Correspondence of MCC gene promoter hypermethylation with loss of RNA or protein expression in cell lines (a and b) and primary colorectal cancers (d and e). (a and d) MSP using primers specific for the methylated (M, 99 bp) and unmethylated (U, 94 bp) MCC promoter and control PCR using MYOD1 primers in bisulfite-treated DNA samples. (b and c) cDNA amplification from colorectal cancer cell lines with primers for MCC and housekeeping genes ALAS1 and GAPDH. Addition of Aza to the control (Con) medium reversed MCC expression in HCT-15, KM12SM and LS411N cell lines, but not in Caco-2 cells, which are unmethylated. (e) Western blotting of primary colorectal cancer (C) and matching normal (N) tissue specimens. Loss or reduced MCC protein expression is evident in patients (P) 3, 4, 6 and in the cell line (CL) HCT-15.