Protein Zero, a Nervous System Adhesion Molecule, Triggers Epithelial Reversion in Host Carcinoma Cells Joseph P. Doyle,* Jerome G. Stempak, II Pamela Cowin, ~ David R. Colman,* and Donatella D'Urso § *The Brookdale Center for Molecular Biology, The Mount Sinai School of Medicine, New York 10029;*Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York 10032;§Neurologische Klinic, Neurochemisches Lab., University of Dfisseldorf, 40225 Dtisseldorf; IIDepartment of Anatomy and Cell Biology, Health Science Center, Brooklyn, New York 11203; and ~Departments of Cell Biology and Dermatology, New York University School of Medicine, New York, New York 10016 Abstract. Protein zero (Po) is the immunoglobulin cells, culminating in suppression of the transformed Downloaded from http://rupress.org/jcb/article-pdf/131/2/465/1265029/465.pdf by guest on 29 September 2021 gene superfamily glycoprotein that mediates the self- state and reversion of the monolayer to an epithelioid adhesion of the Schwann cell plasma membrane that phenotype. Furthermore, this response is apparently yields compact myelin. HeLa is a poorly differentiated accompanied by an increase in mRNA and protein lev- carcinoma cell line that has lost characteristic morpho- els for desmoplakin and N-cadherin which are normally logical features of the cervical epithelium from which it associated with epithelial junctions. Our conclusions originated. Normally, HeLa cells are not self-adherent. are supported by analyses of ten proteins we examined However, when Po is artificially expressed in this line, immunochemically (Po, cingulin, ZO-1, desmoplakin, cells rapidly aggregate, and Po concentrates specifically desmoglein, desmocollin, N-cadherin, a-catenin, vincu- at cell-cell contact sites. Rows of desmosomes are gen- lin, and cytokeratin-18), and by quantitative poly- erated at these interfaces, the plasma membrane local- merase chain reactions to measure relative amounts of ization of cingulin and ZO-1, proteins that have been desmoplakin and N-cadherin mRNAs. shown to be associated with tight junctions, is substan- Po has no known signaling properties; the dramatic tially increased, and cytokeratins coalesce into a cohe- phenotypic changes we observed are highly likely to sive intracellular network. Immunofluorescence pat- have developed in direct response to Po-induced cell terns for the adherens junction proteins N-cadherin, adhesion. More generally, the ability of this "foreign" et-catenin, and vinculin, and the desmosomal polypep- membrane adhesion protein to stimulate desmosome tides desmoplakin, desmocollin, and desmoglein, are and adherens junction formation by augmenting well- also markedly enhanced at the cell surface. Our data studied cadherin-based adhesion mechanisms raises the demonstrate that obligatory cell-cell adhesion, which in possibility that perhaps any bona fide cell adhesion this case is initially brought about by the homophilic as- molecule, when functionally expressed, can engage sociation of Po molecules across the intercellular cleft, common intracellular pathways and trigger reversion of triggers pronounced augmentation of the normally a carcinoma to an epithelial-like phenotype. sluggish or sub-basal cell adhesion program in HeLa ELL--cell contact in a forming epithelial monolayer reinforced by the generation of characteristic junctional is believed to be initiated by cell adhesion mole- complexes composed mainly of tight junctions, subjacent C cules (CAMs) 1 that are Ca ++ dependent, as exem- adherens junctions, and desmosomes (Farquhar and plified by E-cadherin (Gumbiner et al., 1988; Takeichi, Palade, 1963; and see Rodriguez-Boulan and Nelson, 1989). 1991; Wollner et al., 1992). This contact is maintained and The tight junctions provide a permeability barrier at the boundary between the apical and lateral plasma mem- Address all correspondence to Drs. D. D'Urso, Neurologische Klinic, Neurochemisches Lab, University of Diisseldorf, Moorenstr. 5, 40225 brane domains, and the desmosomes and adherens junc- Diisseldorf. Tel.: 49 211 311 89 84. Fax: 49 211 311 84 85; and David R. tions serve to structurally support and stabilize the cell- Colman, Brookdale Center for Molecular Biology, The Mount Sinai cell contacts. School of Medicine, One Gustave Levy Place, Box 1126, New York, NY More than 85% of tumors in humans are carcinomas, 10029. Tel.: (212) 241-5775. Fax: (212) 423-0596. that is, they are epithelial in origin. In many carcinomas in- 1. Abbreviations used in this paper: CAM, cell adhesion molecule; NCAM, timate cell-cell contact is lost and the stereotypic array of neural cell adhesion molecule; PNS, peripheral nervous system. epithelial junctions disappears. The cells become anaplas- © The Rockefeller University Press, 0021-9525/95/10/465/18 $2.00 The Journal of Cell Biology, Volume 131, Number 2, October 1995 465-482 465 tic and pleiomorphic, and may readily metastasize. It has pearance and width of the intercellular gap of the intrape- become clear recently that in these transformed cell popu- riod line of mature peripheral nerve myelin (Raine, 1984). lations, the presence of functional CAMs, that are known We interpreted these data as evidence for pronounced to play roles in the development and maintenance of adhe- cell-cell adhesion induced by the homophilic association sion processes in normal epithelial monolayers, may be in- of the extracellular segments of Po expressed on adjacent versely correlated with the degree of metastatic potential cell surfaces. of the corresponding tumor cells (Birchmeier et al., 1991; In this report, we show that HeLa cells generate a com- Frixen et al., 1991; Vleminckx et al., 1991; Takeichi, 1993). plex response to Po-induced adhesion which greatly aug- For example, low levels of expression of E- and P-cadherin ments the placement of junctional proteins at cell-cell are found in carcinoma cell lines where extensive tumor contacts, thereby compelling the assembly of certain junc- invasiveness has been demonstrated (Frixen et at., 1991; tional complexes, such as desmosomes and adherens junc- Navarro et al., 1991; Vleminckx et al., 1991). From a thera- tions that are characteristic of normal epithelia. To our peutic standpoint therefore, there is reason to believe that knowledge this is the first demonstration that a nervous augmentation of functional Ca÷÷-dependent CAMs in system IgCAM, by obligating cells to adhere to one an- carcinoma cells may be one key to reengaging the normal other, can trigger reversion of host tumor cells to an epi- epithelial cell program through suppression of the trans- thelioid phenotype. Our data raise the possibility that ex- formed state. pression in carcinoma cells of any functional cell adhesion We have been studying the functional properties of Po, a molecule that establishes close membrane apposition ulti- transmembrane glycoprotein (Mr ~28,000) that mediates mately drives the cells to enhance the normal celt adhesion adhesion of Schwann cell plasma membrane surfaces to program such that stabilizing junctions are formed, and Downloaded from http://rupress.org/jcb/article-pdf/131/2/465/1265029/465.pdf by guest on 29 September 2021 form compact myelin in the peripheral nervous system suppression of the transformed phenotype is achieved. (PNS) of vertebrates. Po is a member of the immunoglobu- lin (Ig) gene superfamily of CAMs that contains a single Materials and Methods variable-like region in its extracellular segment (Lai et al., 1987, Lemke et al., 1988). This, and an analysis of its exon- Cell Culture and Transfection intron structure, have led to the suggestion that Po may possess adhesive properties similar to those of a primor- HeLa cells, a cell line derived from a human cervical carcinoma, were rou- tinely cultured in DMEM supplemented with 7.5 % FCS, 100 U/ml penicil- dial IgCAM from which all other members of this large su- lin, 100 mg/ml streptomycin and 2 mM L-glutamine (all supplied by Gibco perfamily may have arisen (Williams and Barclay, 1988). BRL, Gaithersburg, MD). To obtain clonal lines of Po expressors, HeLa According to this view, a general role for an ancestral cells were transfected with a cDNA encoding the full-length Po protein Po-like IgCAM might have been to contribute to intercel- (Lemke and Axel, 1985). The Po cDNA was subcloned into the pECE vec- lular adhesion, essential for the development of multicel- tor (Ellis et al., 1986) and PopECE was introduced into the host HeLa cells along with the plasmid pSV2-neo (Southern and Berg, 1982) by lipo- lular organisms (Williams and Barclay, 1988). A primor- some-mediated transfection as previously described (D'Urso et al., 1990). dial Po-like IgCAM was probably highly adhesive, and The day after transfection cells were split and seeded in complete DMEM homophilic, and in certain tissues of some early organisms containing 400 ixg/ml of Geneticin G418 (Gibco) to select stable transfor- may have formed extraordinarily stable intercellular bonds. mants. Cells transfected with the plasmid pSV2-neo only were used as control populations in all experiments. All stable transformants were The fact that Po expression in contemporary vertebrates is maintained in culture in selection medium. limited to myelinating cells may therefore not be surpris- Unless otherwise noted, in each experiment, control cells and Po ex- ing, since the compact myelin spiral, once formed, is not pressors were treated with 5 mM sodium butyrate, which has been shown disassembled under