Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours

Abstract

Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2 0 -deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5 0 region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5 0 CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours.

Selective silencing of the hypoxia-inducible factor 1 target gene BNIP3 by histone deacetylation and methylation in colorectal cancer

“…A number of previous studies have shown that BNIP3 has a large CpG island spanning its promoter and that methylation of this region plays an important role in the transcriptional silencing of BNIP3 in a number of cancer cell types including pancreatic and gastric tumours (Okami et al, 2004;Murai et al, 2005b). Our results agree with these studies and show that the BNIP3 promoter is methylated in half of the CRC lines that do not hypoxically upregulate BNIP3.…”

“…Our data support and add to two recent reports that find BNIP3 is silenced by DNA methylation in pancreatic and gastric cell lines (Okami et al, 2004;Murai et al, 2005b) and goes further in also highlighting a prominent role for histone de-acetylation in silencing BNIP3. Other mechanisms to by-pass the effects of BNIP3 have been shown, for example, growth factor signalling can protect against BNIP3-mediated cell death in breast cancer cell lines (Kothari et al, 2003).…”

“…However, our study also demonstrates the significance that histone deacetylation can have on the transcriptional status of BNIP3. Whereas previous reports have found that restoration of BNIP3 expression can be achieved by TSA treatment following pre-treatment with the demethylating agent 5aza-dC (Murai et al, 2005b), our study shows for the first time that TSA alone can restore BNIP3 expression in CRC cell lines. In addition, histone deacetylation can be a predominant mechanism of silencing in the presence of promoter methylation as TSA alone can restore BNIP3 expression in SW620 and HCA7, which are also methylated at the BNIP3 promoter.…”

“…PCR was carried out using BNIP3 ChIP primers as described previously (Murai et al, 2005b). PCRs were carried out in triplicate on duplicate independent samples.…”

The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death

“…78 Promoter methylation and downregulation of bnip3 expression is also observed in essential thrombocythemia patients and hematopoietic tumors compared with normal cells. 79,80 Methylation was detected in 15% of primary leukemia samples, 17% of primary acute myloid leukemia samples and 21% of primary MM samples. 80 Normal matched tissue samples from patients with pancreatic, colorectal and gastric tumors did not show any methylation of the bnip3 promoter.…”