Inhibitory effects on the DNA binding of AP-1 transcription factor to an AP-1 binding site modified by benzo[α]pyrene 7,8-dihydrodiol 9,10-epoxide diastereomers (original) (raw)
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Acta biochimica Polonica, 2005
Proteins recognizing DNA damaged by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF) were analyzed in nuclear extracts from rat tissues, using a 36 bp oligonucleotide as a substrate and electrophoretic mobility shift and Southwestern blot assays. One major damage-recognizing protein was detected, whose amount was estimated as at least 10(5) copies per cell. Levels of this protein were similar in extracts from brain, kidney and liver, but much lower in extracts from testis. The affinity of the detected protein for DNA damaged by AAAF was about 70-fold higher than for undamaged DNA. DNA damaged by cis-diamminedichloroplatinum (cis-DDP), benzo(a)pyrene diolepoxide (BPDE) or UV-radiation also bound this protein with an increased affinity, the former more strongly and the latter two more weakly as compared to AAAF-damaged DNA. The detected AAAF/DDP-damaged-DNA-binding (AAAF/DDP-DDB) protein had a molecular mass of about 25 kDa and was distinct from histone H1 or HMGB proteins...
Chemico-Biological Interactions, 2009
Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs), we have studied the phosphorylation of H2AX (H2AX␥). A549 cells were exposed to benzo[a]pyrene diol epoxide [(+)-anti-BPDE] (a bay-region PAH) and dibenzo[a,l]pyrene diol epoxide [(−)-anti-DBPDE] (a fjord-region PAH) and H2AX␥ was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H 2 O 2 ) was used to induce oxidative DNA damage and strand breaks. As showed with single cell gel electrophoresis, neither of the diol epoxides resulted in DNA strand breaks relative to H 2 O 2 . Visualisation of H2AX␥ formation demonstrated that the proportion of cells exhibiting H2AX␥ staining at 1 h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H 2 O 2 treatment, almost all cells demonstrated H2AX␥ at 1 h. Western blot analysis of the H2AX␥ formation also showed concentration and time-dependent response patterns. The kinetics of H2AX␥ formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AX␥ formation and persistence was related to both the number of adducts and their structural features.
Carcinogenesis, 1996
tetrahydrobenzo[a]pyrene (BPDE) is the principal reactive metabolite of the carcinogenic environmental pollutant benzo[a]pyrene. Intensive studies of the distribution of BPDE-induced adduct formation in chromatin DNA compared to that in protein-free DNA have been conducted. However, until recently, investigation of BPDE-induced adduct formation at the nucleotide level in intact mammalian cells has not been feasible. We used ligation-mediated polymerase chain reaction (LMPCR) in conjunction with Escherichia coli UvrABC excinuclease to investigate the distribution of BPDE-induced adducts in the non-transcribed strand of exon 3 of the HPRT gene in normal human fibroblasts at the level of individual nucleotides to single nucleotide resolution using synchronized cell populations. We found that the relative distribution of BPDE adducts in the region of interest was essentially the same in cells treated in early Gl phase, S-phase, late G2/M phase, and in cells blocked at metaphase. Furthermore, for almost all nucleotide positions, the relative distribution of BPDE adducts in the intact cells was very similar to that found when purified DNA was treated with BPDE in vitro. The only exception was that in vivo, adduct formation at a region of six consecutive guanines, i.e. nucleotides 207-212, was strongly enhanced compared with that seen with DNA treated in vitro. No obvious nucleosomal structures or other protein-DNA interaction were detected within the region of interest by in vivo footprinting with micrococcal nuclease and other reagents revealed. In vitro studies mapping BPDE-induced adduct formation using Sequenase and UvrABC excinuclease suggested that this region of six consecutive guanines adopts a special DNA conformation. Therefore, we conclude that rather than reflecting protein-DNA interaction, the enhanced BPDE-induced adduct formation at nucleotides 207-212 in vivo reflects the impact of the physiological environment in the cell nucleus on the local DNA conformation, and that this effect remains constant throughout the cell cycle.
Archives of Biochemistry and Biophysics, 1996
stitutively express a distinct nuclear DNA-binding form of the AhR complex that may result from the pres-2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) supence of an additional protein or a structural variant presses B lymphocyte proliferation and immunoglobuof the AhR. ᭧ 1996 Academic Press, Inc. lin production. We previously reported that the aryl Key Words: dioxin; TCDD; Ah receptor; B lymphohydrocarbon receptor (AhR) complex, composed of the cytes; DNA binding. AhR ligand binding subunit and the Ah receptor nuclear translocator (ARNT), was constitutively present in nuclear extracts from two human B lymphocyte cell lines (Biochem. Biophys. Res. Commun. 212, 27-34, 1995). The present study compared the AhR complex in The prototype halogenated aromatic hydrocarbon the IM-9 and PJS-91 human B lymphocyte and HepG2 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 4 is a perhuman hepatoma cell lines. AhR mRNA levels in the sistent environmental contaminant which produces a two lymphocyte cell lines were substantially lower wide spectrum of adverse effects in laboratory animals, than those in HepG2 cells, as was immunoreactive AhR including tumor promotion, reproductive and developprotein. In contrast, ARNT mRNA and protein were mental toxicity, and immunotoxicity (1). The immunoexpressed at a high level in all three cell lines. TCDD toxicity of TCDD is well documented and includes efinduction of cytochrome P450 1A1 mRNA and protein fects on both cell-mediated and humoral immunity (2, was detected in only the PJS-91 lymphocyte cell line, 3). Decreased antibody responses to T lymphocyte-indeand at a markedly lower level than that in HepG2 cells.
Archives of Biochemistry and Biophysics, 1996
matic hydrocarbons which are widespread environ-The Aryl hydrocarbon receptor (AhR) is known to mental contaminants. The AhR is known to bind to a mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-wide range of planar polycyclic aromatic hydrocarbons. induced toxic effects. Immunocytochemical studies re-Exposure to TCDD results in numerous species-and vealed that AhR in HeLa cells is localized throughout tissue-specific toxic and biological effects, including tuthe cell. Upon TCDD treatment most of the cytoplasmic mor promotion, immunotoxicity, hepatotoxicity, teratoreceptor is translocated into the nucleus in a timegenesis, and enzyme induction (1-4). Induction of sevdependent manner. A significant amount of AhR was eral genes have been reported due to TCDD exposure found to be tightly associated with the nuclear fraction including the most widely studied, cytochrome of untreated HeLa cells. The level of receptor in the P4501A1 and its associated monooxygenase activity nuclear fraction was approximately 16% of the total (5). The AhR, like steroid hormone receptors, is known cellular receptor pool. Further characterization of to be a ligand-dependent transcriptional factor that AhR heterocomplex from the HeLa nuclear fraction modulates gene expression through a high-affinity inby sucrose density gradient analysis revealed that the teraction with responsive elements upstream of ligand AhR was present in the 6 S form, and that the nuclear responsive genes. Unlike steroid hormone receptors, AhR could be coimmunoprecipitated using anti-Arnt both AhR and Arnt represent a novel class of basic mAb. The ability of the AhR to specifically interact region/helix-loop-helix transcriptional factors and with dioxin-responsive elements (DRE) was demonthe activated AhR heterodimerizes with Arnt prior to strated utilizing wild-type and two mutant DREs in gel binding to DNA. Activation or transformation 4 of the shift assays. These results would suggest that, in HeLa AhR is known to occur after cells are exposed to ligand. cells, the AhR-Arnt heterodimer is associated with the Upon ligand binding, the AhR dissociates from the 9 nuclear fraction under normal culture conditions. S complex, followed by heterodimerization with Arnt Therefore, HeLa cells can be used as a model system which probably occurs in the nuclear compartment (6, to study the biochemical and molecular function of the Ah receptor and the process that leads to activation 7). Reversible binding studies with [ 125 I]-2-iodo-7,8-diof the AhR in the absence of exogenous ligand. ᭧ 1996 bromodibenzo-p-dioxin have revealed that Arnt does Academic Press, Inc.
Nucleic Acids Research, 1993
The activity of MHC class 11 promoters depends upon conserved regulatory signals one of which, the extended X-box, contains in its X2 subregion a sequence related to the cAMP response element, CRE and to the TPA response element, TRE. Accordingly, X2 is recognized by the AP-1 factor and by other c-Jun or c-Fos containing heterodimers. We report that the X-box dependent promoter activity of the HLA-DQA1 gene is down-modulated by an array of DNA elements each of which represented twice either in an invertedly or directly repeated orientation. In this frame, we describe a nuclear binding factor, namely DBF, promiscuously interacting with two of these additional signals, 6 and a, and with a portion of the X-box, namely the X-core, devoid of X2. The presence of a single factor recognizing divergent DNA sequences was indicated by the finding that these activities were coeluted from a heparin-Sepharose column and from DNA affinity columns carrying different DNA binding sites as ligands. Competition experiments made with oligonucleotides representing wild type and mutant DNA elements showed that each DNA element specifically inhibited the binding of the others, supporting the contention that DBF is involved in recognition of different targets. Furthermore, we found that DBF also exhibits CRE/TRE binding activity and that this activity can be competed out by addition of an excess of a, 6 and X-core oligonucleotides. Anti-Jun peptide and anti-Fos peptide antibodies blocked not only the binding activity of DBF, but also its X-core and a binding; this blockade was removed by the addition of the Jun or Fos peptides against which the antibodies had been raised. In vitro synthesized Jun/Fos was able to bind to all these boxes, albeit with seemingly different affinities. The cooperativity of DBF interactions may explain the modulation of the X-box dependent promoter activity mediated by the accessory DNA elements described here.