Publications by authors named "Diana Maltseva"

16 Publications

  • Page 1 of 1

Low expression of CD24 is associated with poor survival in colorectal cancer.

Biochimie 2021 Oct 9. Epub 2021 Oct 9.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia; SRC Bioclinicum, Moscow, Russia. Electronic address:

In this study we analyzed expression of CD24 in a cohort of colorectal cancer patients using immunohistochemistry staining of CD24. We found a significant association between absence or low expression of CD24 (10% of membranous and 55% of cytoplasmic staining) and shortened patient survival. Protein localization played a crucial role in the prognosis: membranous form was the major and prognostic one in primary tumors, while cytoplasmic expression was elevated in liver metastases compared to the primary tumors and contained prognostic information. Then, using The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) RNA-seq data, we showed that CD24 mRNA level was two-fold decreased in primary colorectal cancers compared to adjacent normal mucosa. Like the protein staining data, ten percent of patients with the lowest mRNA expression levels of CD24 in primary tumors had reduced survival compared to the ones with higher expression. To explain these findings mechanistically, shRNA-mediated CD24 knockdown was performed in HT-29 colorectal cancer cells. It resulted in the increase of cell migration in vitro, no changes in proliferation and apoptosis, and a slight decrease in cell invasion. As increased cell migration is a hallmark of metastasis formation, this finding corroborates the association of a decreased CD24 expression with poor prognosis. Differential gene expression analysis revealed upregulation of genes involved in cell migration in the group of patients with low CD24 expression, including integrin subunit α3 and α3, β3 subunits of laminin 332. Further co-expression analysis identified SPI1, STAT1 and IRF1 transcription factors as putative master-regulators in this group.
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http://dx.doi.org/10.1016/j.biochi.2021.10.004DOI Listing
October 2021

Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy.

Biochimie 2020 Jul 22;174:107-116. Epub 2020 Apr 22.

Faculty of Biology and Biotechnology, National Research University Higher School of Economics, Myasnitskaya str. 13/4, 117997, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya str. 16/10, 117997, Moscow, Russia; Scientific Research Center Bioclinicum, Ugreshskaya str. 2/85, 115088, Moscow, Russia. Electronic address:

The interaction of tumor cells with the extracellular matrix (ECM) may affect the rate of cancer progression and metastasis. One of the major components of ECM are laminins, the heterotrimeric glycoproteins consisting of α-, β-, and γ-chains (αβγ). Laminins interact with their cell surface receptors and, thus, regulate multiple cellular processes. In this work, we demonstrate that shRNA-mediated knockdown of the α5 laminin chain results in Wnt- and mTORC1-dependent partial dedifferentiation of colorectal cancer cells. Furthermore, we showed that this dedifferentiation involved activation of ER-stress signaling, pathway promoting the sensitivity of cells to 5-fluorouracil.
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http://dx.doi.org/10.1016/j.biochi.2020.04.016DOI Listing
July 2020

Towards embedding Caco-2 model of gut interface in a microfluidic device to enable multi-organ models for systems biology.

BMC Syst Biol 2019 03 5;13(Suppl 1):19. Epub 2019 Mar 5.

SRC BioClinicum, Moscow, Russia.

Background: A cancer cell line originating from human epithelial colorectal adenocarcinoma (Caco-2 cells) serves as a high capacity model for a preclinical screening of drugs. Recent need for incorporating barrier tissue into multi-organ chips calls for inclusion of Caco-2 cells into microperfused environment.

Results: This article describes a series of systems biology insights obtained from comparing Caco-2 models cells grown as conventional 2D layer and in a microfluidic chip. When basic electrical parameters of Caco-2 monolayers were evaluated using impedance spectrometry and MTT assays, no differences were noted. On the other hand, the microarray profiling of mRNAs and miRNAs revealed that grows on a microfluidic chip leads to the change in the production of specific miRNA, which regulate a set of genes for cell adhesion molecules (CAMs), and provide for more complete differentiation of Caco-2 monolayer. Moreover, the sets of miRNAs secreted at the apical surface of Caco-2 monolayers grown in conventional 2D culture and in microfluidic device differ.

Conclusions: When integrated into a multi-tissue platform, Caco-2 cells may aid in generating insights into complex pathophysiological processes, not possible to dissect in conventional cultures.
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http://dx.doi.org/10.1186/s12918-019-0686-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399809PMC
March 2019

Cumulative prognostic power of laminin genes in colorectal cancer.

BMC Med Genomics 2018 02 13;11(Suppl 1). Epub 2018 Feb 13.

SRC Bioclinicum, Ugreshskaya str 2/85, 115088, Moscow, Russia.

Background: Laminins are a major family of extracellular matrix proteins and the main component of basement membranes. Laminins are involved in many if not all stages of cancer progression, and expression of laminin genes has prognostic value in various types of cancer, including colorectal. Only single laminin genes or components of a single laminin trimer with significant differential expression have been regarded as potential biomarkers to date.

Results: Here we compared prognostic power of classifiers constructed from sets of laminin genes with that of any single laminin gene. The analysis showed that cumulative prognostic power of sets of laminin genes was higher and was achieved already with pairs and triples of the genes. Interestingly, components of the pairs and the triples did not belong to any known laminin trimer, but, taken together with the gene weights, suggested higher LAMA4/LAMA5 expression ratio in patients with poor prognosis.

Conclusions: Analysis of the laminin expression profile rather than expression of the single genes or components of laminin trimers is useful for colorectal cancer prognosis in patients. High LAMA4/LAMA5 ratio is associated with increased permeability of basement membranes suggesting that basement membranes produced by colorectal tumors might be an important hindrance to their own dissemination in patients.
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http://dx.doi.org/10.1186/s12920-018-0332-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836818PMC
February 2018

Selectin-independent adhesion during ovarian cancer metastasis.

Biochimie 2017 Nov 14;142:197-206. Epub 2017 Sep 14.

P. Herzen Moscow Oncology Research Institute, Moscow, 125284, Russia. Electronic address:

Purpose: Ovarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well.

Methods: A comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays.

Results: One-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins.

Conclusions: High expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.
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http://dx.doi.org/10.1016/j.biochi.2017.09.009DOI Listing
November 2017

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus.

Molecules 2017 May 15;22(5). Epub 2017 May 15.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood-brain barrier-in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.
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http://dx.doi.org/10.3390/molecules22050808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153759PMC
May 2017

Receptor Mincle promotes skin allergies and is capable of recognizing cholesterol sulfate.

Proc Natl Acad Sci U S A 2017 03 14;114(13):E2758-E2765. Epub 2017 Mar 14.

Laboratory of Gene Engineering of Pathogenic Microorganisms, N. F. Gamaleya Federal Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia.

Sterile (noninfected) inflammation underlies the pathogenesis of many widespread diseases, such as allergies and autoimmune diseases. The evolutionarily conserved innate immune system is considered to play a key role in tissue injury recognition and the subsequent development of sterile inflammation; however, the underlying molecular mechanisms are not yet completely understood. Here, we show that cholesterol sulfate, a molecule present in relatively high concentrations in the epithelial layer of barrier tissues, is selectively recognized by Mincle (Clec4e), a C-type lectin receptor of the innate immune system that is strongly up-regulated in response to skin damage. Mincle activation by cholesterol sulfate causes the secretion of a range of proinflammatory mediators, and s.c. injection of cholesterol sulfate results in a Mincle-mediated induction of a severe local inflammatory response. In addition, our study reveals a role of Mincle as a driving component in the pathogenesis of allergic skin inflammation. In a well-established model of allergic contact dermatitis, the absence of Mincle leads to a significant suppression of the magnitude of the skin inflammatory response as assessed by changes in ear thickness, myeloid cell infiltration, and cytokine and chemokine secretion. Taken together, our results provide a deeper understanding of the fundamental mechanisms underlying sterile inflammation.
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http://dx.doi.org/10.1073/pnas.1611665114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380039PMC
March 2017

miRNome of inflammatory breast cancer.

BMC Res Notes 2014 Dec 4;7:871. Epub 2014 Dec 4.

SRC Bioclinicum, Ugreshskaya str 2/85, 115088 Moscow, Russia.

Background: Inflammatory breast cancer (IBC) is an extremely malignant form of breast cancer which can be easily misdiagnosed. Conclusive prognostic IBC molecular biomarkers which are also providing the perspectives for targeted therapy are lacking so far. The aim of this study was to reveal the IBC-specific miRNA expression profile and to evaluate its association with clinicopathological parameters.

Methods: miRNA expression profiles of 13 IBC and 17 non-IBC patients were characterized using comprehensive Affymetrix GeneChip miRNA 3.0 microarray platform. Bioinformatic analysis was used to reveal IBC-specific miRNAs, deregulated pathways and potential miRNA targets.

Results: 31 differentially expressed miRNAs characterize IBC and mRNAs regulated by them and their associated pathways can functionally be attributed to IBC progression. In addition, a minimal predictive set of 4 miRNAs characteristic for the IBC phenotype and associated with the TP53 mutational status in breast cancer patients was identified.

Conclusions: We have characterized the complete miRNome of inflammatory breast cancer and found differentially expressed miRNAs which reliably classify the patients to IBC and non-IBC groups. We found that the mRNAs and pathways likely regulated by these miRNAs are highly relevant to cancer progression. Furthermore a minimal IBC-related predictive set of 4 miRNAs associated with the TP53 mutational status and survival for breast cancer patients was identified.
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http://dx.doi.org/10.1186/1756-0500-7-871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4289319PMC
December 2014

Exercise immunology meets MiRNAs.

Exerc Immunol Rev 2014 ;20:135-64

A large body of evidence indicates modified expression of protein-coding genes in response to different kinds of physical activity. Recent years have exposed another level of regulation of cellular processes mediated by non-coding RNAs. MicroRNAs (miRNAs) are one of the largest families of non-coding RNAs. MiRNAs mediate post-transcriptional regulation of gene expression. The amount of data supporting the key role of miRNAs in the adaptation of the immune and other body systems to exercise steadily grows. MiRNAs change their expression profiles after exercise and seem to be involved in regulation of exercise-responsive genes in immune and other cell types. Here we discuss existing data and future directions in the field.
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July 2014

Dynamically regulated miRNA-mRNA networks revealed by exercise.

BMC Physiol 2013 Jun 7;13. Epub 2013 Jun 7.

SRC Bioclinicum, Ugreshskaya str 2/85, Moscow 115088, Russia.

Background: MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period.

Results: Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation.

Conclusions: We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.
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http://dx.doi.org/10.1186/1472-6793-13-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681679PMC
June 2013

High-throughput identification of reference genes for research and clinical RT-qPCR analysis of breast cancer samples.

J Clin Bioinforma 2013 Jul 22;3(1):13. Epub 2013 Jul 22.

SRC Bioclinicum, Moscow, Russia.

Background: Quantification and normalization of RT-qPCR data critically depends on the expression of so called reference genes. Our goal was to develop a strategy for the selection of reference genes that utilizes microarray data analysis and combines known approaches for gene stability evaluation and to select a set of appropriate reference genes for research and clinical analysis of breast samples with different receptor and cancer status using this strategy.

Methods: A preliminary search of reference genes was based on high-throughput analysis of microarray datasets. The final selection and validation of the candidate genes were based on the RT-qPCR data analysis using several known methods for expression stability evaluation: comparative ∆Ct method, geNorm, NormFinder and Haller equivalence test.

Results: A set of five reference genes was identified: ACTB, RPS23, HUWE1, EEF1A1 and SF3A1. The initial selection was based on the analysis of publically available well-annotated microarray datasets containing different breast cancers and normal breast epithelium from breast cancer patients and epithelium from cancer-free patients. The final selection and validation were performed using RT-qPCR data from 39 breast cancer biopsy samples. Three genes from the final set were identified by the means of microarray analysis and were novel in the context of breast cancer assay. We showed that the selected set of reference genes is more stable in comparison not only with individual genes, but also with a system of reference genes used in commercial OncotypeDX test.

Conclusion: A selection of reference genes for RT-qPCR can be efficiently performed by combining a preliminary search based on the high-throughput analysis of microarray datasets and final selection and validation based on the analysis of RT-qPCR data with a simultaneous examination of different expression stability measures. The identified set of reference genes proved to be less variable and thus potentially more efficient for research and clinical analysis of breast samples comparing to individual genes and the set of reference genes used in OncotypeDX assay.
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http://dx.doi.org/10.1186/2043-9113-3-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726509PMC
July 2013

Passing the anaerobic threshold is associated with substantial changes in the gene expression profile in white blood cells.

Eur J Appl Physiol 2012 Mar 30;112(3):963-72. Epub 2011 Jun 30.

Department of Molecular Physiology, Russian Research Institute of Physical Education and Sport, Elizavetinsky lane 10, Moscow, Russia.

High and moderate intensity endurance exercise alters gene expression in human white blood cells (WBCs), but the understanding of how this effect occurs is limited. To increase our knowledge of the nature of this process, we investigated the effects of passing the anaerobic threshold (AnT) on the gene expression profile in WBCs of athletes. Nineteen highly trained skiers participated in a treadmill test with an incremental step protocol until exhaustion (ramp test to exhaustion, RTE). The average total time to exhaustion was 14:40 min and time after AnT was 4:50 min. Two weeks later, seven of these skiers participated in a moderate treadmill test (MT) at 80% peak O(2) uptake for 30 min, which was slightly below their AnTs. Blood samples were obtained before and immediately after both tests. RTE was associated with substantially greater leukocytosis and acidosis than MT. Gene expression in WBCs was measured using whole genome microarray expression analysis before and immediately after each test. A total of 310 upregulated genes were found after RTE, and 69 genes after MT of which 64 were identical to RTE. Both tests influenced a variety of known gene pathways related to inflammation, stress response, signal transduction and apoptosis. A large group of differentially expressed previously unknown small nucleolar RNA and small Cajal body RNA was found. In conclusion, a 15-min test to exhaustion was associated with substantially greater changes of gene expression than a 30-min test just below the AnT.
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http://dx.doi.org/10.1007/s00421-011-2048-3DOI Listing
March 2012

Killer cell immunoglobulin-like receptors and exercise.

Exerc Immunol Rev 2011 ;17:150-63

Department of Molecular Physiology, Russian Research Institute of Physical Education and Sport, Moscow, Russia.

Exercise can alter human health in both beneficial (e. g. reduced risk of infection and of atherosclerosis) and adverse (e. g. anaphylaxis, exercise-induced asthma, and exacerbation of chronic illness) ways. Hitherto, the mechanisms linking exercise and health are not fully understood, but may rest on the capability of exercise to both increase circulating immune cells and modulate their activity. Natural killer (NK) cells, a major component of innate immunity, are one of the most sensitive populations of immune cells to exercise stress. NK cells play an important role in the detection and elimination of tumours and virus-infected cells. To mediate NK cell functions, there is an array of activating and inhibitory receptors with distinct specificities on their surface. Killer-cell immunoglobulin-like receptors (KIRs) which bind to MHC class I are a key example of receptors expressed by NK cells. The combination of MHC class I and KIR variants influences resistance to infections, susceptibility to autoimmune diseases, as well as complications of pregnancy. It is suggested that KIRs may also determine a considerable part of the effects of physical activity on human health. In this review we discuss KIRs in more detail, their role in the onset of human diseases, and the influence of acute exercise on KIR gene expression.
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April 2011

Impact of 7,8-dihydro-8-oxoguanine on methylation of the CpG site by Dnmt3a.

Biochemistry 2009 Feb;48(6):1361-8

Chemistry Department and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia.

7,8-Dihydro-8-oxoguanine (8-oxoG) is a ubiquitous oxidative DNA lesion resulting from injury to DNA via reactive oxygen species. 8-oxoG lesions may play a role in the formation of aberrant DNA methylation patterns during carcinogenesis. In this study, we assessed the effects of 8-oxoG on methylation and complex formation of nine 30-mer oligodeoxynucleotide duplexes by the catalytic domain of murine Dnmt3a DNA methyltransferase (Dnmt3a-CD). The effects of 8-oxoG on the methylation rate of hemimethylated duplexes varied from a 25-fold decrease to a 1.8-fold increase, depending on the position of the lesion relative to the Dnmt3a-CD recognition site (CpG) and target cytosine (C). The most significant effect was observed when 8-oxoG replaced guanine within the recognition site immediately downstream of the target cytosine. Fluorescence polarization experiments with fluorescein-labeled duplexes revealed that two molecules of Dnmt3a-CD bind per duplex, generating sigmoid binding curves. Duplexes exhibiting the highest apparent binding cooperativity formed the least stable 1:2 complexes with Dnmt3a-CD and were methylated at the lowest rate. Kinetic analyses disclosed the formation of very stable nonproductive enzyme-substrate complexes with hemimethylated duplexes that act as suicide substrates of Dnmt3a-CD. The presence of 8-oxoG within the CpG site downstream of the target cytosine markedly diminished productive versus nonproductive binding. We propose that 8-oxoG located adjacent to the target cytosine interferes with methylation by weakening the affinity of DNA for Dnmt3a-CD, thereby favoring a nonproductive binding mode.
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http://dx.doi.org/10.1021/bi801947fDOI Listing
February 2009

The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases.

FEBS J 2007 Apr 27;274(8):2121-34. Epub 2007 Mar 27.

Chemistry Department, Moscow State University, Russia.

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.
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http://dx.doi.org/10.1111/j.1742-4658.2007.05754.xDOI Listing
April 2007

Impact of benzo[a]pyrene-2'-deoxyguanosine lesions on methylation of DNA by SssI and HhaI DNA methyltransferases.

Biochemistry 2006 May;45(19):6142-59

Chemistry Department, Moscow State University, Moscow, 119992, Russia.

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.
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http://dx.doi.org/10.1021/bi0511639DOI Listing
May 2006
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