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DNA damage caused by the binding of the tumorigen 7R,8S-diol 9S,10R-epoxide (B[a]PDE), a metabolite of bezo[a]pyrene, to guanine in CpG dinucleotide sequences could affect DNA methylation and, thus, represent a potential epigenetic mechanism of chemical carcinogenesis. In this work, we investigated the impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P-N(2)-dG adducts (B(+) and B(-)) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and M.HhaI. These two methyltransferases recognize CpG and GCGC sequences, respectively, and transfer a methyl group to the C5 atom of cytosine (C). A series of 18-mer unmethylated or hemimethylated oligodeoxynucleotide duplexes containing trans-anti-B[a]P-N(2)-dG adducts was generated. The B(+) or B(-) residues were introduced either 5' or 3' adjacent or opposite to the target 2'-deoxycytidines. The B[a]PDE lesions practically produced no effect on M.SssI binding to DNA but reduced M.HhaI binding by 1-2 orders of magnitude. In most cases, the benzo[a]pyrenyl residues decreased the methylation efficiency of hemimethylated and unmethylated DNA by M.SssI and M.HhaI. An absence of the methylation of hemimethylated duplexes was observed when either the (+)- or the (-)-trans-anti-B[a]P-N(2)-dG adduct was positioned 5' to the target dC. The effects observed may be related to the minor groove conformation of the bulky benzo[a]pyrenyl residue and to a perturbation of the normal contacts of the methyltransferase catalytic loop with the B[a]PDE-modified DNA. Our results indicate that a trans-anti-B[a]P-N(2)-dG lesion flanking a target dC in the CpG dinucleotide sequence on its 5'-side has a greater adverse impact on methylation than the same lesion when it is 3' adjacent or opposite to the target dC.

Benzo[a]pyrene (B[a]P) is a well-characterized environmental polycyclic aromatic hydrocarbon pollutant. In living organisms, B[a]P is metabolized to the genotoxic anti-benzo [a]pyrene diol epoxide that reacts with cellular DNA to form stereoisomeric anti-B [a]PDE-N 2 -dG adducts. In this study, we explored the effects of adduct stereochemistry and position in double-stranded DNA substrates on the functional characteristics of the catalytic domain of murine de novo DNA methyltransferase Dnmt3a (Dnmt3a-CD). A number of 18-mer duplexes containing sitespecifically incorporated (+)-and (−)-trans-anti-B[a]PDE-N 2 -dG lesions located 3′-and 5′-adjacent to and opposite the target cytosine residue were prepared. The Dnmt3a-CD binds cooperatively to the DNA duplexes with an up to 5-fold greater affinity as compared to the undamaged DNA duplexes. Methylation assays showed a 1.7-6.3 fold decrease of the methylation reaction rates for the damaged duplexes. B[a]PDE modifications stimulated a non-productive binding and markedly favoured substrate inhibition of Dnmt3a-CD independently of DNA methylation status. The latter effect was sensitive to the position and stereochemistry of the B[a]PDE-N 2 -dG adducts. The overall effect of trans-anti-B [a]PDE-N 2 -dG adducts on Dnmt3a-CD was less detrimental than in the case of the prokaryotic methyltransferases we previously investigated.Benzo [a]pyrene (B[a]P) is an ubiquitous and harmful pollutant that is abundant in car exhaust and tobacco smoke (1), and is metabolically activated to the biologically active benzo [a]pyrene-7,8-diol-9,10-epoxides with predominant formation of benzo [a]pyrene-7R, 8S-dihydrodiol-9S,10R-epoxide ((+)-anti-B[a]PDE). In the anti-orientation of B[a]PDE, the 7-OH and 9,10-epoxide groups are on opposite sites of the planar polycyclic aromatic ring system (2,3). Both the (+)-and (−)-anti-enantiomers of B[a]PDE bind covalently to the exocyclic amino group of guanine, with trans or cis opening of the epoxide ring. The most abundant adduct is (+)-trans-anti-B[a]PDE-N 2 -dG, both in vivo (4) and in vitro (about 90%) (5), with lower amounts of the (+)-cis-and (−)-trans-anti-B [a] The following buffers (B1-B7) were used: B1, 50 mM sodium phosphate (pH 6.0), 1 M NaCl, 10 mM mercaptoethanol, 10% (v/v) glycerol, 0.1% Triton X-100; B2, buffer B1 containing 17 μg/ml phenylmethanesulfonyl chloride, 5 μg/ml leupeptin and 1 μg/ml pepstatin A; B3, B4, and B5, buffer B2 containing 10 mM, 20 mM and 150 mM imidazoleHCl, respectively; B6, 20 mM Tris-HCl (pH 7.4), 0.2 mM EDTA, 2 mM dithiothreitol, 5% (v/v) glycerol; B7, 20 mM HEPES-NaOH (pH 7.0), 100 mM KCl, 1 mM EDTA, 0.2 mM dithiothreitol. Enzyme Expression and PurificationThe N-terminal His 6 tag fusion catalytic domain of Dnmt3a was expressed in E. coli BL21 (DE3) cells (Novagen) using the pET28a plasmid containing Dnmt3a-CD as a vector, as described previously (28). Cells were grown in LB medium at 32°C with intensive aeration until A 600 ~ 0.7 was attained. Protein expression was initiated by the addition of 1 mM i...

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Glycolysis is a main catabolic pathway of glucose metabolism, accompanied by ATP synthesis. More than 30 enzymes are involved in glycolysis, and genes that encode them can be considered housekeeping genes due to the high conservatism and evolutionary antiquity of the process. We studied the expression of these genes in kidney papillary cancer and planocellular lung cancer via the bioinformatic analysis of transcriptome database and method of quantitative real time PCR. Quantitative analysis of mRNA level demonstrated that only a part ofgenes that encode glycolysis enzymes maintain relatively stable mRNA level, including the HK1, ADPGK, GPI, PGK1, and PKM2 genes in kidney papillary cancer and the ADPGK, ALDOA, GAPDH, PGK1, BPGM, ENO1, and PKM2 genes in planocellular lung cancer. The frequent increase in the mRNA expression of PFKP, ALDOA, and GAPDH genes in kidney cancer, as well as the GPI gene in lung cancer, were detected for the first time by real time PCR. For other genes, their differential expression was demonstrated; the cases of both a decrease and increase in the mRNA level were detected. Thus, several genes that can be used as control genes in transcriptome analysis by real time PCR in kidney and lung cancer, as well as a number of differentially expressed genes that can be potential oncomarkers, were identified.