Inverse correlation between FBXW7/hCDC4-β mRNA expression and promoter methylation in primary breast tumor specimens. (a) Representative analysis of FBXW7/hCDC4-β promoter methylation in normal breast (N), breast tumor specimens (T), immortalized breast epithelial cells (IME) and a cancer cell line (HeLa). Genomic DNA was incubated with (+) or without (-) McrBc enzyme and the promoter was PCR amplified (1.6 kb). The cleavage pattern indicates differential methylation in tumor samples (T1, T2; methylated and T3, T4; unmethylated) and a lack of methylation normal breast (N1, N2). (b) Relationship between FBXW7/hCDC4-β mRNA expression levels and promoter methylation in primary breast tumors. Samples were stratified by relative methylation status (UM; unmethylated group, N = 68, M; methylated group, N = 71, see materials and methods for classification of methylation). FBXW7/hCDC4-β expression levels (normalized to GAPDH mRNA) were lower in the methylated group compared to the unmethylated group (Mann-Whitney U test, P = 0.0002). Bars from each box extend to largest and smallest expression levels. Outliers are plotted as asterisks (*).