Identification of mutagenic site of c-H-ras oncogene damaged by N-acetoxyacetylaminofluorene(AAAF).
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A molecularly cloned human cellular H-ras (c-H-ras) oncogene(pbc N1 plasmid) was treated with N-acetoxyacetylaminofluorene (AAAF) in vitro and subcloned into E.coli. This was done to identify the mutational changes at specific codons of the gene. Guanine nucleotides were identified as the major AAAF binding site of the DNA adduct formed. Base changes in codons 12 and 61 were determined by the analysis of restriction fragment length polymorphism (RFLP) and site specific oligonucleotide hybridization. RFLP was observed due to the loss of the Hpall recognition site at codon 11 and 12 of AAAF-treated c-H-ras gene. Hybridization of AAAF treated c-H-ras with 32P-labeled oligonucleotide probes for the mutant alleles of codon 61 showed no substitutions at codon 61. From these results, it is assumed that AAAF treatment in vitro caused mutation at codon 12 but not at codon 61 of the c-H-ras oncogene and that codon 12 is the primary target of mutation by AAAF. (+info)
Mitochondrial transcription factor A is the major protein in rodent hepatocytes that recognizes DNA lesions induced by N-acetoxy-acetylaminofluorene.
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Extracts from rodent liver cells contain an abundant protein that recognizes DNA adducts induced by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF). This protein also has a strong affinity for DNA damaged by cisplatin (DDP), but not by benzo(a)pyrene diolepoxide or UV-radiation, and has been termed AAAF/DDP-DDB. Here we purified this protein from rat tissue and analyzed it by mass spectrometry and identified it as mitochondrial transcription factor A (TFAM). Experiments with bacterially expressed recombinant TFAM confirmed its high affinity for DNA damaged by AAAF. Assuming its abundance and specificity for AAAF induced lesions, TFAM may significantly impede recognition and repair of DNA adducts induced by AAAF and other derivatives of 2-aminofluorene. (+info)
GenoMass--a computer software for automated identification of oligonucleotide DNA adducts from LC-MS analysis of DNA digests.
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Clinical symptoms and DNA repair characteristics of xeroderma pigmentosum patients from Germany.
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Sixty-one xeroderma pigmentosum (XP) patients living in the Federal Republic of Germany were investigated. Clinical symptoms were correlated with DNA repair parameters measured in fibroblasts grown from skin biopsies. Classification according to the international complementation groups revealed that of the 61 patients 3 belonged to group A, 26 to group C, 16 to group D, 3 to group E, and 2 to group F; 11 were of the XP variant type. A striking clinical aspect was the frequency of histogenetically different skin tumors varying from one XP complementation group to the other: squamous and basal cell carcinomas predominated in XP group C; lentigo maligna melanomas were most frequent in group D; basal cell carcinomas occurred preferentially in group E and XP variants. Three DNA repair parameters were determined for 46 fibroblast strains: colony-forming ability (D0); DNA repair synthesis (G0); and DNA-incising capacity (E0). Dose-response experiments with up to 13 dose levels were performed throughout to achieve sufficient experimental accuracy. DNA-damaging treatments included UV light, the "UV-like" carcinogen N-acetoxy-2-acetylaminofluorene, and the alkylating carcinogens methyl methanesulfonate and N-methyl-N-nitrosourea. Comparison of clinical signs and repair data was made on the basis of D0, G0, and E0 values of both individual cell strains and weighted means of XP complementation groups. Despite considerable clinical and biochemical heterogeneity within complementation groups distinctive features emerged. In general, D0, G0, and E0 values of all XP strains investigated, including XP variants, were found to be reduced upon treatment with UV light or N-acetoxy-2-acetylaminofluorene. After treatment with UV light or N-acetoxy-2-acetylaminofluorene, cell strains in which DNA-incising capacity was reduced also showed a similar reduction in both colony-forming ability and DNA repair synthesis. Consequently, the weighted mean D0, G0, and E0 values of XP complementation groups and XP variants correlated with each other. Furthermore, the onset of both early dermatological symptoms of XP and tumor growth correlated with the extent of DNA repair defects. Of 45 XP fibroblast strains checked for colony-forming ability after treatment with methyl methanesulfonate only 3 cell strains from group D were found to be more sensitive than normal controls, suggesting that overall repair in XP strains was equal to that in controls. Weighted means of DNA repair synthesis of XP complementation groups, however, showed reductions hinting at impaired excision of distinct alkylated bases.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Mutagenesis of Chinese hamster cells in vitro by combination treatments with methyl methanesulfonate and N-acetoxy-2-acetylaminofluorene.
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Mutational synergism was examined in Chinese hamster V79 cells exposed to methyl methanesulfonate followed by N-acetoxy-2-acetylaminofluorene (AcAAF) at different time intervals. Treatment with 500 micron methyl methanesulfonate resulted in 95% survival of cloning ability and induced approximately 4 azaguanine-resistant mutants/10(5) survivors. Seven micron AcAAF produced 10 times as many mutants, and the survival was 7%. Lethal synergism was observed for methyl methanesulfonate treatments followed by 7 micron AcAAF, and the resulting lethality was unaffected by increasing the time interval between treatments from 1 to 48 hr. However, no significant changes in the mutant frequency from that induced by AcAAF alone were found for treatment intervals of 1 to 63 hr. This result contrasts with the 6-fold enhancement of the AcAAF-induced transformation of Syrian hamster embryo cells exposed to the same combination with a 48-hr interval between treatments, as previously reported (Chem.-Biol. Interactions, 9: 351-364, 1974). The difference in the response of these two cell types demonstrates the difficulties in attempting to extrapolate the known correlation between individual mutagen and carcinogen treatments to combination treatments, with different cell types for the two cellular responses. (+info)
Oxidative stress induces DNA damage and inhibits the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene in human peripheral mononuclear leukocytes.
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Human mononuclear leukocytes were exposed to prooxidants such as H2O2, phorbol-12-myristate-13-acetate, and 4-nitroquinoline-N-oxide, and the effects on induction of DNA damage and repair were evaluated. ADP ribosylation was activated by prooxidant exposure and the response was bimodal with peaks of activation occurring at about 30 min and 4-5 h. Other evidence for prooxidant-induced DNA damage was provided by nucleoid sedimentation assays. Unscheduled DNA synthesis (UDS) was only slightly induced by prooxidant exposure which suggested that either the DNA lesions were repaired by a short patch mechanism involving little UDS, or the repair process was inhibited by prooxidant exposures, or some combination of both. This point was clarified by the fact that the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene, an inducer of large patch DNA repair, was inhibited in a dose-dependent manner by exposure to H2O2 and the inhibition was dependent on ADP ribosylation. In contrast, the repair of DNA strand breaks induced by prooxidant exposures as identified above were complete within about 8 h and the repair was independent of ADP ribosylation. Both ADP ribosylation and N-acetoxy-2-acetylaminofluorene-induced UDS were shown to be up- and down-regulated by the redox state of human mononuclear leukocytes indicating a unique mechanism of cellular control over DNA repair. (+info)
The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.
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