No abstract

Dipeptidyl peptidase IV (DPPIV) is a serine exoproteinase expressed at high levels in epithelial cells of kidney, liver and small intestine. Recently Watanabe, Kohima & Fujimoto [(1987) Experientia 43, 400-401] and Gossrau et al. [(1990) Histochem. J. 22, 172-173] reported that Fischer 344 rats are deficient in this enzyme. We have examined DPPIV expression in Fischer 344 rats available from U.S. and German suppliers and find that livers of the U.S. Fischer rats, in contrast with their German counterparts, express active DPPIV (D+). Northern analysis of liver RNA showed comparable levels of 3.4 kb and 5.6 kb DPPIV transcripts in both D+ rats from the U.S. and German (D-) rats. Monoclonal antibody (MAb) 236.3 to DPPIV immunoprecipitated at 150 kDa enzymically active (105 kDa, denatured) protein from surface-labelled D+ hepatocytes and reacted with canalicular and sinusoidal membranes (as shown by immunofluorescence microscopy). MAb 236.3 failed to immunoprecipitate a labelled peptide from D- cell extract or to stain D- liver sections. Polyclonal antibody (PAb) specific for DPPIV immunoprecipitated an enzymically active peptide from D+ hepatocyte extracts and a smaller, inactive peptide from D- hepatocyte extracts. Peptide maps of DPPIV immunoprecipitated from D+ extracts with MAb 236.3 and PAb were identical, but differed from that of the D- hepatocyte component recognized by PAb. The molecular basis of the DPPIV deficiency in the D- rats thus appears to be the translation of an enzymically inactive protein missing the epitope recognized by MAb 236.3. We have exploited these D- rats as hosts for syngeneic transplantation of liver cells from D+ Fischer rats. DPPIV expression is stable in the transplanted cells and allows them to be readily distinguished from the surrounding D- tissue.

One of the cell-adhesion molecules (CAMs) responsible for rat hepatocyte aggregation has been described as a glycoprotein having an Mr of 105,000 (cell-CAM105). The Mr and localization of cell-CAM105 in liver membranes are very similar to those of liver ecto-ATPase, an ATPase with its nucleotide-hydrolysing site localized on the outside of the cell membrane. The protein sequence of the ecto-ATPase has been deduced from cDNA cloning. Structural analysis of the sequence indicates that the ecto-ATPase has immunoglobulin-like domains and is a member of the immunoglobulin superfamily. Since a group of proteins in the immunoglobulin superfamily has been shown to have functions related to cell adhesion, the structural characteristics of the ecto-ATPase further led to the possibility that the ecto-ATPase may have functions related to cell adhesion. In this paper, using the cDNA for the ecto-ATPase, the anti-peptide antibodies produced against peptides derived from the ecto-ATPase cDNA sequence and monoclonal antibodies against the cell-CAM105, we present evidence of identity between cell-CAM105 and ecto-ATPase. First, in Western immunoblots, two anti-cell-CAM105 monoclonal antibodies cross-reacted with the purified ecto-ATPase. Secondly, in immunodepletion experiments, antibodies against the ecto-ATPase depleted the same protein recognized by the anti-cell-CAM105 antibodies. Thirdly, in two-dimensional gel-electrophoretic analysis, anti-peptide antibodies generated against an extracellular N-terminal peptide and the intracellular C-terminal peptides of the ecto-ATPase immunoprecipitated proteins of similar isoelectric points and Mr values to those of the cell-CAM105. Fourthly, proteins immunoprecipitated by anti-ecto-ATPase antibodies and anti-cell-CAM105 antibodies have similar V8-proteinase-digest peptide maps. Finally, monoclonal antibodies against the cell-CAM105 specifically recognized the protein expressed in COS cells transfected with the ecto-ATPase cDNA. These results indicate that the ecto-ATPase cDNA codes for a protein that is identical with the cell-CAM105. Since the ecto-ATPase has structural features of immunoglobulin domains, the identity of cell-CAM105 with ecto-ATPase leads to the conclusion that this liver CAM, similarly to neuronal CAM, is also a member of the immunoglobulin supergene family. Furthermore, immunological studies indicate that the cell-CAM105/ecto-ATPase is composed of two isoforms of different C-terminal sequences. The association of ATPase activity with cell-CAM105 raises the possibility that extracellular nucleotides may play important roles in regulating cell adhesion.

An hepatocyte cell-adhesion molecule (cell-CAM105) was recently shown to be identical with the liver plasma-membrane ecto-ATPase. This protein has structural features of the immunoglobulin superfamily and is homologous with carcinoembryonic antigen proteins. We have cloned a cDNA encoding a new form of the cell-CAM105 which is a variant of the previously isolated clone. In addition to having a shorter cytoplasmic domain, the new isoform also has substitutions clustered in the first 130 amino acids of the extracellular domain. Both of these isoforms are expressed on the surface of hepatocytes with the shorter variant being the predominant form. The previously isolated cell-CAM105 (long form) has more potential phosphorylation sites than does the new isoform (short form). Both isoforms are found to be phosphorylated after incubation with [32P]phosphate in vitro, with the long form being phosphorylated to a significantly higher extent. This observed differential phosphorylation could be one of the mechanisms for the regulation of isoform functions. Using antipeptide antibodies specific for the long form and antibodies that are reactive with both isoforms, we have shown that both isoforms are localized in the canalicular domain of hepatocytes. The sequence differences between these two isoforms suggest that they are probably derived from different genes rather than from alternative splicing.

There was an error published in Development 133, 4269-4279.The Acknowledgements should have read 'Sponsored by grants CA42715, CA93840 and P20RR017695'. The authors apologise to readers for this mistake.

No abstract

Carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) is a member of the CEA family of immunoglobulin-like adhesion molecules with two major splice variants, CEACAM1 a -4L and CEACAM1 b -4S, differing in the length of their COOH-terminal cytoplasmic tail. Both forms are down-regulated in prostate and liver carcinomas relative to normal tissues. We have previously shown in a nude mouse xenograft model that restoration of CEACAM1 a -4L expression in human prostate carcinoma cells (PC-3) suppresses tumorigenicity, an effect observed with carcinomas from several other tissues but never established for hepatocellular carcinomas. In this report, we have examined the effect of CEACAM1 a -4L on tumorigenicity of 1682A, a rat hepatocellular carcinoma that grows on the omentum when injected into the peritoneal cavity. Results show that restoration of CEACAM1 a -4L expression at levels 13-and 0.45-fold compared with negative controls or normal hepatocytes, respectively, completely suppressed the formation of 1682A tumor nodules on the omentum at 3 weeks after injection. In contrast, 1682A cells infected with CEACAM1 b -4S or an empty retroviral vector formed multiple clusters of tumor nodules. Although tumor nodules of 1682A cells positive and negative for CEACAM1 a -4L did not display significant differences in histologic organization, aggregates formed in vitro by 1682A-L were smaller in size and displayed enlarged intercellular spaces relative to their 1682A-V counterparts. Restoration of CEACAM1 a -4L expression did not elevate levels of apoptosis but seemed to cause an increase in the length of G 1 . This is the first demonstration of CEACAM1 a -4L-induced tumor suppression in liver carcinomas using a quantifiable i.p. syngeneic transplantation model.

In this paper, we have characterized the structure, evolutionary origin, and function of rat and human carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) multifunctional Ig-like cell adhesion proteins that are expressed by many epithelial tissues. Restriction enzyme digestion reverse transcriptase-PCR analysis identified three cDNAs encoding novel CEACAM1 N-domains. Comparative sequence analysis showed that human and rat CEACAM1 N-domains segregated into two groups differing in similarity to rat CEACAM1 a -4L and human CEACAM1. Sequence variability analysis indicated that both human and rat Ndomains possessed two variable regions, and one contained a major adhesive epitope. Recombination analysis showed that the group of rat but not human N-domains with high sequence similarity was derived at least in part by recombination. Binding assays revealed that three monoclonal antibodies with strong reactivity for the CEACAM1 a -4L N-domain showed no reactivity with CEACAM1 b -4S, an allele with a different N-domain sequence. CEACAM1 b -4S displayed adhesive activity efficiently blocked by a synthetic peptide corresponding to the adhesive epitope in CEACAM1 a -4L. Blocking analysis also showed that the adhesive epitope for rat CEACAM1 was located downstream from the equivalent human and mouse epitopes. Glycosylation analysis demonstrated O-linked sugars on rat CEACAM1 b -4S from COS-1 cells. However, this was not the alteration responsible for the lack of monoclonal antibody reactivity. When considered together with previous studies, our findings suggest an inverse relationship between functionality and amino acid sequence similarity to CEACAM1. Like IgG, the N-domain of CEACAM1 appears to tolerate 10 -15% sequence diversification without loss of function but begins to show either altered specificity or diminished functionality at higher levels.Carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) 1 is a member of a large family of multifunctional Ig-like cell adhesion molecules (CAMs) structurally related to carcinoembryonic antigen (CEA) (2, 3). CEACAM1 from both rodents and humans is composed of an ectodomain with an N-terminal Ig V-like domain (N-domain), three Ig-like C-domains, a single transmembrane domain, and a cytoplasmic (cyto) domain that through differential splicing varies in length from 6 to 71 amino acids (4 -6). Multiple genes with unique N-domain sequences and a variety of splice variants have been reported in both rodents and humans (2, 3, 5-12). The major splice variants in rodents and humans have from 2 to 4 Ig-like domains and cyto domains with either 70 -71 (L forms) or 9 -10 amino acids (S forms) (1-3, 5, 6). In rodents, allelic variants (Ceacam1 a and 1 b ) 2 or separate genes differing in both the nucleotide and amino acid sequence of their N-terminal Ig domains (rats and mice) have also been described (1, 13).Interest in the role of CEACAM1 in cancer has blossomed since early reports showed that this gene was lost or greatly down-regulated in rodent hepatocellular ...