RESUMO
The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low µM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.
Assuntos
Clorambucila/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Quadruplex G/efeitos dos fármacos , Aminoquinolinas/metabolismo , Linhagem Celular , Adutos de DNA/química , Adutos de DNA/metabolismo , Humanos , Ligantes , Ácidos Picolínicos/metabolismoRESUMO
Synthetic lethality is a genetic concept in which cell death is induced by the combination of mutations in two sensitive genes, while mutation of either gene alone is not sufficient to affect cell survival. Synthetic lethality can also be achieved "chemically" by combination of drug-like molecules targeting distinct but cooperative pathways. Previously, we reported that the small molecule pyridostatin (PDS) stabilizes G-quadruplexes (G4s) in cells and elicits a DNA damage response by causing the formation of DNA double strand breaks (DSB). Cell death mediated by ligand-induced G4 stabilization can be potentiated in cells deficient in DNA damage repair genes. Here, we demonstrate that PDS acts synergistically both with NU7441, an inhibitor of the DNA-PK kinase crucial for nonhomologous end joining repair of DNA DSBs, and BRCA2-deficient cells that are genetically impaired in homologous recombination-mediated DSB repair. G4 targeting ligands have potential as cancer therapeutic agents, acting synergistically with inhibition or mutation of the DNA damage repair machinery.
Assuntos
DNA de Neoplasias/genética , Quadruplex G , Neoplasias/genética , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromonas/química , Cromonas/farmacologia , Quebras de DNA , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Morfolinas/química , Morfolinas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacologia , Relação Estrutura-AtividadeRESUMO
There is considerable interest in the structure and function of G-quadruplex nucleic acid secondary structures, their cellular functions, and their potential as therapeutic targets. G-Quadruplex sequence motifs are prevalent in gene promoter regions and it has been hypothesized that G-quadruplex structure formation is associated with the transcriptional status of the downstream gene. Using a functional cell-based assay, we have identified two novel G-quadruplex ligands that reduce the transcription of a luciferase reporter driven from the G-quadruplex-containing c-KIT promoter. We have further shown that endogenous c-KIT expression in a human gastric carcinoma cell line is also reduced on treatment with these molecules. Biophysical analysis using surface plasmon resonance has shown that these molecules preferentially bind with high affinity to one of the two G-quadruplex sequences in the c-KIT promoter over double-stranded DNA. This work highlights the utility of cell-based reporter assays to identify new G-quadruplex binding molecules that modulate transcription and identifies benzo[a]phenoxazine derivatives as potential antitumor agents.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Quadruplex G , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Oxazinas/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Sítios de Ligação , Humanos , Estrutura Molecular , Oxazinas/química , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
Tamoxifen elevates the risk of endometrial tumours in women and alpha-(N(2)-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with alpha-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N(2)-tamoxifen and dG-N(2)-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both alpha-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for alpha-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only alpha-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC-->TA transversions. Application of the LwPy53 algorithm to the alpha-acetoxytamoxifen spectrum predicted strong GC-->TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N(2)-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.
Assuntos
Adutos de DNA/análise , Endométrio/efeitos dos fármacos , Antagonistas de Estrogênios/toxicidade , Genes p53 , Mutagênese , Tamoxifeno/análogos & derivados , Algoritmos , Animais , Animais Geneticamente Modificados , Linhagem Celular , Análise Mutacional de DNA , Endométrio/química , Endométrio/citologia , Feminino , Genes Supressores , Humanos , RNA de Transferência/genética , Ratos , Tamoxifeno/análise , Tamoxifeno/toxicidadeRESUMO
Ethylene oxide (EO) is a widely used chemical intermediate also formed endogenously from ethylene metabolism. Despite conflicting epidemiological evidence, EO is classified by the IARC as a human carcinogen. The mutagenicity and carcinogenicity of EO is attributed to direct reaction with DNA and formation of multiple 2-hydroxyethyl (HE) DNA adducts. However, the actual lesions responsible for the reported mutagenicity of EO have not been established. This study used the supF mutation assay to investigate the biological relevance of low levels of EO-induced DNA adducts in human Ad293 cells, with respect to the type and level of each HE adduct present. Initial experiments were conducted using pSP189 plasmid containing up to 290 N7-HEGuanine (N7-HEG) adducts/10(6) nucleotides, which far exceeds that typically detected in human DNA. No other HE-lesions were detectable using our validated LC-MS/MS assay. Replication in cells failed to produce a statistically significant increase in relative mutation frequency, above background rates in the solvent control. Furthermore, the mutation spectrum compiled for EO-treated plasmid (10-2000muM) did not differ significantly from the spontaneous distribution, suggesting EO is not strongly mutagenic in this system. Under refined reaction conditions using higher EO concentrations capable of inducing detectable levels of N1-HEdA, O(6)-HEdG and N3-HEdU along with N7-HEG, there was a significant dose-related increase in relative mutation frequency above background (3.76- and 5.30-fold at 10 and 30mM, respectively). EO treatment appeared associated with an elevated frequency of GC-->CG mutations and the occurrence of substitutions at AT base pairs. Additionally, there was a distinct GC-->TA mutational hotspot in the 10mM EO spectrum. Overall, the results suggest a certain level of promutagenic adducts must be attained before mutations become detectable above background, indicating that N7-HEG is not a promutagenic lesion, and support a role for the minor products of DNA hydroxyethylation in the generation of base substitutions by EO.
Assuntos
Adutos de DNA/metabolismo , Óxido de Etileno/toxicidade , Mutagênicos/toxicidade , Sequência de Bases , Linhagem Celular , Dano ao DNA , Relação Dose-Resposta a Droga , Genes Supressores , Vetores Genéticos , Humanos , Dados de Sequência Molecular , RNA de Transferência/químicaRESUMO
We have developed and validated a novel site-specific mutagenesis assay, termed SSMA-MS, which incorporates MALDI-ToF mass spectrometry (MALDI-MS) analysis as a means of determining the mutations induced by a single DNA adduct. The assay involves ligating an adducted deoxyoligonucleotide into supF containing pSP189 plasmid. The plasmid is transfected into human Ad293 kidney cells allowing replication and therefore repair or a mutagenic event to occur. Escherichia coli indicator bacteria are transformed with recovered plasmid and plasmids containing the insert are identified colormetrically, as they behave as frameshift mutations. The plasmid is then amplified and digested using a restriction cocktail of Mbo11 and Mnl1 to yield 12 bp deoxyoligonucleotides, which are characterized by MALDI-MS. MALDI-MS takes advantage of the difference in molecular weight between bases to identify any induced mutations. This analysis method therefore provides qualitative and quantitative information regarding the type and frequency of mutations induced. This assay was developed and validated using an O(6)-methyl-2'-deoxyguanosine adduct, which induced the expected GC-->AT substitutions, when replicated in human or bacterial cells. This approach can be applied to the study of any DNA adduct in any biologically relevant gene sequence (e.g. p53) in human cells and would be particularly amenable to high-throughput analysis.
Assuntos
Mutagênese Sítio-Dirigida/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linhagem Celular , Adutos de DNA/análise , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Escherichia coli/genética , Vetores Genéticos , Humanos , Oligodesoxirribonucleotídeos/análise , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/normas , Plasmídeos/genética , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , TransfecçãoRESUMO
Our interest in benzene-DNA adduct formation and their consequence has led us to develop a number of sensitive methods for their analysis. A HPLC method for the analysis of 32P-postlabelled benzene-DNA adducts was developed and used to detect adducts formed from the reaction of DNA or individual deoxynucleotides with the metabolites para-benzoquinone (p-BQ) and hydroquinone (HQ). Reaction of DNA with BQ yielded four adducts, the major product being a deoxycytidine adduct. HQ formed a single detectable deoxyguanosine DNA adduct, which was a minor product of the reaction of DNA with p-BQ. The supF forward mutation assay was used to assess the mutagenicity of p-BQ and HQ after transfection of treated plasmid in the human kidney cell line, Ad293. Single base substitution mutations at GC base pairs (bp) predominated for each treatment. However, when the mutation spectra achieved for each treatment were compared they were shown to be significantly different (p=0.004). These results may suggest either a possible role for the minor benzene-deoxyguanosine adducts in benzene genotoxicity or that HQ is causing DNA modification via a different mechanism, such as oxidative damage.
Assuntos
Benzoquinonas/toxicidade , Adutos de DNA , Hidroquinonas/toxicidade , Benzeno/metabolismo , Benzoquinonas/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Genes Supressores , Humanos , Hidroquinonas/metabolismo , Testes de Mutagenicidade , Radioisótopos de Fósforo , RNA de Transferência/genéticaRESUMO
Benzene, a ubiquitous environmental pollutant and occupational hazardous chemical, is a recognised human leukaemogen and rodent carcinogen. The mechanism by which benzene exerts its carcinogenic effects is to date unknown but it is considered that mutations induced by benzene-DNA adducts may play a role. The benzene metabolite, para-benzoquinone (p-BQ) following reaction in vitro with DNA, forms four major adducts, which include two adducts on 2'-deoxyguanosine 3'-monophosphate (dGp). Reaction of DNA with the benzene metabolite hydroquinone (HQ) results in only one major DNA adduct, which corresponds to one of the dGp adducts formed following reaction with p-BQ. The mutagenicity of the adducts formed from these two benzene metabolites was investigated using the supF forward mutation assay. Metabolite-treated plasmid (pSP189) containing the supF gene was replicated in human Ad293 cells before being screened in indicator bacteria. Treatment with 5-20 mM p-BQ gave a 12 to 40-fold increase in mutation rate compared to 5-20 mM HQ treatment, a result reflected in the level of DNA modification observed (8 to 26-fold increase compared to HQ treatment). Treatment with p-BQ gave equal numbers of GC --> TA transversions and GC --> AT transitions, whereas treatment with HQ gave predominantly GC-->AT transitions. The spectra of mutations achieved for the two individual treatments were shown to be significantly different (P = 0.004). A combination of both treatments also resulted in a high level of GC --> AT transitions and a synergistic increase in the number of multiple mutations, which again predominated as GC --> AT transitions. Sites of mutational hotspots were observed for both individual treatments and one mutational hotspot was observed in the multiple mutations for the combined treatment. These results suggest that the dGp adducts formed from benzene metabolite treatment may play an important role in the mutagenicity and myelotoxicity of benzene.
Assuntos
Benzeno/toxicidade , Benzoquinonas/toxicidade , Adutos de DNA , Hidroquinonas/toxicidade , RNA de Transferência/genética , Sequência de Bases , Primers do DNA , Genes Supressores , Humanos , Dados de Sequência Molecular , Testes de MutagenicidadeRESUMO
We report here on the screening of a fragment library against a G-quadruplex element in the human c-MYC promoter. The ten fragment hits had significant concordance between a biophysical assay, in silico modelling and c-MYC expression inhibition, highlighting the feasibility of applying a fragment-based approach to the targeting of a quadruplex nucleic acid.
Assuntos
Quadruplex G , Proteínas Proto-Oncogênicas c-myc/genética , Simulação por Computador , Humanos , Modelos Moleculares , Regiões Promotoras Genéticas/genéticaRESUMO
The human population is continually exposed to benzene due to its presence in complex environmental mixtures and exposure has been linked to a range of haematotoxic effects, including an increased risk of leukaemia. Several hypotheses have been postulated on how benzene exerts its toxic and carcinogenic effects, one idea being that following metabolism to more reactive species it can react with DNA to form adducts which subsequently give rise to mutations. Previously, we have demonstrated the formation of four major DNA adducts from the reaction of DNA with the benzene metabolites hydroquinone (HQ) and p-benzoquinone (p-BQ) and the mutagenicity of these adducts when analysed using the supF forward mutation assay after replication in a human kidney cell line. This study demonstrates a potential role in the carcinogenicity of benzene for the DNA adducts formed on 2'-deoxyguanosine 3'-monophosphate. As a continuation of this work, benzene metabolite-treated plasmid pSP189 containing the supF reporter gene was transfected into human nucleotide excision repair (NER)-proficient and NER-deficient (xeroderma pigmentosum, complementation group A) fibroblast cells to determine the method of adduct repair. For all metabolite treatments in both cell lines the majority of mutations were single base substitutions occurring at GC base pairs, predominantly GC-->TA transversions and GC-->AT transitions. Comparison of mutation frequencies showed a similarity for the HQ treatment for the two cell lines, whereas for the treatments involving p-BQ, an overall higher mutation frequency was observed in the NER-deficient cells compared with the NER-proficient cells. Mutation spectra were significantly different following treatment with HQ in the two cell lines (P = 0.0004). No difference was observed for the control, p-BQ or the combined treatment. The results suggest the involvement of a different repair mechanism for HQ-induced DNA damage and further highlights the potential different roles for the two benzene metabolites in benzene mutagenicity.
Assuntos
Benzoquinonas/toxicidade , Dano ao DNA , DNA/efeitos dos fármacos , Hidroquinonas/toxicidade , Mutagênicos/toxicidade , Sequência de Bases , Linhagem Celular Transformada , Genes Supressores , Humanos , Dados de Sequência Molecular , Mutação , RNA de Transferência/genéticaRESUMO
Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.
Assuntos
Adutos de DNA/toxicidade , Reparo do DNA/genética , Predisposição Genética para Doença/genética , Mutagênese , Tamoxifeno/análogos & derivados , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologia , Sequência de Bases , Linhagem Celular Transformada , Análise Mutacional de DNA/métodos , Genes Supressores , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Radioisótopos de Fósforo/metabolismo , Plasmídeos , RNA de Transferência/genética , Tamoxifeno/toxicidade , TransfecçãoRESUMO
The drug tamoxifen, used to treat breast cancer, causes liver cancer in rats and endometrial cancer in women. Tamoxifen forms liver DNA adducts in both short- and long-term dosing of rodents, and DNA adducts have also been reported in tissues of women undergoing tamoxifen therapy. It is not known if the induction of endometrial cancer in women is through these DNA adducts or through the estrogenic nature of the drug. In this study, we have investigated the mutagenicity of two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM). These form the same DNA adducts as those found in tamoxifen-treated rats. The two compounds were used to treat the pSP189 plasmid containing the supF gene, which was replicated in Ad293 cells before being screened in indicator bacteria. Plasmid reacted with 4-OHtamQM was more likely to be mutated (2-7-fold increase) than that reacted with alpha-acetoxytamoxifen, despite having a lower level of DNA damage (12-20-fold less), as assayed by (32)P-postlabeling. The two compounds induced statistically different mutation spectra in the supF gene. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC -->TA transversions while GC-->AT transitions were formed in 4-OHtamQM-treated plasmid. 4-OHTamQM-treated DNA induced a larger proportion of multiple mutations and large deletions compared to alpha-acetoxytamoxifen. Sites of mutational hotspots were observed for both compounds. In conclusion, the quantitatively minor DNA adduct of tamoxifen (dG-N(2)-4-hydroxytamoxifen) is more mutagenic than the major tamoxifen DNA adduct (dG-N(2)-tamoxifen).
Assuntos
Adutos de DNA , Tamoxifeno , Tamoxifeno/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Carcinógenos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Genes Supressores , Vetores Genéticos , Humanos , Isomerismo , Dados de Sequência Molecular , Plasmídeos , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Tamoxifeno/farmacologiaRESUMO
We have shown previously that UVC irradiation of benzo[a]pyrene diol epoxide (BPDE)-adducted DNA (BPDE/UVC) induces an increase in mutation frequency in the supF gene greater than the calculated additive value derived from either treatment alone, with a greater absolute increase in the level of BPDE signature transversions. Possible explanations were that (i) the BPDE adducts are photoactivated to a more mutagenic lesion or (ii) the presence of UV-induced DNA damage enhanced the mutagenicity of BPDE adducts elsewhere on the DNA. In the present study, to determine which of these mechanisms is responsible for the enhanced mutagenicity of the combined treatment, plasmid pSP189 containing supF was treated with UVC radiation before BPDE treatment (UVC/BPDE). If BPDE adducts were being modified by UV irradiation to more mutagenic species, then reversing the order of exposure would be predicted to lower the mutation frequency and the number of transversions. Conversely, if merely the presence of UV damage influences the mutagenicity of BPDE adducts (or vice versa), the observed mutagenicity should be independent of the order of exposure. Previously, treatment with BPDE/UVC increased the mutation frequency by >400% over the calculated additive value derived from the individual BPDE and UV exposures. In the present study, treatment with UVC followed by BPDE increased the mutation frequency by only approximately 60%, compared with the corresponding calculated additive value. However, whilst this shows that the order of treatment affects the mutation frequency, there was little change in the percentage of base substitutions in the two spectra. Hence, whilst the change in mutation frequency is consistent with UVC directly enhancing the mutagenicity of the BPDE adducts, the similarity in the types of mutations induced by BPDE/UVC and UVC/BPDE suggests that the mechanism may not be that simple.