The CD133 promoter is hypomethylated in malignant endometrial tumors. Panel A is a schematic depicting the methylation status of the Regions 1-3 of the CD133 promoter in normal endometrium (n = 3) and malignant tumors (samples T7-T9). Ten DNA clones were analyzed from each sample and individual CpG sites are represented by circles. The methylation of any specific CpG sites is indicated by a filled circle. Panel B is a histogram illustrating the relative percentage of methylated CpG sites detected in the analysis shown in panel A. The percentage of methylated CpG sites in Region 1 of the CD133 promoter was significantly decreased (p < 0.01) in malignant endometrial tumors compared to benign endometrium. CD133 mRNA levels in a subset of the same tumor (samples T8 and T9) and normal endometrium (N1 and N2) samples were analyzed by RT-PCR (panel C). Expression of CD133 mRNA was increased in the tumor samples relative to the benign endometrium, consistent with the methylation patterns depicted in panel A. β-actin expression was used to confirm equivalent RNA concentrations in all samples.