Institute of Molecular Biology and Biophysics SB RAMS

Novosibirsk, Russia

Institute of Molecular Biology and Biophysics SB RAMS

Novosibirsk, Russia
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Kazantseva Y.A.,Institute of Molecular Biology and Biophysics SB RAMS | Yarushkin A.A.,Institute of Molecular Biology and Biophysics SB RAMS | Pustylnyak V.O.,Novosibirsk State University
Toxicology | Year: 2014

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments. © 2014 Elsevier Ireland Ltd.


Kazantseva Y.A.,Institute of Molecular Biology and Biophysics SB RAMS | Yarushkin A.A.,Institute of Molecular Biology and Biophysics SB RAMS | Pustylnyak V.O.,Novosibirsk State University
Toxicology and Applied Pharmacology | Year: 2013

Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p'-DDT (85%) and o,p'-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CAR and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45β, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45β. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the β-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers. © 2013 Elsevier Inc..


Mostovich L.A.,Institute of Molecular Biology and Biophysics SB RAMS | Prudnikova T.Y.,Institute of Molecular Biology and Biophysics SB RAMS | Kondratov A.G.,NASU Institute of Molecular Biology and Genetics | Gubanova N.V.,RAS Institute of Cytology and Genetics | And 9 more authors.
Epigenetics | Year: 2012

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/β-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/β-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/β-catenin mRNA levels and activated TCF4/β-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/β-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and β-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/β-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/β-catenin complex activity. © 2012 Landes Bioscience.


Yarushkin A.,Institute of Molecular Biology and Biophysics SB RAMS | Kachaylo E.,Institute of Molecular Biology and Biophysics SB RAMS | Pustylnyak V.,Novosibirsk State University
British Journal of Pharmacology | Year: 2013

Background and Purpose The dual role of the constitutive androstane receptor (CAR) as both a xenosensor and a regulator of endogenous energy metabolism (lipogenesis and gluconeogenesis) has recently gained acceptance. Here, we investigated the effects of 4-[(4R,6R)-4,6-diphenyl-1,3-dioxan-2-yl]-N, N-dimethylaniline (transpDMA), an effective CAR activator, on the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in rat livers. Experimental Approach The effects of transpDMA were investigated in normal and high-fat diet-fed Wistar rats using real-time PCR, Western blotting, chromatin immunoprecipitation assays (ChIP), glucose tolerance test and insulin tolerance test. Key Results The expression of the gluconeogenic enzymes PEPCK and G6Pase was repressed by transpDMA treatment under fasting conditions. Long-term CAR activation by transpDMA significantly reduced fasting blood glucose levels and improved glucose homeostasis and insulin sensitivity in high-fat diet-fed rats. The metabolic benefits of CAR activation by transpDMA may have resulted from the inhibition of hepatic gluconeogenic genes. ChIP assays demonstrated that transpDMA prevented the binding of forkhead box O1 (FOXO1) to insulin response sequences in the PEPCK and G6Pase gene promoters in rat livers. Moreover, transpDMA-activated CAR inhibited hepatocyte nuclear factor-4α (HNF4α) transactivation by competing with HNF4α for binding to the specific binding element (DR1-site) in the gluconeogenic gene promoters. Conclusions and Implications Our results provide evidence to support the conclusion that transpDMA inhibits the gluconeogenic genes PEPCK and G6Pase through suppression of HNF4α and FOXO1 transcriptional activity. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.


Perepechaeva M.,Institute of Molecular Biology and Biophysics SB RAMS
Journal of physiology and biochemistry | Year: 2011

Cytochrome P4501A (the CYP1A1 and CYP1A2 enzymes) is known to metabolize anthropogenic xenobiotics to carcinogenic and mutagenic compounds. CYP1A1 transcriptional activation is regulated via the aryl hydrocarbon receptor (AhR)-dependent signal transduction pathway. CYP1A2 activation may occur through the AhR-dependent or AhR-independent signal transduction pathways. We used male Wistar rats to explore possible mechanisms of CYP1A activation induced by exposure to cold and the effects of the protein-tyrosine kinase inhibitors genistein, herbimycin A, and geldanamycin on the properties of hepatic CYP1A1 and CYP1A2 proteins following exposure to cold and to classic CYP1A inducers. The molecular mechanisms of cold-induced CYP1A1 and CYP1A2 activation are different. The CYP1A2 activation apparently occurs at the post-transcriptional level. The CYP1A1 activation, whether caused by exposure to cold or by classic CYP1A inducers, is AhR-dependent and occurs at the transcriptional level. Protein tyrosine kinase inhibitors have no effect on benzo(a)pyrene-induced CYP1A expression but alter cold-induced CYP1A1 activity and the CYP1A1 mRNA level. Thus, treatment with herbimycin A or geldanamycin leads to an increase in CYP1A1 activity, while treatment with genistein increases CYP1A1 mRNA expression and decreases CYP1A2 activity. These data elucidate the molecular mechanisms of cold-induced CYP1A activation and the role of protein kinases in the regulation of CYP1A during exposure to cold. Our results can also help identify the differences between the molecular mechanisms underlying the effects of the classic CYP1A inducers and the effects of cooling.


Perepechaeva M.,Institute of Molecular Biology and Biophysics SB RAMS | Kolosova N.,RAS Institute of Cytology and Genetics | Grishanova A.,Institute of Molecular Biology and Biophysics SB RAMS
Journal of Physiology and Biochemistry | Year: 2011

Cytochrome P4501A (the CYP1A1 and CYP1A2 enzymes) is known to metabolize anthropogenic xenobiotics to carcinogenic and mutagenic compounds. CYP1A1 transcriptional activation is regulated via the aryl hydrocarbon receptor (AhR)-dependent signal transduction pathway. CYP1A2 activation may occur through the AhR-dependent or AhR-independent signal transduction pathways. We used male Wistar rats to explore possible mechanisms of CYP1A activation induced by exposure to cold and the effects of the protein-tyrosine kinase inhibitors genistein, herbimycin A, and geldanamycin on the properties of hepatic CYP1A1 and CYP1A2 proteins following exposure to cold and to classic CYP1A inducers. The molecular mechanisms of cold-induced CYP1A1 and CYP1A2 activation are different. The CYP1A2 activation apparently occurs at the post-transcriptional level. The CYP1A1 activation, whether caused by exposure to cold or by classic CYP1A inducers, is AhR-dependent and occurs at the transcriptional level. Protein tyrosine kinase inhibitors have no effect on benzo(a)pyrene-induced CYP1A expression but alter cold-induced CYP1A1 activity and the CYP1A1 mRNA level. Thus, treatment with herbimycin A or geldanamycin leads to an increase in CYP1A1 activity, while treatment with genistein increases CYP1A1 mRNA expression and decreases CYP1A2 activity. These data elucidate the molecular mechanisms of cold-induced CYP1A activation and the role of protein kinases in the regulation of CYP1A during exposure to cold. Our results can also help identify the differences between the molecular mechanisms underlying the effects of the classic CYP1A inducers and the effects of cooling. © 2011 University of Navarra.


Pustylnyak V.O.,Novosibirsk State University | Lisachev P.D.,Russian Academy of Sciences | Shtark M.B.,Institute of Molecular Biology and Biophysics SB RAMS
Neural Plasticity | Year: 2015

Gene expression plays an important role in the mechanisms of long-term potentiation (LTP), which is a widely accepted experimental model of synaptic plasticity. We have studied the expression of at least 50 genes that are transcriptionally regulated by p53, as well as other genes that are related to p53-dependent processes, in the early phase of LTP. Within 30 min after Schaffer collaterals (SC) tetanization, increases in the mRNA and protein levels of Bax, which are upregulated by p53, and a decrease in the mRNA and protein levels of Bcl2, which are downregulated by p53, were observed. The inhibition of Mdm2 by nutlin-3 increased the basal p53 protein level and rescued its tetanization-induced depletion, which suggested the involvement of Mdm2 in the control over p53 during LTP. Furthermore, nutlin-3 caused an increase in the basal expression of Bax and a decrease in the basal expression of Bcl2, whereas tetanization-induced changes in their expression were occluded. These results support the hypothesis that p53 may be involved in transcriptional regulation during the early phase of LTP. We hope that the presented data may aid in the understanding of the contribution of p53 and related genes in the processes that are associated with synaptic plasticity. © 2015 Vladimir O. Pustylnyak et al.


PubMed | Novosibirsk State University, Russian Academy of Sciences and Institute of Molecular Biology and Biophysics SB RAMS
Type: | Journal: Neural plasticity | Year: 2015

Gene expression plays an important role in the mechanisms of long-term potentiation (LTP), which is a widely accepted experimental model of synaptic plasticity. We have studied the expression of at least 50 genes that are transcriptionally regulated by p53, as well as other genes that are related to p53-dependent processes, in the early phase of LTP. Within 30min after Schaffer collaterals (SC) tetanization, increases in the mRNA and protein levels of Bax, which are upregulated by p53, and a decrease in the mRNA and protein levels of Bcl2, which are downregulated by p53, were observed. The inhibition of Mdm2 by nutlin-3 increased the basal p53 protein level and rescued its tetanization-induced depletion, which suggested the involvement of Mdm2 in the control over p53 during LTP. Furthermore, nutlin-3 caused an increase in the basal expression of Bax and a decrease in the basal expression of Bcl2, whereas tetanization-induced changes in their expression were occluded. These results support the hypothesis that p53 may be involved in transcriptional regulation during the early phase of LTP. We hope that the presented data may aid in the understanding of the contribution of p53 and related genes in the processes that are associated with synaptic plasticity.


PubMed | Institute of Molecular Biology and Biophysics SB RAMS
Type: Journal Article | Journal: Cell and tissue research | Year: 2015

Glycosylation changes occur widely in colon tumours, suggesting glycosylated molecules as potential biomarkers for colon cancer diagnostics. In this study, proteoglycans (PGs) expression levels and their transcriptional patterns are investigated in human colon tumours in vivo and carcinoma cells in vitro. According to RT-PCR analysis, normal and cancer colon tissues expressed a specific set of PGs (syndecan-1, perlecan, decorin, biglycan, versican, NG2/CSPG4, serglycin, lumican, CD44), while the expression of glypican-1, brevican and aggrecan was almost undetectable. Overall transcriptional activity of the PGs in normal and cancer tissues was similar, although expression patterns were different. Expression of decorin and perlecan was down-regulated 2-fold in colon tumours, while biglycan and versican expression was significantly up-regulated (6-fold and 3-fold, respectively). Expression of collagen1A1 was also increased 6-fold in colon tumours. However, conventional HCT-116 colon carcinoma and AG2 colon cancer-initiating cells did not express biglycan and decorin and were versican-positive and -negative, respectively, demonstrating an extracellular origin of the PGs in cancer tissue. Selective expression of heparan sulfate (HS) proteoglycans syndecan-1 and perlecan in the AG2 colon cancer-initiating cell line suggests these PGs as potential biomarkers for cancer stem cells. Overall transcriptional activity of the HS biosynthetic system was similar in normal and cancer tissues, although significant up-regulation of extracellular sulfatases SULF1/2 argues for a possible distortion of HS sulfation patterns in colon tumours. Taken together, the obtained results suggest versican, biglycan, collagen1A1 and SULF1/2 expression as potential microenvironmental biomarkers and/or targets for colon cancer diagnostics and treatment.


PubMed | Institute of Molecular Biology and Biophysics SB RAMS and Novosibirsk State University
Type: | Journal: Toxicology | Year: 2014

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments.

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