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Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor y chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.Carcinoembryonic antigen (CEA), a large cell membrane glycoprotein of molecular weight about 180,000 (40), is produced in a high proportion of human tumors arising at the most common sites including colon, breast, and lung (37). CEA is also produced during human embryogenesis, while a related but distinctly different series of antigens termed CEA-cross-reactive antigens can be produced by a variety of adult normal cells (17). The consistent presence of CEA in tumors has led to its wide use as a clinical assay for prognosis and management of colon carcinomas. The normal function, role in carcinogenesis, and molecular basis of production of CEA in tumors, however, are completely unknown. As a first step in the solution of these questions, we have initiated a study of the molecular genetics of CEA, beginning with the isolation of a family of CEA cDNA clones.We now report the molecular cloning, nucleotide sequence, and structural analysis of a number of functional full-length CEA cDNA clones. These clones reveal a basic structure of common 5' and 3' ends embracing a number (usually three) of strikingly similar relatively long repeating units. The repeats include amino acid sequences which show significant homology with several members of the rat immunoglobulin family and, in particular, with...

: Human cancers express organ-specific neoaptigens (OSNs) which elicit specific cellular immune responses in the cancer patient, as demonstrated by leukocyte adherence inhibition (LAI), an in vitro immune response assay. A purified protein of MW 40,000 (p40) exhibiting OSN (colon specific) activity was cleaved into specific peptide fragments and their partial amino acid sequences determined. This information was used in the polymerase chain reaction (PCR) to obtain a 992 by cDNA clone (PCR-992) from a human colon adenocarcinoma cell line (LS-180). By comparison of the predicted amino acid sequence of PCR-992 with the known sequence of p40 peptides, PCR-992 was shown to correspond to almost the entire coding region of p40. Nucleotide sequence analysis suggested that the protein was mycoplasmal in origin due to its high A H-T content (76%) and the presence of five in frame TGA termination codons ; at least two of the latter are actually read as tryptophan, a known feature of mycoplasma translation. We have confirmed this origin by direct isolation of a contaminating mycoplasma species from the LS-180 cell line and demonstration that it could be hybridized with the PCR-992 probe. Northern and PCR analysis of RNA preparations from the contaminated LS-180 cell line showed that p40 was part of the high affinity transport system operon of Mycoplasma hyorhinis (Dudler et al, EMBO J., 7: 3963-3970, 1988). Total protein lysates of Mycoplasma hyorhinis cultivated without animal cells could elicit positive LAI responses when incubated with cancer patient leukocytes but not with normal patient leukocytes. The organ-specific nature of the response was, however, not observed indicating that host cell-mycoplasmal interactions may play a role in determining the organspecific nature of p40 seen with the LAI. The significance of these findings will be discussed in the context of previous thinking regarding the origin of OSNs.

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