DNA cross-linking and promoting cell-cycle arrest at the G2/M phase. This study demonstrates that phenyl N-mustard-4-anilinoquinoline conjugates are generally more potent than phenyl N-mustard-4-anilinoquinazoline conjugates against the cell growth of various tumor cell-lines. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Mitsunobu mischief: Neighbor-directed histidine N(π)–alkylation provides access to peptides containing selectively functionalized imidazolium heterocycles

Science.gov (United States)

Qian, Wen-Jian

2015-01-01

There are few methodologies that yield peptides containing His residues with selective N(π), N(π)-bis-alkylated imidazole rings. We have found that, under certain conditions, on-resin Mitsunobu coupling of alcohols with peptides having a N(π)-alkylated His residue results in selective and high-yield alkylation of the imidazole N(π) nitrogen. The reaction requires the presence of a proximal phosphoric, carboxylic or sulfonic acid, and proceeds through an apparent intramolecular mechanism involving Mitsunobu intermediates. These transformations have particular application to phosphopeptides, where “charge masking” of one phosphoryl anionic charge by the cationic histidine imidazolium ion is now possible. This chemistry opens selective access to peptides containing differentially functionalized imidazolium heterocycles, which provide access to new classes of peptides and peptide mimetics. PMID:25739367

Inhibition of mutagenicity of N-methyl-N-nitrosourea by ellagic acid

International Nuclear Information System (INIS)

Dixit, R.; Gold, B.

1986-01-01

Ellagic acid (EA), a plant phenol present in a variety of soft fruits and vegetables, has been shown to possess antimutagenic and anticarcinogenic properties against bay region diol epoxide of polycyclic aromatic hydrocarbons. It is suggested that EA forms an adduct with diol epoxide of benzo (α) pyrene and thus prevents its binding to DNA. To better understand the mechanism of reactivity and inhibition properties of EA, we studied the effect of EA on mutagenicity and DNA alkylation of carcinogenic N-nitroso compounds, including N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-Nnitrosoguanidine (MNNG). MNU and MNNG are direct-acting mutagens requiring no metabolic activation. MNU showed a linear dose response between the concentration range of 50 to 400 nmole in an Ames/Salmonella mammalian mutagenicity test. EA at concentrations of 100, 250, 500, and 1,000 nmole inhibited the mutagenicity of MNU (400 nmole) by 3, 13, 45,and 60%, respectively. MNNG produced a nonlinear dose response in mutagenicity between the concentrations of 0.5 to 4 nmole. EA showed no appreciable inhibition of MNNG mutagenicity. Inhibition of DNA alkylation by MNU and MNNG by EA was studied by preincubating 50 to 200 nmole of EA with 200 nmole of ( 3 H)-MNU or ( 3 H)-MNNG for 10 min at 37 0 c, followed by incubation of polymer deoxyguanosine: deoxycytosine (poly dG:dC) (1 unit) overnight. EA caused no inhibitory effect on MNNG alkylation of poly dG:dC. Experiments on the effect of EA on alkylation of DNA and formation of nucleoside adducts by MNU are in progress, and results will be discussed with reference to MNU and MNNG mutagenicity and EA inhibition

Effects of Photo-chemically Activated Alkylating Agents of the FR900482 Family on Chromatin

Science.gov (United States)

Subramanian, Vidya; Ducept, Pascal; Williams, Robert M.; Luger, Karolin

2011-01-01

SUMMARY Bioreductive alkylating agents are an important class of clinical antitumor antibiotics that cross-link and mono-alkylate DNA. Here we use a synthetic photochemically activated derivative of FR400482 to investigate the molecular mechanism of this class of drugs in a biologically relevant context. We find that the organization of DNA into nucleosomes effectively protects it against drug-mediated cross-linking, while permitting mono-alkylation. This modification has the potential to form covalent cross-links between chromatin and nuclear proteins. Using in vitro approaches, we found that interstrand cross-linking of free DNA results in a significant decrease in basal and activated transcription. Finally, cross-linked plasmid DNA is inefficiently assembled into chromatin. Our studies suggest new pathways for the clinical effectiveness of this class of reagents. PMID:17524986

the influence of acidity of zeolite H-BEA catalyzed isobutane/n-butene alkylation

NARCIS (Netherlands)

Nivarthy, G.S.; Seshan, Kulathuiyer; Lercher, J.A.

1998-01-01

The influence of the concentration of acid sites for isobutane/n-butene alkylation on zeolite BEA with varying degrees of Na+ ion exchange is reported. All catalysts studied showed complete n-butene conversion over a significant time-on-stream. Isooctanes were the dominating products over H-BEA,

Force induced DNA melting

International Nuclear Information System (INIS)

Santosh, Mogurampelly; Maiti, Prabal K

2009-01-01

When pulled along the axis, double-strand DNA undergoes a large conformational change and elongates by roughly twice its initial contour length at a pulling force of about 70 pN. The transition to this highly overstretched form of DNA is very cooperative. Applying a force perpendicular to the DNA axis (unzipping), double-strand DNA can also be separated into two single-stranded DNA, this being a fundamental process in DNA replication. We study the DNA overstretching and unzipping transition using fully atomistic molecular dynamics (MD) simulations and argue that the conformational changes of double-strand DNA associated with either of the above mentioned processes can be viewed as force induced DNA melting. As the force at one end of the DNA is increased the DNA starts melting abruptly/smoothly above a critical force depending on the pulling direction. The critical force f m , at which DNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the DNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base pairs. The fraction of Watson-Crick base pair hydrogen bond breaking as a function of force does not show smooth and continuous behavior and consists of plateaus followed by sharp jumps.

Investigation of J-shaped dose-responses induced by exposure to the alkylating agent N-methyl-N-nitrosourea.

Science.gov (United States)

Chapman, Katherine E; Hoffmann, George R; Doak, Shareen H; Jenkins, Gareth J S

2017-07-01

Hormesis is defined as a biphasic dose-response where biological effects of low doses of a stressor demonstrate the opposite effect to high-dose effects of the same stressor. Hormetic, or J-shaped, dose-response relationships are relatively rarely observed in toxicology, resulting in a limited understanding and even some skepticism of the concept. Low dose-response studies for genotoxicity endpoints have been performed at Swansea University for over a decade. However, no statistically significant decreases below control genotoxicity levels have been detected until recently. A hormetic-style dose-response following a 24h exposure to the alkylating agent N-methyl-N-nitrosourea (MNU) was observed in a previous study for HPRT mutagenesis in the human lymphoblastoid cell line AHH-1. A second recent study demonstrated a J-shaped dose-response for the induction of micronuclei by MNU in a 24h treatment in a similar test system. Following mechanistic investigations, it was hypothesized that p53 may be responsible for the observed hormetic phenomenon. As genotoxic carcinogens are a major causative factor of many cancers, consideration of hormesis in carcinogenesis could be important in safety assessment. The data examined here offer possible insights into hormesis, including its estimated prevalence, underlying mechanisms and lack of generalizability. Copyright © 2017 Elsevier B.V. All rights reserved.

Pd@[nBu4][Br] as a Simple Catalytic System for N-Alkylation Reactions with Alcohols

Directory of Open Access Journals (Sweden)

Bastien Cacciuttolo

2016-08-01

Full Text Available Palladium nanoparticles, simply and briefly generated in commercial and cheap onium salts using supercritical carbon dioxide, have been found to be an effective catalytic system for additive free N-alkylation reaction using alcohols via cascade oxidation/condensation/reduction steps.

Williamson alkylation approach to the synthesis of poly(alkyl vinyl ether) copolymers

International Nuclear Information System (INIS)

Markova, D.; Christova, D.; Velichkova, R.

2008-01-01

A method for synthesis of poly(alkyl vinyl ether-co-vinyl alcohol) copolymers was developed based on the Williamson's alkylation of poly(vinyl acetate) (PVAc) with alkyl iodides. The influence of the alkylating agent and the reaction conditions on the efficiency of the modification reaction was investigated. The copolymers obtained were characterized by means of 1 H NMR and GPC. It was proved that by applying the proposed method copolymers of different composition and properties containing methyl vinyl ether, ethyl vinyl ether as well as n-butyl vinyl ether units could be prepared. Poly(methyl vinyl ether-co-vinyl alcohol)s of high degree of methylation exhibit sharp temperature response at 38-39 deg C in aqueous solution typical of the so-called smart polymers. (authors)

The proofreading 3'→5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

International Nuclear Information System (INIS)

Khare, Vineeta; Eckert, Kristin A.

2002-01-01

The 3'→5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase γ (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'→5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity

N-alkylvaline levels in globin as a new type of biomarker in risk assessment of alkylating agents.

Science.gov (United States)

Lewalter, J

1996-01-01

Adducts with the N-terminal valine of erythrocyte globin can serve as individual biomarkers of systemic and cellular exposure to endogenous and exogenous alkylating agents. In contrast to "detoxification markers" of this kind of mecapturic acids derived from alkylation of glutathione, individual N-alkylations of valine in globin reflect the formally "toxifying" part of the stress due to alkylating agents transformed into the ultimate toxicant. Thus, in contrast to the traditional methods of biological monitoring this approach enables a better evaluation of systemic exposure to reactive agents, adapted more sensibly to the exposure situation over the whole life span of erythrocytes, and it can serve as a specific biomarker of exposure for the purpose of health surveillance in occupational medicine. An individual evaluation of exposures in comparison with the range of corresponding background levels is discussed from the point of view of supplementary risk assessment in medical surveillance of occupationally exposed persons.

Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents

International Nuclear Information System (INIS)

Kaina, B.; Lohrer, H.; Karin, M.; Herrlich, P.

1990-01-01

Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions

Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Kaina, B.; Lohrer, H.; Karin, M.; Herrlich, P. (Kernforschungszentrum Karlsruhe, Karlsruhe (Germany, F.R.))

1990-04-01

Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions.

Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.

Science.gov (United States)

Kaina, B; Lohrer, H; Karin, M; Herrlich, P

1990-01-01

Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions. Images PMID:2320583

N-acetyl cysteine protects against ionizing radiation-induced DNA damage but not against cell killing in yeast and mammals

Energy Technology Data Exchange (ETDEWEB)

Reliene, Ramune [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Medicine, Center for Human Nutrition, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Pollard, Julianne M. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Biomedical Physics Interdepartmental Program, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Sobol, Zhanna; Trouiller, Benedicte [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Gatti, Richard A. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Schiestl, Robert H., E-mail: rschiestl@mednet.ucla.edu [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Biomedical Physics Interdepartmental Program, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Radiation Oncology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Environmental Health Sciences, School of Public Health, University of California Los Angeles, Los Angeles, CA 90095 (United States)

2009-06-01

Ionizing radiation (IR) induces DNA strand breaks leading to cell death or deleterious genome rearrangements. In the present study, we examined the role of N-acetyl-L-cysteine (NAC), a clinically proven safe agent, for it's ability to protect against {gamma}-ray-induced DNA strand breaks and/or DNA deletions in yeast and mammals. In the yeast Saccharomyces cerevisiae, DNA deletions were scored by reversion to histidine prototrophy. Human lymphoblastoid cells were examined for the frequency of {gamma}-H2AX foci formation, indicative of DNA double strand break formation. DNA strand breaks were also measured in mouse peripheral blood by the alkaline comet assay. In yeast, NAC reduced the frequency of IR-induced DNA deletions. However, NAC did not protect against cell death. NAC also reduced {gamma}-H2AX foci formation in human lymphoblastoid cells but had no protective effect in the colony survival assay. NAC administration via drinking water fully protected against DNA strand breaks in mice whole-body irradiated with 1 Gy but not with 4 Gy. NAC treatment in the absence of irradiation was not genotoxic. These data suggest that, given the safety and efficacy of NAC in humans, NAC may be useful in radiation therapy to prevent radiation-mediated genotoxicity, but does not interfere with efficient cancer cell killing.

S-alkylation of soft scorpionates.

Science.gov (United States)

Rajasekharan-Nair, Rajeev; Moore, Dean; Chalmers, Kirsten; Wallace, Dawn; Diamond, Louise M; Darby, Lisa; Armstrong, David R; Reglinski, John; Spicer, Mark D

2013-02-11

The alkylation reactions of soft scorpionates are reported. The hydrotris(S-alkyl-methimazolyl)borate dications (alkyl = methyl, allyl, benzyl), which were prepared by the reaction of Tm(Me) anion and primary alkyl halides, have been isolated and structurally characterised. The reaction is, however, not universally successful. DFT analysis of these alkylation reactions (C=S versus B-H alkylation) indicates that the observed outcome is driven by kinetic factors. Extending the study to incorporate alternative imine thiones (mercaptobenzothiazole, bz; thiazoline, tz) led to the structural characterisation of di[aquo-μ-aquohydrotris(mercaptobenzothiazolyl)boratosodium], which contains sodium atoms in the κ(3)-S,S,S coordination mode. Alkylation of Na[Tbz] and Na[tzTtz] leads to decomposition resulting in the formation of the simple S-alkylated heterocycles. The analysis of the species involved in these reactions shows an inherent weakness in the B-N bond in soft scorpionates, which has implications for their use in more advanced chemistry. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065

International Nuclear Information System (INIS)

Zsido, T.J.; Woynarowski, J.M.; Baker, R.M.; Gawron, L.S.; Beerman, T.A.

1991-01-01

CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand break. The authors have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, They observe thermally induced breakage of [ 14 C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [ 3 H]thymidine-labeled untreated cells with the [ 14 C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10 6 base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The results of this study demonstrate that CC-1065 does generate heat-labile sites with the cellular DNA of intact cells and suggest that a mechanism of cytotoxic action of CC-1065 involves formation of covalent adducts to DNA

[DNA-dependent DNA polymerase induced by herpes virus papio (HVP) in producing cells].

Science.gov (United States)

D'iachenko, A G; Beriia, L Ia; Matsenko, L D; Kakubava, V V; Kokosh, L V

1980-11-01

A new DNA polymerase was found in the cells of suspension lymphoblastoid cultures, which produce lymphotropic baboon herpes virus (HVP). The enzyme was isolated in a partially purified form. In some properties the enzyme differs from other cellular DNA polymerases. The HVP-induced DNA polymerase has the molecular weight of 1,6 x 10(5) and sedimentation coefficient of about 8S. The enzyme is resistant to high salt concentrations and N-ethylmaleimide, but shows a pronounced sensitivity to phosphonoacetate. The enzyme effectively copies "activated" DNA and synthetic deoxyribohomopolymers. The attempts to detect the DNA polymerase activity in HVP virions were unsuccessful.

The alkylation of imine anions formation of enamines

NARCIS (Netherlands)

Heiszwolf, G.J.; Kloosterziel, H.

1970-01-01

The ambident anions derived from imines were alkylated using a variety of solvents and alkylating agents. Under reactive conditions enamines (N-alkylation) are formed as the main products instead of the usually obsd. homologous imines (C-alkylation). The influence of the type of imine, solvent, and

Molecular structure, supramolecular organization and thermotropic phase behavior of N-acylglycine alkyl esters with matched acyl and alkyl chains.

Science.gov (United States)

Reddy, S Thirupathi; Swamy, Musti J

2017-11-01

N-Acylglycines (NAGs), the endogenous single-tailed lipids present in rat brain and other mammalian tissues, play significant roles in cell physiology and exhibit interesting pharmacological properties. In the present study, a homologous series of N-acylglycine alkyl esters (NAGEs) with matched chains were synthesized and characterized. Results of differential scanning calorimetric studies revealed that all NAGEs exhibit a single sharp phase transition and that the transition enthalpy and entropy show a linear dependence on the N-acyl and ester alkyl chain length. The structure of N-myristoylglycine myristyl ester (NMGME), solved by single-crystal X-ray diffraction, showed that the molecule adopts a linear geometry and revealed that the structure of N-myristoyl glycyl moiety in NMGME is identical to that in N-myristoylglycine. The molecules are packed in layers with the polar functional groups of the ester and amide functionalities located at the center of the layer. The crystal packing is stabilized by NH⋯O hydrogen bonds between the amide CO and NH groups of adjacent molecules as well as by CH⋯O hydrogen bonds between the amide carbonyl and the methylene CH adjacent to the ester carbonyl of neighboring molecules as well as between ester carbonyl and methylene group of the glycine moiety of adjacent molecules. Powder X-ray diffraction studies showed a linear dependence of the d-spacings on the acyl chain length, suggesting that all NAGEs adopt a structure similar to the packing exhibited in the crystal lattice of NMGME. Copyright © 2017 Elsevier B.V. All rights reserved.

Binding of radiation-induced phenylalanine radicals to DNA

International Nuclear Information System (INIS)

Schans, G.P. van der; Rijn, C.J.S. van; Bleichrodt, J.F.

1975-11-01

When an aqueous solution of double-stranded DNA of bacteriophage PM2 containing phenylalanine and saturated with N 2 O is irradiated with γ-rays, radiation-induced phenylalanine radicals are bound covalently. Under the conditions used about 25 phenylalanine molecules may be bound per lethal hit. Also for single-stranded PM2 DNA, most of the phenylalanine radicals bound are non-lethal. Evidence is presented that in double-stranded DNA an appreciable fraction of the single-strand breaks is induced by phenylalanine radicals. Radiation products of phenylalanine and the phenylalanine bound to the DNA decrease the sensitivity of the DNA to the induction of single-strand breaks. There are indications that the high efficiency of protection by radiation products of phenylalanine is due to their positive charge, which will result in a relatively high concentration of these compounds in the vicinity of the negatively charged DNA molecules

N-O chemistry for antibiotics: discovery of N-alkyl-N-(pyridin-2-yl)hydroxylamine scaffolds as selective antibacterial agents using nitroso Diels-Alder and ene chemistry.

Science.gov (United States)

Wencewicz, Timothy A; Yang, Baiyuan; Rudloff, James R; Oliver, Allen G; Miller, Marvin J

2011-10-13

The discovery, syntheses, and structure-activity relationships (SAR) of a new family of heterocyclic antibacterial compounds based on N-alkyl-N-(pyridin-2-yl)hydroxylamine scaffolds are described. A structurally diverse library of ∼100 heterocyclic molecules generated from Lewis acid-mediated nucleophilic ring-opening reactions with nitroso Diels-Alder cycloadducts and nitroso ene reactions with substituted alkenes was evaluated in whole cell antibacterial assays. Compounds containing the N-alkyl-N-(pyridin-2-yl)hydroxylamine structure demonstrated selective and potent antibacterial activity against the Gram-positive bacterium Micrococcus luteus ATCC 10240 (MIC(90) = 2.0 μM or 0.41 μg/mL) and moderate activity against other Gram-positive strains including antibiotic resistant strains of Staphylococcus aureus (MRSA) and Enterococcus faecalis (VRE). A new synthetic route to the active core was developed using palladium-catalyzed Buchwald-Hartwig amination reactions of N-alkyl-O-(4-methoxybenzyl)hydroxylamines with 2-halo-pyridines that facilitated SAR studies and revealed the simplest active structural fragment. This work shows the value of using a combination of diversity-oriented synthesis (DOS) and parallel synthesis for identifying new antibacterial scaffolds.

Boron-Catalyzed N-Alkylation of Amines using Carboxylic Acids.

Science.gov (United States)

Fu, Ming-Chen; Shang, Rui; Cheng, Wan-Min; Fu, Yao

2015-07-27

A boron-based catalyst was found to catalyze the straightforward alkylation of amines with readily available carboxylic acids in the presence of silane as the reducing agent. Various types of primary and secondary amines can be smoothly alkylated with good selectivity and good functional-group compatibility. This metal-free amine alkylation was successfully applied to the synthesis of three commercial medicinal compounds, Butenafine, Cinacalcet. and Piribedil, in a one-pot manner without using any metal catalysts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alkylation of amide linkages and cleavage of the C chain in the enzyme-activated-substrate inhibition of alpha-chymotrypsin with N-nitrosamides

International Nuclear Information System (INIS)

Donadio, S.; Perks, H.M.; Tsuchiya, K.; White, E.H.

1985-01-01

Active-site-directed N-nitrosamides inhibit alpha-chymotrypsin through an enzyme-activated-substrate mechanism. In this work, the activation results in the release--in the active site--of benzyl carbonium ions, which alkylate and inhibit the enzyme. The final ratio of benzyl groups to enzyme molecules is 1.0, but the alkyl groups are scattered over a number of sites. Reduction and alkylation of the inhibited enzyme generate peptides insoluble in most media. Guanidine hydrochloride at 6 M proved a good solvent, and its use as an eluant on G-75 Sephadex permitted separation of the peptides. In the case of 14 C-labeled enzyme, such an approach has shown that all of the alkylation occurs on the C chain of the enzyme, the chain of which the active site is constructed. Chemical modification of the peptides with ethylenediamine and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide rendered them soluble in dilute acid, permitting high-performance liquid chromatographic separation. Model studies have shown that the benzyl carbonium ions are highly reactive, alkylating amide linkages at both oxygen and nitrogen. Chromatography of this mixture and also 13 C NMR spectroscopy of the intact inhibited enzyme have shown that three major N-alkylations have occurred. Tryptic digestion of the C chain of chymotrypsin, which contains all of the alkylation sites, provides evidence that the stable N sites are principally located between residue 216 and residue 230

Thermodynamic calculation of simultaneous reactions of n-butane isomerization and isobutane alkylation with ethylene

Energy Technology Data Exchange (ETDEWEB)

Batyrshin, N.N.; Beresneva, L.D.; Sidorov, V.A.

1981-08-01

Industrial production of ethylene alkylate has gained further development in connection with worldwide ecological problems and the planned changeover of automobile transport to unleaded gasolines, but the scale of production is still substantially less than that of sulfuric acid or hydrogen fluoride alkylates. This is due both to the instability of market prices for ethylene and the shortage of isobutane - a raw material for these large-tonnage production processes and for the synthetic rubber industry. The latter difficulty can be overcome by combining processes of isomerization of n-butane and alkylation of the resultant isobutane with ethylene in a single reaction unit. The possibility of combining these reactions using AlCl/sub 3/-based catalysts has been pointed out previously but in the literature there are no theoretical developments of technology or thermodynamic substantiation of a combined process. We have made a thermodynamic calculation of the consecutive (series-parallel) reactions of isomerization and alkylation with the goal of determining suitable technological conditions for carrying them out simultaneously and establishing the expected equilibrium yields of target products and the compositions of the reaction mixture.

Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

Science.gov (United States)

Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

2014-02-01

Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

Pandemic influenza 1918 H1N1 and 1968 H3N2 DNA vaccines induce cross-reactive immunity in ferrets against infection with viruses drifted for decades

DEFF Research Database (Denmark)

Bragstad, Karoline; Martel, Cyril; Thomsen, Joakim S.

2011-01-01

Please cite this paper as: Bragstad et al. (2010) Pandemic influenza 1918 H1N1 and 1968 H3N2 DNA vaccines induce cross-reactive immunity in ferrets against infection with viruses drifted for decades. Influenza and Other Respiratory Viruses 5(1), 13-23. Background Alternative influenza vaccines...... and vaccine production forms are needed as the conventional protein vaccines do not induce broad cross-reactivity against drifted strains. Furthermore, fast vaccine production is especially important in a pandemic situation, and broader vaccine reactivity would diminish the need for frequent change...... in the vaccine formulations. Objective In this study, we compared the ability of pandemic influenza DNA vaccines to induce immunity against distantly related strains within a subtype with the immunity induced by conventional trivalent protein vaccines against homologous virus challenge. Methods Ferrets were...

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

Science.gov (United States)

Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

An adaptive response to alkylating agents in Aspergillus nidulans.

Science.gov (United States)

Hooley, P; Shawcross, S G; Strike, P

1988-11-01

A simple method is described for demonstrating adaptation to alkylation damage in Aspergillus nidulans. One wild type, two MNNG-sensitive, and one MNNG-resistant strain all showed improvement in colony growth when challenged with MNNG following appropriate inducing pretreatments. Other alkylating agents (MMS, EMS) could also adapt mycelium to later MNNG challenge, while 4NQO and UV could not. The inducible effect was not transmissible through conidia. A standard reversion assay based upon methG proved impractical for studying mutation frequencies during alkylation treatments owing to variations in MNNG resistance amongst revertants.

Exposure of E. coli to DNA-methylating agents impairs biofilm formation and invasion of eukaryotic cells via down regulation of the N-acetylneuraminate lyase NanA

Directory of Open Access Journals (Sweden)

Pamela eDi Pasquale

2016-02-01

Full Text Available DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analogue acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.

DNA Base Excision Repair (BER) and Cancer Gene Therapy: Use of the Human N-mythlpurien DNA Glycosylase (MPG) to Sensitize Breast Cancer Cells to Low Dose Chemotherapy

National Research Council Canada - National Science Library

Harvey, Tia

2003-01-01

The DNA Base Excision Repair (PER) pathway is responsible for the repair of alkylation and oxidative DNA damage resulting in protection against the deleterious effects of endogenous and exogenous agents encountered on a daily basis...

Apoptin's functional N- and C-termini independently bind DNA

NARCIS (Netherlands)

Leliveld, S. R.; Dame, R.T.; Rohn, J. L.; Noteborn, M. H. M.; Abrahams, J. P.

2004-01-01

Apoptin induces apoptosis specifically in tumour cells, where Apoptin is enriched in the DNA-dense heterochromatin and nucleoli. In vitro, Apoptin interacts with dsDNA, forming large nucleoprotein superstructures likely to be relevant for apoptosis induction. Its N- and C-terminal domains also have

Differences in the regulation by poly(ADP-ribose) of repair of DNA damage from alkylating agents and ultraviolet light according to cell type

Energy Technology Data Exchange (ETDEWEB)

Cleaver, J.E.; Bodell, W.J.; Morgan, W.F.; Zelle, B.

1983-08-10

Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cells influenced the responses to DNA damage from methyl methanesulfonate, but not from ultraviolet light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but only stimulated repair replication in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects may be due to disturbance of precursor pathways and irrelevant to the role of poly(ADP-ribose) in repair. Previous claims that 3-aminobenzamide stimulates repair synthesis after exposure to UV light are probably artifacts, because the stimulations are only observed in lymphocytes in the presence of a high concentration of hydroxyurea that itself inhibits repair. The initial inhibition of semiconservative DNA synthesis and the excision of the major alkylation products and pyrimidine dimers were unaffected by 3-aminobenzamide. In general poly(ADP-ribose) synthesis appears to be uniquely involved in regulating the ligation stage of repair of alkylation damage but not ultraviolet damage. By regulating the ligation efficiency, poly(ADP-ribosylation) modulates the dynamic balance between incision and ligation, so as to minimize the frequency of DNA breaks. The ligation stage of repair of UV damage appears different and is not regulated by poly(ADP-ribosylation).

Nitric oxide donors attenuate clongenic potential in rat C6 glioma cells treated with alkylating chemotherapeutic agents.

Science.gov (United States)

Yang, Jir-Jei; Yin, Jiu-Haw; Yang, Ding-I

2007-05-11

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) kills tumor cells via multiple actions including alkylation and carbamoylation. Previously, we have reported that formation of S-nitrosoglutathione (GSNO) in glioma cells overexpressing inducible nitric oxide synthase (iNOS) contributed to nitric oxide (NO)-dependent carbamoylating chemoresistance against BCNU. To further characterize the effects of NO on alkylating cytotoxicity, colony formation assay was applied to evaluate the effects of various NO donors on rat C6 glioma cells challenged with alkylating agents. We demonstrate that NO donors including GSNO, diethylamine NONOate (DEA/NO), and sodium nitroprusside (SNP) substantially reduced the extent of colony formation in glioma cells treated with alkylating agents, namely methyl methanesulfonate (MMS), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU). Without alkylating agents these NO-releasing agents alone had no effects on clongenic potential of rat C6 glioma cells. Among these three NO donors used, the effectiveness in potentiating alkylating cytotoxicity is in the order of "GSNO>DEA/NO>SNP" when applied at the same dosages. GSNO also exerted similar synergistic actions reducing the extents of colony formation when co-administrated with 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazine (compound #1), another alkylating agent that mimics the chloroethylating action of BCNU. Together with our previous findings, we propose that NO donors may be used as adjunct chemotherapy with alkylating agents for such malignant brain tumors as glioblastoma multiforme (GBM). In contrast, production of NO as a result of iNOS induction, such as that occurring after surgical resection of brain tumors, may compromise the efficacy of carbamoylating chemotherapy.

Ionic conductivity of N-alkyl pyridinium halides mesophases

International Nuclear Information System (INIS)

Meftah, Ahmed

1980-01-01

The quasi anhydrous N-alkyl pyridinium halides undergo at a temperature T c a phase transition from a crystalline isolating state to a conducting mesophase (σ = 3.10 -2 Ω -1 cm -1 ). The transition temperature depends on the nature on counter-ion and on the aliphatic chain length. The present study is devoted to the N-alkyl pyridinium chlorides, bromides and iodides varying the number of carbon atoms in the chain from ten to twenty two. The transition temperatures T c were found to increase from 30 deg. C up to 110 deg. C by a step of 10 deg. C for two added carbon atoms in the chain. The electrical measurements have shown that the conductivity of the mesophases which is ionic in origin is due to a large mobility of counter-ions in hydrophilic parts. At high frequencies (F > 10 3 Hz) ionic conductivity predominates in the bulk and does not depend on frequency. At low frequencies (F 3 Hz) the most important are interface phenomena depending on the square root of inverse frequency (ω -1/2 ) and being due to an electronic exchange limited by diffusion velocity of counter-ions. The electrical conductivity depends weekly on the chain length and the mesophases textures. The most conducting mesophase is the optically isotropic. The conductivity increases with increasing water content of the system and decreases with increasing atomic number of counter-ion. The diffusion measurements by radioactive tracers confirm the ionic character of charge carriers although the diffusion factors obtained by this method are largely higher than the calculated ones from the conductivity values. (author) [fr

UV laser-induced histone-DNA crosslinking proceeds via the N-terminal tails

International Nuclear Information System (INIS)

Stefanovski, V.; Dimitrov, S.; Angelov, D.; Keskinova, E.; Pashev, I.

1990-01-01

The covalent crosslinking of histones to DNA by UV laser irradiation is accomplished solely via the N-terminal part of the molecule. Irradiated isolated calfthymus nuclei are treated with clostripain. The crosslinked protein-DNA complexes are isolated and the presence of each core histone analyzed by dot-immunoassay using antibodies, specific to the central globular domain of the respective histone. The reaction is negative for all core histones i.e. the globular domain is absent. It means that this domain has not been crosslinked to DNA and, once cleaved by clostripain, it has been stripped from DNA during the centrigugation in CsCl. This peculiar property of the crosslinked procedure makes it particularly useful in addressing some yet unanswered questions concerning histone-DNA interactions, such as the interaction of the N-terminal tails with linker DNA, the effect of the transient postsynthetic histone acetylation on its interaction with DNA, etc. These questions are now under study. 1 fig., 6 refs

Synthesis and pharmacology of N-alkylated derivatives of the excitotoxin ibotenic acid

DEFF Research Database (Denmark)

Madsen, U; Dumpis, M A; Bräuner-Osborne, Hans

1998-01-01

Three amino-alkylated derivatives of the naturally occurring excitatory amino acid (EAA) receptor agonist ibotenic acid (Ibo) have been synthesized and tested pharmacologically. N-Methyl-Ibo (1a) and N-ethyl-Ibo (1b) were shown to be agonists at NMDA receptors (EC50 = 140 and 320 micro......-c and the potent NMDA agonist 2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid (AMAA) in order to elucidate the observed structure-activity data....

Literature Survey and Further Studies on the 3-Alkylation of N-Unprotected 3-Monosubstituted Oxindoles. Practical Synthesis of N-Unprotected 3,3-Disubstituted Oxindoles and Subsequent Transformations on the Aromatic Ring.

Science.gov (United States)

Kókai, Eszter; Simig, Gyula; Volk, Balázs

2016-12-26

The paper provides a comprehensive review of the base-catalysed C3-alkylation of N-unprotected-3-monosubstituted oxindoles. Based on a few, non-systematic studies described in the literature using butyllithium as the deprotonating agent, an optimized method has now been elaborated, via the corresponding lithium salt, for the selective C3-alkylation of this family of compounds. The optimal excess of butyllithium and alkylating agent, and the role of the halogen atom in the latter (alkyl bromides vs. iodides) were also studied. The alkylation protocol has also been extended to some derivatives substituted at the aromatic ring. Finally, various substituents were introduced into the aromatic ring of the N-unprotected 3,3-dialkyloxindoles obtained by this optimized method.

Literature Survey and Further Studies on the 3-Alkylation of N-Unprotected 3-Monosubstituted Oxindoles. Practical Synthesis of N-Unprotected 3,3-Disubstituted Oxindoles and Subsequent Transformations on the Aromatic Ring

Directory of Open Access Journals (Sweden)

Eszter Kókai

2016-12-01

Full Text Available The paper provides a comprehensive review of the base-catalysed C3-alkylation of N-unprotected-3-monosubstituted oxindoles. Based on a few, non-systematic studies described in the literature using butyllithium as the deprotonating agent, an optimized method has now been elaborated, via the corresponding lithium salt, for the selective C3-alkylation of this family of compounds. The optimal excess of butyllithium and alkylating agent, and the role of the halogen atom in the latter (alkyl bromides vs. iodides were also studied. The alkylation protocol has also been extended to some derivatives substituted at the aromatic ring. Finally, various substituents were introduced into the aromatic ring of the N-unprotected 3,3-dialkyloxindoles obtained by this optimized method.

Enhanced mutagenicity of low doses of alkylating agents and UV-light by inhibition of ribonucleotide reductase

International Nuclear Information System (INIS)

Jenssen, D.

1986-01-01

Monofunctional alkylating agents and UV-light are potent inducers of gene mutations in mammalian cells. Most data on these agent are supporting the idea that 0/sup 6/-alkylguanine is the dominating lesion responsible for the mutations induced by the alkylating agents and thymine-dimers in the case of UV-light. However, little is known about the mutagenic fate of these lesions during the replicative process. This is an essential issue to investigate not the least because of quantitative aspects. By investigating the factors affecting the mutagenic yield of these lesions, they hope to get further information on the mechanisms(s) involved. To study this, a system was applied which involves synchronized V79 Chinese hamster cells and inhibitors of the replication process. By applying hydroxyurea (HU), as inhibitor of the ribonucleotide reductase (RNR) step in DNA synthesis, the effect of nucleotide pool imbalance has been studied at the HGPRT-locus using V79 Chinese hamster cells

Dynamic studies of poly(di-n-alkyl itaconate)s

CERN Document Server

Arrighi, V; Gagliardi, S; McEwen, I J; Telling, M T F

2002-01-01

We report a preliminary dynamic study of poly(di-n-alkyl itaconate)s with varying side chain length n. QENS measurements were carried out on two backscattering spectrometers, IRIS at ISIS and IN10 at the ILL in the temperature range of 4 to 350 K. We show that molecular motion can be detected well below the polymer glass transition for all samples. It is possible to distinguish different dynamic processes. The temperature range over which these are observed is dependent on the length of the side chain, n. The intermediate scattering function, I(Q,t), was determined from the IRIS and found to obey time-temperature superposition. We show that the I(Q,t) data at different temperatures can be overlapped using the same time-scale shift factors, indicating that the relaxation process is common to all the polymers investigated. (orig.)

DNA alkylating agents alleviate silencing of class II transactivator gene expression in L1210 lymphoma cells.

Science.gov (United States)

Murphy, Shawn P; Holtz, Renae; Lewandowski, Nicole; Tomasi, Thomas B; Fuji, Hiroshi

2002-09-15

MHC class II (Ia) Ag expression is inversely correlated with tumorigenicity and directly correlated with immunogenicity in clones of the mouse L1210 lymphoma (1 ). Understanding the mechanisms by which class II Ag expression is regulated in L1210 lymphoma may facilitate the development of immunotherapeutic approaches for the treatment of some types of lymphoma and leukemia. This study demonstrates that the variation in MHC class II Ag expression among clones of L1210 lymphoma is due to differences in the expression of the class II transactivator (CIITA). Analysis of stable hybrids suggests that CIITA expression is repressed by a dominant mechanism in class II-negative L1210 clones. DNA-alkylating agents such as ethyl methanesulfonate and the chemotherapeutic drug melphalan activate CIITA and class II expression in class II negative L1210 cells, and this effect appears to be restricted to transformed cell lines derived from the early stages of B cell ontogeny. Transient transfection assays demonstrated that the CIITA type III promoter is active in class II(-) L1210 cells, despite the fact that the endogenous gene is not expressed, which suggests that these cells have all of the transacting factors necessary for CIITA transcription. An inverse correlation between methylation of the CIITA transcriptional regulatory region and CIITA expression was observed among L1210 clones. Furthermore, 5-azacytidine treatment activated CIITA expression in class II-negative L1210 cells. Collectively, our results suggest that 1) CIITA gene expression is repressed in class II(-) L1210 cells by methylation of the CIITA upstream regulatory region, and 2) treatment with DNA-alkylating agents overcomes methylation-based silencing of the CIITA gene in L1210 cells.

Measure of O(6)-Alkylguanine-DNA Alkyltransferase Activity in Normal Human Epidermal Keratinocytes in Culture and Effects of Bis-(2-chloroethyl)sulfide in the Activity

National Research Council Canada - National Science Library

Kurt, Ellen

1999-01-01

...)-alkylguanine in DNA. AGT may be important in DNA repair following injury induced by bis-(2-chloroethyl)sulfide (sulfur mustard, HD), since O(sup 6)-alkylguanine is one of the HD alkylation products...

Radiation-induced DNA damage as a function of DNA hydration

International Nuclear Information System (INIS)

Swarts, S.G.; Miao, L.; Wheeler, K.T.; Sevilla, M.D.; Becker, D.

1995-01-01

Radiation-induced DNA damage is produced from the sum of the radicals generated by the direct ionization of the DNA (direct effect) and by the reactions of the DNA with free radicals formed in the surrounding environment (indirect effect). The indirect effect has been believed to be the predominant contributor to radiation-induced intracellular DNA damage, mainly as the result of reactions of bulk water radicals (e.g., OH·) with DNA. However, recent evidence suggests that DNA damage, derived from the irradiation of water molecules that are tightly bound in the hydration layer, may occur as the result of the transfer of electron-loss centers (e.g. holes) and electrons from these water molecules to the DNA. Since this mechanism for damaging DNA more closely parallels that of the direct effect, the irradiation of these tightly bound water molecules may contribute to a quasi-direct effect. These water molecules comprise a large fraction of the water surrounding intracellular DNA and could account for a significant proportion of intracellular radiation-induced DNA damage. Consequently, the authors have attempted to characterize this quasi-direct effect to determine: (1) the extent of the DNA hydration layer that is involved with this effect, and (2) what influence this effect has on the types and quantities of radiation-induced DNA damage

Pyrrolizidine alkaloid-derived DNA adducts are common toxicological biomarkers of pyrrolizidine alkaloid N-oxides.

Science.gov (United States)

He, Xiaobo; Xia, Qingsu; Woodling, Kellie; Lin, Ge; Fu, Peter P

2017-10-01

There are 660 pyrrolizidine alkaloids (PAs) and PA N-oxides present in the plants, with approximately half being possible carcinogens. We previously reported that a set of four PA-derived DNA adducts is formed in the liver of rats administered a series of hepatocarcinogenic PAs and a PA N-oxide. Based on our findings, we hypothesized that this set of DNA adducts is a common biological biomarker of PA-induced liver tumor formation. In this study, we determined that rat liver microsomal metabolism of five hepatocarcinogenic PAs (lasiocarpine, retrorsine, riddelliine, monocrotaline, and heliotrine) and their corresponding PA N-oxides produced the same set of DNA adducts. Among these compounds, lasiocarpine N-oxide, retrorsine N-oxide, monocrotaline N-oxide, and heliotrine N-oxide are for first time shown to be able to produce these DNA adducts. These results further support the role of these DNA adducts as potential common biomarkers of PA-induced liver tumor initiation. Copyright © 2017. Published by Elsevier B.V.

Pyrrolizidine alkaloid-derived DNA adducts are common toxicological biomarkers of pyrrolizidine alkaloid N-oxides

Directory of Open Access Journals (Sweden)

Xiaobo He

2017-10-01

Full Text Available There are 660 pyrrolizidine alkaloids (PAs and PA N-oxides present in the plants, with approximately half being possible carcinogens. We previously reported that a set of four PA-derived DNA adducts is formed in the liver of rats administered a series of hepatocarcinogenic PAs and a PA N-oxide. Based on our findings, we hypothesized that this set of DNA adducts is a common biological biomarker of PA-induced liver tumor formation. In this study, we determined that rat liver microsomal metabolism of five hepatocarcinogenic PAs (lasiocarpine, retrorsine, riddelliine, monocrotaline, and heliotrine and their corresponding PA N-oxides produced the same set of DNA adducts. Among these compounds, lasiocarpine N-oxide, retrorsine N-oxide, monocrotaline N-oxide, and heliotrine N-oxide are for first time shown to be able to produce these DNA adducts. These results further support the role of these DNA adducts as potential common biomarkers of PA-induced liver tumor initiation.

High-affinity monoclonal antibodies specific for deoxynucleosides structurally modified by alkylating agents: Applications for immunoanalysis

International Nuclear Information System (INIS)

Adamkiewicz, J.; Ahrens, O.; Rajewsky, M.F.

1984-01-01

So far the results of attempts to use monoclonal antibodies for the demonstration of carcinogen-DNA adducts in cells by immunostaining have been promising. Thus the authors have established a standardized procedure for the quantitation of specific alkyl-deoxynucleosides in the nuclear DNA of individual cells by direct immunofluorescence, using tetramethylrhodamine isothiocyanate-labeled monoclonal antibodies and a computer-based image analysis of electronically intensified fluorescence signals. With a fluorescent anti-(O/sup 6/-EtdGuo) monoclonal antibody, the present detection limit for O/sup 6/-Etd-Guo in the nuclei of individual cells previously exposed to an ethylating N-nitroso compound (e.g., N-ethyl-N-nitrosourea) is -- 700 O/sup 6/-EtdGuo molecules per diploid genome, i.e., similar to the detection limit for the same ethylation product in a hydrolysate of (O/sup 6/-EtdGuo)-containing DNA analyzed by competitive RIA

Current study on ionizing radiation-induced mitochondial DNA damage and mutations

International Nuclear Information System (INIS)

Zhou Xin; Wang Zhenhua; Zhang Hong

2012-01-01

Current advance in ionizing radiation-induced mitochondrial DNA damage and mutations is reviewed, in addition with the essential differences between mtDNA and nDNA damage and mutations. To extent the knowledge about radiation induced mitochondrial alterations, the researchers in Institute of Modern Physics, Chinese Academy of Sciences developed some technics such as real-time PCR, long-PCR for accurate quantification of radiation induced damage and mutations, and in-depth investigation about the functional changes of mitochondria based on mtDNA damage and mutations were also carried out. In conclusion, the important role of mitochondrial study in radiation biology is underlined, and further study on mitochondrial study associated with late effect and metabolism changes in radiation biology is pointed out. (authors)

4-Alkyl radical extrusion in the cytochrome P-450-catalyzed oxidation of 4-alkyl-1,4-dihydropyridines

International Nuclear Information System (INIS)

Lee, J.S.; Jacobsen, N.E.; Ortiz de Montellano, P.R.

1988-01-01

Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4,-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine matabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when [4- 2 H]DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl[4- 2 H]DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms

Temozolomide-Induced Myelodysplasia

Directory of Open Access Journals (Sweden)

Ethan A. Natelson

2010-01-01

Full Text Available A patient who had received temozolomide (TMZ as a single agent in treatment of malignant glioma developed therapy-induced myelodysplasia (T-MDS. TMZ is an orally active imidazotetrazine which methylates guanine residues in DNA, ultimately causing single and double-strand DNA breaks leading to apoptotic cell death. TMZ does not chemically cross-link DNA and is considered a nonclassical alkylating agent, similar in structure and activity to dacarbazine. Observations on this patient, and on similarly treated others, suggest that the cumulative dose threshold (CDT for TMZ that predisposes to T-MDS and which may potentially lead to acute myeloid leukemia (T-AML is around 18000 to 20000 mg/sq m. Although the incidence of T-MDS and the predisposing CDT of TMZ may differ from that of other potentially leukemogenic compounds currently and formerly used as chemotherapeutic agents, all alkylating agents, including TMZ, should be considered potentially leukemogenic when administered long term.

Photoinduced, copper-catalyzed alkylation of amides with unactivated secondary alkyl halides at room temperature.

Science.gov (United States)

Do, Hien-Quang; Bachman, Shoshana; Bissember, Alex C; Peters, Jonas C; Fu, Gregory C

2014-02-05

The development of a mild and general method for the alkylation of amides with relatively unreactive alkyl halides (i.e., poor substrates for SN2 reactions) is an ongoing challenge in organic synthesis. We describe herein a versatile transition-metal-catalyzed approach: in particular, a photoinduced, copper-catalyzed monoalkylation of primary amides. A broad array of alkyl and aryl amides (as well as a lactam and a 2-oxazolidinone) couple with unactivated secondary (and hindered primary) alkyl bromides and iodides using a single set of comparatively simple and mild conditions: inexpensive CuI as the catalyst, no separate added ligand, and C-N bond formation at room temperature. The method is compatible with a variety of functional groups, such as an olefin, a carbamate, a thiophene, and a pyridine, and it has been applied to the synthesis of an opioid receptor antagonist. A range of mechanistic observations, including reactivity and stereochemical studies, are consistent with a coupling pathway that includes photoexcitation of a copper-amidate complex, followed by electron transfer to form an alkyl radical.

Some features of formation and dissolution of a series of Pu(IV) and Zr alkyl and butyl alkyl phosphates in the system TBP -n-dodecane - nitric acid - water

International Nuclear Information System (INIS)

Markov, G.S.; Moshkov, M.M.; Kokina, S.A.

1990-01-01

The formation and composition of salts produced on interaction of a series of alkyl- and butylalkylphosphoric acids having alkyl radical chain lengths from C 4 to C 1 0 with Pu(IV) and Zr in organic and aqueous phases of the system TBP - n-dodecane -nitric acid - water were studied. The composition of compounds was found to depend on the conditions of their formation, defined first of all by the HNO 3 concentration in aqueous and organic phases. (author) 12 refs.; 4 figs.; 1 tab

Smiles Rearrangement Based Practical One-pot Synthesis of N-Alkyl/aryl-6-aminoquinolines from 6-Hydroxylquinoline

Energy Technology Data Exchange (ETDEWEB)

Xie, Yongsheng; Vijaykumar, B. V. D.; Jang, Kiwan; Choi, Kyungmin; Shin, Dongsoo [Changwon National Univ., Changwon (Korea, Republic of); Zuo, Hua [Southwest Univ., Chongqing (Korea, Republic of); Yoon, Yongjin [Gyeongsang National Univ., Chinju (Korea, Republic of)

2013-12-15

The C-N coupling protocol reported herein represents a convenient and practical synthesis of N-alkyl/aryl-6-aminoquinolines in a three-step one-pot manner by simple addition of 6-hydroxyquinoline and N-alkyl/aryl-2-chloroacetamides with Cs{sub 2}CO{sub 3} or K{sub 2}CO{sub 3} in DMF at 150 .deg. C via Smiles rearrangement. An electron donating substituent on the nitrogen counterpart would accelerate the rearrangement process to achieve various anilines in good yields. We currently engaged in making a chemical library including multifarious N-substituted-6-aminoquinolines, to be used in the screening for specific AChEI activity. Furthermore, this work extends the scope of preparing different heterocyclic synthons in drug design for various biological activities. Aminoquinolines and their derivatives are important chemical entities that are widely used as pro-drugs and drugs due to their antimicrobial, cytotoxic and anti-malarial activities etc.

Effect of radiation and alkylating agents on chromatin degradation in normal and malignant lymphoid cells