Germline mutations of the putative tumor suppressor gene APC are associated in high frequency with the familial adenomatous polyposis, predisposing the patients to colorectal neoplasia. Similarly, sequence analyses have revealed that in more than half of patients with sporadic colorectal carcinoma or adenoma, the APC gene was mutated. By employing genomic sequencing, i.e., base-specific analysis of methylated cytosines, we show here that the promoter region of the APC gene is heavily methylated at CpG sites in patients with colorectal carcinoma in comparison with normal colonic mucosa and premalignant adenomas. Our results suggest that cytosine methylation of the regulatory sequences of the APC gene could be involved in the progression of human colorectal cancer. Int. J. Cancer 70:644-648, 1997.r 1997 Wiley-Liss, Inc.The APC (adenomatous Polyposis coli) gene has been convincingly linked to the development of colorectal cancer. The human APC gene located at 5q21 (Cunningham and Dunlop, 1994) and encoding a large protein interacting with the cell adhesion protein b-catenin (Munemitsu et al., 1995) has been shown to be mutated in the germline of patients with familial adenomatous polyposis (Cunningham and Dunlop, 1994). Similar somatic mutations of the APC gene have also been found at very early stages of tumorigenesis in the majority of patients with sporadic colorectal cancer or adenoma (Powell et al., 1992). Most of the mutations of the gene result from premature termination of the translation leading to a truncated protein that can no longer associate with the b-catenin (Powell et al., 1992;Munemitsu et al., 1995). The close association of the mutations of the APC gene with the early stages of colorectal tumorigenesis makes it a strong candidate for a tumor suppressor gene.In addition to the well-documented mutations of the APC gene, the development and progression of colorectal cancer appear to be associated with mutations of the tumor suppressor gene p53, the DCC (deleted in colon cancer) gene and the oncogene ras (Cunningham and Dunlop, 1994). The progression of colorectal carcinogenesis likewise includes epigenetic changes such as altered DNA methylation. Restriction enzyme analyses with a number of gene probes indicated that DNA from both benign polyps and malignant colorectal carcinomas is substantially hypomethylated (Goelz et al., 1985), and quantitative determination of the 5-methylcytosine content of DNA revealed significant hypomethylation with no difference between benign and malignant tumors (Feinberg et al., 1988). Specific hypomethylation at a CCGG site of the third exon of the c-myc oncogene was also shown to be associated with human colorectal neoplasia (Sharrard et al., 1992). Despite the overall genomic hypomethylation, there appears to be regional hypermethylation associated with human colorectal tumorigenesis. The calcitonin gene at 11p15, a locus residing in a number of putative tumor suppressor genes (Ichikawa et al., 1992;Loh et al., 1992), was reported to be hypermethylated in human col...