Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 Page 5 of 10 correlation of the levels of the transcripts between PXR (exon 5-6) and PXR (exon 1a-2) throughout all analyses. Increased mRNA expression by 5-aza-dC treatment In order to determine whether DNA methylation is involved in the transcriptional regulation of these genes, the 6 cell lines were treated with DNA demethylating agent (5-aza-dC). The treatment with 5-aza-dC induced a clear increase in CYP3A4 (28- to 116-fold) and PXR (3- to 10-fold) transcripts in a dose-dependent manner in the low expression cells, but not in the high expres- sion cells (Figure 3). In particular, the CYP3A4 tran- script of the low expression cells eventually reached the levels seen in the high expression cells by 5-aza-dC treatment. In contrast, 5-aza-dC had no marked effect on VDR expression in any of the cell lines (0.5- to 1.2-fold increase). These results suggested that DNA methylation is involved in the regulation of CYP3A4 and PXR, but not VDR, in the low expression cell lines. DNA methylation status of the colon cancer cell lines The bisulfite direct sequencing detected no DNA methylation in the VDR or PRMT1 promoter sequences in the 6 cell lines. Partial methylation of the CYP3A4 5’- distal region and full methylation of the PXR exon 3 region were equally observed among the 6 cell lines. Therefore, the different expression profiles of the two groups cannot be explained by methylation of these sequences. On the other hand, the CpG-rich sequence of the PXR promoter showed a different methylation status among the 6 cell lines. Interestingly, the degree of methylation of the PXR promoter (segments 1 and 2) in Figure 3 Expression profile of colon cancer cell lines after 5-aza-dC treatment. Levels of PXR, VDR and CYP3A4 transcripts in (a), LS180; (b), Caco-2; (c), HCT116; (d), LoVo; (e), HT29; and (f), SW48 cells. Cells were treated with 5-aza-2’-deoxycytidine (0, 0.5 or 5 μM). The vertical axis indicates a relative transcript level (ratio to cells without 5-aza-dC treatment). Numbers in italics indicate relative transcript levels that were over the maximum scale on the vertical axis. The transcripts indicated by * (PXR exon 5-6 transcript in SW48 cells and CYP3A4 transcript in HCT116 cells) were not detected and we estimated the minimum detectable levels among gradually diluted calibration samples.