deregulated expression of the human tumor marker cea and cea family member ceacam6 disrupts tissue architecture and blocks colonocyte differentiation

RESEARCH ARTICLE Neoplasia Vol 4, No 2, 2002, pp 151 – 163 151 www.nature.com/neo Deregulated Expression of the Human Tumor Marker CEA and CEA Family Member CEACAM6 Disrupts Tissue Architecture and Blocks Colonocyte Differentiation1 Christian Ilantzis, Luisa Demarte, Robert A Screaton and Clifford P Stanners McGill Cancer Centre and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6 Abstract Human carcinoembryonic antigen ( CEA ) and the CEA family member CEACAM6 ( formerly nonspecific cross reacting antigen [ NCA ] ) function in vitro, at least, as homotypic intercellular adhesion molecules and, in model systems, can block the terminal differentiation and anoikis of several different cell types We have recently demonstrated that the increased cell surface levels of CEA and CEACAM6 in purified human colonocytes from freshly excised, well to poorly differentiated colon carcinomas are inversely correlated with the degree of cellular differentiation Thus, deregulated expression of CEA / CEACAM6 could directly contribute to colon tumorigenesis by the inhibition of terminal differentiation and anoikis Evidence against this view includes the common observation of increased CEA / CEACAM6 expression as normal colonocytes differentiate in their migration up colonic crypt walls We report here the direct effects of deregulated overexpression of CEA / CEACAM6, at levels observed in colorectal carcinomas, on the differentiation of two human colonic cell lines, SW - 1222 and Caco - Stable transfectants of both of these cell lines that constitutively express 10 - to 30 - fold higher cell surface levels of CEA / CEACAM6 than endogenous levels failed to polarize and differentiate into glandular structures in monolayer or 3D culture or to form colonic crypts in a tissue architecture assay in nude mice In addition, these transfectants were found to exhibit increased tumorigenicity in nude mice These results thus support the contention that deregulated overexpression of CEA and CEACAM6 could provide a tumorigenic contribution to colon carcinogenesis Neoplasia ( 2002 ) 4, 151 – 163 DOI: 10.1038/sj/neo/7900201 Keywords: carcinoembryonic antigen, colon cancer, tissue architecture, colonocyte differentation, cell polarization Introduction The identification and characterization of genetic alterations associated with the acquisition of epithelial tumorigenicity has contributed much to our understanding of neoplastic progression Thus, studies on the development of colorectal cancer [ ] have implicated the stepwise accumulation of multiple genetic lesions as the underlying cause Less attention has been paid to phenotypic changes that are not directly fixed by mutation, though some of these could well represent an essential component of the malignant phenotype Alterations in the expression of carcinoembryonic antigen ( CEA ) family members fall into the latter category as no activating mutations have been found in their corresponding genes [ ] ( although mutations in a subdomain of CEA affecting binding to and clearance by Kupffer cells of the liver have recently been reported [ ] ) They are of special interest because such high proportions of so many different types of tumors ( about 50% of all human tumors ) show deregulated expression of CEA [ ] CEA is the prototypic member of a highly related group of cell surface glycoproteins [ 5,6 ] representing a subfamily of the immunoglobulin gene superfamily; CEA and the closely related family member CEACAM6 ( formerly nonspecific cross - reacting antigen [ NCA ] ) have been found to be overexpressed in a wide variety of epithelial malignancies [ – 15 ] , whereas a third member of the family, CEACAM1 ( formerly biliary glycoprotein [ BGP ] ) , is usually reported to be downregulated [ 16,17 ] CEA is thus widely used clinically as both a blood and tissue tumor marker of epithelial malignancy, especially for tumors of the colon The correlation of elevated blood and tumor levels of CEA and CEACAM6 with so many human tumors has led to the question of a possible instrumental role for these molecules in the development of human cancer [ 18 ] and, indeed, results with model systems, such as the inhibition of myogenic differentiation [ 19 ] and increased tumorigenicity [ 20 ] in rat myoblasts by CEA and CEACAM6, have supported this view Results consistent with these findings have been reported for freshly excised human colorectal carcinomas, in which the cell surface levels of CEA and CEACAM6 on purified tumor colonocytes were shown to be inversely correlated with their degree of differentiation [ ] In addition, CEA and CEAAbbreviations: BGP, biliary glycoprotein; CEA, carcinoembryonic antigen; FBS, fetal bovine serum; FRCC, fetal rat colonic cells; mAb, monoclonal antibody; NCA, nonspecific cross reacting antigen; PBS, phosphate - buffered saline lacking Ca2 + and Mg2 + Address all correspondence to: Clifford P Stanners, McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6 E-mail: stanners@med.mcgill.ca This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Medical Research Council of Canada Received 27 June 2001; Accepted 20 August 2001 Copyright # 2002 Nature Publishing Group All rights reserved 1522-8002/02/$25.00 152 CEA family members block colonocyte differentiation CAM6, but not CEACAM1, have been recently shown to inhibit anoikis, an apoptotic process that maintains tissue architecture by killing cells that lose their anchorage, in many different cell types, including human Caco - colonocytes [ 21 ] Contrary evidence, however, such as the common observation of increased apical expression of CEA [ 22,23 ] and CEACAM6 [ 23 ] as normal adult colonocytes differentiate in the upper third of colonic crypts and increased CEA and CEACAM6 expression in cultured colon carcinoma cell lines, such as Caco - 2, after the cells reach confluence and differentiate [ 24 ] , has challenged this view Also, transgenic mice expressing the human CEA gene have been reported not to show increased colonic tumorigenicity [ 25 ] Although all of this contrary evidence can be argued to be nondefinitive ( see Discussion section ) , we decided to directly test the hypothesis that deregulated overexpression of CEA and CEACAM6 can have tumorigenic effects To mimic the situation pertaining to human colonic cancer as closely as possible, we studied the effects of cell surface overexpression of CEA and CEACAM6 deregulated to be expressed in human colonocytes that still have division potential, because it is cells with division potential at the base of colonic crypts which normally express very low levels of CEA / CEACAM6 that are the normal targets for colonic carcinogenesis The effects of CEA / CEACAM6 overexpression in this context were surprisingly similar to key changes in cell and tissue architecture observed during colonic carcinogenesis in vivo, i.e., a loss of cell polarization and terminal differentiation, and an abrogation of normal colonic crypt glandular architecture Materials and Methods Cell Lines and Culture Conditions Caco - 2cellswereobtainedfromtheAmericanTypeCulture Collection ( Rockville, MD ) The SW - 1222 cell line was obtained from Dr W Bodmer ( ICRF Cancer and Immunogenetics Laboratory, Oxford, UK ) Cultures were maintained in - MEM medium [ 26 ] , supplemented with 10% fetal bovine serum ( FBS; Gibco BRL, Grand Island, NY ) , 100 U / ml penicillin, and 100 g / ml streptomycin ( - MEM / 10% FBS ) , at 378C in a humidified atmosphere of 5% CO2 in air Monoclonal Antibodies D14.6.43 ( IgG1 ) is a monoclonal antibody ( mAb ) specific for CEA [ 27 – 29 ] , and 9A6FR ( IgG1 ) , generously provided by Dr F Grunert ( Institute of Immunobiology, Freiburg ) , is a mAb specific for CEACAM6 [ 30 ] These mAbs not cross - react with other members of the CEA family V34420 ( IgG1 ) is an anti – human villin specific mAb ( Transduction Laboratories, Lexington, KY ) Transfection of SW - 1222 and Caco - cells Full - length cDNAs encoding CEA, CEACAM1 ( CEACAM1 - 4L splice variant, formally BGPa [ 31 ] ) , and a cDNA lacking the major portion of the 30 UT of CEACAM6 were cloned in the sense orientation into the Zn2 + - inducible Ilantzis et al episomal expression vector pML1 containing the mouse metallothionein promoter ( mMT1 ) and the hygromycin - B resistance gene The vector, constructed by replacing the cytomegalovirus promoter cassette of pCEP4 ( Invitrogen, San Diego, CA ) with the mMT1 promoter [ 32 ] , was kindly provided by S M Frisch, The Burnham Institute, La Jolla, CA Vector alone and vector containing the cloned cDNAs were transfected into SW - 1222 and Caco - cells as follows SW - 1222 cells were seeded at 1.0Â106 cells / 100 - mm culture dish and transfected after 24 hours incubation by the calcium phosphate precipitation method with vector alone, or vector containing CEACAM6 cDNA Stably transfected cell populations consisting of hundreds of independent clones were selected in medium containing 200 g / ml of hygromycin - B ( Sigma, St Louis, MO ) Such populations avoid difficulties in interpretation arising from clonal variation in phenotypic properties unrelated to expression of the transfected gene Caco - cells seeded at 2.5Â105 cells / 100 - mm culture dish were transfected as above with vector alone, or vector containing CEA, CEACAM1, and CEA + CEACAM6 cDNAs ( equimolar amounts of both vectors together ) and transfectants selected as described above Unless otherwise stated, transfected cell lines were continuously cultured in the presence of hygromycin - B Cells used for experiments were cultured in medium without hygromycin - B for one passage before use To induce maximal expression of the transfected cDNAs, ZnSO4 was added to the culture medium at a concentration of 100 M, as specified in the experiments Vector - alone controls were also treated with the same concentration of ZnSO4 ( 100 M ) for comparative purposes The nomenclature used for the transfectant populations is as follows: SW ( Hygro ) = vector - alone transfected SW - 1222 cells, SW - CEACAM6" = CEACAM6 transfected SW - 1222 cells, Caco ( Hygro ) = vector - alone transfected Caco - cells, Caco - CEA / CEACAM6" = Caco - cells transfected with both CEA and CEACAM6, Caco - CEA" = CEA transfected Caco - cells, Caco - CEACAM1 = CEACAM1 transfected Caco - cells, Caco - CEA" / - Hygro = Caco - CEA" cells grown without hygromycin - B so as to lose expression of CEA FACS Analysis of Cell Surface CEA and CEACAM6 Expression Cells in monolayer cultures were collected in their mid to late exponential growth phase with or without the addition of ZnSO4 to the culture medium 24 hours before harvesting Cells were analyzed for CEA and CEACAM6 cell surface expression using CEA - specific mAb D14.6.43 and CEACAM6 - specific mAb 9A6FR as primary antibodies, essentially as described previously by Zhou et al [ 33 ] Briefly, cells were incubated with the appropriate primary mAb in 0.5 ml phosphate - buffered saline lacking Ca2 + and Mg2 + ( PBS ) containing 2% FBS ( PBS / FBS ) , washed with PBS / FBS and incubated with fluorescein isothiocyanate ( FITC ) - conjugated F ( ab0 ) goat anti – mouse IgG ( H + L ) ( Jackson Laboratories, West Grove, PA ) in 0.5 ml PBS / FBS Cells were washed again and analyzed by a Becton Dickinson flow Neoplasia Vol 4, No 2, 2002 CEA family members block colonocyte differentiation Ilantzis et al cytometer using LYSYS - II research software (Becton Dickinson, Mississauga, Ontario, Canada ) Growth of Cells in 3D Collagen Gels Collagen gel solutions were prepared using Vitrogen 100 collagen type I ( Collagen Biomaterials, Palo Alto, CA ) according to the manufacturer’s instructions Cells were resuspended at 2Â104 / ml in neutralized collagen gel solution and plated in 24 - well multidish plates ( Nalge Nunc International, Naperville, IL ) and on permeable support filters ( Millicell - CM 12 - mm culture plate inserts, Millipore, Bedford, MA ) Collagen gelation was initiated by incubating the plated cell / gel solutions at 378C and allowed to proceed for 30 minutes Cultures were overlaid ( and underlaid in the case of filter - plated cells ) with - MEM / 10% FBS containing 100 M ZnSO4 and maintained at 378C ( in the presence of 100 M ZnSO4 ) in a humidified atmosphere of 5% CO2 in air Fourteen days later, 200 colonies of each cell line tested were assessed microscopically for glandular morphology, as indicated by the presence of a central lumen Differentiation of Caco - Transfectants Cells ( 3Â105 ) were seeded and cultured in 25 - cm2 flasks in the presence of 100 M ZnSO4, and assessed for dome formation at the indicated days after seeding, as described previously [ 24 ] Millicell - CM 12 - mm culture plate inserts ( Millipore ) were coated with Vitrogen collagen ( Collagen Biomaterials ) as per the manufacturer’s instructions and seeded with 1Â105 and 2Â105 cells per culture well insert The plated cells were cultured for weeks postconfluence in the presence of 100 M ZnSO4 and processed for histologic and immunohistochemical analysis Tissue Architecture Assay Mixed aggregates of dissociated fetal rat colonic cells ( FRCC ) and transfected SW - 1222 cells were formed as described previously [ 34 ] Briefly, 1Â106 FRCC were mixed with either 1Â103 or 2Â103 SW ( Hygro ) or SW - CEACAM6" Zn2 + - induced cells in ml of - MEM / 10% FBS containing 10 g / ml DNAse and 100 M ZnSO4 The cell mixtures were placed in 25 - ml Erlenmeyer flasks, gassed with 5% CO2 in air, and rotated at 378C on a gyratory shaker at 70 rpm for 36 hours As previously reported [ 34 ] , this procedure resulted in the formation of a single large aggregate approximately 1.0 to 1.5 mm in diameter per flask Resultant aggregates ( three per transfectant population per experiment ) were implanted under the kidney capsule of nude mice ( one per mouse ) in accordance with the policies and procedures set forth by the Facility Animal Care Committee and McGill University, as well as those described in the ‘‘Guide to the Care and Use of Experimental Animals’’ prepared by the Canadian Council on Animal Care Surgical procedures required for implantation, approved by the Facility Animal Care Committee, were carried out under sterile conditions as described by Bogden et al [ 35 ] using male homozygous nu / nu CD - mice ( Charles River Canada ) at least weeks postweaning ZnSO4 was added to the drinking water to a final concentration of 25 mM Mice were sacrificed to 10 days later and Neoplasia Vol 4, No 2, 2002 153 the implant - bearing kidney removed for immunohistochemical analysis Identical growth periods in vivo were used throughout each experiment to validate comparisons Tumorigenicity in Nude Mice Caco - CEA / CEACAM6", Caco - CEA", Caco ( Hygro ) , and Caco - CEACAM1 cells ( 5Â106 ) in their logarithmic growth phase were injected subcutaneously into the right and left flanks of - week - old male athymic CD - nude mice ( Charles River Canada, Montreal, Quebec, Canada ) The average latent periods for tumor formation were determined as the time of appearance of a visible mass ( > 0.5 cm in diameter ) The statistical significance of the difference in tumor latency was evaluated using the Student’s t test Immunohistochemistry Collagen gels, implant - bearing kidneys, and filter - grown Caco - transfectants were fixed in 4% paraformaldehyde dissolved in PBS, infused with 0.5 M sucrose in PBS, and frozen in isopentane at À 708C Serial cryostat sections to m thick were taken and immunostained as follows Air - dried sections were incubated with either 9A6FR or D - 14.6.43 followed by incubation either with biotinylated rabbit anti – mouse IgG ( Dako, Santa Barbara, CA ) using normal rabbit serum as a blocking agent or with Dako Envision labelled polymer peroxidase ( Dako ) using Universal Blocker ( Dako ) to block nonspecific staining D - 14.6.43 mAb is absolutely specific for CEA of the human CEA family [ 29 ] and does not react with rat CEA family members ( DeMarte, Beauchemin, and Stanners, unpublished results ) Detection of bound antibodies was carried out using a streptavidin - biotinylated horseradish peroxidase system ( Dako ) and / or visualized by incubating in 0.3% H2O2 containing 0.6 mg / ml 3,30 diaminobenzidine tetrahydrochloride Sections were lightly counterstained with hematoxylin CEA / CEACAM6 and villin immunostaining of SW ( Hygro ) and SW - CEACAM6" monolayer cultures was performed on cells grown on glass slides in medium containing 100 M ZnSO4 Cells were washed twice with PBS and permeabilized with acetone at À 208C for 10 minutes CEA, CEACAM6, and villin were detected using D - 14.6.43, 9A6FR, and V34420 as primary antibodies, respectively, following the procedure outlined above, followed by light counterstaining of the cells with hematoxylin Results To directly test the hypothesis that deregulated overexpression of CEA and CEACAM6 can have tumorigenic effects on human colonocytes, we tested their effects on human colonocyte cell lines, SW - 1222 and Caco - 2, which retain the capacity to polarize, differentiate, and organize into crypt - like glandular structures in culture [ 34,36,37 ] Both of these cell lines produce endogenous CEA and CEACAM6 ( Figure 1AandB ) but, as with normal colonic crypt cells, endogenous expression tends to be positively correlated with polarization and differentiation This is especially the case for Caco - colonocytes where expression of these 154 CEA family members block colonocyte differentiation Ilantzis et al Figure FACS profiles indicating levels of cell surface expression of CEA and CEACAM6 in parental SW - 1222 cells, in transfected SW - 1222 and Caco - cell populations, and in purified epithelial single - cell suspensions prepared from a freshly resected colonic tumor sample and adjacent normal crypts [ ] ( A ) Levels of CEA and CEACAM6 in parental ( untransfected ) SW - 1222 cells ( B ) Levels of CEA and CEACAM6 in control Caco ( Hygro ) cells transfected with vector alone in the presence of Zn2 + ( C ) Cell surface expression of CEA and CEACAM6 in purified epithelial cells prepared from normal colonic crypts [ ] ( D ) CEACAM6 overexpression in uninduced ( four - fold increase in mean level ) and Zn2 + - induced ( nine - fold increase in mean level ) SW - CEACAM6" relative to endogenous control levels in Zn2 + - treated control SW ( Hygro ) cells transfected with vector alone ( E ) Zn2 + - induced levels of CEA and CEACAM6 in Caco - CEA / CEACAM6" cells ( F ) Cell surface expression of CEA and CEACAM6 in purified epithelial cells prepared from tumor colonic crypts [ ] The tumor profiles show levels of both CEA and CEACAM6 that greatly exceed levels in adjacent normal crypts molecules is observed to markedly increase in confluent, differentiated cultures [ 24 ] Both were transfected with CEA and / or CEACAM6 cDNAs in the Zn2 + - inducible episomal expression vector pML1 to isolate stable populations of transfectants that produce higher levels of CEA / CEACAM6 constitutively, i.e., no longer correlated with differentiation, as observed in human colorectal carcinomas Although the inducible expression vector was leaky, in that only about two - fold increases were obtained with Zn2 + addition, final expression levels were increased by about 10 - to 30 - fold, even in exponentially proliferating nondifferentiated cultures ( Figures 1D and E and 4C, inset; Table ) These final levels were quite similar to the cell surface levels of CEA and CEACAM6 found on tumor colonocytes purified from typical freshly excised colorectal carcinomas ( Figure 1F ) All transfectants sought, i.e., expressing increased levels of CEA, CEACAM6 and CEA + CEACAM6, could not be obtained in both cell lines, for undetermined reasons Effect of CEACAM6 Overexpression on Polarization and Differentiation in Cell Monolayers SW - 1222 parental and SW ( Hygro ) control cells, maintained in culture into late stationary phase, accumulated gland - like structures of polarized cells radially arranged around a central lumen, forming ‘‘intercellular cysts’’ [ 38,39 ] that resemble colonic crypts cut in transverse cross section ( Figure 2A ) SW - CEACAM6" cells ( Figure 2B ) , on the other hand, accumulated 95% fewer such intercellular cysts ( Figure 2B, inset ) The nature of these intercellular cysts was examined by immunohistochemical assays for the presence and localization of biochemical markers normally present in polarized colonocytes Figure 2C shows the polarized localization of villin on the lumenal membrane of cysts formed by SW ( Hygro ) control cells, indicating the presence of an apical brush border In contrast, the SW - CEACAM6" cells showed a complete lack of localized expression for villin ( Figure 2D ) These results show that deregulated overexpression of CEACAM6 can block the formation of intercellular cysts, thus blocking cellular polarization and crypt - like glandular tissue architecture Effect of CEACAM6 Overexpression on Differentiation in 3D Collagen Gels When embedded and grown within collagen gels, parental and vector - only transfected SW - 1222 colonocytes can grow into 3D spheroids [ 36 ] , with a relatively high proportion ( Figure 3A ) exhibiting visible central gland - like Neoplasia Vol 4, No 2, 2002 CEA family members block colonocyte differentiation Ilantzis et al 155 Table Summary of Transfectants and Transfectant Controls* Cells Parental Line Change in CEAy Change in CEACAM6y SW ( Hygro ) SW - 1222 no change no change SW - CEACAM6" SW - 1222 no change – - fold increase Caco - CEA" Caco - 32 - fold increasez no change Caco - CEA" Caco - 10 - fold increasex no change Caco - CEA / CEACAM6" Caco - 20 - fold increase 20 - fold increase Caco ( Hygro ) Caco - no change no change Caco - CEA" / - Hygro Caco - no change no change Caco - CEACAM1 Caco - no change no change *Control populations expressing endogenous levels of CEA / CEACAM6 y Fold increase in mean levels obtained by FACS analysis of Zn2 + - treated populations used in all experiments unless otherwise indicated, compared to controls transfected with vector alone z At passages used in dome experiments x At passages used in tumorigenicity experiments lumens surrounded by a radial arrangement of polarized cells, with apical surfaces facing the lumen ( Figure 3B, c ) ; these structures thus resemble closed colonic crypts ( Figure 3B, a ) The radially arranged cells exhibit basal nuclei and predominantly apical immunostaining patterns for both endogenous CEA ( data not shown ) and endogenous Figure Intercellular cysts in control ( A ) and ( C ) versus SW - CEACAM6" cells ( B ) and ( D ) in monolayer culture at late stationary phase ( A ) Live phase contrast appearance of control SW ( Hygro ) cells showing the formation of numerous intercellular cysts ( arrows ) ( B ) Phase contrast appearance of live SW CEACAM6" cells at late stationary phase demonstrating a lack of appreciable intercellular cysts; ( inset ) quantitation of cysts per square centimeter per 105 cells Values shown are the mean of three experiments comparing SW ( Hygro ) and SW - CEACAM6" cells; error bars, SD ( C ) Immunolocalization of villin to the apical membrane domain ( arrows ) of cells lining intercellular cysts in monolayer culture of control SW ( Hygro ) cells counterstained with hematoxylin ( D ) Delocalized immunohistochemical demonstration of villin in monolayer culture of SW - CEACAM6" cells counterstained with hematoxylin Bars: 10 m Neoplasia Vol 4, No 2, 2002 156 CEA family members block colonocyte differentiation Ilantzis et al Figure Glandular differentiation of SW - 1222 cells and transfected populations grown in 3D collagen gels ( A ) Quantitation of glandular differentiation as assessed by the percentage of spheroid colonies with identifiable central gland - like lumens Values shown represent the mean of three experiments; error bars, SD ( B ) Comparison of control SW ( Hygro ) ( a and c ) and SW - CEACAM6" spheroid colonies ( b, d, and e ) : ( a ) Phase contrast appearance of live unsectioned SW ( Hygro ) spheroid colonies in collagen gel Cells are organized around a central lumen indicating glandular differentiation ( arrows ) ( b ) Phase contrast appearance of live unsectioned SW - CEACAM6" spheroid colonies in collagen gel SW - CEACAM6" cells show a more irregular and undifferentiated pattern of growth ( c ) Immunostained section of well - differentiated SW ( Hygro ) spheroid showing well - oriented nuclei polarized to the periphery and endogenous CEACAM6 expression localized to the apical pole ( arrow ) at the cellular level ( counterstained with hematoxylin ) ( d ) Hematoxylin - stained section of a poorly differentiated SW - CEACAM6" spheroid colony showing disorganized growth ( e ) Immunostained section of a poorly differentiated SW - CEACAM6" spheroid colony showing intense staining of delocalized CEACAM6 ( counterstained with hematoxylin ) Bars: 12 m CEACAM6 ( Figure 3B, c ) , indicative of a well - developed gland - like morphology In contrast, dramatically fewer SW CEACAM6" - derived spheroids appeared to exhibit gland like morphologies ( Figure 3A and B, b ) In addition, a significant proportion of the relatively few SW - CEACAM6" spheroids scored as having central lumens were more irregular and dysplastic in appearance than the majority of positive control spheroids ( data not shown ) Sectioned SW -CEACAM6" spheroids typically revealed poorly formed structures of disorganized cells ( Figure 3B, d ) with delocalized expression of both endogenous CEA ( data not shown ) and total ( endogenous plus transfected ) CEACAM6 ( Figure 3B, e ) , indicating a complete lack of polarization at the cellular level; CEACAM6 was localized both cytoplasmically and circumferentially at the membrane surface of SW - CEACAM6" cells and could also be observed at the peripheral edge of the spheroids in contact with the collagen matrix These results show that deregulated overexpression of CEACAM6 prevents the formation of gland - like structures by SW - 1222 colonocytes in collagen gels and results in spheroid colonies of unpolarized, disorganized cells Neoplasia Vol 4, No 2, 2002 CEA family members block colonocyte differentiation Ilantzis et al Effects of CEA / CEACAM6 Overexpression on Caco - Differentiation In Vitro Domes When grown in vitro under standard conditions, the human colonocyte cell line Caco - undergoes spontaneous postconfluent enterocytic differentiation [ 37 ] The early stage of the differentiation process is characterized morphologically by the presence of short brush border microvilli on the upper surface of the monolayer and the presence of tight junctions between the lateral membranes of adjacent cells, typical of well polarized epithelial cells At late confluence, the differentiation process is functionally complete with high 157 levels of brush border – associated enzyme activities and the appearance of ‘‘domes,’’ local displacements of the monolayer from the underlying support due to the vectorial transport of electrolytes and fluid [ 40,41 ] , which are typical of transporting polarized epithelial monolayers When assessed for dome formation, control Caco ( Hygro ) cells, after having reached confluence on day 6, consistently produced numerous domes with a peak number at about day ( Figure 4A; andB ) In contrast, Caco -CEA / CEACAM6" cells produced far fewer domes ( approximately 80% less ) under the same conditions ( Figure 4A; andB ) The time in culture for Caco -CEA / Figure Dome formation of control Caco ( Hygro ) cells versus Caco - CEA / CEACAM6 transfectants in monolayer culture ( A, left ) Phase contrast micrograph showing numerous domes formed by postconfluent monolayer of control Caco ( Hygro ) cells ( A, right ) Phase contrast micrograph of postconfluent Caco - CEA / CEACAM6" cells showing dramatic reduction in dome formation Bars: 60 m ( B ) Kinetics of dome formation for Caco ( Hygro ) versus Caco - CEA / CEACAM6" cells Values shown represent the mean of three experiments; error bars, SD ( C ) Kinetics of dome formation for Caco - CEA" versus Caco - CEA" cells that have completely lost CEA overexpression ( Caco - CEA" / - Hygro ) Values shown represent the mean of two experiments; error bars, SD Relative cell surface levels of CEA ( inset ) are indicated Neoplasia Vol 4, No 2, 2002 158 CEA family members block colonocyte differentiation CEACAM6" transfectants was extended by an additional days to allow for the possibility of a lag in appearance of domes corresponding to their - day lag in reaching confluence but no significant increase was observed ( Figure 4B ) To test for both the reproducibility and reversibility of this effect, an independent transfectant population of Caco - cells overexpressing CEA alone, Caco - CEA", was tested both before and after losing CEA overexpression As with the previous CEA / CEACAM6 transfectant population, essentially no domes were seen in this population but when, by culture in the absence of hygromycin selection for just two passages, all CEA overexpression was lost ( Figure 4C, inset ) , dome formation returned to high levels ( Figure 4C ) Thus, the inability to form domes and, by implication, to polarize was specifically dependent on the presence of high cell surface levels of CEA Monolayer tissue architecture Caco ( Hygro ) control colonocytes were grown to confluence on collagen - coated filter supports to assess their tissue architecture Sections of Ilantzis et al these cultures perpendicular to the plane of the support revealed a monolayer of cells ( Figure 5A ) with immunohistochemically detected endogenous CEA ( Figure 5C ) and CEACAM6 ( data not shown ) on the upper cellular surface, indicating the presence of a palisade layer of polarized cells with apical membrane microvilli facing the medium, characteristic of a morphologically differentiated colonic epithelium Under the same conditions, Caco - CEA / CEACAM6" colonocytes failed completely to establish a polarized monolayer; in this case, cells were found piled up in multilayered sheets of two to three cells in thickness ( Figure 5B ) In addition, expression of total ( endogenous plus transfected ) CEA ( Figure 5D ) and CEACAM6 ( data not shown ) was seen distributed over the entire surface of cells, at cell – cell borders and on cell surfaces in contact with the underlying matrix support, consistent with a lack of cellular polarization and strikingly reminiscent of the distorted tissue architecture observed in colonic crypts of human colorectal carcinomas [ ] These results, therefore, show that deregulated overexpression of CEA and CEACAM6 blocks Caco - differentiation by preventing cellular polarization Figure Comparison of postconfluent control Caco ( Hygro ) ( A and C ) and Caco - CEA / CEACAM6" cells ( B and D ) grown on collagen - coated filters ( Millicell CM, Millipore ) sectioned perpendicular to the plane of support ( A ) Section of control cells stained with hematoxylin showing a single layer of Caco ( Hygro ) cells in contact with the underlying filter support directly beneath ( B ) Section of hematoxylin stained Caco - CEA / CEACAM6" cells revealing multilayering ( C ) Immunohistochemical demonstration of CEA localized to the upper monolayer surface ( arrow ) of Caco ( Hygro ) control cells indicating cellular polarization ( D ) Intense delocalized expression of CEA in immunostained section of CEA / CEACAM6" cells showing high levels of CEA over the entire surface of cells, in adjacent areas between cells, and on membrane surfaces in contact with the underlying support, consistent with a lack of cellular polarization Bars: m Neoplasia Vol 4, No 2, 2002 CEA family members block colonocyte differentiation Ilantzis et al 159 Figure Comparison of architecture adopted by control SW ( Hygro ) and SW - CEACAM6" test cells detected by CEA immunoreactivity using tissue architecture assay ( A, C, and E ) Photomicrographs of immunostained sections of in vivo grown SW ( Hygro ) / FRCC control mixed aggregates showing well - organized SW ( Hygro ) crypt - like structures with polarized staining of CEA ( arrows ) localized to the apical pole of cells facing the crypt lumen Nuclei well - oriented to the basal pole of CEA positive cells can be seen Note the presence of CEA - negative crypt structures in ( A ) derived from FRCC ( star ) ( B, D, and F ) Photomicrographs of immunostained sections of in vivo grown SW - CEACAM6" / FRCC mixed aggregates showing poorly organized and highly dysplastic foci of SW - CEACAM6" cells ( thick arrows ) with delocalized staining of CEA Micrographs are representative of serial sections obtained from three separate independent experiments Bars: 10 m and configuring the cells into a multilayered tumor - like tissue architecture To determine whether these observed effects of CEA / CEACAM6 overexpression on Caco - could promote tumorigenic behavior in vivo, the tumorigenic potential of Caco - CEA" and Caco - CEA / CEACAM6" colonocytes versus Caco control transfectants [ Caco ( Hygro ) and Caco CEACAM1 ] was tested in nude mice The average latency for the overexpressing group was significantly reduced ( P < 04 ) by a period of 5.5 weeks compared to that of the control group ( Figure 5B, inset ) In addition, the overexpressing tumors consistently showed greater rates of growth post_latency ( observed increase in tumor mass over time ) when compared to control tumors ( data not shown ) indicative of a more rapid proliferative rate and / or enhancement of cell survival Neoplasia Vol 4, No 2, 2002 Effect of CEACAM6 Overexpression on SW - 1222 Differentiation In Vivo As a further test of the effects of CEACAM6 deregulated overexpression on colonocyte phenotype in vivo, we tested SW ( Hygro ) control versus SW - CEACAM6" test colonocytes for their ability to conform to normal colonic crypt architecture in nude mice This was achieved by application of a recently developed tissue architecture assay [ 34 ] , in which a low proportion of human colonic epithelial test cells are mixed with dissociated FRCC and allowed to reaggregate in vitro; resultant aggregates are implanted under the kidney capsule of nude mice and allowed to grow for a period of to 10 days, then sectioned and stained immunohistochemically for CEA and CEACAM6 to identify the test human colonocytes This assay provides a more valid assessment of the malignant phenotype of test cells in the context of their 160 CEA family members block colonocyte differentiation Ilantzis et al Figure Model for oncogenic effect of deregulated overexpression of CEA / CEACAM6" in colonic epithelial crypt cells with proliferative potential normal location in whole tissue and mimics tumor development in vivo [ 34 ] With this assay, the control SW ( Hygro ) cells formed well - differentiated colonic crypt - like structures with polarized cells exhibiting basally oriented nuclei and apical CEA ( Figure 6A; C and E ) and CEACAM6 ( data not shown ) expression In dramatic contrast, the SW CEACAM6" cells produced poorly formed foci of disorganized cells without recognizable crypt structure ( Figure 6B; D and F ) Also, the SW - CEACAM6" cells exhibited CEA and CEACAM6 ( data not shown ) expression all over their surfaces and at cell – cell borders, further demonstrating their lack of polarity ( Figure 6B and F ) Thus, deregulated expression of CEACAM6 caused a dramatic distortion of tissue architecture and a loss of cell polarity and differentiation in vivo Discussion The above results demonstrate that deregulated overexpression of CEA / CEACAM6 in human colonocytes can inhibit differentiation, block cellular polarization and abort normal tissue architecture both in vitro and in vivo Because these cell surface glycoproteins are overexpressed in as many as 50% of all human cancers, this could represent a clinically significant observation There are, however, some serious counter - observations to be considered before this conclusion can be accepted Negative Findings First, although most groups have reported elevated levels of tumor - associated CEACAM6, not all groups agree that CEA levels are similarly elevated, especially at the mRNA level [ 42 – 44 ] There seems to be general agreement that mRNA levels show a more consistent increase for CEACAM6 than for CEA [ 45 ] However, it is tumor cell surface protein levels that will determine biologic effects and, where measured, these have been observed to be markedly increased for both CEACAM6 and CEA [ ] Also, because of field effects that impart tumor - like properties to normal adjacent tissues as far away as 10 cm from tumor tissue ( in the case of colorectal carcinomas ) [ 46,47 ] , tumor - normal comparisons should involve more distant and therefore more normal tissue This has not been the case in many studies Second, how could CEA and CEACAM6 expression cause loss of cell polarization, cell differentiation, and tissue architecture when, in vivo, it is precisely in polarized, differentiated, and normal epithelia at the ends of colonic crypts that the expression of these molecules is seen to increase [ 22,23 ] ? Reconciliation of this seemingly opposite observation can be provided by the common finding that the biologic effects of expression of many active molecules can depend radically on the cellular context, e.g., c - myc [ 20 ] and TGF - [ 48 ] In the case of CEA and CEACAM6, we suggest that deregulated overexpression, in cells that still possess division potential and have not yet differentiated, is required for tumorigenic effect The human Caco - colonocytes used in this study normally express CEA and CEACAM6 after becoming polarized and differentiated late in the growth cycle [ 24 ] , just as observed in normal colonocytes in vivo They produce very little CEA / CEACAM6 in the proliferative phase of the growth cycle but when transfected with CEA / CEACAM6 cDNA driven by promoters giving constitutive expression at all phases of the growth cycle, as occurs in most colorectal carcinomas [ 24 ] , dramatic tumorigenic effects are seen Neoplasia Vol 4, No 2, 2002 CEA family members block colonocyte differentiation Ilantzis et al Third, mice not possess genes determining the glycophosphatidyl inositol ( GPI ) - linked members of the CEA family such as CEA and CEACAM6, and when the human gene for CEA was inserted into mice, the transgenic animals showed no statistically significant increase in tumor formation, even after crossing with Min mice harboring a mutation in the mouse APC gene [ 49 ] Aside from the fact that negative results in such experimental situations are never definitive, e.g., the mice still not have the gene for CEACAM6 that is also deregulated in human carcinomas, the human CEA gene showed normal human tissue – specific patterns of expression in the mice and would therefore be expected to be normally regulated with differentiation in colonic crypts The actual observation of human CEA expression in the cytoplasm of colonocytes and in cells at the base of the crypts reported in this work [ 25 ] is presumably due to an increase in the sensitivity of detection of CEA by immunohistochemistry because the method of fixation employed in this study appears to be a determinant factor in setting the threshold level required for visualization [ 28,46,50 ] Positive Findings Aside from the observations of inhibition of differentiation in model systems, the inverse correlation between cell surface levels of these molecules and differentiation status of tumors, and the general inhibition of anoikis induced by CEA / CEACAM6 overexpression alluded to in the introduction, there is a generally positive correlation between CEA expression and the propensity for metastasis by colorectal carcinomas — in fact, CEA expression is invariably high in liver metastases ( A Fuks, personal communication ) Also, expression of tumorigenic Ras has been reported to result in upregulation of CEA and loss of cellular polarization in HD6 colon carcinoma cells [ 51 ] In breast, the correlation of CEA / CEACAM6 expression with cancer is even more definitive in that normal breast epithelial cells show absolutely no expression of these molecules, whereas 40% to 70% of carcinomas show such expression [ ] , which is correlated positively with stage [ 52 – 55 ] Although the perturbation of the colonocyte systems utilized in this study, involving the effects of single added genes, is very specific, a molecular rationale for the observed effects would be desirable Our evidence to date indicates the necessity for self - binding of the extracellular domains of CEA / CEACAM6, consistent with their demonstrated function as homotypic intercellular adhesion molecules [ 18,56,57 ] , and membrane anchorage by the specific GPI anchors of these molecules [ 58 ] As a consequence of such self - binding, the alteration of cell surface levels or functional status of specific integrins1,2 and the inhibition of anoikis [ 21 ] have been documented The specific integrins implicated, Ordon˜ez C, Screaton RA, Ilantzis C, Fan M, and Stanners CP Carcinoembryonic antigen, a human tumor marker, inhibits cell differentiation and apoptosis by perturbing the function of the integrin receptor Submitted for publication Malette B, and Stanners CP, Carcinoembryonic antigen, a human tumor marker, inhibits retinoic acid - induced neurogenic differentiation of P19 embryonal carcinoma cells by disrupting integrin regulation Submitted for publication Neoplasia Vol 4, No 2, 2002 161 51 and v3, have been shown to have the effects we observe on myogenic differentiation [ 59 – 62 ] and, in the case of 51, to operate through inhibition of MAP kinase [ 61,63 ] How can these observations be integrated into proposed schemes for tumorigenesis in the colon [ ] ? In this study, we have utilized human colorectal carcinoma cell lines that retain considerable differentiation capacity, i.e., cells that could be considered less progressed along the pathway of tumorigenesis, as delineated in Figure Both Caco - cells and SW - 1222 cells are known to possess inactivating mutations in the APC gene [ 64 ] Additionally, Caco - cells exhibit chromosomal instability [ 65 ] , are mutant for p53 [ 66 ] , and contain a heterozygous mutation of the - catenin gene [ 64 ] , yet are still able to differentiate and are poorly tumorigenic when injected into nude mice [ 67,68 ] CEA and CEACAM6 expression have been shown to be upregulated at the microadenoma ( early dysplastic ) stage in colons of FAP patients with APC mutations [ ] and in adenomas [ 69 – 72 ] We therefore propose that, as a very early event, CEA and CEACAM6 are upregulated in cells retaining division potential and lead to a progressive loss of cellular polarization, a distortion of tissue architecture and an inhibition of differentiation This resultant cell population, held in a G0 like state with proliferative potential, would represent an expanded target for the acquisition of the further genetic lesions frequently found in colorectal carcinomas ( Figure ) CEA and CEACAM6 could thus be considered to provide ‘‘fertile soil’’ [ 20 ] for the action of the three known colon tumor mutators: chromosomal instability [ 73 ] , mismatch repair deficiency [ 74 ] , and p53 inactivation [ 75 ] Because CEA and CEACAM6 are overexpressed in so many different, prevalent human cancers, the reversal of these effects represents an appealing novel approach for cancer treatment Acknowledgements We thank Serge Jothy, Vincent Gigue`re, and Mark Featherstone for critical reading of the manuscript References [1] Kinzler KW, and Vogelstein B ( 1996 ) Lessons from hereditary colorectal cancer Cell 87, 159 – 70 [2] Boucher D, Cournoyer D, Stanners CP, and Fuks A ( 1989 ) Studies on the control of gene expression of the carcinoembryonic antigen family in human tissue Cancer Res 49, 847 – 52 [3] Zimmer R, and Thomas P ( 2001 ) Mutations in the carcinoembryonic antigen gene in colorectal cancer patients: implications on liver metastasis Cancer Res 61, 2822 – 26 [4] Chevinsky AH ( 1991 ) CEA in tumors of other than colorectal origin Semin Surg Oncol 7, 162 – 66 ¨ brink B ( 1997 ) CEA adhesion molecules: multifunctional proteins [5] O with signal - regulatory properties Curr Opin Cell Biol 9, 616 26 [6] Hammarstroăm S, Olsen A, Teglund S, and Baranov V ( 1998 ) The Nature and Expression of the Human CEA Family: Basic and Clinical Perspectives In Cell Adhesion and Communication Mediated by the CEA Family ( 1st ed ) , Vol CP Stanners ( Ed ) Hardwood Academic Publishers, Amsterdam pp – 30 [7] Ilantzis C, Jothy S, Alpert LC, Draber, P, and Stanners CP ( 1997 ) 162 [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] CEA family members block colonocyte differentiation Cell - surface levels of human carcinoembryonic antigen are inversely correlated with colonocyte differentiation in colon carcinogenesis Lab Invest 76, 703 – 16 Sheahan K, O’Brien MJ, Burke B, Dervan PA, O’Keane JC, Gottlieb LS, and Zamcheck N ( 1990 ) Differential reactivities of carcinoembryonic antigen ( CEA ) and CEA - related monoclonal and polyclonal antibodies in common epithelial malignancies Am J Clin Pathol 94, 157 – 64 Zimmermann W, Weber B, Ortlieb B, Rudert F, Schempp W, Fiebig HH, Shivel JE, von Kleist, S, and Thompson JA ( 1988 ) Chromosomal localization of the carcinoembryonic antigen gene family and differential expression in various tumors Cancer Res 48, 2550 – 54 Sheibani K, Battifora H, and Burke JS ( 1986 ) Antigenic phenotype of malignant mesotheliomas and pulmonary adenocarcinomas An immunohistologic analysis demonstrating the value of Leu M1 antigen Am J Pathol 123, 212 – 19 Albers GH, Fleuren G, Escribano MJ, and Nap M ( 1988 ) Immunohistochemistry of CEA in the human pancreas during development, in the adult, chronic pancreatitis, and pancreatic adenocarcinoma Am J Clin Pathol 90, 17 – 22 Banks ER, and Cooper PH ( 1991 ) Adenosquamous carcinoma of the skin: a report of 10 cases J Cutaneous Pathol 18, 227 – 34 Athanassiadou P, Athanassiades P, Lazaris D, Kyrkou K, Petrakakou E, and Aravantinos D ( 1994 ) Immunocytochemical differentiation of reactive mesothelial cells and adenocarcinoma cells in serous effusions with the use of carcinoembryonic antigen and fibronectin Acta Cytol 38, 718 – 22 Sanders DS, Ferryman SR, Bryant FJ, and Rollason TP ( 1993 ) Patterns of CEA - related antigen expression in invasive squamous carcinoma of the cervix J Pathol 171, 21 – 26 Kim J, Kaye FJ, Henslee JG, Shively JE, Park JG, Lai SL, Linnoila RI, Mulshine JL, and Gazdar AF ( 1992 ) Expression of carcinoembryonic antigen and related genes in lung and gastrointestinal cancers Int J Cancer 52, 718 – 25 Neumaier M, Paululat S, Chan A, Matthaes P, and Wagener C ( 1993 ) Biliary glycoprotein, a potential human cell adhesion molecule, is down - regulated in colorectal carcinomas Proc Natl Acad Sci USA 90, 10744 – 48 Kleinerman DI, Troncoso P, Lin SH, Pisters LL, Sherwood ER, Brooks T, von Eschenbach AC, and Hsieh JT ( 1995 ) Consistent expression of an epithelial cell adhesion molecule ( C - CAM ) during human prostate development and loss of expression in prostate cancer: implication as a tumor suppressor Cancer Res 55, 1215 – 20 Benchimol S, Fuks A, Jothy S, Beauchemin N, Shirota K, and Stanners CP ( 1989 ) Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule Cell 57, 327 – 34 Eidelman FJ, Fuks A, DeMarte L, Taheri M, and Stanners CP ( 1993 ) Human carcinoembryonic antigen, an intercellular adhesion molecule, blocks fusion and differentiation of rat myoblasts J Cell Biol 123, 467 – 75 Screaton RA, Penn LZ, and Stanners CP ( 1997 ) Carcinoembryonic antigen, a human tumor marker, cooperates with Myc and Bcl - in cellular transformation J Cell Biol 137, 939 – 52 Ordon˜ez C, Screaton RA, Ilantzis C, and Stanners, CP ( 2000 ) Human carcinoembryonic antigen functions as a general inhibitor of anoikis Cancer Res 60, 3419 – 24 Ahnen DJ, Nakane PK, and Brown WR ( 1982 ) Ultrastructural localization of carcinoembryonic antigen in normal intestine and colon cancer: abnormal distribution of CEA on the surfaces of colon cancer cells Cancer 49, 2077 – 90 Baranov V, Yeung MM, and Hammarstrom S ( 1994 ) Expression of carcinoembryonic antigen and nonspecific cross - reacting 50 - kDa antigen in human normal and cancerous colon mucosa: comparative ultrastructural study with monoclonal antibodies Cancer Res 54, 3305 – 14 Hauck W, and Stanners CP ( 1991 ) Control of carcinoembryonic antigen gene family expression in a differentiating colon carcinoma cell line, Caco - Cancer Res 51, 3526 – 33 Eades - Perner A M, van der Putten H, Hirth A, Thompson J, Neumaier M, von Kleist S, and Zimmermann W ( 1994 ) Mice transgenic for the human carcinoembryonic antigen gene maintain its spatiotemporal expression pattern Cancer Res 54, 4169 – 76 Stanners CP, Eliceiri GL, and Green H ( 1971 ) Two types of ribosome in mouse – hamster hybrid cells Nature ( London ) New Biol 230, 52 – 54 Haggarty A, Legler C, Krantz MJ, and Fuks A ( 1986 ) Epitopes of carcinoembryonic antigen defined by monoclonal antibodies prepared from mice immunized with purified carcinoembryonic antigen or HCT 8R cells Cancer Res 46, 300 – 309 Ilantzis et al [28] Jothy S, Brazinsky SA, Chin ALM, Haggarty A, Krantz MJ, Cheung M, and Fuks A ( 1986 ) Characterization of monoclonal antibodies to carcinoembryonic antigen with increased tumor specificity Lab Invest 54, 108 – 17 [29] Zhou H, Stanners CP, and Fuks A ( 1993 ) Specificity of anti carcinoembryonic antigen monoclonal antibodies and their effects on CEA - mediated adhesion Cancer Res 53, 3817 – 22 [30] Daniel S, Nagel G, Johnson JP, Lobo FM, Hirn M, Jantscheff P, Kuroki M, von Kleist S, and Grunert F ( 1993 ) Determination of the specificities of monoclonal antibodies recognizing members of the CEA family using a panel of transfectants Int J Cancer 55, 303 – 10 [31] Barnett TR, Kretschmer A, Austen DA, Goebel SJ, Hart JT, Elting JJ, and Kamarck ME ( 1989 ) Carcinoembryonic antigens: alternative splicing accounts for the multiple mRNAs that code for novel members of the carcinoembryonic antigen family J Cell Biol 108, 267 – 76 [32] Lukashev ME, Sheppard D, and Pytela R ( 1994 ) Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed beta integrin cytoplasmic domain J Biol Chem 269, 18311 – 14 [33] Zhou H, Fuks A, Alcaraz G, Bolling TJ, and Stanners CP ( 1993 ) Homophilic adhesion between Ig superfamily carcinoembryonic antigen molecules involves double reciprocal bonds J Cell Biol 122, 951 – 60 [34] Ilantzis C, and Stanners CP ( 1997 ) A colonic tissue architecture assay applied to human colon carcinoma cells In Vitro Cell Dev Anim 33, 50 – 61 [35] Bogden AE, Kelton DE, Cobb WR, and Esber HJ ( 1978 ) A rapid screening method for testing chemotherapeutic agents against human tumor xenographs In Proceedings of the Symposium on the Use of Athymic ( Nude ) Mice in Cancer Research DP Houchens and AA Ovejera ( Eds ) Gustav Fischer, New York pp 231 – 50 [36] Pignatelli M, and Bodmer WF ( 1988 ) Genetics and biochemistry of collagen binding - triggered glandular differentiation in a human colon carcinoma cell line Proc Natl Acad Sci USA 85, 5561 – 65 [37] Pinto M, Robine - Leon S, Appay MD, Keinger M, Triadou N, Dussaulx E, Lacroix B, Simon - Assmann P, Haffen K, Fogh J, and Zweibaum A ( 1983 ) Enterocyte - like differentiation and polarization of the human colon carcinoma cell line Caco - in culture Biol Cell 47, 323 – 30 [38] Remy L, Marvaldi J, Rua S, Secchi J, and Lechene de la Porte P ( 1984 ) The role of intracellular lumina in the repolarization process of a colonic adenocarcinoma cell line Virchows Archiv B 46, 297 – 305 [39] Le Bivic A, Hirn M, and Reggio H ( 1988 ) HT - 29 cells are an in vitro model for the generation of cell polarity in epithelia during embryonic differentiation Proc Natl Acad Sci USA 85, 136 – 40 [40] Ramond MJ, Martinot - Peignoux M, and Erlinger S ( 1985 ) Dome formation in the human colon carcinoma cell line Caco - in culture Influence of ouabain and permeable supports Biol Cell 54, 89 – 92 [41] Dharmsathaphorn K, McRoberts JA, Mandel KG, Tisdale LD, and Masui H ( 1984 ) A human colonic tumor cell line that maintains vectorial electrolyte transport Am J Physiol 246, G204 – 208 [42] Hinoda Y, Takahashi H, Higashide T, Nakano T, Arimura Y, Yoshimoto M, Tsujisaki M, Imai K, and Yachi A ( 1991 ) Correlated expression of mRNAs of carcinoembryonic antigen and nonspecific cross - reacting antigen genes in malignant and nonmalignant tissues of the colon Am J Physiol 21, 75 – 81 [43] Thompson J, Seitz M, Chastre E, Ditter, M, Aldrian C, Gespach C, and Zimmermann W ( 1997 ) Down - regulation of carcinoembryonic antigen family member expression is an early event in colorectal tumorigenesis Cancer Res 57, 1776 – 84 [44] Scholzel S, Zimmermann W, Schwarzkopf G, Grunert F, Rogaczewski B, and Thompson J ( 2000 ) Carcinoembryonic antigen family members CEACAM6 and CEACAM7 are differentially expressed in normal tissues and oppositely deregulated in hyperplastic colorectal polyps and early adenomas Am J Pathol 156, 595 – 605 [45] Chi KF, Jessup JM, and Frazier ML ( 1991 ) Predominant expression of mRNA coding for nonspecific cross - reacting antigen in colorectal carcinomas Tumour Biol 12, 298 – 308 [46] Jothy S, Slesak B, Harlozinska A, Lapinska J, Adamiak J, and Rabczynski J ( 1996 ) Field effect of human colon carcinoma on normal mucosa: relevance of carcinoembryonic antigen expression Tumour Biol 17, 58 – 64 [47] Guadagni F, Roselli M, Cosimelli M, Spila A, Cavaliere F, Arcuri R, D’Alessandro R, Fracasso PL, Casale V, Vecchione A, Casciani CU, Greiner JW, and Schlom J ( 1997 ) Quantitative analysis of CEA expression in colorectal adenocarcinoma and serum: lack of correlation Int J Cancer 72, 949 – 54 [48] Gold LI ( 1999 ) The role for transforming growth factor - beta ( TGF beta ) in human cancer Crit Rev Oncog 10, 303 – 60 Neoplasia Vol 4, No 2, 2002 CEA family members block colonocyte differentiation Ilantzis et al [49] Thompson JA, Eades - Pernier AM, Ditter M, Muller WJ, and Zimmermann W ( 1997 ) Expression of transgenic carcinoembryonic antigen ( CEA ) in tumor - prone mice: an animal model for CEA - dierected tumor immunotherapy Int J Cancer 72, 197 – 202 [50] Primus FJ, Clark CA, and Goldenburg DM ( 1981 ) Immunoperoxidase localization of carcinoembryonic antigen in normal human intestinal mucosa J Natl Cancer Inst 67, 1031 – 39 [51] Yan Z, Deng X, Chen M, Xu Y, Ahram M, Sloane BF, and Friedman E ( 1997 ) Oncogenic c - Ki - ras but not oncogenic c - Ha - ras up regulates CEA expression and disrupts basolateral polarity in colon epithelial cells J Biol Chem 272, 27902 – 907 [52] Hayes DF, Zurawski VR Jr, and Kufe DW ( 1986 ) Comparison of circulating CA15 - and carcinoembryonic antigen levels in patients with breast cancer J Clin Oncol 4, 1542 – 50 [53] Safi F, Kohler I, Rottinger E, Suhr P, and Beger HG ( 1989 ) Comparison of CA 15 - and CEA in diagnosis and monitoring of breast cancer Int J Biol Markers 4, 207 – 14 [54] Dnistrian AM, Schwartz MK, Greenberg EJ, Smith CA, and Schwartz DC ( 1991 ) Evaluation of CA M26, CA M29, CA 15 - and CEA as circulating tumor markers in breast cancer patients Tumour Biol 12, 82 – 90 [55] Pavesi F, Lotzniker M, Scarabelli M, Mauro E, Visconti G, Nicolato E, and Moratti R ( 1994 ) Circulating CA 549 and other associated antigens in breast cancer patients Oncology 51, 18 – 21 [56] Zhou H, Fuks A, and Stanners CP ( 1990 ) Specificity of intercellular adhesion mediated by various members of the immunoglobulin supergene family Cell Growth Differ 1, 209 – 15 [57] Oikawa S, Inuzuka C, Kuroki M, Matsuoka Y, Kosaki G, and Nakazato H ( 1989 ) Cell adhesion activity of non - specific cross - reacting antigen ( NCA ) and carcinoembryonic antigen ( CEA ) expressed on CHO cell surface: homophilic and heterophilic adhesion Biochem Biophys Res Commun 164, 39 – 45 [58] Screaton RA, DeMarte L, Draber P, and Stanners CP ( 2000 ) The specificity for the differentiation blocking activity of carcinoembryonic antigen resides in its glycophosphatidyl - inositol anchor J Cell Biol 150, 613 – 26 [59] Gullberg D, Sjoberg G, Velling T, and Sejersen T ( 1995 ) Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation Exp Cell Res 220, 112 – 23 [60] Yang JT, and Hynes RO ( 1996 ) Fibronectin receptor functions in embryonic cells deficient in alpha beta integrin can be replaced by alpha V integrins Mol Biol Cell 7, 1737 – 48 [61] Sastry SK, Lakonishok M, Thomas DA, Muschler J, and Horwitz AF ( 1996 ) Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation J Cell Biol 133, 169 – 84 Neoplasia Vol 4, No 2, 2002 163 [62] Sastry SK, Lakonishok M, Wu S, Truong TQ, Huttenlocher A, Turner CE, and Horwitz AF ( 1999 ) Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal J Cell Biol 144, 1295 – 309 [63] Huang Y, Li J, Zhang Y, and Wu C ( 2000 ) The roles of integrin - linked kinase in the regulation of myogenic differentiation J Cell Biol 150, 861 – 72 [64] Ilyas M, Tomlinson IP, Rowan A, Pignatelli M, and Bodmer WF ( 1997 ) Beta - catenin mutations in cell lines established from human colorectal cancers Proc Natl Acad Sci USA 94, 10330 – 34 [65] Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JKV, Markowitz SD, Kinzler KW, and Vogelstein B ( 1998 ) Mutations of mitotic checkpoint genes in human cancers Nature 392, 300 – 303 [66] Djelloul S, Forgue - Lafitte ME, Hermelin B, Mareel M, Bruyneel E, Baldi A, Giordano A, Chastre E, and Gespach C ( 1997 ) Enterocyte differentiation is compatible with SV40 large T expression and loss of p53 function in human colonic Caco - cells Status of the pRb1 and pRb2 tumor suppressor gene products FEBS Lett 406, 234 – 42 [67] Rousset M, Dussaulx E, Chevalier G, and Zweibaum A ( 1980 ) Growth - related glycogen levels of human intestine carcinoma cell lines grown in vitro and in vivo in nude mice J Natl Cancer Inst 65, 885 – 89 [68] Trainer DL, Kline T, McCabe FL, Faucette LF, Feild J, Chaikin M, Anzano M, Rieman D, Hoffstein S, Li DJ, et al ( 1988 ) Biological characterization and oncogene expression in human colorectal carcinoma cell lines Int J Cancer 41, 287 – 96 [69] Amanti C, Midiri G, Benedetti M, Campisi C, Di Tondo U, Castagna G, Peronace L, Santeusanio G, and Di Paola M ( 1985 ) Tissue CEA detection by immunoperoxidase ( PAP ) test in colorectal polyps: correlations with the degree of dysplasia J Surg Oncol 28, 222 – 26 [70] Au FC, Stein B, and Tang CK ( 1986 ) Carcinoembryonic antigen levels in colonic lesions Am J Surg 151, 61 – 64 [71] Turani H, Kessler E, and Lurie B ( 1988 ) The value of tissue mucin changes and CEA content in evaluation of benign colonic adenomas Dis Colon Rectum 31, 123 – 25 [72] Fischbach W, and Mossner J ( 1988 ) Tissue concentrations of CEA and CA 19 - in the carcinogenesis of colorectal carcinoma exemplified by the adenoma – carcinoma sequence Res Exp Med 188, 101 – 14 [73] Lengauer C, Kinzler KW, and Vogelstein B ( 1997 ) Genetic instability in colorectal cancers Nature 386, 623 – 27 [74] Chung DC, and Rustgi AK ( 1995 ) DNA mismatch repair and cancer Gastroenterology 109, 1685 – 99 [75] Levine AJ ( 1997 ) p53, the cellular gatekeeper for growth and division Cell 88, 323 – 31  ... levels of CEA and CEACAM6 in Caco - CEA / CEACAM6" cells ( F ) Cell surface expression of CEA and CEACAM6 in purified epithelial cells prepared from tumor colonic crypts [ ] The tumor profiles... determine whether these observed effects of CEA / CEACAM6 overexpression on Caco - could promote tumorigenic behavior in vivo, the tumorigenic potential of Caco - CEA" and Caco - CEA / CEACAM6" colonocytes... test the hypothesis that deregulated overexpression of CEA and CEACAM6 can have tumorigenic effects on human colonocytes, we tested their effects on human colonocyte cell lines, SW - 1222 and