CEA as a cell adhesion molecule

SELECTED SUMMARIES

July 1990

observations, it is difficult not to speculate about therapeutic implications for ras oncogene-associated tumors. If isoprenylation of the C-terminal cysteine of the CAAX box is a critical event for ras protein activity, can this step be inhibited, thus controlling the expression of ras oncogenes? Because isoprenoids are derived from

mevalonate synthesis, agents such as the inhibitor mevinolin could potentially be useful in anticancer therapy by decreasing the availability of this key substrate required for membrane binding and activity of ras oncogene proteins. In fact, mevinolin has been shown to block cell growth in the Gl phase and the GYM phase (J Cell Physiol 1987;125:540-558; J Cell Physiol 1988;137:133-140). In addition, in this study by Hancock et al., mevinolin did prevent correct processing and membrane association of ras proteins. These observations obviously await further study, but they may provide interesting leads to the development of effective alternative chemotherapeutic agents. E. B. CHANG, M.D.

CEA AS A CELL ADHESION

MOLECULE

Benchimol S, Fuks A, lothy S, et al. (Cancer Center, and Departments of Biochemistry and Pathology, McGill University, Montreal, Quebec, Canada). Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule. Cell 1989;57:327-334. Aggregation of LS-180 and 86/8 cells [two lines derived from the same human colon carcinoma) was tested in an in vitro adhesion assay. Carcinoembryonic antigen (CEA) production by 86/8 and LS-180 cells was 0.1 and 1000 ng CEA/mg protein, respectively. After 2 hours, -90% of 86/8 cells remained single and unaggregated, and -70% of LS-180 cells had aggregated. Aggregation by LS-180 cells was prevented by the presence of Fab’ fragments of rabbit anti-CEA antibodies. To confirm that CEA was responsible for aggregation, a Chinese hamster ovary cell line, LR-73, that does not express the mRNA for CEA [and therefore cannot synthesize the protein] was transfected with the full length CEA cDNA. Untransfected LR-73 cells remained single in the adhesion assay, but the cells transfected with CEA cDNA, and secreting the protein, aggregated. Aggregation of the transfected cells was prevented by Fab’ fragments of anti-CEA antibodies. When fluorescent-labeled transfected cells were mixed with unlabeled untransfected LR-73 cells, the resulting aggregates consisted almost entirely of the former, indicating that adhesion was homotypic. To determine whether CEA, like other intercellular adhesion molecules, can mediate specific cell sorting, fluorescentlabeled LR-73 cells transfected with CEA cDNA were mixed with equivalent numbers of Syrian hamster embryo fibroblasts, which, unlike untransfected LR-73, aggregate in suspension. Aggregates formed and were homogeneous, consisting either of -90% transfected LR-73 cells or -90% hamster embryo fibroblasts. These results were confirmed in similar mixing experiments with Chinese hamster embryo fibroblasts, which are from the same species as LR-73 cells. Finally, by immunohistochemical and immunoelectron microscopic methods, the distribution of CEA in human colonic epithelium was examined. The CEA was confined to the apical membranes of cells from the normal adult colon and was absent from the basolateral membranes that form

277

the borders between adjacent cells. In the embryo before about 20 weeks’ gestation and in adult colonic adenocarcinomas, CEA was distributed all around the cell periphery, including the borders between adjacent cells. The distribution pattern of CEA correlated with epithelial organization: in the multilayered epithelia of early fetal normal colon and adult adenocarcinomas, CEA was abundant along intercellular borders, in contrast to its confinement to the nonbordering apical membranes of cells in the single-layered epithelium of normal adult colon. The authors conclude that CEA can act as an intercellular adhesion molecule and mediate homotypic cell sorting in heterogeneous populations of cells. Comment. A role for the family of glycoproteins that constitute CEA as a clinically useful marker of early or recurrent colorectal cancer is now largely defunct, but there is much interest in the functions of CEA as a member of the immunoglobulin superfamily (Proc Nat1 Acad Sci USA 1987;84:920-924); besides immunoglobulins, members include T-cell receptors and intercellular adhesion molecules. Through isolation of cDNA clones, the nucleotide sequences of the mRNAs that encode CEA and the closely related nonspecific cross-reaching antigen (NCA) are known (Proc Nat1 Acad Sci USA 1987;84:2960; J Biol Chem 1988;263:3203-3207). The CEA and related genes are apparently located on chromosome 19 (Cancer Res 1988;48:2550-2554). The nucleotide sequences of the coding regions of CEA and NCA show 90% or more similarity, indicating that the predicted amino acid sequences must be virtually identical. A total of four species of CEA and NCA mRNA have been identified in Northern analyses of a wide variety of normal and malignant human tissues. In the normal colon, 2.6- (an mRNA for NCA) and 3.0- (an mRNA for CEA) kilobase (kb) transcripts are expressed (Cancer Res 1988:48:2550-2554; Cancer Res 1988;48:31533157). The adenocarcinomatous colon and leukocytes from patients with chronic myelogenous leukemia express additional CEA transcripts of 3.5 and 2.3 kb, respectively. Chronic myelogenous leukemia leukocytes lack the 3.0- and 3.5-kb transcripts, which probably differ only in the lengths of their 3’untranslated regions. The limited number of CEA and NCA mRNA transcripts (and, therefore, peptide sequences) contrasts with the larger repertoire (heterogeneity) of CEA epitopes that has been identified with monoclonal antibodies (Cancer Res 1985;45:5769-5780). Thus, the heterogeneity of CEA in normal and malignant tissues is thought to be attributable largely to variations in posttranscriptional modification, particularly different glycosylation patterns, rather than to generation of different mRNA transcripts. The present study has demonstrated convincingly that CBA can act as a homotypic intercellular adhesion molecule on the surface of cultured human colonic adenocarcinoma cells. Application of this observation to elucidation of the roles that CEA may play in normal development and carcinomatous degeneration of the human colon is necessarily more speculative. That CEA is distributed on the basolateral and apical membranes of malignant colonocytes but only on the apical membranes of normal colonocytes is well known (Cancer 1982;49:2077-2090). although it is not acknowledged in this study. One fruitful area for further study might be the epithelium of the small bowel. It has been suggested that goblet cells but not columnar (absorptive) cells of the normal human small bowel produce CEA, in contrast to the large bowel, where CEA is produced by both cell types (Cancer 1982;49:2077-2090). In any event, it remains to be proven that the functions fulfilled by CEA in cultured cells accurately represent the situation in vivo. Location on the cell surface and possession of multiple glycosylation sites equip CEA appropriately for functions related to cell recognition, which may be mediated principally through the saccha-

278 SELECTED SUMMARIES

GASTROENTEROLOGY

ride chains of glycoproteins (Trends Biochem Sci 1989;145:272-2763. Logically, an understanding of putative functions such as homotypic intercellular adhesion on the part of CEA will therefore require detailed analysis of its saccharide components. Polyclonal antihodies of the type used in this study may not distinguish qualitative

differences between CEAs with quite different functional properties. In future studies, the combined application of monoclonal antibodies to CEA with different epitope specificities and experimental manipulations of CEA glycosylation. e.g., by oligosaccharidedirected mutagenesis of glycosylation sites, should help explain what CEA does. The present study provides a very promising start. M. P. LANCE, M.B.. M.R.C.P.

CENTRAL REGULATION OF DUDODENAL BICARBONATE SECRETION Lenz HJ, Brown MR. (Department of Medicine, University of Hamburg, Hamburg, Federal Republic of Germany, and Departments of Medicine and Surgery, University of California, San Diego, California]. Cerebroventricular calcitonin gene-related peptide inhibits rat duodenal bicarbonate secretion by release of norepinephrine and vasopressin. J Clin Invest 1990;85:25-32 (January]. Duodenal bicarbonate secretion is an important factor protecting the mucosa against acid damage. Decreased duodenal bicarbonate secretion at rest and in response to acid is observed in duodenal ulcer patients (N Engl J Med 1987;316:374-379). This study examines a possible role for calcitonin gene-related peptide (CGRP) in the central nervous system regulation of duodenal bicarbonate secretion in the unanesthetized rat. The objectives were threefold: (a) to examine the pharmacological properties of CGRP inhibition of resting and stimulated duodenal bicarbonate secretion, (b) to determine the peripheral pathways involved in mediating the central nervous system effects of CGRP, and (c) to study the peripheral effects of the presumed transmitters released by CGRP. The results in this study indicate that cerebroventricular administration of CGRP inhibits both basal duodenal bicarbonate secretion and secretion induced by diverse stimuli such as vasoactive intestinal peptide, neurotensin, misoprostol (a prostaglandin E, analogue), and hydrochloric acid. This central inhibitory effect of CGRP appears to be neurally mediated via sympathetic efferents because it could be abolished by ganglionic blockage with chlorisondamine, attenuated by noradrenergic blockage with bretylium, and enhanced by vagotomy. Furthermore, inhibition of duodenal bicarbonate secretion by CGRP coincided with increases in the plasma levels of norepinephrine and vasopressin. Pretreatment of the animals with the combination of an cY-adrenergic antagonist, phentolamine, and the vasopressin V, antagonist l-deaminopenicillamine 2-[Omethyl] Tyr, 8-Arg vasopressin abolished the central inhibitory effect of CGRP. When administered alone, neither antagonist was able to completely reverse the effect of the neuropeptide. Finally, intraperitoneal administration of vasopressin or norepinephrine decreased duodenal bicarbonate secretion; their effects were additive and could be prevented by their respective antagonists. The authors conclude that cerebroventricular CGRP inhibits rat duodenal bicarbonate secretion by activation of sympathetic efferents with consequent release of norepinephrine and vasopressin.

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These peripheral mediators then act on a-adrenergic and vasopressin receptors, causing an inhibition of duodenal bicarbonate secretion. Comment. A decreased ability to secrete bicarbonate from the duodenum appears to be an important determinant in the pathogenesis of duodenal ulcers. Hence an understanding of the factors that regulate duodenal bicarbonate secretion in health may improve our ability to pharmacologically modify bicarbonate secretion in disease. However, regulation of duodenal bicarbonate secretion is a complex and interactive process. Locally, there may be luminal and basolateral regulators of the duodenal epithelial cells. From the luminal side, hydrochloric acid from the stomach appears to be the most potent stimulus. From the basolateral side, prostaglandins, certain peptides, and neurotransmitters, including prostaglandin E,, vasoactive intestinal polypeptide, and neurotensin, can also contribute to the regulation of bicarbonate secretion. Some of these basolateral factors, such as prostaglandin E, and vasoactive intestinal polypeptide, have been implicated as the mediators released by luminal acid to stimulate bicarbonate secretion. It is likely that other stimulatory factors remain to be recognized. Candidates for such factors might include local paracrine and endocrine controls, neurally mediated controls [including those arising from the local, peripheral, and central nervous systems], and substances derived from immunologic effector cells. Of course, inhibitory controls are just as important as the stimulatory ones, and such inhibitory controls are the focus of the study described. In this paper, the authors convincingly demonstrate that cerebroventricular administration of CGRP inhibits bot$ resting duodenal bicarbonate secretion and a stimulated state of secretion induced by the activating stimuli described above. The authors have carefully shown that this effect is likely to be caused by activation of sympathetic efferents that release norepinephrine and vasopressin. These secondary mediators then inhibit duodenal biocarbonate secretion, although the mechanisms involved in this inhibitory process remain to be investigated. Similarly, it is still unknown whether norepinephrine and vasopressin act directly on the secretory epithelial cells or whether a tertiary level of messengers is involved. Duodenal mucosal integrity represents a fine balance between aggressive forces (such as gastric acid and others) and protective forces [such as bicarbonate and others]. One may assume that the paracrine, neurocrine, endocrine, and immune systems control the relative contributions of aggressive and protective factors at the local level. Central regulation via the central and peripheral nervous systems provides another layer of control, which may additionally interact with local controlling mechanisms. Central nervous system controls are usually mediated by peptides or neurotransmitters that can either directly affect secretory cell function or indirectly affect secretory cells via actions on other systems, such as the blood supply for example. In summary, the paper by Lenz and Brown makes two contributions. First, it significantly enhances our understanding of the regulatory physiology of duodenal bicarbonate secretion. Second, it suggests that we should look not only to increased gastric acid secretion, but also possibly to decreased duodenal bicarbonate secretion, to explain duodenal mucosal damage associated with stress or other central nervous system pathology. K. DHARMSATHAPHORN, K. E. BARRETT,

M.D. PH.D.

Helicobacter pylori GASTRITIS: A ‘NORMAL’ PHENOMENON Dooley CP, Cohen H, Fitzgibbons PL, et al. (Departments Medicine and Pathology, Los Angeles County-University

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