JOURNAL OF CELLULAR PHYSIOLOGY 143468-474 (1990)
Impaired Carcinoembryonic Antigen Release During the Process of Suramin-Induced Differentiation of the Human Colic Adenocarcinoma Cell Clone HT29-D4 JACQUES FANTINI,* JEAN-BAPTISTE ROGNONI, MAGALI THEVENIAU, GILBERT POMMIER, AND JACQUES MARVALDI UniversitP d'Aix-Marseille I, Centre National de la Recherche Scientifique, URA 202, 13331 Marseille (I.F., I.-B.R., M.T., 1.M.) and FacultP de Medecine de la Timone, 73005 Marseille (C.P.), France
The establishment of a differentiated state of the human colic adenocarcinoma cell clone HT29-D4 can be obtained by two ways: 1) the removal of glucose and its replacement by galactose in the culture medium (Fantini et al.: Biology of the Cell 65: 163-1 69, 1989); 2) the addition of suramin, a polyanionic compound, in the glucose-containing medium (Fantini et al.: lournal of Biological Chemistry 264:10282-10286, 1989). We investigated the release of CEA in the culture medium of glucose-deprived HT29-D4 cells (HT29-D4-Gal) and studied its alteration in suramin-treated HT29-D4 cells (HT29-D4-S). The amount of CEA released in the medium in function of time in culture of undifferentiated HT29D4-Glu cells was very low (5 to 8 ng/106 cells/24 hours) and almost constant throughout the experiment whereas it increased sharply during differentiation of HT29-D4-Gal cells (380 ng/106 cells/24 hours after 9 days in culture). Surprisingly the amount of CEA released by differentiated HT29-D4-S cells remained very low and comparable with the one of HT29-D4-Glu cells. Moreover suramin, when added to CEA-producing HT29-D4-Gal cells, strongly inhibited its release. Radioiodination of cell surface proteins followed by immunoprecipitation using an anti-CEA monoclonal antibody showed the presence of a 180 kDa polypeptide, i.e., CEA, predominantly labeled in HT29-D4-Gal and -S cells. The total CEA cellular content was higher in HT29-D4-Glu and HT29-D4-S cells than in HT29-D4-Gal cells. When HT29-D4-Gal or -S cells were treated with the bacterial phosphatidylinositol phospholipase C (PI-PLC) a similar level of CEA was released suggesting a similar type of CEA anchorage. The present data demonstrate that a decrease in CEA release (i.e., in HT29-D4-Glu and -S cells) corresponds to an increase in its overall cellular expression. These results are in favour of a regulatory mechanism, impaired by suramin, which determines the balance between the soluble and the membrane bound forms of CEA.
T h e carcinoembryonic a n t i g e n (CEA) was f i r s t described as a colon t u m o r associated a n t i g e n (Gold and Freedman, 1965). I t i s a 180 kDa highly glycosylated p r o t e i n s t r u c t u r a l l y r e l a t e d t o a family o f molecules such as non-specific cross-reacting a n t i g e n ( N C A ) ( V o n K l e i s t e t al., 1972), biliary glycoprotein (BGP) (Svenberg, 1976), and n o r m a l fecal a n t i g e n (NFA) ( M a t suoka e t al., 1978). T h e a m i n o acid sequence o f CEA demonstrates a consistent s i m i l a r i t y with various members o f t h e i m m u n o g l o b u l i n superfamily (Paxton e t al., 1987, 1989; S h i v e l y e t al., 1989). T h e C E A m o l ecule is anchored t o t h e cell m e m b r a n e by a glycophospholipid-containing phosphatidylinositol ( H e f t a e t al., 1988) w h i c h c a n b e cleaved f r o m t h e m e m b r a n e by a bacterial phosphatidylinositol-phospholipaseC (PIP L C ) (Takami e t al., 1988; J e a n e t al., 1988; Sack e t al., 1988) or by proteases (Sack e t al., 1988). L i k e other 0 1990 WILEY-LISS, INC.
members o f t h e i m m u n o g l o b u l i n superfamily, anchored in t h e m e m b r a n e by a glycolipid complex, such as N-CAM 120 ( H e e t al., 19861, C E A does n o t bind an anti-cross-reacting d e t e r m i n a n t antibody (anti-CRD) (Jean e t al., 1988). CEA i s an i m p o r t a n t human colorectal cancer m a r k e r and i s used in t h e diagnosis and management o f t h i s malignant disease. However, c l i n i c a l significance o f c i r c u l a t i n g CEA l e v e l i s l i m i t e d because it i s n o t a l w a y s correlated with t h e presence o f tumors. A m o n g t h e events that determine t h e l e v e l of circulating C E A one m u s t t a k e i n t o account t h e mechanisms of release and clearance o f t h i s molecule. T h e colonic adenocarcinoma cell l i n e HT29-D4 exReceived November 1, 1989; accepted February 2, 1990. *To whom reprint requestskorrespondence should be addressed.
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presses CEA and can be induced to differentiate when glucose is substituted by galactose in the culture medium (Fantini et al., 1986, 1988, 1989a). During the process of cell differentiation of this cell line, CEA was overexpressed at the cell surface, segregated a t the apical membrane domain, and exclusively released in the luminal side of the monolayer (Fantini et al., 1989b). Recently we have shown that the anti-helmintic drug, suramin (Hawking, 1978), which antagonizes the effects of several growth factors (Coffey et al., 19871, is able to induce the differentiation of HT29-D4 cells even in the presence of glucose in the culture medium (Fantini et al., 1989~ ). The state of differentiation obtained under these conditions is achieved more than in glucose-free culture condition (Fantini et al., 1990). It should also be emphasized that growth factors such as TGFP stimulate the release of CEA from human colon carcinoma cells with a concurrent induction of a more differentiated phenotype (Chakrabarty et al., 1988, 1989). In this report we have investigated the release of CEA in suramin-treated cells (HT29-D4-S) compared with control cells (HT29-D4-Glu) and with cells grown in glucose-free medium (HT29-D4-Gal). It appears from our results that the levels of CEA released by differentiated HT29-D4-S cells were similar to the ones for undifferentiated HT29-D4-Glu cells; by contrast, a very consistent release of CEA was monitored for differentiated HT29-D4-Gal cells. We concluded that suramin interferes with the mode of release of CEA in HT29-D4 cells. MATERIALS AND METHODS Materials Suramin (obtained from Specia, Paris, France) was prepared as a sterile stock solution of 100 mg/ml in distilled water and stored a t -20°C. The anticarcinoembryonic antigen monoclonal antibody MAC 601 was purchased from Biosys (Compiegne, France). This antibody recognizes the epitope 4930 on the CEA molecule and does not cross-react with NCA-1, NCA-2, and BGP-1. The f luorescein-conjugated antimouse IgG antibodies were from Sigma Chemical Co. (Saint-Louis, Mo). The phosphatidylinositol-phospholipaseC was purified in our laboratory from Bacillus thuringiensis cultures. We checked that the purified enzyme was devoid of any proteolytic activity. Cell culture The human colon adenocarcinoma cell line HT29 was cloned by limit dilution technique (Fantini et al., 1986), and the resulting clone HT29-D4 (passage 1020) was grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 25 mM glucose. These cells will be referred to as HT29D4-Glu cells. Differentiation of HT29-D4 cells was achieved by culturing the cells in glucose-free DMEM containing 5 mM galactose (HT29-D4-Gal cells) or by adding suramin (100 pg/ml) in DMEM containing 25 mM glucose (HT29-D4-S cells) as previously described (Fantini et al., 1986, 1989a). CEA measurement Cell culture media was collected daily, centrifuged 10 min a t 10,OOOg and stored a t -20°C before dosing.
The amount of CEA was determined with the ABBOTT CEA-EIA Monoclonal One-Step kit (Abbott Laboratories, Chicago). CEA immunodetection in HT29-D4 cell e x t racts Radiolabeling detection of CEA. Cell monolayer was rinsed twice with PBS, and the cell surface was radioiodinated in PBS with 50 p1 of lactoperoxidase (2 mg/ml), 500 pCi 1251Na,and H 2 0 2 added sequentially according to Goding (1986). The reaction was stopped with tyrosine 1 mM. Cell monolayer was solubilized with the same buffer as below and CEA was immunoprecipitated using the MAC 601 antibody and protein A-Sepharose. The total amount of protein A-antibody complex was diluted in sample buffer and submitted to 10%PAGE in presence of SDS. Western blot detection of CEA. HT29-D4 cells either grown in glucose, or glucose plus suramin-containing medium, or in glucose-free medium were solubilized in Tris-HC1 50 mM buffer pH 8 containing MgC1, 1 mM, NaCl 150 mM, benzamidine 2 mM, PMSF 150 FM, and a2-macroglobuline 0.1 mg/ml. The lysate was clarified by centrifugation at 10,OOOg for 10 min and aliquots were diluted in SDS-PAGE sample buffer and submitted to 10% PAGE in the presence of SDS. The gel was transferred onto NitroscreenTMmembranes (NEN) and CEA was immunodetected using monoclonal antibody MAC 601 and 1251-proteinA as a revealing agent. Fuji X-ray films were used for autoradiographic studies for both techniques. Immunofluorescence technique Staining of the apical membrane of HT29-D4 cells was performed using the mouse anti-CEA monoclonal antibody MAC 601. Cells were seeded on glass coverslips a t a density of lo5 cells per cm2. Immunofluorescence studies were performed on intact cells (i.e., without any fixation) 1 week after cell cultures reached confluency. Cells were treated a t 4°C with the primary antibody for 2 h and then with the secondary antimouse IgG antibody-FITC conjugate for 45 min. Before mounting the coverslips with glycerol, cells were extensively rinsed in PBS containing 1% BSA. Effect of PI-PLC on CEA release In order to investigate if suramin interfered with the mode of anchorage of CEA in HT29-D4 cell membranes, three kinds of experiments were performed. Effect of PI-PLC on C E A release in long-term suramin-treated cells. HT29-D4 cells were cultured in DMEM containing 10% FCS, 25 mM glucose, and suramin (100 pg/ml). Cell cultures were used 1 week after confluency, and starved of serum for 16 h. Just before the experiment, cell cultures were washed twice with PBS (CA' 0.1 mM; Mg+ 1 mM). The effect of PI-PLC was tested in 2 ml of DMEM containing glucose (25 mM) deprived of serum and supplemented with protease inhibitors (PMSF, 500 pM; leupeptine, 2 pg/ml; aprotinine, 10 unitdml). Fourteen microliters of PIPLC (25 IU/ml) was added and a similar amount of PI-PLC was added again in the incubation medium 1 and 2 h after the initial addition and the incubation was stopped 3 h after the initial PI-PLC input. Proteins in the collected media were precipitated with cold ace+
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tone, resuspended in sample buffer, and analyzed by SDS-PAGE and Western blot using an anti-CEA monoclonal antibody. Effect of PI-PLC on CEA release in HT29-D4-Gal cells. HT29-D4-Gal cells were cultured in glucose-free DMEM containing 5 mM galactose and 10%FCS. The action of PI-PLC was assayed in 2 ml of glucose-free DMEM containing 5 mM galactose and protease inhibitors as described above. Effect of suramin o n PI-PLC-induced CEA release in HT29-D4-Gal cells. HT29-D4-Gal cells were grown in glucose-free DMEM containing 5 mM galactose and 10% FCS. The action of PI-PLC was tested in 2 ml of glucose-free DMEM supplemented with 5 mM galactose and 100 pg/ml suramin in the presence of protease inhibitors as described above. RESULTS
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CEA release during the process of cell differentiation in glucose-free medium or in the presence of suramin HT29-D4 cells were seeded in glucose-containing medium and transferred in suramin-containing medium or in glucose-free medium 2 or 5 days later, respectively, and the medium was changed daily. The amount of CEA released in the different culture media was monitored every day using an enzymatic immunoassay. As already described (Fantini et al., 1989b) the amount of CEA released in the culture medium from undifferentiated HT29-D4-Glu cells was very low (5-8 ng/106cells/ 24 h) and almost stable in function of time. On the contrary the level of CEA increased sharply during the differentiation of HT29-D4-Gal cells and reached 380 ng/106 cells/24 h after 9 days of culture following the medium change. Surprisingly the amount of CEA released in the culture medium by differentiated HT29-D4-S cells was comparable with the one of HT29-D4-Glu cells (Fig. 1).In order to discard an artifactual interference of suramin on the quantitation of CEA, we have tested the influence of suramin addition in a CEA-containing culture medium from HT29D4-Gal cells. The data from Figure 2 (histogram D) demonstrate that no difference can be detected in the amount of CEA measured by immunoassay, following the addition of suramin.
Fig. 1. CEA measurement during the process of cell differentiation induced by glucose starvation or suramin. HT29-D4 cells were plated in 25 cm2 flasks and grown in glucose-containing medium. Two days later suramin (100 +g/ml) was added in the culture medium for one part of the culture (arrow). For another part of the cell culture the medium was replaced by a glucose-free medium (asterisk-arrow).The medium was changed every day, and the amount of CEA was measured using the Abbott enzyme immunoassay and expressed as mean & SD (n = 3). 0-0, CEA amount in glucose-containing medium; A-A; CEA amount in suramin and glucose-containing medium; 0 - 0 ; CEA amount in glucose free galactose-containing medium.
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Suramin prevents the release of CEA by HT29-D4-Gal cells On account of the above observation it was of interest to investigate the effect of suramin on the release of CEA by HT29-D4-Gal cells. Fourteen days after seeding suramin was added or not added in HT29-D4-Gal cell cultures. The medium was changed every day. At days 19, 21, and 24, the medium was removed and tested for CEA. The data are shown in Figure 2 (histograms A, B, C). It is clear that suramin depressed the release of CEA in dome-forming differentiated HT29D4-cells whatever the time of the sampling.
Fig. 2. Effect of suramin in the release of CEA by HT29-D4-Gal cells A,-C). HT29-D4-Gal cells were seeded in glucose-free medium in 25 cm2 flask. Fourteen days later suramin (100 kgiml) was added or not added to the culture medium. The medium was changed every day. At days 19 (A), 21 (B), and 24 (C) the medium from HT29-D4-Gal cells (stippled columns) or from suramin-treated HT29-D4-Gal cells (hatched columns) was tested for CEA using the Abbott enzyme immunoassay. In D, suramin was added (hatched) or not added (stippled) to the culture medium of HT29-D4-Gal cells just before CEA measurement. Values are means 2 SD (n = 3).
Cell surface expression of CEA in HT29-D4-S cells Due to the differential release of CEA by HT29-D4Gal compared to HT29-D4-S cells, it was of importance
to investigate the presence of CEA a t the cell surface of both types of differentiated cells. HT29-D4-Glu, -Gal, or -S cells were cultured in glucose-containing medium on glass coverslips in order to perform indirect immunof luorescence experiments us-
CEA RELEASE AND SURAMIN-INDUCEDDIFFERENTIATION
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Fig. 3. Indirect immunofluorescence staining with a n anti-CEA antibody. A HT29-D4-Glu cells (cultured for 10 days). B: HT29-D4-Gal cells cultured in glucose-free galactose-containing medium for 14 days. C: HT29-D4-S cells grown in the presence of suramin (100pgiml) for 14 days. The cells were stained with a n anti-CEA monoclonal antibody as described in “Materials and Methods.” Bar: 20 wm.
ing an anti-CEA monoclonal antibody. A part of the coverslips were incubated in glucose free medium 5 days after seeding whereas control cells were cultured in parallel in the presence of glucose. Twelve days later the cells were tested for CEA cell surface expression. HT29-D4-Glu cells displayed a faint but non negligible fluorescence. The periphery of the cell was quite visible indicating a uniform labeling of the cell membrane (Fig. 3A). On the contrary large clusters of differentiated HT29-D4-Gal and S cells were strongly labeled exhibiting a punctated fluorescence interpreted as an apical membrane labeling at the level of microvilli viewed en face. No basolateral staining can be observed (Fig. 3B,C) even when other planes of focus were chosen. No difference in cell surface expression could be observed in both types of cells. Radioiodination of the cell surface was performed on HT29-D4 cells grown under the three conditions of culture (i.e., in the presence of glucose, glucose + suramin, or glucose-free medium + galactose). After labeling, the cells were lysed in the presence of triton XlOO and the lysate was immunoprecipitated with an anti-CEA monoclonal antibody. The immunoprecipitated material was analyzed by SDS-PAGE. The autoradiogram of the dried gel is shown in Figure 4A. A 180 kDa polypeptide was labeled in the three types of cells, however, the immunoprecipitated molecule was much more labeled in HT29-D4-Gal and -S cells. These data demonstrated that cell surface expression of CEA was not altered by suramin. Cellular CEA characterization in HT29-D4-S cells Since CEA was only released during the establishment of the differentiation state of HT29-D4-Gal cells we looked at the CEA cellular content during the process of differentiation induced by suramin or by glucose removal. HT29-D4-Glu, -Gal, and -S cells were lysed in the presence of triton XlOO and a same amount of protein from each lysate was analysed by SDS-PAGE. Proteins were then transferred to NitroscreenTM membranes
Fig. 4. A: Immunoprecipitation of CEA after eel1 surface radiolabeling. HT29-D4-Glu, -Gal or, -S cells were radioiodinated in the presence of [‘2sIl Na and lactoperoxidase. After solubilization with Triton X100, CEA was immunoprecipitated as described in “Materials and Methods.” B: Western Blot analysis of total CEA. HT29-D4-Glu, -Gal, or -S cells were lysed with Triton X100.The same amount of proteins (100 pg) was analysed by SDS-PAGE and transferred to NitroscreenTMmembrane. CEA was revealed with an anti-CEA monoclonal antibody and by ‘251-proteinA. The arrow indicate the migration of a 180 Kda polypeptide, i.e., CEA. Data are from one experiment representative of three.
and Western blot analysis was performed using an anti-CEA monoclonal antibody and lZ5I-labeledprotein A as revealing agent. The autoradiogram is shown in Figure 4B. A low amount of CEA was reproducibly seen in HT29-D4-Gal cells while similar but higher quantities of CEA were revealed in HT29-D4-Glu and -S cells. The corresponding scanning of autoradiograms derived
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Fig. 5. Effect of PI-PLC on CEA release in HT29-D4-Gal cells (A) or HT29-D4-S cells (B). Fourteen microliters of PI-PLC (25 IUlml) was sequentially injected (at 0, 1, and 2 h) or not injected (control) in glucose-free DMEM containing 5 mM galactose (A) or in DMEM containing 25 mM glucose (B).The incubation was stopped 3 h after the first injection. The media were collected and the proteins precipitated with acetone, resuspended in sample buffer, and analyzed by SDSPAGE and Western blot. CEA was evidenced using a monoclonal antibody (MAC 601,Biosys) and '"%protein A. A unique band was typically observed at 180 kDa after autoradiography of the NitroscreenTMfilter. Lane l: Lysate of PI-PLC-treated cells. L a n e 2: PI-PLC-untreated cells medium. L a n e 3: PI-PLC-treated cells medium. Lane 4 lysate of PI-PLC-untreated cells. The autoradiogram from A was overexposed (72 h) compared to the one from B (24 h) due to the low cellular content of HT29-D4-Gal cells.
Fig. 6. Effect of suramin (100Fgiml) on the PI-PLC-induced CEA release in HT29-D4-Gal cells. Fourteen microliters of PI-PLC (25 IUI ml) was sequentially added (0, 1, and 2 h) to glucose-free DMEM containing 5 mM galactose supplemented or not with suramin (100 kg/ml). The assay was stopped 3 h after the first injection. Proteins of collected media were precipitated with cold acetone and resuspended in sample buffer analyzed by SDS-PAGE and Western blot. CEA was evidenced by a monoclonal antibody and lZ5Iprotein A. L a n e 1: Lysate of suramin-treated cells. L a n e 2 Medium of suramin-treated cells. Lane 3 Medium of suramin-untreated cells. Lane 4: Lysate of suramin-untreated cells.
amount of CEA was released after the PI-PLC treatment (Fig. 5A,B, lane 3). The cellular content of CEA from PI-PLC treated or untreated cells was identical (Fig. 5A,B, lanes 1 and 4). These data demonstrated that the mode of CEA anfrom three independent experiments revealed that the chorage in HT29-D4 cells is very likely identical with CEA content of HT29-D4-Galcells never exceed 25% of that of HT29-D4-Gal cells. In order to test if suramin the cellular CEA content of HT29-D4-S or -Glu cells. modulates the enzymatic activity of exogenous PI-PLC Thus, it appears that a low level of CEA release in the during the incubation, an experiment was designed culture medium (i.e., in HT29-D4-Glu and -S cells) is where suramin (100 pg/ml) was added or not added to correlated with an increased cellular CEA content the incubation medium containing PI-PLC of HT29while the elevated CEA release by HT29-D4-Gal cells D4-Gal cells. The recovered medium and the cell lysate were analysed by SDS-PAGE and Western blot using is correlated with a low cellular content. an anti-CEA monoclonal antibody. Release of CEA by phosphatidylinositol-specific Data from Figure 6 show that suramin has virtually phospholipase C (PI-PLC) no effect on the enzymatic activity of the bacterial PIWe have demonstrated that CEA release is impaired PLC (Fig. 6, lanes 2,3). The corresponding CEA celluin suramin-treated cells although differentiated HT29- lar content was identical as shown in Figure 6 lanes 1 D4-S cells synthesize and express CEA at the apical and 4. cell surface just like HT29-D4-Gal cells do. Since CEA DISCUSSION is anchored to the membrane by a glycane phosphatiThe establishment of differentiated state of the dylinositol (Sack et al., 1988; Jean et al., 19881, it was important to determine if the same type of CEA an- clonal HT29-D4 cells can be obtained by two ways: 1) The removal of glucose and the addition of galactose chorage occurred in both types of cells. The ability of PI-PLC to release CEA from intact (Fantini et al., 1989b); 2) The addition of suramin, a HT29 cells (Jean et al., 1988) was used to solubilize the polyanionic compound, in the glucose-containing meCEA molecules from HT29-D4-Glu, -Gal, and -S cells. dium (Fantini et al., 1989~).Important differences in Cells were incubated for 3 h in the presence or absence the differentiation state according to the culture proof PI-PLC in serum-deprived glucose-free medium for tocol were observed a t the morphological as well as at HT29-D4-Gal cells or in serum-deprived glucose-con- the electrophysiological level, the most elaborated taining medium for HT29-D4-S cells. The medium was state being obtained in the presence of suramin (Fancollected, precipitated with cold acetone, and proteins tini et al., 1990). This unique model gives us the opportunity to inveswere analyzed by SDS-PAGE transferred to NitroscreenTMmembranes and revealed with an anti-CEA tigate the process of CEA release according to the way monoclonal antibody, and lZ5I-proteinA. In parallel, cells were induced to differentiate. In this report, we cells treated or not treated with PI-PLC were lysed in demonstrate that HT29-D4-.Gal cells release steeply the presence of Triton XlOO and proteins were ana- the CEA during the process of cell differentiation inlyzed by SDS-PAGE and immunoblotted under the duced by the removal of glucose from the culture medium while HT29-D4-S cells were unable to do that. same conditions as for the collected medium. The autoradiogram of the Western blot is shown in These later cells behaved just like the undifferentiated Figure 5. For the two types of cells the medium from HT29-D4-Glu cells did. The impairment of CEA release during the process of PI-PLC untreated cells contained a very low amount of solubilized CEA (Fig. 5A,B, lane 2) while an important differentiation induced by suramin was not due to an
CEA RELEASE AND SURAMIN-INDUCED DIFFERENTIATION
interference of the drug with the CEA radioimmunoassay, because the addition of suramin in a CEA-containing medium did not alter the measured amount of soluble CEA. Moreover suramin was unable to inhibit the recognition of blotted CEA by an anti-CEA monoclonal antibody (data not shown). We can conclude that suramin did not affect the interaction of CEA with an anti-CEA antibody. The expression of CEA at the apical cell membrane was identical in HT29-D4-Gal or HT29-D4-S cells as shown by indirect immunof luorescence staining or immunoprecipitation of radioiodinated apical cell surface CEA. We can conclude that suramin did not prevent the normal polarized expression of CEA. These data are consistent with those obtained with HT29-D4-Gal and -S cells grown on permeable filters which demonstrated the exclusive localization of CEA at the apical membrane domain (Fantini et al., 1989b, 1990). The defect of CEA release observed after suramin treatment could be due to a different mode of CEA anchorage in the cellular membrane. However, our results demonstrate that the artificial release of CEA by exogenous PI-PLC can be achieved for HT29-D4-Gal as well as HT29-D4-Scells. Moreover suramin was unable to activate or inhibit the bacterial enzyme added during the experiment. These data support the idea that CEA molecules from both types of cells were probably anchored in the cell membrane by a glycolipid containing phosphatidylinositol as already demonstrated for the non-cloned HT29 cells (Jean et al., 1988). The failure of HT29-D4-S cells to release CEA is yet due to the action of suramin because the addition of the drug to HT29-D4-Gal cell culture sharply depressed the release of CEA in the culture medium. It is of course tempting to postulate that suramin inhibits the endogenous mechanisms involved in the release of CEA. These physiological processes are so far not understood and are currently under investigation. When the release of CEA was depressed (i.e., in HT29-D4-Glu and -S cells), by opposition the cellular content of CEA became very high when compared to that of HT29-D4Gal cells which released a large quantity of CEA. This latter information is in favour of a regulatory mechanism which would determine the balance between soluble and membrane bound molecular forms of CEA. The finding that colic mucosa contained a very low amount of CEA in spite of its active production (Kuroki et al., 1988) strongly suggests that CEA is rapidly released into the lumen of the digestive tract. This observation is in favour of our hypothesis. It appears from our data that suramin could modulate these regulatory mechanisms either by its action on external growth factors (Garrett et al., 1984; Coffey et al., 1987) and/or by its lysosomotropic effect (Kielian et al., 1982; Sjolund and Thyberg, 1989). ACKNOWLEDGMENTS We would like to thank the expert technical assistance of Fernand Gianellini and Jean-Jacques Roccabianca. We also thank Dr Genevieve Rougon for critical reading of the manuscript. This work was supported by grants from the Federation Nationale des Centres de Lutte Contre le Cancer, the G.E.F.L.U.C, and the Association pour la Recherche contre le Cancer.
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LITERATURE CITED Chakrabarty, S., Tobon, A,, Varani, J., and Brattain, M.G. (1988) Induction of carcinoembryonic antigen secretion and modulation of protein secretion/expression and fibronectidlaminin expression in human colon carcinoma cells by transforming growth factor-p. Cancer Res., 48:4059-4064. Chakrabarty, S., Jan, Y., Brattain, M.G., Tobon, A., and Varani, J. (1989) Diverse cellular responses elicited from human colon carcinoma cells by transforming growth factor-p. Cancer Res., 49t21122117. Coffey, R.J., Jr, Leof, E.B., Shipley, G.D., and Moses, H.L. (1987) Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-PB cells. J. Cell. Physiol., 132.143-148. Fantini, J., Abadie, B., Tirard, A., Remy, L., Ripert, J.P., El Battari, A., and Marvaldi, J. (1986) Spontaneous and induced dome formation by two clonal cell populations derived from a human adenocarcinoma cell line, HT29. J. Cell Si., 83:235-249. Fantini, J., Martin, J.M., Luis, J., =my, L., Tirard, A,, Marvaldi, J., and Pichon, J. (1988) Restricted localization of functional vasoactive intestinal peptide receptors (VIP) in in vitro differentiated human colonic adenocarcinoma cells (HT29-D4). Eur. J. Cell Biol., 46:458-465. Fantini, J . , Verrier, B., Marvaldi, J., and Mauchamp, J . (1989a) In vitro differentiated HT29-D4 clonal cell line generates leakproof and electrically active monolayers when cultured in porous-bottom culture dishes. Biol. Cell, 65r163-169. Fantini, J.,Rognoni, J.B., Culouscou, J.M., Pommier, G., Marvaldi, J., and Tirard, A. (1989b) Induction of polarized apical expression and vectorial release of carcinoemhryonic antigen (CEA)during the process of differentiation of HT29-D4 cells. J . Cell. Physiol., 141t126134. Fantini, J., Rognoni, J.B., Roccabianca, M., Pommier, G., and Marvaldi, J . ( 1 9 8 9 ~Suramin ) inhibits cell growth, glycolytic activity and triggers differentiation of human colic adenocarcinoma cell clone HT29-D4. J. Biol. Chem., 264:10282-10286. Fantini, J.,Rognoni, J.B., Verrier, B., Lehmann, M., Roccabianca, M., Mauchamp, J., and Marvaldi, J . (1990) Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose. Eur. J. Cell Biol. 51:llO-119. Garrett, J.S., Coughlin, S.R., Niman, H.L., Tremble, P.M., Giels, G.M., and Williams L.J. (1984) Blockade of autocrine stimulation in simian sarcoma virus transformed cell reverses down-regulation of platelet derived growth factor receptors. Roc. Natl. Acad. Sci. U.S.A., 81r7466-7470. Goding, J.W. (1986) Monoclonal Antibodies: Principles and Practice, 2nd Edition. Academic Press Inc., London, pp. 154-156. Gold, P., and Freedman, S.O. (1965) Demonstration of tumor specific antigen in human colonic carcinomata by immunological tolerance and absorption techniques. J. Exp. Med., 121t439-462. Hawking, F. (1978) Suramin: With special reference to onchocerciasis. Adv. Pharmacol. Chemother., 15:289-322. He, H.T., Barhet, J., Chaix, J.C., and Goridis, C. (1986) Phosphatidylinositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule. EMBO J., 5.2489-2494. Hefta, S.A., Hefta, L.J.F., Lee, T.D., Paxton, R.J., and Shively, J.E. (1988) Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosylphosphatidylinositolmoiety: Identification of the ethanolamine linkage site. Proc. Natl. Acad. Sci. U.S.A., 85t4648-4652. Jean, F., Malapert, P., Rougon, G., and Barhet, J . (1988) Cell membrane, but not circulating carcinoembryonic antigen is linked to a phosphatidylinositol-containinghyrophobic domain. Biochem. Biophys. Res. Commun., 155:794-800. Kielian, M.C., Steinman, R.M., and Cohn, Z.A. (1982) Intralysosomal accumulation of polyanions. I- Fusion of pinocytic and phagocytic vacuoles with secondary lysosomes. J . Cell Biol., 93:866-874. Kuroki, M., Arakawa, F., Yamamoto, H., Shimura, H., Ikehara, Y . , and Matsuoka, Y. (1988) Active production and membrane anchoring of carcinoembryonic antigen observed in normal colon mucosa. Cancer Lett., 43t151-157. Matsuoka, Y.,Koga, Y . ,Makuta, H., Yoshino, M., and Tsuru, E (1978) Proteolytic release of antigenic fragment corresponding to a normal fecal antigen and non-specific cross-reacting antigen from carcinoembryonic antigen. Int. J. Cancer, 21t604-610. Paxton, R.J., Mooser, G., Pande, H., Lee, T.D., and Shively, J.E. (1987) Sequence analysis of carcinoembryonic antigen: Identification of
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glycosylation sites and homology with the immunoglobulin supergene family. Proc. Natl. Acad. Sci. U.S.A., 84:920-924. Paxton, R.J., Hefta, S.A., Hefta, L.J.F., Hinoda, Y., Lee, T.D., and Shively, J.E. (1989) Structural studies of the carcinoembryonic antigen family: Sequence analysis and posttranslation modifications. In: The Carcinoembryonic Antigen Gene Family. A. Yachi and J.E. Shively, ed. Elsevier Science Publishers BV, Amsterdam, pp. 23-36. Sack, T.L., Gum, J.R., Low, M.G., and Kim, YS. (1988) Release of carcinoembryonic antigen from human colon cancer cells by phosphatidyl specific phospholipase C. J. Clin. Invest., 82:586-593. Shively, J.E., Hinoda, Y., Hefta, L.J.F., Neumaier, M., Hefta, S.A., Shively, L., Paxton, R.J., and Riggs, A.D. (1989) Molecular cloning of members of the carcinoembryonic antigen gene family. In: The Carcinoembryonic Antigen Gene Family. A. Yachi and J.E. Shively, ed. Elsevier Science Publishers BV, Amsterdam, pp. 97-110.
Sjolund, M., and Thyberg, J. (1989) Suramin inhibits binding and degradation of platelet-derived growth factor in arterial smooth muscle but does not interfere with autocrine stimulation of DNA synthesis. Cell Tissue Res., 256:35-43. Svenberg, T. (1976) Carcinoembryonic antigen-like substances of human bile. Isolation and partial characterization. Int. J. Cancer, 17: 588-596. Takami, N., Misumi, Y., Kuroki, M., Matsuoka, Y., and Ikehara, P. (1988) Evidence for carboxyl-terminal processing and glycolipidanchoring of human carcinoembryonic antigen. J . Biol. Chem., 263: 12716-12720. Von Kleist, S., Chavanel, G., and Burtin, P. (1972) Identification of an antigen from normal human tissue that crossreacts with the carcinoembryonic antigen. Proc. Natl. Acad. Sci. U.S.A., 692492-2494.
Impaired Carcinoembryonic Antigen Release During the Process of Suramin-Induced Differentiation of the Human Colic Adenocarcinoma Cell Clone HT29-D4 JACQUES FANTINI,* JEAN-BAPTISTE ROGNONI, MAGALI THEVENIAU, GILBERT POMMIER, AND JACQUES MARVALDI UniversitP d'Aix-Marseille I, Centre National de la Recherche Scientifique, URA 202, 13331 Marseille (I.F., I.-B.R., M.T., 1.M.) and FacultP de Medecine de la Timone, 73005 Marseille (C.P.), France
The establishment of a differentiated state of the human colic adenocarcinoma cell clone HT29-D4 can be obtained by two ways: 1) the removal of glucose and its replacement by galactose in the culture medium (Fantini et al.: Biology of the Cell 65: 163-1 69, 1989); 2) the addition of suramin, a polyanionic compound, in the glucose-containing medium (Fantini et al.: lournal of Biological Chemistry 264:10282-10286, 1989). We investigated the release of CEA in the culture medium of glucose-deprived HT29-D4 cells (HT29-D4-Gal) and studied its alteration in suramin-treated HT29-D4 cells (HT29-D4-S). The amount of CEA released in the medium in function of time in culture of undifferentiated HT29D4-Glu cells was very low (5 to 8 ng/106 cells/24 hours) and almost constant throughout the experiment whereas it increased sharply during differentiation of HT29-D4-Gal cells (380 ng/106 cells/24 hours after 9 days in culture). Surprisingly the amount of CEA released by differentiated HT29-D4-S cells remained very low and comparable with the one of HT29-D4-Glu cells. Moreover suramin, when added to CEA-producing HT29-D4-Gal cells, strongly inhibited its release. Radioiodination of cell surface proteins followed by immunoprecipitation using an anti-CEA monoclonal antibody showed the presence of a 180 kDa polypeptide, i.e., CEA, predominantly labeled in HT29-D4-Gal and -S cells. The total CEA cellular content was higher in HT29-D4-Glu and HT29-D4-S cells than in HT29-D4-Gal cells. When HT29-D4-Gal or -S cells were treated with the bacterial phosphatidylinositol phospholipase C (PI-PLC) a similar level of CEA was released suggesting a similar type of CEA anchorage. The present data demonstrate that a decrease in CEA release (i.e., in HT29-D4-Glu and -S cells) corresponds to an increase in its overall cellular expression. These results are in favour of a regulatory mechanism, impaired by suramin, which determines the balance between the soluble and the membrane bound forms of CEA.
T h e carcinoembryonic a n t i g e n (CEA) was f i r s t described as a colon t u m o r associated a n t i g e n (Gold and Freedman, 1965). I t i s a 180 kDa highly glycosylated p r o t e i n s t r u c t u r a l l y r e l a t e d t o a family o f molecules such as non-specific cross-reacting a n t i g e n ( N C A ) ( V o n K l e i s t e t al., 1972), biliary glycoprotein (BGP) (Svenberg, 1976), and n o r m a l fecal a n t i g e n (NFA) ( M a t suoka e t al., 1978). T h e a m i n o acid sequence o f CEA demonstrates a consistent s i m i l a r i t y with various members o f t h e i m m u n o g l o b u l i n superfamily (Paxton e t al., 1987, 1989; S h i v e l y e t al., 1989). T h e C E A m o l ecule is anchored t o t h e cell m e m b r a n e by a glycophospholipid-containing phosphatidylinositol ( H e f t a e t al., 1988) w h i c h c a n b e cleaved f r o m t h e m e m b r a n e by a bacterial phosphatidylinositol-phospholipaseC (PIP L C ) (Takami e t al., 1988; J e a n e t al., 1988; Sack e t al., 1988) or by proteases (Sack e t al., 1988). L i k e other 0 1990 WILEY-LISS, INC.
members o f t h e i m m u n o g l o b u l i n superfamily, anchored in t h e m e m b r a n e by a glycolipid complex, such as N-CAM 120 ( H e e t al., 19861, C E A does n o t bind an anti-cross-reacting d e t e r m i n a n t antibody (anti-CRD) (Jean e t al., 1988). CEA i s an i m p o r t a n t human colorectal cancer m a r k e r and i s used in t h e diagnosis and management o f t h i s malignant disease. However, c l i n i c a l significance o f c i r c u l a t i n g CEA l e v e l i s l i m i t e d because it i s n o t a l w a y s correlated with t h e presence o f tumors. A m o n g t h e events that determine t h e l e v e l of circulating C E A one m u s t t a k e i n t o account t h e mechanisms of release and clearance o f t h i s molecule. T h e colonic adenocarcinoma cell l i n e HT29-D4 exReceived November 1, 1989; accepted February 2, 1990. *To whom reprint requestskorrespondence should be addressed.
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presses CEA and can be induced to differentiate when glucose is substituted by galactose in the culture medium (Fantini et al., 1986, 1988, 1989a). During the process of cell differentiation of this cell line, CEA was overexpressed at the cell surface, segregated a t the apical membrane domain, and exclusively released in the luminal side of the monolayer (Fantini et al., 1989b). Recently we have shown that the anti-helmintic drug, suramin (Hawking, 1978), which antagonizes the effects of several growth factors (Coffey et al., 19871, is able to induce the differentiation of HT29-D4 cells even in the presence of glucose in the culture medium (Fantini et al., 1989~ ). The state of differentiation obtained under these conditions is achieved more than in glucose-free culture condition (Fantini et al., 1990). It should also be emphasized that growth factors such as TGFP stimulate the release of CEA from human colon carcinoma cells with a concurrent induction of a more differentiated phenotype (Chakrabarty et al., 1988, 1989). In this report we have investigated the release of CEA in suramin-treated cells (HT29-D4-S) compared with control cells (HT29-D4-Glu) and with cells grown in glucose-free medium (HT29-D4-Gal). It appears from our results that the levels of CEA released by differentiated HT29-D4-S cells were similar to the ones for undifferentiated HT29-D4-Glu cells; by contrast, a very consistent release of CEA was monitored for differentiated HT29-D4-Gal cells. We concluded that suramin interferes with the mode of release of CEA in HT29-D4 cells. MATERIALS AND METHODS Materials Suramin (obtained from Specia, Paris, France) was prepared as a sterile stock solution of 100 mg/ml in distilled water and stored a t -20°C. The anticarcinoembryonic antigen monoclonal antibody MAC 601 was purchased from Biosys (Compiegne, France). This antibody recognizes the epitope 4930 on the CEA molecule and does not cross-react with NCA-1, NCA-2, and BGP-1. The f luorescein-conjugated antimouse IgG antibodies were from Sigma Chemical Co. (Saint-Louis, Mo). The phosphatidylinositol-phospholipaseC was purified in our laboratory from Bacillus thuringiensis cultures. We checked that the purified enzyme was devoid of any proteolytic activity. Cell culture The human colon adenocarcinoma cell line HT29 was cloned by limit dilution technique (Fantini et al., 1986), and the resulting clone HT29-D4 (passage 1020) was grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 25 mM glucose. These cells will be referred to as HT29D4-Glu cells. Differentiation of HT29-D4 cells was achieved by culturing the cells in glucose-free DMEM containing 5 mM galactose (HT29-D4-Gal cells) or by adding suramin (100 pg/ml) in DMEM containing 25 mM glucose (HT29-D4-S cells) as previously described (Fantini et al., 1986, 1989a). CEA measurement Cell culture media was collected daily, centrifuged 10 min a t 10,OOOg and stored a t -20°C before dosing.
The amount of CEA was determined with the ABBOTT CEA-EIA Monoclonal One-Step kit (Abbott Laboratories, Chicago). CEA immunodetection in HT29-D4 cell e x t racts Radiolabeling detection of CEA. Cell monolayer was rinsed twice with PBS, and the cell surface was radioiodinated in PBS with 50 p1 of lactoperoxidase (2 mg/ml), 500 pCi 1251Na,and H 2 0 2 added sequentially according to Goding (1986). The reaction was stopped with tyrosine 1 mM. Cell monolayer was solubilized with the same buffer as below and CEA was immunoprecipitated using the MAC 601 antibody and protein A-Sepharose. The total amount of protein A-antibody complex was diluted in sample buffer and submitted to 10%PAGE in presence of SDS. Western blot detection of CEA. HT29-D4 cells either grown in glucose, or glucose plus suramin-containing medium, or in glucose-free medium were solubilized in Tris-HC1 50 mM buffer pH 8 containing MgC1, 1 mM, NaCl 150 mM, benzamidine 2 mM, PMSF 150 FM, and a2-macroglobuline 0.1 mg/ml. The lysate was clarified by centrifugation at 10,OOOg for 10 min and aliquots were diluted in SDS-PAGE sample buffer and submitted to 10% PAGE in the presence of SDS. The gel was transferred onto NitroscreenTMmembranes (NEN) and CEA was immunodetected using monoclonal antibody MAC 601 and 1251-proteinA as a revealing agent. Fuji X-ray films were used for autoradiographic studies for both techniques. Immunofluorescence technique Staining of the apical membrane of HT29-D4 cells was performed using the mouse anti-CEA monoclonal antibody MAC 601. Cells were seeded on glass coverslips a t a density of lo5 cells per cm2. Immunofluorescence studies were performed on intact cells (i.e., without any fixation) 1 week after cell cultures reached confluency. Cells were treated a t 4°C with the primary antibody for 2 h and then with the secondary antimouse IgG antibody-FITC conjugate for 45 min. Before mounting the coverslips with glycerol, cells were extensively rinsed in PBS containing 1% BSA. Effect of PI-PLC on CEA release In order to investigate if suramin interfered with the mode of anchorage of CEA in HT29-D4 cell membranes, three kinds of experiments were performed. Effect of PI-PLC on C E A release in long-term suramin-treated cells. HT29-D4 cells were cultured in DMEM containing 10% FCS, 25 mM glucose, and suramin (100 pg/ml). Cell cultures were used 1 week after confluency, and starved of serum for 16 h. Just before the experiment, cell cultures were washed twice with PBS (CA' 0.1 mM; Mg+ 1 mM). The effect of PI-PLC was tested in 2 ml of DMEM containing glucose (25 mM) deprived of serum and supplemented with protease inhibitors (PMSF, 500 pM; leupeptine, 2 pg/ml; aprotinine, 10 unitdml). Fourteen microliters of PIPLC (25 IU/ml) was added and a similar amount of PI-PLC was added again in the incubation medium 1 and 2 h after the initial addition and the incubation was stopped 3 h after the initial PI-PLC input. Proteins in the collected media were precipitated with cold ace+
+
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tone, resuspended in sample buffer, and analyzed by SDS-PAGE and Western blot using an anti-CEA monoclonal antibody. Effect of PI-PLC on CEA release in HT29-D4-Gal cells. HT29-D4-Gal cells were cultured in glucose-free DMEM containing 5 mM galactose and 10%FCS. The action of PI-PLC was assayed in 2 ml of glucose-free DMEM containing 5 mM galactose and protease inhibitors as described above. Effect of suramin o n PI-PLC-induced CEA release in HT29-D4-Gal cells. HT29-D4-Gal cells were grown in glucose-free DMEM containing 5 mM galactose and 10% FCS. The action of PI-PLC was tested in 2 ml of glucose-free DMEM supplemented with 5 mM galactose and 100 pg/ml suramin in the presence of protease inhibitors as described above. RESULTS
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CEA release during the process of cell differentiation in glucose-free medium or in the presence of suramin HT29-D4 cells were seeded in glucose-containing medium and transferred in suramin-containing medium or in glucose-free medium 2 or 5 days later, respectively, and the medium was changed daily. The amount of CEA released in the different culture media was monitored every day using an enzymatic immunoassay. As already described (Fantini et al., 1989b) the amount of CEA released in the culture medium from undifferentiated HT29-D4-Glu cells was very low (5-8 ng/106cells/ 24 h) and almost stable in function of time. On the contrary the level of CEA increased sharply during the differentiation of HT29-D4-Gal cells and reached 380 ng/106 cells/24 h after 9 days of culture following the medium change. Surprisingly the amount of CEA released in the culture medium by differentiated HT29-D4-S cells was comparable with the one of HT29-D4-Glu cells (Fig. 1).In order to discard an artifactual interference of suramin on the quantitation of CEA, we have tested the influence of suramin addition in a CEA-containing culture medium from HT29D4-Gal cells. The data from Figure 2 (histogram D) demonstrate that no difference can be detected in the amount of CEA measured by immunoassay, following the addition of suramin.
Fig. 1. CEA measurement during the process of cell differentiation induced by glucose starvation or suramin. HT29-D4 cells were plated in 25 cm2 flasks and grown in glucose-containing medium. Two days later suramin (100 +g/ml) was added in the culture medium for one part of the culture (arrow). For another part of the cell culture the medium was replaced by a glucose-free medium (asterisk-arrow).The medium was changed every day, and the amount of CEA was measured using the Abbott enzyme immunoassay and expressed as mean & SD (n = 3). 0-0, CEA amount in glucose-containing medium; A-A; CEA amount in suramin and glucose-containing medium; 0 - 0 ; CEA amount in glucose free galactose-containing medium.
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Suramin prevents the release of CEA by HT29-D4-Gal cells On account of the above observation it was of interest to investigate the effect of suramin on the release of CEA by HT29-D4-Gal cells. Fourteen days after seeding suramin was added or not added in HT29-D4-Gal cell cultures. The medium was changed every day. At days 19, 21, and 24, the medium was removed and tested for CEA. The data are shown in Figure 2 (histograms A, B, C). It is clear that suramin depressed the release of CEA in dome-forming differentiated HT29D4-cells whatever the time of the sampling.
Fig. 2. Effect of suramin in the release of CEA by HT29-D4-Gal cells A,-C). HT29-D4-Gal cells were seeded in glucose-free medium in 25 cm2 flask. Fourteen days later suramin (100 kgiml) was added or not added to the culture medium. The medium was changed every day. At days 19 (A), 21 (B), and 24 (C) the medium from HT29-D4-Gal cells (stippled columns) or from suramin-treated HT29-D4-Gal cells (hatched columns) was tested for CEA using the Abbott enzyme immunoassay. In D, suramin was added (hatched) or not added (stippled) to the culture medium of HT29-D4-Gal cells just before CEA measurement. Values are means 2 SD (n = 3).
Cell surface expression of CEA in HT29-D4-S cells Due to the differential release of CEA by HT29-D4Gal compared to HT29-D4-S cells, it was of importance
to investigate the presence of CEA a t the cell surface of both types of differentiated cells. HT29-D4-Glu, -Gal, or -S cells were cultured in glucose-containing medium on glass coverslips in order to perform indirect immunof luorescence experiments us-
CEA RELEASE AND SURAMIN-INDUCEDDIFFERENTIATION
47 1
Fig. 3. Indirect immunofluorescence staining with a n anti-CEA antibody. A HT29-D4-Glu cells (cultured for 10 days). B: HT29-D4-Gal cells cultured in glucose-free galactose-containing medium for 14 days. C: HT29-D4-S cells grown in the presence of suramin (100pgiml) for 14 days. The cells were stained with a n anti-CEA monoclonal antibody as described in “Materials and Methods.” Bar: 20 wm.
ing an anti-CEA monoclonal antibody. A part of the coverslips were incubated in glucose free medium 5 days after seeding whereas control cells were cultured in parallel in the presence of glucose. Twelve days later the cells were tested for CEA cell surface expression. HT29-D4-Glu cells displayed a faint but non negligible fluorescence. The periphery of the cell was quite visible indicating a uniform labeling of the cell membrane (Fig. 3A). On the contrary large clusters of differentiated HT29-D4-Gal and S cells were strongly labeled exhibiting a punctated fluorescence interpreted as an apical membrane labeling at the level of microvilli viewed en face. No basolateral staining can be observed (Fig. 3B,C) even when other planes of focus were chosen. No difference in cell surface expression could be observed in both types of cells. Radioiodination of the cell surface was performed on HT29-D4 cells grown under the three conditions of culture (i.e., in the presence of glucose, glucose + suramin, or glucose-free medium + galactose). After labeling, the cells were lysed in the presence of triton XlOO and the lysate was immunoprecipitated with an anti-CEA monoclonal antibody. The immunoprecipitated material was analyzed by SDS-PAGE. The autoradiogram of the dried gel is shown in Figure 4A. A 180 kDa polypeptide was labeled in the three types of cells, however, the immunoprecipitated molecule was much more labeled in HT29-D4-Gal and -S cells. These data demonstrated that cell surface expression of CEA was not altered by suramin. Cellular CEA characterization in HT29-D4-S cells Since CEA was only released during the establishment of the differentiation state of HT29-D4-Gal cells we looked at the CEA cellular content during the process of differentiation induced by suramin or by glucose removal. HT29-D4-Glu, -Gal, and -S cells were lysed in the presence of triton XlOO and a same amount of protein from each lysate was analysed by SDS-PAGE. Proteins were then transferred to NitroscreenTM membranes
Fig. 4. A: Immunoprecipitation of CEA after eel1 surface radiolabeling. HT29-D4-Glu, -Gal or, -S cells were radioiodinated in the presence of [‘2sIl Na and lactoperoxidase. After solubilization with Triton X100, CEA was immunoprecipitated as described in “Materials and Methods.” B: Western Blot analysis of total CEA. HT29-D4-Glu, -Gal, or -S cells were lysed with Triton X100.The same amount of proteins (100 pg) was analysed by SDS-PAGE and transferred to NitroscreenTMmembrane. CEA was revealed with an anti-CEA monoclonal antibody and by ‘251-proteinA. The arrow indicate the migration of a 180 Kda polypeptide, i.e., CEA. Data are from one experiment representative of three.
and Western blot analysis was performed using an anti-CEA monoclonal antibody and lZ5I-labeledprotein A as revealing agent. The autoradiogram is shown in Figure 4B. A low amount of CEA was reproducibly seen in HT29-D4-Gal cells while similar but higher quantities of CEA were revealed in HT29-D4-Glu and -S cells. The corresponding scanning of autoradiograms derived
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Fig. 5. Effect of PI-PLC on CEA release in HT29-D4-Gal cells (A) or HT29-D4-S cells (B). Fourteen microliters of PI-PLC (25 IUlml) was sequentially injected (at 0, 1, and 2 h) or not injected (control) in glucose-free DMEM containing 5 mM galactose (A) or in DMEM containing 25 mM glucose (B).The incubation was stopped 3 h after the first injection. The media were collected and the proteins precipitated with acetone, resuspended in sample buffer, and analyzed by SDSPAGE and Western blot. CEA was evidenced using a monoclonal antibody (MAC 601,Biosys) and '"%protein A. A unique band was typically observed at 180 kDa after autoradiography of the NitroscreenTMfilter. Lane l: Lysate of PI-PLC-treated cells. L a n e 2: PI-PLC-untreated cells medium. L a n e 3: PI-PLC-treated cells medium. Lane 4 lysate of PI-PLC-untreated cells. The autoradiogram from A was overexposed (72 h) compared to the one from B (24 h) due to the low cellular content of HT29-D4-Gal cells.
Fig. 6. Effect of suramin (100Fgiml) on the PI-PLC-induced CEA release in HT29-D4-Gal cells. Fourteen microliters of PI-PLC (25 IUI ml) was sequentially added (0, 1, and 2 h) to glucose-free DMEM containing 5 mM galactose supplemented or not with suramin (100 kg/ml). The assay was stopped 3 h after the first injection. Proteins of collected media were precipitated with cold acetone and resuspended in sample buffer analyzed by SDS-PAGE and Western blot. CEA was evidenced by a monoclonal antibody and lZ5Iprotein A. L a n e 1: Lysate of suramin-treated cells. L a n e 2 Medium of suramin-treated cells. Lane 3 Medium of suramin-untreated cells. Lane 4: Lysate of suramin-untreated cells.
amount of CEA was released after the PI-PLC treatment (Fig. 5A,B, lane 3). The cellular content of CEA from PI-PLC treated or untreated cells was identical (Fig. 5A,B, lanes 1 and 4). These data demonstrated that the mode of CEA anfrom three independent experiments revealed that the chorage in HT29-D4 cells is very likely identical with CEA content of HT29-D4-Galcells never exceed 25% of that of HT29-D4-Gal cells. In order to test if suramin the cellular CEA content of HT29-D4-S or -Glu cells. modulates the enzymatic activity of exogenous PI-PLC Thus, it appears that a low level of CEA release in the during the incubation, an experiment was designed culture medium (i.e., in HT29-D4-Glu and -S cells) is where suramin (100 pg/ml) was added or not added to correlated with an increased cellular CEA content the incubation medium containing PI-PLC of HT29while the elevated CEA release by HT29-D4-Gal cells D4-Gal cells. The recovered medium and the cell lysate were analysed by SDS-PAGE and Western blot using is correlated with a low cellular content. an anti-CEA monoclonal antibody. Release of CEA by phosphatidylinositol-specific Data from Figure 6 show that suramin has virtually phospholipase C (PI-PLC) no effect on the enzymatic activity of the bacterial PIWe have demonstrated that CEA release is impaired PLC (Fig. 6, lanes 2,3). The corresponding CEA celluin suramin-treated cells although differentiated HT29- lar content was identical as shown in Figure 6 lanes 1 D4-S cells synthesize and express CEA at the apical and 4. cell surface just like HT29-D4-Gal cells do. Since CEA DISCUSSION is anchored to the membrane by a glycane phosphatiThe establishment of differentiated state of the dylinositol (Sack et al., 1988; Jean et al., 19881, it was important to determine if the same type of CEA an- clonal HT29-D4 cells can be obtained by two ways: 1) The removal of glucose and the addition of galactose chorage occurred in both types of cells. The ability of PI-PLC to release CEA from intact (Fantini et al., 1989b); 2) The addition of suramin, a HT29 cells (Jean et al., 1988) was used to solubilize the polyanionic compound, in the glucose-containing meCEA molecules from HT29-D4-Glu, -Gal, and -S cells. dium (Fantini et al., 1989~).Important differences in Cells were incubated for 3 h in the presence or absence the differentiation state according to the culture proof PI-PLC in serum-deprived glucose-free medium for tocol were observed a t the morphological as well as at HT29-D4-Gal cells or in serum-deprived glucose-con- the electrophysiological level, the most elaborated taining medium for HT29-D4-S cells. The medium was state being obtained in the presence of suramin (Fancollected, precipitated with cold acetone, and proteins tini et al., 1990). This unique model gives us the opportunity to inveswere analyzed by SDS-PAGE transferred to NitroscreenTMmembranes and revealed with an anti-CEA tigate the process of CEA release according to the way monoclonal antibody, and lZ5I-proteinA. In parallel, cells were induced to differentiate. In this report, we cells treated or not treated with PI-PLC were lysed in demonstrate that HT29-D4-.Gal cells release steeply the presence of Triton XlOO and proteins were ana- the CEA during the process of cell differentiation inlyzed by SDS-PAGE and immunoblotted under the duced by the removal of glucose from the culture medium while HT29-D4-S cells were unable to do that. same conditions as for the collected medium. The autoradiogram of the Western blot is shown in These later cells behaved just like the undifferentiated Figure 5. For the two types of cells the medium from HT29-D4-Glu cells did. The impairment of CEA release during the process of PI-PLC untreated cells contained a very low amount of solubilized CEA (Fig. 5A,B, lane 2) while an important differentiation induced by suramin was not due to an
CEA RELEASE AND SURAMIN-INDUCED DIFFERENTIATION
interference of the drug with the CEA radioimmunoassay, because the addition of suramin in a CEA-containing medium did not alter the measured amount of soluble CEA. Moreover suramin was unable to inhibit the recognition of blotted CEA by an anti-CEA monoclonal antibody (data not shown). We can conclude that suramin did not affect the interaction of CEA with an anti-CEA antibody. The expression of CEA at the apical cell membrane was identical in HT29-D4-Gal or HT29-D4-S cells as shown by indirect immunof luorescence staining or immunoprecipitation of radioiodinated apical cell surface CEA. We can conclude that suramin did not prevent the normal polarized expression of CEA. These data are consistent with those obtained with HT29-D4-Gal and -S cells grown on permeable filters which demonstrated the exclusive localization of CEA at the apical membrane domain (Fantini et al., 1989b, 1990). The defect of CEA release observed after suramin treatment could be due to a different mode of CEA anchorage in the cellular membrane. However, our results demonstrate that the artificial release of CEA by exogenous PI-PLC can be achieved for HT29-D4-Gal as well as HT29-D4-Scells. Moreover suramin was unable to activate or inhibit the bacterial enzyme added during the experiment. These data support the idea that CEA molecules from both types of cells were probably anchored in the cell membrane by a glycolipid containing phosphatidylinositol as already demonstrated for the non-cloned HT29 cells (Jean et al., 1988). The failure of HT29-D4-S cells to release CEA is yet due to the action of suramin because the addition of the drug to HT29-D4-Gal cell culture sharply depressed the release of CEA in the culture medium. It is of course tempting to postulate that suramin inhibits the endogenous mechanisms involved in the release of CEA. These physiological processes are so far not understood and are currently under investigation. When the release of CEA was depressed (i.e., in HT29-D4-Glu and -S cells), by opposition the cellular content of CEA became very high when compared to that of HT29-D4Gal cells which released a large quantity of CEA. This latter information is in favour of a regulatory mechanism which would determine the balance between soluble and membrane bound molecular forms of CEA. The finding that colic mucosa contained a very low amount of CEA in spite of its active production (Kuroki et al., 1988) strongly suggests that CEA is rapidly released into the lumen of the digestive tract. This observation is in favour of our hypothesis. It appears from our data that suramin could modulate these regulatory mechanisms either by its action on external growth factors (Garrett et al., 1984; Coffey et al., 1987) and/or by its lysosomotropic effect (Kielian et al., 1982; Sjolund and Thyberg, 1989). ACKNOWLEDGMENTS We would like to thank the expert technical assistance of Fernand Gianellini and Jean-Jacques Roccabianca. We also thank Dr Genevieve Rougon for critical reading of the manuscript. This work was supported by grants from the Federation Nationale des Centres de Lutte Contre le Cancer, the G.E.F.L.U.C, and the Association pour la Recherche contre le Cancer.
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