The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias

Abstract

Background: The Ras-association family (RASSF) of tumour suppressor genes (TSGs) contains 10 members that encode proteins containing Ras-association (RA) domains. Several members of the RASSF family are frequently epigenetically inactivated in cancer, however, their role in leukaemia has remained largely uninvestigated. Also, RASSF10 is a predicted gene yet to be experimentally verified. Here we cloned, characterised and demonstrated expression of RASSF10 in normal human bone marrow. We also determined the methylation status of CpG islands associated with RASSF1-10 in a series of childhood acute lymphocytic leukaemias (ALL) and normal blood and bone marrow samples.

Results: COBRA and bisulphite sequencing revealed RASSF6 and RASSF10 were the only RASSF members with a high frequency of leukaemia-specific methylation. RASSF6 was methylated in 94% (48/51) B-ALL and 41% (12/29) T-ALL, whilst RASSF10 was methylated in 16% (8/51) B-ALL and 88% (23/26) T-ALL. RASSF6 and RASSF10 expression inversely correlated with methylation which was restored by treatment with 5-aza-2'deoxycytidine (5azaDC).

Conclusion: This study shows the hypermethylation profile of RASSF genes in leukaemias is distinct from that of solid tumours and represents the first report of inactivation of RASSF6 or RASSF10 in cancer. These data show epigenetic inactivation of the candidate TSGs RASSF6 and RASSF10 is an extremely frequent event in the pathogenesis of childhood leukaemia. This study also warrants further investigation of the newly identified RASSF member RASSF10 and its potential role in leukaemia.

Figure 1

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 = TaqI 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 TaqI 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.

Figure 2

Characterisation of the RASSF10 gene. A, RT-PCR analysis showing expression of RASSF10 in normal human bone marrow. B, The 5' region of the RASSF10 transcript was amplified from normal human bone marrow using 5'RACE. Arrowed is the major product of 5'RACE which was cloned and sequenced to determine the transcription start point and open reading frame of the RASSF10 gene. C, Schematic summarising the structure of the RASSF10 gene and the open reading frame encoding a shortened protein of 507amino acids that does not contain the in silico predicted N-terminal 108 amino acids that are divergent from the RASSF7–9 proteins. D, ClustalW alignments of RASSF7–10 proteins including both the in silico predicted 615 amino acid RASSF10 [GenBank:NM_001080521] and the shorter 507 amino acid RASSF10 confirmed in this study suggesting the divergent N-terminal 108 amino acids is not likely to be part of the RASSF10 protein.

Figure 3

RASSF10 is hypermethylated in childhood leukaemias. A, COBRA analysis showing methylation status of RASSF10 in leukaemia cell lines and normal bone marrow, normal blood, primary T-ALL and primary B-ALL. No methylation was observed in normal blood or bone marrow (BM). U = undigested PCR product; T = TaqI digested PCR product. B = BstUI digested PCR product. B, Direct bisulphite sequencing of normal blood, normal bone marrow, leukaemia cell lines and primary T-ALL leukaemias. Top shows a schematic of the region amplified by COBRA PCR (black bar) relative to the RASSF10 gene and CpG island region (hatched bar). Each horizontal line represents the total DNA amplified from the sample whilst the circles represent single CpG dinucleotides. Filled black circle represents a completely methylated CpG dinucleotide, open circle represents a completely unmethylated CpG dinucleotide and filled gray circle represents a partially methylated CpG dinucleotide. The dashed vertical line indicates the CpG dinucleotides within TaqI (TCGA) and BstUI (CGCG) restriction sites used to assay for methylation. C, Methylation of the RASSF10 CpG island correlates with loss or downregulation of RASSF10 expression. The partially methylated cell line THP-1 and the completely methylated cell line SUP-T1 were cultured in the presence or absence of 5azaDC and TSA. RT-PCR analysis showed loss or downregulation of RASSF10 expression correlates with the methylation status of the RASSF10 CpG island (A and B). RASSF10 expression is restored following 5azaDC and TSA treatment. GAPDH was used as a control for RNA integrity and equal loading. Ct = control mock treated; RT = reverse transcriptase.