Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 expression is also observed in human intestine. Interest- ingly, a report using paired tissue samples of livers and small intestines indicated no observed correlation between the hepatic and small intestine CYP3A4 expres- sion levels [5]. Another research group reported that a majority of CYP3A4 resided in the proximal region of the small intestine, and that the CYP3A4 protein levels decreased dramatically in the distal small intestine [6]. These observations suggest the CYP3A4 is regulated through tissue specific epigenetic regulation in normal tissue. The aim of the present study is to find possible and not yet fully elucidated mechanisms which regulate heterogeneous basal PXR and CYP3A4 expression and activity in cancerous tissues as well. Indeed, several stu- dies previously found a fraction of genes that exhibited inter-individual differences in transcript levels associated with DNA methylation status [7,8]. DNA methylation of the CpG-rich sequence around exon 3 of the PXR gene is involved in the epigenetic regulation of PXR in human neuroblastoma [9]. How- ever, epigenetic regulation of PXR and CYP3A4 in human gut is poorly understood. In order to determine whether epigenetic mechanisms function in PXR and CYP3A4 regulation and intestinal metabolism, we exam- ined DNA methylation and mRNA expression of several candidate genes on the PXR/CYP3A4 regulatory path- way in human colon cancer cell lines and tissues. Methods Cell lines and tissue samples Human colorectal cancer cell lines LS180, Caco-2, HT29, HCT116, DLD-1, LoVo, SW48 and SW620 were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). LS180 cells were cultured in E-MEM medium (Invitrogen Corp., Carlsbad, CA) at 37°C under an atmosphere of 5% CO2. The other cells were cultured under conditions described elsewhere [10]. Eighteen pairs of cancerous and adjacent normal mucosa were excised from surgical specimens of color- ectal cancers. The cancerous and normal epithelia were separated from stroma using crypt isolation [11]. All samples were selected from the same series of cancers as we used in a previous study [10]. All 18 patients with colorectal cancers did not receive chemotherapy before surgical resection. The study protocol was approved by ethics committee of Iwate Medical University (molecular analysis of gastrointestinal tumors and the surrounding mucosa; reference number, H21-140). Treatment with 5-aza-2’-deoxycytidine LS180, LoVo, Caco-2, HCT116, HT29 and SW48 cells were seeded at a concentration of 1 × 105 cells on a 100 mm dish. The next day, treatment of cells with 0, 0.5 or 5 μM 5-aza-2’-deoxycytidine (5-aza-dC) (Sigma Page 2 of 10 Chemical, St. Louis, MO) was started, and 5-aza-dC was removed by changing the medium 24 h later. The cells were harvested 4 days after removal of 5-aza-dC for DNA and RNA extraction. Quantitative real-time PCR analysis of basal CYP3A4, PXR and VDR mRNA levels Total RNA extraction, cDNA synthesis and real-time PCR were carried out on the cells prepared above, by the same methods as described previously [10]. mRNA levels of the CYP3A4 (exon 3-4, Hs01546612_m1), PXR (exon 5-6, Hs00243666_m1) and vitamin D receptor (VDR) (exon 10-11, Hs01045840_m1) were evaluated by TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). In addition, the mRNA level of the PXR splicing variants (exon 1a-2) was also examined by SYBR Green assays using the following primers: 5’-GATTGTTCAAAGTG- GACCCC-3’(forward) and 5’-TCCAGGAACAGACTC TGTGT-3’. The mRNA level of the above target genes was normalized to b-actin mRNA [10]. All samples were analyzed in duplicate and average quantities of the gene transcripts were used for calculation. Deviation of the mRNA level of each sample was within 7% of the average. DNA methylation analysis of the 6 colon cancer cell lines and 18 colon cancer samples We found CpG islands within the PXR (around exon 3 region), VDR (promoter region) and protein arginine methyltrasferase 1(PRMT1) (promoter region) genes using the CpG Island Searcher program [12,13]. A CpG island was also detected in the 5’ untranslated region (UTR) of the CYP3A4 gene (approximately 25 kb distal to the transcription start site). We also found a CpG- rich sequence in the promoter region of the PXR gene, although this sequence did not strictly satisfy the criteria for a CpG island [13,14]. The location of all CpG sequences examined in this study are shown in Figure 1. Genomic DNA extracted from the 6 cell lines and the 18 pairs of normal and colon cancer tissue samples was modified by sodium bisulfite, and then each segment including a CpG island or CpG-rich sequence was amplified by PCR and subjected to direct sequencing. The methylation status of the PXR promoter sequence was also estimated by bisulfite sequencing on at least 7 individual DNA strands after subcloning of PCR pro- ducts into the pCR4-TOPO vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen). A relative methylation level of the PXR promoter was visually determined by the density of each HpyCH4IV-digested band using combined bisulfite restriction analysis (COBRA) [15]. The methylation status of the PXR exon 3 region was also examined by the COBRA assay using an HhaI digestion. All primer sequences are listed in Table 1.