KLF4, DAPK1 and SPG20 promoter methylation is not affected by DNMT1 silencing and hypomethylating drugs in lymphoma cells

Abstract

Promoter methylation represents one of the major epigenetic mechanisms responsible for the regulation of gene expression. Hypomethylating drugs are currently approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia, and some studies have recently been carried out on diffuse large B cell lymphoma (DLBCL). DLBCL is a type of Non-Hodgkin lymphoma. The aim of the present study was to assess the role of DNA methyltransferase (DNMT)1 in mediating the epigenetic regulation of some key targets previously emerged as hypermethylated in Non-Hodgkin lymphoma. Reverse transcription-quantitative PCR, genome-wide arrays and methylation-specific PCR were used to determine the level of methylation of specific targets. Gene silencing, gene expression and immunoblotting were used to investigate the role of DNMT1 and DNMT3a in lymphoma cells. The present study showed that lymphoma cell lines displayed a completely different methylation profile on selected targets compared with primary B lymphocytes and peripheral blood mononuclear cells. 5′-aza-cytidine (5AZA) and 5′-aza-2-deoxycitidine (decitabine) exerted their activity through, at least in part, mechanisms independent of DNMT1 downregulation. Despite a global hypomethylating effect of 5AZA and decitabine, DNMT1 was not found to be necessary to maintain the hypermethylation of Krüppel-like factor 4 (KLF4), death associated protein 1 (DAPK1) and spastic paraplegia 20 ( SPG20). SPG20 was found to be a completely methylated target in all the tested cell lines, but not in peripheral blood mononuclear cells, suggesting its association with malignancy. The highest methylation was clustered upstream of the transcription starting site in a panel of 28 DLBCL cell lines and the results were unaffected by the silencing of DNMT1 expression. These data demonstrated the epigenetic regulation of SPG20 in lymphoid cells and identified a number of novel markers associated with lymphomas that deserve further investigation.