Methylation of SFRP promoters in AML cell lines and childhood patients with AML. (A) MSP (top) and real-time RT-PCR (bottom) analysis of SFRP promoter methylation and expression in 6 AML cell lines. M and U represent PCR products with primers specific for the methylated and unmethylated sequences, respectively. SFRP mRNA levels were normalized to GAPDH and compared with the mean expression levels of 3 normal BM samples. Results are presented as mean ± SE from triplicate assays. Note that SFRP promoter methylation was associated with mRNA down-regulation in these cell lines. (B) Semiquantitative RT-PCR analysis of SFRP expression in MV4-11 cells after treatment with the DNA demethylating agent 5′-AZA. Amplification of GAPDH cDNA served as internal control. (C) Representative MSP analysis of the 4 SFRP promoters in 5 childhood patients with AML (AML-1 to -5). The results of positive (LP1 and HeLa cell lines) and negative (NPB, normal peripheral blood) controls are also shown.