Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 Page 6 of 10 the high expression cells (LS180 and LoVo) was lower than that in the low expression cells (Caco-2, HT29, HCT116 and SW48) (Figure 4). Therefore, the details of the methylation status of the PXR promoter were esti- mated on individual DNA strands after subcloning (Figure 5). We found that an HpyCH4IV site within seg- ment 2 was a suitable marker for the COBRA assay to assess PXR promoter methylation, because the degree of methylation of this site showed inverse correlation with the levels of CYP3A4 and PXR expression. The COBRA assay demonstrated that the treatment with 5-aza-dC resulted in decreased amounts of methylation of the PXR promoter in a dose-dependent manner in the low expression cells (Figure 6a). Therefore, the magnitude of methylation was likely to be associated with the decreased levels of CYP3A4 and PXR gene expression in all 6 cell lines. These results suggest that the PXR gene was tran- scriptionally silenced by methylation of the promoter CpG sites and that the downregulation of the PXR protein resulted in decreased expression of the CYP3A4 gene. The PXR promoter methylation was not associated with the profile of microsatellite instability (MSI) or other methylated genes (Table 2). This suggested that altered PXR methylation was accumulated during Figure 4 DNA methylation profile of PXR as detected by direct sequencing. Methylation status of the PXR gene in the 6 colon cancer cell lines and a cancerous tissue sample (255C) and its paired adjacent normal tissue sample (255N) detected by bisulfite direct sequencing. Open and closed circles represent unmethylated and fully methylated CpG sites, respectively. Half-closed circles represent partially methylated CpG sites. The methylation status of the CpG island (around exon 3) and CpG-rich promoter sequence (segments 1, 2, 3 and 4) are shown in the upper and lower panels, respectively.