Carol A. Toth

The structure, metabolism and function of the carcinoembryonic antigen gene family

II. CEA structure and heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 A. Structure of the polypeptide backbone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 B. Structure of the oligosaccharide chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 C. The domain model for carcinoembryonic antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 D. Relationship to the immunoglobulin supergene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Metastatic potential of human colon cancer cell lines: relationship to cellular differentiation and carcinoembryonic antigen production

The relationship between cellular differentiation and carcinoembryonic antigen (CEA) production by human colorectal tumor cells and their ability to form hepatic metastases was studied. Eight human colon cancer cell lines were injected into athymic mice using different routes of administration to characterize their metastatic potential. The four poorly differentiated, non or low CEA producing cell lines were poorly metastatic to the liver after intrasplenic injection. After intraperitoneal implantation the same cell lines were highly tumorigenic, and subsequently metastastic to the liver. In contrast, the four moderate to well-differentiated cell lines that produced moderate to high levels of CEA were highly metastatic to the liver following intrasplenic injection. After intraperitoneal implantation they were less tumorigenic, and metastatic to the liver. We conclude that in this system poorly differentiated non or low CEA producing colorectal cell lines have a lower metastatic capacity compared to the well-differentiated high CEA producing colorectal cell lines. These data correlate directly with the pattern of metastatic spread and clinical course observed in patients with these tumors, suggesting that degree of differentiation and level of CEA production may play a role in development of site-specific metastases.

A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on kupffer cells

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases.

Carcinoembryonic antigen binding to Kupffer cells is via a peptide located at the junction of the N-terminal and first loop domains.

A 11kD glycopeptide has been isolated by pepsin digestion of carcinoembryonic antigen (CEA) that is rapidly endocytosed by isolated rat Kupffer cells and lung alveolar macrophages. Uptake of this glycopeptide by the isolated cells can be inhibited by excess unmodified CEA. Removal of the N-linked oligosaccharide chains by N-glycanase did not alter cellular uptake but reduced the MW to approximately 5500. A seventeen amino acid N-terminal sequence locates this peptide at the junction of the N-terminal and first loop domain of CEA. It is suggested that the recognition of a peptide sequence in this area of CEA is responsible for its clearance from the circulation.

Kupffer cell/tumor cell interactions and hepatic metastasis in colorectal cancer

The degree of interaction with Kupffer cells of two moderately well differentiated cell lines, CX-1 and CCl-188 of high metastatic potential (61%) were compared to two poorly differentiated cell lines, MIP-101 and Clone A of low metastatic potential (6%) in the intrasplenic injection model for liver metastasis. MIP-101 and Clone A bound significantly better to mouse Kupffer cells in vitro than either CX-1 or CCL-188. We also identified specific cell surface proteins mediating attachment of colorectal carcinoma cells to murine Kupffer cells. Kupffer cells were radiolabelled and their surface proteins incubated with MIP-101 and CX-1. Two radiolabelled proteins from murine Kupffer cells of 14 and 34 kDa were identified consistently binding to the tumor cells. Binding of both proteins was inhibited by asialofetuin but not by fetuin. This suggests that the major binding proteins between Kupffer cells and colorectal cancer cells are galactose binding lectins.

A carcinoembryonic antigen (CEA) binding protein from ascites influences CEA uptake by macrophages

A variant of CEA which is less readily endocytosed by macrophages has been isolated from malignant ascites. In vivo, CEA is cleared more slowly by the liver (t1/2 = 15.1 minutes) than CEAs isolated from hepatic metastases (t1/2 = 3.1 minutes). In vitro, rat and human Kupffer cells and rat alveolar macrophages endocytose this CEA less effectively. This slow clearing form of CEA is associated with a smaller (45kD) acidic glycoprotein (CORA) with which it forms a stable complex. CORA can be visualized on reducing gels but not on non reducing gels or by HPLC run under non reducing conditions. This suggests a non-covalent complex between the two glycoproteins. Analysis of protein conformation by circular dichroism revealed changes in the ascites CEA consistent with binding of CORA to the molecule. Western blot showed that CORA crossreacts with antisera to alpha 1-acid glycoprotein and double immunodiffusion demonstrated cross-reactivity but not identify. Sequencing of CNBr peptides showed sequence homology with alpha 1-acid glycoprotein but areas of unique sequence were also found. It is suggested that binding of CORA to CEA blocks the macrophage receptor binding of CEA.

Hepatic clearance and metabolism in the rat of a human breast cancer associated glycoprotein (GCDFP-15)

SummaryGross Cystic Disease Fluid Protein (GCDFP-15) is a 60,000 dalton glycoprotein isolated from human breast cyst fluid, composed of four 15,000 dalton monomers. Carbohydrate analysis indicates that each monomer has a single carbohydrate chain of the complex type. GCDFP-15 intravenously injected into rats showed a rapid circulatory clearance, the rate of clearance being faster in female animals [t1/2 = 12.8 (± 2.0) min. females, and 16.7 (± 2.6) min. males]. The major organs of clearance were the liver (70%) and kidneys (15%). Immunoperoxidase staining showed localization in Kupffer cells and the proximal convoluted tubules of the kidney. Removal of sialic acid from GCDFP-15 resulted in a more rapid clearance (t1/2 = 2.2 min) by the liver (85%). This clearance was inhibited by coinjection of asialo alpha1 acid glycoprotein. About 3% of GCDFP-15 was excreted in bile with a transit time through the liver of 38 min. Examination of the uptake of GCDFP-15 by isolated rat Kupffer cells showed that yeast mannan, fucosylated BSA, and carcinoembryonic antigen (CEA) failed to inhibit uptake, though the binding of GCDFP-15 was clearly saturable. This suggests that a novel receptor system on the rat Kupffer cell may be responsible for GCDFP-15 clearance.