Figure 1.

BRMS1 promoter activity, mRNA and protein levels are regulated by promoter methylation. (A) Schematic illustration of the CpG island in BRMS1 promoter. Arrow, transcription start site (TSS); empty box, exon 1; small bars, positions of primers for methylation-specific PCR (MSP). (B) Lung cancer (H157 and A549) and NHBE cells were treated or not with 5-Aza (5 μM) for 5 days. Genomic DNAs were extracted. Quantitative MSP was done by using methylated (M)- and unmethylated (U)-specific primers in the CpG island of BRMS1 promoter. (C) H157, A549 and NHBE cells were treated or not with 5-Aza (5 μM) for 5 days; (lower panels) BRMS1 mRNA levels determined by QRT–PCR; (upper panel) BRMS1 protein levels detected by western blot. (D) Methylation inhibits BRMS1 promoter activity. (Left panel) 20 μg BRMS1 promoter reporter was treated with CpG methyltransferase M. SssI (20 U) and digested with BglII/HindIII, as described in Materials and methods. The completeness of methylation was visualized by 2% agarose gel electrophoresis after digestion with BstUI. (Centre panel) pGL3 basic empty vector and pGL3–BRMS1 were treated with SssI or buffer only, as described above, and then were transiently transfected into 293T cells. Luciferase activity was determined. (Right panel) pGL3–BRMS1 was treated with SssI or buffer only, as described above, and then was transiently transfected into H157 and H1299 NSCLC cells. Luciferase activity was determined.