Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 Page 8 of 10 Figure 6 Methylation status of the PXR promoter and exon 3 regions, as detected by the COBRA assay. Unmethylated and methylated DNAs are shown as U and M, respectively. PCR products that were not cut by the restriction enzymes are shown as UC. (a) PXR promoter methylation was examined in the 6 cell lines after treatment with 5-aza-2’-deoxycytidine (0, 0.5 or 5 μM). (b) Methylation status of the CpG island of the PXR exon 3 region in 6 of the 18 primary colorectal cancers (numbers are those for particular cases). DNA samples from normal and cancerous tissue are shown as N and C, respectively. Note that a high degree of methylation was detected in cancerous, but not normal, tissue. (c) Methylation status of the CpG-rich sequence of the PXR promoter region in 8 primary colorectal cancers. Note that a lower degree of methylation was detected in cancerous tissue compared to normal tissue. Table 2 Profile of Microsatellite instability (MSI), mismatch repair (MMR) deficiency and promoter methylation in 6 colon cancer cell lines MMR deficiency* Methylation** Cell lines MSI MLH1 MSH2 MLH1 p16 PXR LS180 LoVo Caco-2 HT29 HCT116 SW48 + + - - + + + - - - + + - + - - - - M U ? U U M ? M ? M M M U U M M M M Summary of previous studies [27,28] and the present study (PXR methylation). *Mismatch repair deficiency was associated with mutations or defects in mRNA transcripts. **Methylation status was defined as unmethylated (U) or methylated (M). level of the CYP3A4 transcripts in a dose-dependent manner [23]. CpG islands in the exon 3 region were fully methylated throughout the cancer cell lines and most cancer tissues. Even after treatment of the cell lines with 5-aza-dC, no increase in the PXR mRNA levels was observed in the high-PXR expressing cell lines, LS180 and LoVo. This strongly suggests that in human colon cancer cells, the methylated CpG islands in the exon 3 play a much less role in the epigenetic regulation of PXR, instead, promo- ter methylation plays a pivotal role in its regulation. In contrast, Misawa et al. previously demonstrated a dis- tinct methylation profile of neuroblastoma cells, in which mRNA expression of the PXR splicing variant (exon 1a-2) was specifically regulated by the methylation of the exon 3 region rather than promoter methylation [9]. We