RASSF6 is hypermethylated in childhood leukaemias. A, COBRA analysis showing methylation of RASSF6 in leukaemia cell lines. No methylation was observed in normal blood or bone marrow (BM). The methylation index (MI) for each is shown below the gel image. U = undigested PCR product; T = Taq I digested PCR product. B, COBRA analysis of B-ALL and T-ALL childhood leukaemias. C, Cloning and bisulphite sequencing of the RASSF6 CpG island. Top left shows a schematic of the region analysed relative to the RASSF6 gene. Exon 1 of RASSF6A [GenBank:NM_177532] and RASSF6B [GenBank:NM_201431] is shown relative to the CpG island region (hatched bar), the region amplified by COBRA PCR (black bar) and the location of COBRA primers (F, FN and R). The remaining 5 panels show representative data from primary B-ALL and T-ALL leukaemias 27 and 6 respectively with leukaemia cell lines CEM, U937 and THP-1. Each horizontal line represents an individual allele whilst the circles represent single CpG dinucleotides. Filled circle represents a methylated CpG dinucleotide whereas an open circle represents an unmethylated CpG dinucleotide. Indicated by the arrow is the transcription start site of RASSF6A. The dashed vertical line indicates the CpG dinucleotide within the Taq I restriction site (TCGA) used to assay for methylation. D, Methylation of the RASSF6 CpG island correlates with loss or downregulation of RASSF6 expression. Methylated cell lines CEM, THP-1 and the unmethylated cell line U937 were cultured in the presence or absence of 5azaDC and TSA. RT-PCR analysis showed loss or downregulation of RASSF6 expression correlates with the methylation status of the RASSF6 CpG island (A and C). RASSF6 expression is restored following 5azaDC and TSA treatment. GAPDH was used as a control for RNA integrity and equal loading. E, RASSF6 protein expression is lost or downregulated in methylated leukaemia cell lines. We used an antibody towards human RASSF6 protein to investigate expression before and after 5azaDC and TSA treatment. α-Tubulin was used as a control for equal loading.