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Expression of Complementary DNA and Genomic Clones for Carcinomebryonic Antigen and Nonspecific Cross-Reacting Antigen in Chinese Hamster Ovary and Mouse Fibroblast Cells and Characterization of the Membrane-expressed Products¹

Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010 [L. J. F. H., J. E. S.]; Institut für Immunbiologie, Universitat Freiberg, Stefan-meier-Strasse 8, D-7800 Freiberg, Federal Republic of Germany [H. S., J. A. T.]; and Suntory Institute for Biomedical Research, Shimamoto-cho, Mishima-gun, Osaka 618, Japan [S. O., H. N.]

Complementary DNA clones coding for both carcinoembryonic antigen (CEA), a well characterized colonic tumor marker, and nonspecific cross-reacting antigen (NCA), a related antigen, were expressed in Chinese hamster ovary (CHO) cells and L-cells (mouse fibroblasts). A genomic clone coding for CEA was also expressed in CHO cells. Positive clones were identified by fluorescence flow cytometry and enzyme-linked immunosorbent assay. Membrane location of the recombinant CEA and NCA was confirmed by indirect immunofluorescence labeling of the transfectants, followed by visualization under a fluorescence microscope. The apparent molecular weight of the expressed CEA and NCA were 180,000 and 96,000, respectively, for both cell lines, as determined by immunoblot analysis. The CEA and NCA expressed on CHO cells were sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the CEA and NCA proteins on L-cells were resistant to removal by PI-PLC. Unlike NCA, which contains three methionine residues, the only methionine in CEA is in the C-terminal hydrophobic domain. This domain in CEA was shown to be removed and replaced by a phosphatidylinositol glycan (PI-G) anchor (Hefta et al., Proc. Natl. Acad. Sci. USA, 85: 4648–4652, 1988). The recombinant CEA from both CHO cells and L-cells could be labeled with [³H]-ethanolamine (a component of the PI-G anchor) but not with [³⁵S] methionine, whereas the recombinant NCA could be labeled with both [³H]ethanolamine and [³⁵S]methionine. The labeling studies and PI-PLC treatment results are consistent with the CEA and NCA expressed on CHO cells possessing a PI-G anchor. The CEA expressed on the L-cell transfectants may contain a PI-G anchor which is resistant to cleavage by PI-PLC. In addition, the membrane-bound and secreted levels of CEA from the CHO and L-cell transfectants were determined.

¹ We gratefully acknowledge the support of a fellowship for L. J. F. H. from Hoffman LaRoche. In addition, this work was supported by Grant CA-37808 from the USPHS, NIH.

² To whom requests for reprints should be addressed.

Received 8/17/89. Revised 12/11/89.

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