ISOLATION AND CHARACTERIZATION OF GENES CODING FOR CARCINOMA-ASSOCIATED ANTIGENS

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Monoclonal antibodies (MAbs) have defined several antigens associated with human carcinomas. Two of the most widely studied antigens are carcinoembryonic antigen (CEA) and TAG-72. CEA is a 180,000-kd glycoprotein and TAG-72 is a high- molecular-weight mucin. Recent studies have demonstrated that CEA belongs to a multigene family related to the immunoglobulin supergene family. These include CEA, normal cross-reacting antigen (NCA), biliary glycoprotein (BGP), and human pregnancy-specific beta 1-glycoprotein (SP1). A 550-base pair CEA probe derived from cloned complementary DNA was used to carry out Southern analysis of the DNA isolated from normal and colon tumor cell lines. At high stringency, the CEA probe detected seven BamHI fragments in all DNAs analyzed. Results of the Southern analysis demonstrate no amplification or rearrangement of the CEA genes in tumor cells. Methylation-sensitive restriction endonucleases, Hpa(II) and Hha(I), were used to compare the degrees of methylation of the CEA family of genes in normal and colon tumor cell lines. The results demonstrate that the CEA family of genes exists in a state of hypermethylation in normal cells. In contrast, the CEA gene(s) are relatively hypomethylated in the tumor cell lines, suggesting a correlation between the state of methylation and degree of expression of the CEA gene(s). Using a CEA cDNA probe, we have investigated the phenomenon of up-regulation of CEA expression in human tumor cell lines by recombinant human gamma-interferon (Hu-IFN-gamma). An increase in MAb binding was shown to be accompanied by a 6- to 11-fold increase in the steady-state levels of three CEA transcripts with sizes of 4.2, 3.5, and 2.8 kb. Studies are also in progress to identify and characterize the gene(s) encoding the TAG-72 mucin antigen. A number of mucin genes representing several tissue types have been cloned. These include the genes encoding the epitopes for breast cancer, HMFG2, DF3 and H23 and the pancreatic antigen DuPAN-2. Nucleic acid sequence analysis of these isolated genes has shown them to be identical and that the difference resides in the sugar epitopes detected by the MAb. The dominant feature of the breast and pancreatic mucin clones was an extremely GC-rich, 60-bp tandem repeat sequence that encoded a repeating peptide unit rich in serine, threonine, and proline.