Differential expression of FBXW7/hCDC4-β mRNA and promoter methylation in tumor cell lines. (a) Semi-quantitative RT-PCR analysis of FBXW7/hCDC4 isoforms, left, in normal breast, immortalized mammary epithelial cells and breast cancer cell lines, right, in human cancer lines from different origins. GAPDH was amplified as an internal control. (b) Schematic map of the FBXW7/hCDC4-β promoter region around the transcription start site (TSS). CpG dinucleotides are depicted. PCR primers used for amplification of the promoter are indicated as arrows (FP1 and RP1). The translational start codon (ATG), centromere (Cen) and telomere (Tel) are marked. (c) real-time PCR showing the relationship between FBXW7/hCDC4-β expression (top) and promoter methylation (bottom); Methylation is presented as the percentage of methylated CpG sites based on bisulphate sequence analysis of genomic DNA from different cancer cell lines. The degrees of methylated cytosines or unmethylated cytosines are indicated by black or white boxes, respectively. (d) Representative gel analysis of PCR product (1.6 kb) from McrBc digested (+) genomic DNA in comparison with undigested (-) control DNA. Note the lack of amplification of McrBc digested DNA from methylated cell lines (HeLa and DLD1) in comparison to unmethylated cell lines (IME and T-ALL).