Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 Page 7 of 10 Figure 5 Detailed DNA methylation profile of PXR segment 2. Detailed methylation profile 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). Methylation of each CpG site was estimated by bisulfite sequencing on 7 or 8 individual DNA strands after subcloning. Open and closed circles represent unmethylated and methylated CpG sites, respectively. A ‘TATA’ indicates a putative TATA box. A curved arrow indicates a transcription start site. ‘HpyCH4IV’ indicates restriction site using the COBRA assay. colorectal tumorigenesis, independent of these genetic and epigenetic events. DNA methylation status of colon cancer tissue samples No or slight methylation of the PXR exon 3 region was detected in the normal colon tissue samples by direct sequencing and the COBRA assay. The levels of methy- lation in the cancer tissues were mostly higher than those in the paired adjacent normal tissues (Figure 6b). By contrast, the CpG-rich sequence of the PXR promo- ter was partially methylated in normal tissues, and the degree of methylation was decreased in the paired can- cer tissues (Figure 6c). The decreased level of the PXR promoter methylation suggested increased expression of the PXR gene during colorectal carcinogenesis. There were no differences in the clinicopathological findings between the colorectal cancers with PXR methylation and those without methylation. Discussion In the present study, 6 colon cancer cell lines showed heterogeneous mRNA expression profiles and were able to be classified into two groups with respect to their basal levels of the PXR/CYP3A4 transcripts (high expression cells, LS180 and LoVo; low expression cells Caco-2, HT29, HCT116, and SW48). These results are consistent with previous studies, in which LS180 and Caco-2 cells were characterized as PXR-sufficient and PXR-deficient cells, respectively [16,17]. Genetic polymorphisms in the regions that regulate transcription are often a major cause of inter-individual variability in the levels of transcripts. However, such polymorphisms have not been frequently observed in the human PXR or CYP3A4 genes, implying that certain epigenetic mechanisms are involved in the regulation of PXR and CYP3A4 expression. We found that the CpG- rich sequence within the PXR promoter region is methy- lated to different levels in high and low expression cells. Importantly, the magnitude of this promoter methyla- tion was inversely associated with the levels of PXR and CYP3A4 expression. Furthermore, the levels of the PXR and CYP3A4 transcripts in low expression cells were mostly restored when DNA methylation was reversed by treatment with 5-aza-dC. Although this CpG-rich sequence did not strictly satisfy the criteria for a CpG island, the most affected CpG sites were located in a highly restricted region (segments 1 and 2) and these CpG sites were proximal to several putative transcrip- tion factor binding sites (such as Sp1 and hepatocyte nuclear factor 4 alpha) [18-20]. Therefore, PXR gene expression is most likely transcriptionally regulated by methylation of these promoter CpG sites. CYP3A4 is transactivated by functional interplays with VDR-RXRa or PXR-PRMT1 [20-22]. CpG-island methy- lation of the VDR or PRMT1 promoter was not detected in these cell lines and the mRNA expression of VDR was not affected by 5-aza-dC treatment. These observa- tions imply that DNA methylation of PXR, but not VDR or PRMT1, resulted in downregulation of the CYP3A4 mRNA in these colon cancer cells. It is still uncertain whether re-expression of the CYP3A4 by 5-aza-dC treatment was due to the re- expression of some other genes than PXR. However, PXR must be a candidate for methylation and reduced expression of the PXR by promoter methylation, even if partially, contributes to downregulation of the CYP3A4. Indeed, several studies demonstrated that selective downregulation of the PXR by siRNA reduces the basal