Suppression of RAR-β
expression by BPDE. (A) Northern blotting. Esophageal cancer cell lines TE-3 and TE-12 were grown and treated with 1 μM BPDE for up to 24 h. RNA was then isolated from the cells and subjected to Northern blotting analysis of RAR-β2 expression. (B) MSP. Esophageal cancer cell lines TE-3 and TE-12 were grown and treated with 1 μM BPDE for up to 24 h. Genomic DNA was then isolated from the cells and subjected to PCR analysis of RAR-β2 gene promoter methylation. M, methylated RAR-β2 gene promoter; U, unmethylated RAR-β2 gene promoter. (C) Sequencing histogram (matching GenBank accession number X56849) of a partial RAR-β2 gene promoter. TE-3 cells were treated with or without 1 μM BPDE for 12 h, and genomic DNA was extracted and subjected to MSP analysis. The PCR product was then cloned into a TA cloning vector. The clones carrying RAR-β2 gene promoter were then sequenced in our institutional DNA sequencing facility. Compared with the controls, BPDE-treated cells showed two BPDE-methylated sites. Note: Bisulfite converted all C to T, but methylated C cannot be converted, which is the principle of the MSP assay.