Molecular dosimetry for sister-chromatid exchange induction and cytotoxicity by monofunctional and bifunctional alkylating agents

The induction of sister-chromatid exchanges (SCEs) and cytotoxicity in 9L cells treated with monofunctional and bifunctional alkylating agents has been investigated. Three classes of monofunctional and bifunctional agents were studied: nitrosoureas, mustards and epoxides. Independent of class the bifunctional agents were 55–630-fold more effective at inducing SCEs and 300–2400-fold more effective at inducing cellular cytotoxicity than the corresponding monofunctional agents. Comparing the induction of SCEs and cytotoxicity by these agents showed that these two cellular responses to DNA damage are highly correlated. The extent of DNA alkylation in cells treated with 1-ethyl-1-nitrosourea (ENU) or 1-(2-chloro-ethyl)-1-nitrosourea (CNU) was similar indicating that the increased effectiveness of CNU to induce SCEs and cytotoxicity was not due to increased DNA alkylation. Molecular dosimetry calculations indicate that for CNU and ENU treatment of 9L cells there are 116 and 8500 alkylations per SCE induced and 2.6 × 10⁴ and 4.6 × 10⁶ alkylations at the dose required to reduce survival of 9L cells by 90%. Comparison of the DNA alkylation products produced by CNU and ENU treatment of 9L cells suggests that the formation of the intrastrand crosslink N⁷-bis(guanyl)ethane the interstrand crosslink 1-(3-deoxycytidyl)-2-(1-deoxyguanosinyl)ethane by CNU is responsible for the increased effectiveness of CNU treatment at both induction of SCEs and cytotoxicity.     

Differential sensitivities of transformed and untransformed murine cell lines to DNA cross-linking agents relative to repair of O6-methylguanine

Sensitivities of several murine cell strains to killing by the DNA cross-linking agents 1-(2-chloroethyl)-1-nitrosourea (CNU), cis-diamminedichloroplatinum (II) (Cis-Pt) and mitomycin C (MMC) were measured by post-treatment colony-formation. Virally-transformed murine cells were usually more sensitive to cell killing by these agents than were the parental 3T3 cell strains. The hypersensitivity to CNU of some virally-transformed murine cell strains correlated well with the reduced ability to repair O6-methylguanine (O6mGua), a phenomenon similar to that in human cells. The loss of ability to repair O6mGua, as well as the increased sensitivity of transformed strains to cell killing, may not be due to a mutation but rather due to a change of gene expression associated with transformation by viruses or activation of oncogenes.     

Structural basis for the inhibition of human alkyladenine DNA glycosylase (AAG) by 3,N4-ethenocytosine-containing DNA

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.     

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

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 (MGMT or AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity of O(6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the mismatch repair system (MMR) and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks. Therefore, 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 are complex and require complex repair mechanisms. Thus, primary chloroethyl adducts at O(6)-G are repaired by AGT, while the secondary highly cytotoxic interstrand cross-links (ICLs) require nucleotide excision repair factors (e.g. XPF-ERCC1) for incision and homologous recombination to complete repair. Recently, Escherichia coli protein AlkB and human homologues were shown to be oxidative demethylases that repair cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues. Numerous AlkB homologues are found in viruses, bacteria and eukaryotes, including eight human homologues (hABH1-8). These have distinct locations in subcellular compartments and their functions are only starting to become understood. Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological effects of environmental mutagens, as well as understanding the mechanism of action and resistance to alkylating drugs require a detailed understanding of DNA repair processes.     

Regulation of the activity and expression of ERK8 by DNA damage

We have investigated the agonists that activate transfected extracellular signal-regulated kinase 8 (ERK8) in cells, and have found that the most potent activators are hydrogen peroxide, DNA alkylating and cross-linking agents and the poly (ADP-ribose) polymerase inhibitor KU-0058948. The feature shared by all these agents is that they lead to the accumulation of single strand breaks in DNA, suggesting a role for ERK8 in the response to, or repair of, DNA single strand breaks. The DNA alkylating agent MMS also induced the disappearance of endogenous ERK8 by a proteasome-dependent mechanism.     

The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers

The genotoxicity of 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers selected from IARC (International Agency for Research on Cancer) groups 1, 2A, and 2B was evaluated in eight mouse organs with the alkaline single cell gel electrophoresis (SCGE) (comet) assay. Groups of four mice were treated once intraperitoneally at the dose at which micronucleus tests had been conducted, and the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and/or 24 h later. All chemicals were positive in the SCGE assay in at least one organ. Of the 22 mono-functional alkylating agents, over 50% were positive in all organs except the brain and bone marrow. The two subsets of mono-functional alkylating agents differed in their bone marrow genotoxicity: only 1 of the 9 dialkyl N-nitrosoamines was positive in bone marrow as opposed to 8 of the 13 other alkylating agents, reflecting the fact that dialkyl N-nitrosoamines are poor micronucleus inducers in hematopoietic cells. The two groups of mono-functional alkylating agents also differ in hepatic carcinogenicity in spite of the fact that they are similar in hepatic genotoxicity. While dialkyl N-nitrosoamines produce tumors primarily in mouse liver, only one (styrene-7,8-oxide) out of 10 of the other type of mono-functional alkylating agents is a mouse hepatic carcinogen. Taking into consideration our previous results showing high concordance between hepatic genotoxicity and carcinogenicity for aromatic amines and azo compounds, a possible explanation for the discrepancy might be that chemicals that require metabolic activation show high concordance between genotoxicity and carcinogenicity in the liver. A high percent of the 10 DNA crosslinkers were positive in the SCGE assay in the gastrointestinal mucosa, but less than 50% were positive in the liver and lung. In this study, we allowed 10 min alkali-unwinding to obtain low and stable control values. Considering that DNA crosslinking lesions can be detected as lowering of not only positive but also negative control values, low control values by short alkali-treatment might make it difficult to detect DNA crosslinking lesions. In conclusion, although both mono-functional alkylating agents and DNA crosslinkers are genotoxic in mouse multiple organs, the genotoxicity of DNA crosslinkers can be detected in the gastrointestinal organs even though they were given intraperitoneally followed by the short alkali-treatment.     

Cross-linking of 2-deoxyribonolactone and its beta-elimination product by base excision repair enzymes

2-Deoxyribonolactone (3) is produced in DNA as a result of reaction with a variety of DNA damaging agents. The lesion undergoes beta-elimination to form a second metastable electrophilic product (4). In this study, DNA containing 2-deoxyribonolactone (3) and its beta-elimination product (4) are generated at specific sites using a photolabile nucleotide precursor. 2-Deoxyribonolactone is not incised by any of the 8 AP lyases tested. One enzyme, Escherichia coli endonuclease III, cross-links to 3, and the lesion strongly inhibits excision of typical abasic sites by this enzyme. Two of the enzymes, FPG and NEIL1 known to cleave normal abasic sites (1) by effecting beta,delta-elimination form cross-links to the butenolide lesion (4). The observed results are ascribable to characteristics of the enzymes and the lesions. These enzymes are also important for the removal of oxidative base lesions. These results suggest that high concentrations of 3 and 4 may exert significant effects on the repair of normal AP site and oxidative base lesions in cells by reducing the cellular activity of these BER enzymes either via cross-linking or competing with binding to the BER enzymes.     

Effect of oxidative stress on homer scaffolding proteins

Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.     

DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase. Effects of protein and DNA alkylation on complex stability

The mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O(6)-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we report properties of the interaction between AGT and short DNA oligonucleotides. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This strong cooperative interaction is only slightly perturbed by active site mutation of AGT or by alkylation of either AGT or DNA. The stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged by several active site mutations or by protein or DNA alkylation. These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair.     

Adaptive resistance of Saccharomyces cerevisiae to chronic treatment with mutagens being due to a dominant mutation

The exposure of mammalian cells or tumors for weeks or months to low non-lethal doses of cytostatic drugs may induce multi-drug resistance, which can be enhanced by a variety of DNA-damaging agents. In yeast multi-drug resistance to a variety of drugs has been observed. DNA-damaging agents have not yet been tested. As the appearance of resistance is the result of long-term exposure, we decided to extend the application of test substances to a period of up to 400 days. In such long-term experiments S. cerevisiae MP1 adapted to treatment with low doses of mutagens. Consistent results were obtained for both alkylating and non-alkylating mutagenic substances. Furthermore, the adaptive resistance to the alkylating agent also adapted cells to the non-alkylating agent, which implies that there may be a single pathway for mutagens with different modes of action. Random spore analysis of adapted yeast cells and the back-cross to the parental wild type indicates that a single dominant mutation is responsible for the adaptive resistance.     

Involvement of Escherichia coli DNA polymerase IV in tolerance of cytotoxic alkylating DNA lesions in vivo

Escherichia coli PolIV, a DNA polymerase capable of catalyzing synthesis past replication-blocking DNA lesions, belongs to the most ubiquitous branch of Y-family DNA polymerases. The goal of this study is to identify spontaneous DNA damage that is bypassed specifically and accurately by PolIV in vivo. We increased the amount of spontaneous DNA lesions using mutants deficient for different DNA repair pathways and measured mutation frequency in PolIV-proficient and -deficient backgrounds. We found that PolIV performs an error-free bypass of DNA damage that accumulates in the alkA tag genetic background. This result indicates that PolIV is involved in the error-free bypass of cytotoxic alkylating DNA lesions. When the amount of cytotoxic alkylating DNA lesions is increased by the treatment with chemical alkylating agents, PolIV is required for survival in an alkA tag-proficient genetic background as well. Our study, together with the reported involvement of the mammalian PolIV homolog, Polkappa, in similar activity, indicates that Y-family DNA polymerases from the DinB branch can be added to the list of evolutionarily conserved molecular mechanisms that counteract cytotoxic effects of DNA alkylation. This activity is of major biological relevance because alkylating agents are continuously produced endogenously in all living cells and are also present in the environment.     

Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase.

Escherichia coli expresses two DNA repair methyltransferases (MTases) that repair the mutagenic O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT) DNA lesions; one is the product of the inducible ada gene, and here we confirm that the other is the product of the constitutive ogt gene. We have generated various ogt disruption mutants. Double mutants (ada ogt) do not express any O6MeG/O4MeT DNA MTases, indicating that Ada and Ogt are probably the only two O6MeG/O4MeT DNA MTases in E. coli. ogt mutants were more sensitive to alkylation-induced mutation, and mutants arose linearly with dose, unlike ogt+ cells, which had a threshold dose below which no mutants accumulated; this ogt(+)-dependent threshold was seen in both ada+ and ada strains. ogt mutants were also more sensitive to alkylation-induced killing (in an ada background), and overexpression of the Ogt MTase from a plasmid provided ada, but not ada+, cells with increased resistance to killing by alkylating agents. The induction of the adaptive response was normal in ogt mutants. We infer from these results that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the Ogt MTase also protects cells from killing by alkylating agents. We also found that ada ogt E. coli had a higher rate of spontaneous mutation than wild-type, ada, and ogt cells and that this increased mutation occurred in nondividing cells. We infer that there is an endogenous source of O6MeG or O4MeT DNA damage in E. coli that is prevalent in nondividing cells.     

The contribution of alkylation to the activity of quinone antitumor agents

Studies have shown that the quinone group can produce tumor cell kill by a mechanism involving active oxygen species. This cytotoxic activity can be correlated with the induction of DNA double strand breaks and is enhanced by the ability of the quinone compound to bind to DNA by alkylation. The cytotoxic activity and the production of DNA damage by model quinone antitumor agents were compared in L5178Y cells, sensitive and resistant to alkylating agents, to assess the contribution of alkylation to the activity of these agents. The resistant L5178Y/HN2 cells were found to be two fold and six fold more resistant to the alkylating quinones, benzoquinone mustard and benzoquinone dimustard, respectively, than parent L5178Y cells. In contrast, the L5178Y/HN2 cells showed no resistance to the nonalkylating quinones, hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone. The alkylating quinones produced approximately two fold less cross-linking in L5178Y/HN2 cells compared with L5178Y sensitive cells. DNA double strand break formation by hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone was not significantly different in sensitive and resistant cells. However, the induction of double strand breaks by the alkylating quinones benzoquinone mustard and benzoquinone dimustard was reduced by 5-fold and 15-fold, respectively, in L5178Y/HN2 cells. These results show that the alkylating activity of the alkylating quinones cannot directly explain all of the enhanced cytotoxic activity of these agents. Furthermore, they provide strong evidence that the enhanced formation of DNA double strand breaks by alkylating quinone agents is directly related to the ability of these agents to bind to DNA. This increased formation of strand breaks may account for the enhanced cytotoxic activity of the alkylating quinones.     

Alkyltransferase-like proteins

Recent in silico analysis has revealed the presence of a group of proteins in pro and lower eukaryotes, but not in Man, that show extensive amino acid sequence similarity to known O(6)-alkylguanine-DNA alkyltransferases, but where the cysteine at the putative active site is replaced by another residue, usually tryptophan. Here we review recent work on these proteins, which we designate as alkyltransferase-like (ATL) proteins, and consider their mechanism of action and role in protecting the host organisms against the biological effects of O(6)-alkylating agents, and their evolution. ATL proteins from Escherichia coli (eAtl, transcribed from the ybaz open reading frame) and Schizosaccharomyces pombe (Atl1) are able to bind to a range of O(6)-alkylguanine residues in DNA and to reversibly inhibit the action of the human alkyltransferase (MGMT) upon these substrates. Isolated proteins were not able to remove the methyl group in O(6)-methylguanine-containing DNA or oligonucleotides, neither did they display glycosylase or endonuclease activity. S. pombe does not contain a functional alkyltransferase and atl1 inactivation sensitises this organism to a variety of alkylating agents, suggesting that Atl1 acts by binding to O(6)-alkylguanine lesions and signalling them for processing by other DNA repair pathways. Currently we cannot exclude the possibility that ATL proteins arose through independent mutation of the alkyltransferase gene in different organisms. However, analyses of the proteins from E. coli and S. pombe, are consistent with a common function.     

Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene.

Dual expression vectors derived from pSV2gpt and encoding all or part of the Escherichia coli ada+ gene have been constructed. Following transformation into an E. coli ada strain or transfection and stable integration into the genome of Chinese hamster ovary (CHO) cells, plasmid vectors containing the whole ada+ gene conferred resistance to both killing and mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thus, the bacterial DNA repair gene was functionally expressed in the mammalian cells. Plasmids containing an N-terminal fragment of the ada+ gene which encoded only one of the two methyltransferase activities of the Ada protein did not significantly protect E. coli or CHO cells against MNNG. These results are consistent with the central role of the intact ada+ gene in controlling the adaptive response to alkylating agents in E. coli. However, the data further suggest that some alkylation lesions in DNA, such as O6-methylguanine, may exert partly different biological effects in E. coli and mammalian cells.     

Cloning and study of the function of recA-like gene of Yersinia pestis in Escherichia coli cells

A 2 kb fragment of Yersinia pestis genome cloned in Escherichia coli cells of the strain HB101 contains a gene able to complement the recA-dependent deficiency of E. coli cells in UV-resistance, resistance to alkylating agents, UV- and MNNG-induced mutability. Cellular capability for homologous recombination in crosses with HfrH donor, derepressed synthesis of bacteriocins (colicin E1 and pesticin 1) is also complemented by the fragment in E. coli recA-strains. The obtained data suggest the functional homology of the cloned recA-like gene product with the product of E. coli recA-gene.     

An aza-nucleoside,fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG)

The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG.