Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia

BMC Cancer

Research Article

Katrin Luckert¹  Oliver Pötz¹  Thomas O Joos¹  Kamil D Kaplanov²  Olga B Kalashnikova²  Jörg D Hoheisel³  Achim Stephan³  Marita Schrenk³  Evgeny A Moskalev⁴  Aleena A Gladkikh⁵  Ivan A Vorobjev⁶  Sergey E Mastitsky⁷ 

[1] Biochemistry Department, NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, 72770, Reutlingen, Germany;Department of Haematology, Volgograd Regional Clinical Oncological Dispensary No.1, Zemlyachki str. 78, 400138, Volgograd, Russia;Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany;Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany;Diagnostic Molecular Pathology, Institute of Pathology, University of Erlangen-Nürnberg, Krankenhausstr. 8-10, 91054, Erlangen, Germany;Functional Morphology of Hemablastoses, National Hematology Research Centre of Russian Academy of Medical Sciences, Novy Zykovsky passage 4a, 125167, Moscow, Russia;Functional Morphology of Hemablastoses, National Hematology Research Centre of Russian Academy of Medical Sciences, Novy Zykovsky passage 4a, 125167, Moscow, Russia;A.N. Belozersky Institute and Biological Faculty, Moscow State University, Leninskie Gory 1, 119991, Moscow, Russia;Theoretical Bioinformatics, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany;

关键词: B cell chronic lymphocytic leukaemia;    Wnt/β-catenin pathway;    Inhibitor genes;    DNA hypermethylation;    Epigenetic silencing;    β-catenin;   

DOI  :  10.1186/1471-2407-12-213

 received in 2012-02-14, accepted in 2012-06-06,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundThe Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes – CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 – in the cell lines EHEB and MEC-1 as well as patient samples.MethodsQuantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.ResultsFor 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.ConclusionsThe results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

【 授权许可】

CC BY   
© Moskalev et al.; licensee BioMed Central Ltd. 2012