We initially used RT–PCR to evaluate BNIP3 expression in a panel of 20 haematopoietic tumour cell lines (). Expression of BNIP3 was readily detectable in normal bone marrow and lymph nodes, as well as in 15 of the cell lines tested. Of the remaining five lines, two (TALL1 and Raji) expressed only negligible levels of BNIP3, and three (SupT1, PEER and KHM1B) expressed none at all. The earlier findings that under hypoxic conditions expression of BNIP3 can be induced by the transcription factor HIF-1α
; Guo et al, 2001
; Sowter et al, 2001
) prompted us to evaluate the extent to which expression of BNIP3 in haematopoietic tumour cell lines could be induced by hypoxia. Using real-time PCR with cDNA prepared from Jurkat and SupT1 cells incubated under hypoxic conditions, we found that hypoxia leads to increased expression of BNIP3 in Jurkat cells but not in SupT1 cells (). When we carried out a Western blot analysis to determine whether the absence of BNIP3 expression in SupT1 cells was caused by impaired HIF-1α
function, we found that hypoxia induced HIF-1α
expression in both Jurkat and Supt1 cells; thus, the absence of BNIP3 was not caused by a HIF-1α
Figure 1 Expression of BNIP3 in haematopoietic tumour cell lines. (A) A panel of haematopoietic tumours cell lines was analysed for BNIP3 expression by RT–PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for the integrity (more ...)
Using Blast (http://www.ncbi.nlm.nih.gov/BLAST/
) and CpG island Searcher (http://www.uscnorris.com/cpgislands/
), we found that the 5′ region of BNIP3 contains a CpG-rich region that satisfies the criteria for a CpG island (CpG
GpC=0.65, GC%=55%; ). Then, to explore the role of BNIP3 methylation in haematopoietic tumours, we first used COBRA, a semiquantitative methylation analysis, to examine the methylation status of the region around the transcription start site in a panel of haematopoietic tumour cell lines. Aberrant methylation of BNIP3 was detected in all five cell lines (SupT1, PEER, TALL1, Raji and KHM1B) that either did not express BNIP3 at all or expressed it only to a negligible degree (). By contrast, methylation of BNIP3 was not detected in cell lines that expressed BNIP3, although NAGL1 showed a low level of methylation but expressed BNIP3, nevertheless. Notably, expression of BNIP3 could be restored in the five methylated cell lines by treating them with the methyltransferase inhibitor 5-aza-dC, which strongly suggests that BNIP3 was epigenetically silenced by methylation in these cells ().
Figure 2 Analysis of BNIP3 methylation in a panel of haematopoietic tumour cell lines. (A) CpG island of BNIP3; CpG sites are shown by vertical bars. The region analysed by COBRA is shown by a solid bar. Exon 1 is shown by a solid box on a solid line. The transcription (more ...)
To examine the methylation status of each CpG dinucleotide within the BNIP3 CpG island, bisulphite-sequencing was carried out in six cell lines (BALL1, CCRF-CEM, KHM1B, SupT1, PEER and TALL1). In BALL1 and CCRF-CEM cells, which COBRA showed to be unmethylated, virtually no methylation was detected within the region analysed (). Conversely, in SupT1, PEER and TALL1 cells, which COBRA showed to be highly methylated, virtually all of the CpG sites within the region examined were methylated (). KHM1B cells showed a more a heterogeneous methylation pattern ().
Figure 3 Bisulphite-sequencing of BNIP3. (A) Representative results of bisulphite-sequencing. Amplified PCR products were purified from gels and then directly sequenced. The cell lines examined are shown below the column; U, unmethylated CpG sites; M, methylated (more ...)
To determine the extent to which the silencing of BNIP3 affected hypoxia-mediated apoptosis, we examined the effect of 5-aza-dC on the incidence hypoxia-mediated cell death among TALL1 cells, which do not otherwise express BNIP3 as a result of the gene's methylation (). We found that hypoxia readily induced cell death among control Jurkat cells, which do express BNIP3. Among TALL1 cells, pretreatment with 5-aza-dC had no significant effect on the incidence of cell death under normoxic conditions. Under hypoxic conditions, by contrast, significant numbers of apoptotic cells were detected following pretreatment with 5-aza-dC, which is indicative of the role played by epigenetic silencing of BNIP3 in protecting cells from hypoxia-mediated apoptosis.
Methylation-dependent gene silencing is reportedly associated with altered chromatin structure involving deacetylation of histone (Jones and Baylin, 2002
). Therefore, to assess the role of histone deacetylation in the silencing of BNIP3, we treated the methylated SupT1 cell line with 5-aza-dC and/or TSA, a histone deacetylase inhibitor (). Treating the cells with a low dose (0.2μM
) of 5-aza-dC induced only a small amount of BNIP3 expression. However, addition of TSA (300
) to the 5-aza-dC had a synergistic effect that markedly increased gene expression, indicating a role for histone deacetylation in silencing of BNIP3 gene expression. We then examined the acetylation status of the BNIP3 CpG island in more detail using ChIP assays with anti-histone H3 antibody and found that acetylation of histone H3 in the region around the transcription start site correlated directly with gene expression and inversely with DNA methylation ().
Figure 5 The role of histone deacetylation in silencing BNIP3 gene expression. (A) Effects of DNA methyltransferase and/or histone deacetylase inhibitor on the expression of BNIP3. SupT1 cells, which show BNIP3 methylation, were treated with 0.2μ (more ...)
Finally, we determined the extent to which BNIP3 is methylated in primary tumours by examining the methylation status of BNIP3 in a panel of primary leukaemia specimens. Using COBRA, methylation of BNIP3 was detected in five of 34 (15%) ALL specimens, in six of 35 (17%) AML specimens and in three of 14 (21%) multiple myeloma specimens (). These findings were then confirmed by bisulphite-sequencing of the amplified PCR products. All of the cases found to be methylated using COBRA were found to be methylated at all of the CpG sites examined ().
Figure 6 Analysis of BNIP3 methylation in a panel of primary haematopoietic tumours. (A) Methylation of BNIP3 examined using COBRA. Tumour type is shown above the gels. Percentages of methylated alleles were calculated by densitometry and are shown below the gels. (more ...)