RUNX3 P2 promoter was unmethylated in AML. (A) Schematic diagram of the human RUNX3 gene structure. ■ and □ represent coding and noncoding regions, respectively. The location of the RUNX3 P1 and P2 promoters is shown. Below the diagram are representative results of MSP analysis of 3 consecutive regions in the RUNX3 P2 promoter in AML samples. The size of each analyzed region is indicated. Lanes: 1, MV4-11; 2, Kasumi-1; 3, CMK; 4, ME-1; 5-6, 2 diagnostic childhood AML BM; 7-8, 2 diagnostic adult AML BM; 9, normal BM; 10, Reh (positive control). M and U represent PCR using primers specific for the methylated and unmethylated sequences, respectively. (B) Results of nucleotide sequencing of MSP products from regions 1 and 2 in 3 AML cell lines (MV4-11, Kasumi-1, and CMK), 3 diagnostic childhood AML BM, a normal BM, and the 697 cell line. Each row of circles represents one PCR clone. Open and filled circles indicate unmethylated and methylated CpG dinucleotides, respectively. (C) MSP (top panel) and RT-PCR (bottom panel) analysis of RUNX3 P2 methylation and expression in 4 ALL and 2 CML cell lines. Note that RUNX3 P2 methylation is associated with RUNX3 silencing in these leukemic cell lines. (D) Treatment with the DNA demethylating agent 5′-AZA induced RUNX3 mRNA expression in the methylated Reh cell line but not in unmethylated Kasumi-1 and CMK cell lines. RUNX3 mRNA levels were determined by real-time RT-PCR and normalized using GAPDH. Results are presented as relative RUNX3/GAPDH level by comparing the normalized RUNX3 level in treatment groups with that in the respective control group (DMSO treated).