Carcinoembryonic Antigen Release Research Articles

Regulation of carcinoembryonic antigen release from colorectal cancer cells

Regulation of carcinoembryonic antigen release from colorectal cancer cells

Regulation of carcinoembryonic antigen release from colorectal cancer cells

Clinical and experimental evidence suggest that circulating carcinoembryonic antigen (CEA) released from tumor cells has an instrumental role in colorectal cancer-liver metastasis. However, the precise mechanism of the regulation of the CEA release from cancer cells is not known. We investigated if the rate of CEA and another GPI-anchored protein, alkaline phosphatase (AP) release is correlated with cellular glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) expression. We also evaluated the effects of phosphatidic acid (PA), a compound known to inhibit GPI-PLD activity, on the CEA and AP release from colon cancer cells. The expression of CEA, GPI-PLD, and AP in five colon carcinoma cells (LS180, Caco2, SW742, SW1116, and HT29/219) was verified by immunoblot and real-time RT-PCR analysis. The amounts of CEA and AP released into cell culture media were determined using ELISA and a colorimetric assay, respectively. We examined the effects of PA (20-100μM) on CEA and AP release from LS180 cells. All five cancer cell lines analyzed expressed GPI-PLD protein. While there was a positive relationship between AP release and the levels of GPI-PLD transcript expression, we found no direct correlation between CEA released from cancer cells and the GPI-PLD mRNA expression level. However, the rate of CEA release was positively associated with the level of CEA transcript expression. In comparison to controls, the release of GPI-anchored CEA and AP, but not CA19-9 was inhibited significantly by both crude and pure phosphatidic acid (by 56 and 54.5%, respectively). Using PA for inhibiting CEA release from cancer cells may have therapeutic application in preventing CRC-liver metastasis.

Pattern of carcinoembryonic antigen drop after laparoscopic radiofrequency ablation of liver metastasis from colorectal carcinoma

Laparoscopic radiofrequency ablation (RFA) is being increasingly used for local control of hepatic metastasis from colorectal carcinoma (CRC). After surgical resection of colorectal liver metastasis, carcinoembryonic antigen (CEA) values fall within 2 weeks, making this a useful parameter to follow shortly after surgery. Little is known, however, about the expected pattern of the CEA drop after RFA. From September 1998 to October 2002, RFA to CRC liver metastasis was performed on 144 patients. A subset of 17 patients were studied who had no evidence of extrahepatic disease preoperatively, had all detectable tumor ablated intraoperatively, and who on long-term follow-up (up to 15 months) had no evidence of recurrent disease. Serum CEA was determined preoperatively, on the first postoperative day, at 1 week, and every 3 months afterwards. A computed tomography (CT) scan of the abdomen and pelvis was obtained every 3 months postoperatively. Ten (58.8%) patients showed an increase in CEA on postoperative Day 1, averaging 38.3% compared with the preoperative value. CEA then fell to 50% of the preoperative value, on average, on Day 7 postoperatively and only reached its nadir at 3 months. Unlike resection patients, those undergoing ablation show an initial rise in CEA, probably due to release from the ablated tissue. Although heating of RFA would be expected to destroy CEA, the initial rise and slow drop postoperatively argue for a release of immunoreactive CEA from the ablated zone. This slow decline in CEA indicates that several months should pass before assessing the extent of potential residual disease.

Evidence for CEA release from human colon cancer cells by an endogenous GPI-PLD enzyme

Elevated carcinoembryonic antigen (CEA) blood levels are found in a wide variety of epithelial neoplasms. The precise mechanism of the spontaneous CEA release from normal and cancer cells has not been established yet. In this study we investigated ‘in vitro’ the role of an endogenous glycosylphosphatidyl inositol phospholipase D (GPI-PLD) in spontaneous CEA release from human colon carcinoma cells. We detected GPI-PLD-specific transcript expression in four human colorectal tumor cell lines, LS180, HT29, HT29/219, and SW742 by RT-PCR. Furthermore, CEA release could be activated and inhibited by incubation of LS180 cells with suramin and 1,10-phenanthroline, compounds known to activate and inhibit GPI-PLD activity, respectively. The results suggest a mechanism for the involvement of an endogenous GPI-PLD in spontaneous CEA release from human colon cancer cells.

Determination of carcinoembryonic antigen levels in peripheral and draining venous blood in patients with colorectal carcinoma

The problem of the relationship between blood carcinoembryonic antigen (CEA) levels and tissue CEA content in colorectal carcinoma, and the mechanisms for CEA release from tumor cells in tissue adjacent to the neoplasm is important to understanding the biology of colorectal carcinoma. It has not been adequately explained whether CEA in the peripheral blood is drained mainly by portal system blood or by the lymphatic system, or indeed by both systems. To study the behavior of CEA levels in peripheral blood (CEA-p) and venous effluent blood (CEA-d) among patients with colorectal tumors, who underwent curative operation. A total of 28 patients were studied (12 male [42.9%] and 16 female [57.1%], mean age 66.1 years [range: 43 - 84]). Immediately after laparotomy, peripheral venous blood was extracted by antecubital venous puncture and venous effluent blood was collected from the main drainage vein of the lesions. Values of CEA-p, CEA-d and the gradient between CEA-d and CEA-p that were less than 5.0 ng/mL were considered normal. Eight (28.6%) patients were stage A in Duke's classification, nine (32.1%) stage B and 11 (39.3%) stage C. The neoplasm was located in the rectum of 14 patients (50.0%), in the transverse colon in five (17.9%), in the sigmoid in four (14.3%), in the cecum and/or ascending colon in three (10.7%), and in the descending colon in two (7.1%). The histopathological examination revealed well-differentiated adenocarcinoma in all the patients. Only one patient (3.6%), Duke's classification stage C, presented neoplasm with venous invasion. The gradient between the CEA-p and CEA-d levels were normal in 25 patients (88.3%) and high in three (10.7%). The mean value for CEA-p was 3.8 +/- 4.1 ng/mL (0.1-21.1 ng/mL) and for the drained CEA (CEA-d) it was 4.5 +/- 4.3 ng/mL (0.3-20.2 ng/mL), without significant difference between these values. There was a significant difference between the mean value for CEA-p and CEA-d levels greater than 5 ng/mL. The CEA-p and CEA-d levels in the colorectal carcinoma patients were not shown to be different. The results from this study suggest that, in colorectal neoplasm without venous invasion, there may not be notable CEA drainage from the tumor by the portal vein effluent blood.

Multidrug-Resistant Phenotype Influences the Differentiation of a Human Colon Carcinoma Cell Line

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiatein vitroby replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60–90%) of CEA release.

Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope.

Growth inhibition, enhancement of intercellular adhesion, and increased expression of carcinoembryonic antigen by overexpression of phosphoinositides-specific phospholipase C beta 1 in LS174T human colon adenocarcinoma cell line.

By using a retrovirus‐derived system we generated derivatives of the human colon adenocarcinoma cell line LS174T (ATCC CL 188) that stably overexpress a full‐length cDNA encoding the β1 isoform of bovine phosphoinositides‐specific phospholipase C (PI‐PLC). This was confirmed by the elevated levels of catalytic activity to release phosphoinositides from phosphatidylinositol (PI‐PLC) or phosphatidylinositol‐bis‐phosphate (PIP2‐PLC), and the enhanced expressions of messenger RNA and protein. PI‐PLC β1 overexpresser clones grew to form cell clumps floating in liquid medium, whereas the pMV7‐introduced control clones displayed morphologic characteristics that were very similar to those of the parent LS174T cell line. Three individual PI‐PLC β1 overexpresser cell lines displayed increased doubling time (18.0 h, 21.5 h, and 23.8 h) when compared with 4 individual pMV7‐introduced control cell lines (13.1 h, 10.7 h, 12.9 h, and 9.3 h). Anchorage‐independent growth ability in soft agar medium was dramatically suppressed by overexpression of PLC β1, and the ability of PLC‐overproducer clones to form aggregates when cultured in liquid medium was dramatically enhanced when compared with that of pMV7‐introduced control clones. Tumorigenicity of PLC β1‐overproducers was much weaker than that of vector‐transduced control clones. The spontaneous release of carcinoembryonic antigen from PLC β1‐overproducer clones was much higher than that from pMV7 control clones. The ability of PLC β1‐overproducer clones to form aggregates during suspension culture was much stronger than that of the control clones. These results provide the first evidence that elevated levels of endogenous PI‐PLC β1 suppress tumor cell growth, but enhance the ability to form cell aggregates and to release carcinoembryonic antigen, an intercellular adhesion molecule.

Modulation of carcinoembryonic antigen release by glucosylceramide

Previous work suggested that glucosylceramide (GlcCer) plays a role in the regulation of cell differentiation of HT29 human colon tumor cells. In the present study, we investigated the role of GlcCer in the cellular release of carcinoembryonic antigen (CEA), a marker for cell differentiation. This was done by modulating the intracellular level of the glycolipid, according to two different approaches. The cells were treated with D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which resulted in a specific lowering of the cellular GlcCer pool. Alternatively, by exogenous addition of a short-chain analog of the lipid, hexanoyl(C6)-GlcCer, the cellular pool was enhanced. The results demonstrate that PDMP causes an increase in the release of CEA, while exogenous C6-GlcCer suppresses its release. Furthermore, the enhanced release of CEA in the presence of PDMP, could be completely reversed upon exogenous addition of C6-GlcCer. Control experiments reveal that a potential interference of the well-known modulator of cell physiology, ceramide (Cer), can be excluded. Long-term depletion of GlcCer resulted in a change in a morphological feature of differentiation of the cells, i.e. an increase in apical membrane surface with microvilli brush borders, accompanied by an enhanced expression of the cytoskeletal protein villin. These results, together with the observations on modulation of the differentiation marker CEA by GlcCer, provide support for the conclusion that GlcCer interferes with the differentiation of HT29 cells.

Augmentation of carcinoembryonic antigen release from intact, viable tumor cells by a factor in human serum

Measurement of carcinoembryonic antigen (CEA) levels in human serum is frequently used to detect tumor recurrence in patients with resected primary colorectal cancers. These levels are highly variable from patient to patient, and the mechanism that determines these levels is still poorly understood. Using a 6-h in vitro CEA-release assay, we determined that a factor in human and fetal bovine sera significantly augments the release of CEA from the tumor cell surface into cell culture supernatants. As little as 1% serum admixed with tumor cells results in CEA release up to 200% greater than that of serum-free controls. It is not inhibited by 1,10-phenanthroline or heat inactivation (of serum) but is calcium dependent. The electrophoretic mobility and membrane linkage of CEA released by serum appear to be identical to those of CEA released by bacterial phospholipase C. Because bacterial phospholipase C is known specifically to cleave the phosphoinositol (PI) glycan moiety that anchors CEA to the tumor cell surface, a mechanism of action for serum cleaving this anchor is suggested. The large range of CEA levels observed in patients with colorectal cancers may be related to differential sensitivity of the CEA membrane anchor to the CEA-releasing factor in serum.

Non-proteolytic release of carcinoembryonic antigen from normal colonic epithelial cells cultured in collagen gel: [formula omitted], Mo. Kuroki∗, T. Yamanaka, K. Kido, T. Sakai, T. Maekawa, T. Hideshima, Y. Yamashita., Y. Matsuoka∗ and T. Shirakusa. 2nd. Dep. of Surgery, Biochemistry∗, School of Medicine Fukuoka Univ.

Non-proteolytic release of carcinoembryonic antigen from normal colonic epithelial cells cultured in collagen gel: [formula omitted], Mo. Kuroki∗, T. Yamanaka, K. Kido, T. Sakai, T. Maekawa, T. Hideshima, Y. Yamashita., Y. Matsuoka∗ and T. Shirakusa. 2nd. Dep. of Surgery, Biochemistry∗, School of Medicine Fukuoka Univ.

Non‐proteolytic release of carcinoembryonic antigen from normal human colonic epithelial cells cultured in collagen gel

Recent studies have shown that, even with a minimal content of carcinoembryonic antigen (CEA), normal human colonic epithelial cells express substantial amounts of CEA mRNA and colonic mucosal fragments cultured in vitro produce CEA quite actively, indicating that CEA should no longer be considered to be of an oncofetal nature. To understand the basis of the usefulness of CEA as a tumor marker, we analyzed the release of CEA, a glycosyl-phosphatidylinositol (GPI)-anchored protein, from colonic epithelial cells, by culturing isolated colonic crypts in collagen gel. The crypts appeared to preserve their morphological and biochemical integrity in the gel for at least 16 hr, and released CEA spontaneously. Three forms of CEA--spontaneously released CEA, CEA liberated with phosphatidylinositol-specific phospholipase C (PI-PLC) and CEA in cell lysates--were indistinguishable on SDS-PAGE. This is in contrast to recombinant CEA spontaneously released from CHO transfectants, which showed a smaller molecular mass than that of PI-PLC-cleaved recombinant CEA. By phase separation using Triton X-114, CEA in the cell lysates of crypts was separated mostly into the detergent phase, while the spontaneously released and the PI-PLC-cleaved CEA were separated into the aqueous phase. When the cells were metabolically labeled with the precursors of the GPI-anchor, 3H-ethanolamine but not 3H-palmitic acid was found in the spontaneously released CEA. These findings suggest that, in contrast to the proteolysis-like release of the recombinant CEA from CHO cells, CEA in normal colonic epithelial cells is released by a non-proteolytic cleavage, which probably occurs through the action of some endogenous phospholipase.

Release of Carcinoembryonic Antigen from Human Tumor Cells by Phosphatidylinositol-Specific Phospholipase C: Highly Effective Extraction and Upregulation from LS-174T Colonic Adenocarcinoma Cells

Carcinoembryonic antigen (CEA), produced by gastrointestinal tumor cells, is anchored to cell membrane by a glycosyl-phosphatidylinositol moiety which can be cleaved with phosphatidylinositol-specific phospholipase C (PI-PLC). We studied the extraction of CEA from living human colon carcinoma (LS-174T, HT-29, COLO-205, and HRT-18) and pancreatic carcinoma (CAPAN) cells by PI-PLC from Bacillus cereus. The total CEA content of LS-174T cells, quantitated by Triton X-1 14 extraction followed by radioimmunoassay or by immunohistochemistry, was 3.5-fold higher than that of other cells ( P < 0.001). The spontaneous release of CEA from LS-174T cells into culture medium was also higher than that from other cells ( P < 0.00 1), reaching 620 ng/107 cells (≈28% of cellular content) after 24 h. Overall, living LS-174T cells were highly susceptible to CEA extraction by PI-PLC, which was dependent on PI-PLC dose and on treatment time, leading in optimal conditions to the solubilization of 4100 ng/10 7 cells after 24 h (≈75% of total CEA). After 24 h treatment at the highest PI-PLC dose, cell lines remained viable and growing, and membrane CEA expression was not exhausted but only reduced as compared to untreated cells. At the same time, the amount of CEA solubilized by P1-PLC exceeded the CEA reduction in membranes, suggesting that enzyme treatment increased CEA turnover. This was particularly true for LS- 174T cells which maintained 54% of the expression of untreated cells, whereas the amount of CEA extracted by PI-PLC reached 190% of this expression. Growing LS-174T cells thus constitute an effective material for producing high quantities of CEA by PI-PLC cleavage, especially since these cells probably "regenerate" be cause of enhanced turnover during PI-PLC action, thus allowing continuous CEA production. These experimental conditions also provide an interesting model for studying the modulation of CEA expression and release.

Des-(1-3)-IGF-I, an insulin-like growth factor analog used to mimic a potential IGF-II autocrine loop, promotes the differentiation of human colon-carcinoma cells.

HT29-D4 human colon-carcinoma cells have been shown to secrete insulin-like growth factor (IGF)-II and to simultaneously express type-I IGF receptors. However, the sequestration of IGF-II by several molecular forms of IGF-binding proteins (IGFBP) in the culture medium prevents the establishment of an operative IGF-II autocrine loop. IGFBPs secreted by HT29-D4 cells (HT29-D4 IGFBP) comprise isoforms of IGFBP-4 (25, 27 and 30 kDa) and 2 unidentified forms (34.5 and 32-34 kDa). This latter does not bind 125I-IGF-I. The net affinity of HT29-D4 IGFBP is about 12 times stronger for IGF-II (KD approx. 10(-10) M) than for IGF-I. All the HT29-D4 IGFBP molecular forms are unable to bind the N-terminally truncated IGF-I analog, des-(1-3)-IGF-I. In contrast, HT29-D4 cell-surface type-I IGF receptors bind IGF-I and des-(1-3)-IGF-I identically (KD approx. 5 x 10(-10) M). We have taken advantage of these particular binding properties to use des-(1-3)-IGF-I to mimic a potential IGF autocrine loop and to observe its biological consequences. Nanomolar concentrations of des-(1-3)-IGF-I induce HT29-D4 cells to develop into a differentiated phenotype, as judged by a substantial carcinoembryonic antigen release and the induction of numerous intercellular cysts with well-organized microvilli. In the same way, des-(1-3)-IGF-I early induces a slight inhibition of HT29-D4 cell proliferation. Based on these findings, we conclude that the type-I IGF receptor primarily controls the differentiation of these colonic cells, and that HT29-D4 cancer cells remain in an undifferentiated state because of their inability to use endogenous IGF-II as an autocrine regulatory factor.

Vectorial release of carcinoembryonic antigen induced by IFN-γ in human colon cancer cells cultured in serum-free medium

Confluent monolayers of intestinal cell lines are useful models for studies of intestinal epithelial structure and function. Three cell lines have retained morphological and functional properties of intestinal epithelial cells compatible with such studies: Caco-2, T84 and HT29. However, the requirement of fetal bovine serum for the culture of these cells does not facilitate the design of experiments dealing with growth factors or hormonal regulation. The clonal intestinal cell line HT29-D4 can be cultured as fully differentiated epithelial monolayers in a synthetic medium containing transferrin, selenous acid, epidermal growth factor and suramin, a potent differentiation inducer. In the present study it is shown that HT29-D4 cells grown on permeable substratum in this synthetic medium developed electrically active monolayers consisting of columnar cells with morphological characteristics of normal enterocytes. After metabolic labelling with [35S]-methionine, HT29-D4 monolayers released most of their radiolabelled secretory proteins preferentially in the basal compartment of the cell culture chamber. However, the carcinoembryonic antigen, shown to be present in the apical plasma membrane, was exclusively released apically. This oriented release was stimulated by recombinant gamma-interferon (IFN-γ) added only in the basal chamber, suggesting a basolateral restriction for IFN-γ receptors.

Calculation of half-life of carcinoembryonic antigen after lung tumour resection: a case report

Since little is known about carcinoembryonic antigen (CEA) half-life in humans, we followed changes in CEA levels after curative resection of a small bronchial adenocarcinoma in a 54 yr old man with an unusually high preoperative serum CEA level of 1,199 ng.ml-1. Serum CEA followed a four phase curve with an initial increase, attributed to release of CEA from the tumour, a rapid fall during four days (CEA half-life 3.2 days), a slower exponential decay to normal levels during almost three months (CEA half-life 11 days) and eventually maintenance of normal levels for 14 months. These results are consistent with data from animal experiments and a single report on CEA half-life in humans, but CEA half-life seems longer in man than in animals. This observation also suggests that a very high serum CEA level should not in itself preclude curative surgical resection of a primary bronchogenic carcinoma.

Impaired carcinoembryonic antigen release during the process of suramin‐induced differentiation of the human colic adenocarcinoma cell clone HT29‐D4

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 65:163-169, 1989); 2) the addition of suramin, a polyanionic compound, in the glucose-containing medium (Fantini et al.: Journal 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 HT29-D4-Glu cells was very low (5 to 8 ng/10(6) cells/24 hours) and almost constant throughout the experiment whereas it increased sharply during differentiation of HT29-D4-Gal cells (380 ng/10(6) 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 (Pl-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.

The Effects of Adverse Growth Conditions on the Shedding of Carcinoembryonic Antigen from Cultured LS180 Colon Cancer Cells

The synthesis and release of the tumor marker carcinoembryonic antigen (CEA) from the colon cancer cell line LS180 has previously been reported to be enhanced during the later stages of in vitro culture after growth has stopped. It has been suggested that CEA expression was inversely related to the growth rate for these cells (Kahan, B.D.; Rutzky, L.P.; Legrue, S.J.; Tom, B.H. Methods Cancer Res. 18:197-275; 1979 and Shi, Z.R.; Tsao, D.; Kim, Y.S. Cancer Res. 43:4045-4049; 1983). Our studies indicate, however, that while certain environmental perturbations that halt growth (e.g., glucose starvation and elevated temperatures) do indeed stimulate CEA expression and release; other growth-arresting conditions, such as oxygen starvation, have no effect. Replacement of spent or conditioned medium with fresh medium during the later culture stages resulted in a 10-fold decrease in CEA release, indicating that either depleted nutrients or accumulating cellular products (such as lactate or ammonium) trigger enhanced CEA production.

Increase in carcinoembryonic antigen release from cancer cells by combined treatment with retinoic acid and low-temperature hyperthermia

The growth of a human gastric adenocarcinoma cell line, MKN-45, was inhibited and the amount of carcinoembryonic antigen (CEA) in both the culture medium and the cell extract was increased in the presence of retinoic acid at a concentration of 75 (1.5x)-125 microM (1.9x), which did not substantially affect cell survival. Treatment using a combination of retinoic acid (125 microM) and low-temperature hyperthermia (40 degrees C, 30 min) was more effective in increasing CEA compared with retinoic acid alone (extracellular 1.9-2.4x, intracellular 1.5-1.9x). The inhibition of cell growth was reversed after the retinoic acid was removed from the medium. Cells treated with both retinoic acid and (low-temperature) hyperthermia, however, could be induced to release a significant amount of CEA at about 48 h after retinoic acid removal. The induced CEA increase in the cells, but not in the medium, was suppressed by actinomycin D (1 ng/ml) or cyclohexamide (0.2 microgram/ml). These results suggest that retinoic acid, used alone or in combination with hyperthermia, enhances the production and release of CEA in human gastric cancer cells.

Induction of polarized apical expression and vectorial release of carcinoembryonic antigen (CEA) during the process of differentiation of HT29‐D4 cells

HT29-D4 clonal cells can be induced to differentiate by a simple alteration of the culture medium, that is, by the replacement of glucose by galactose [Fantini, J., et al. (1986) J. Cell Sci., 83:235-249] as reported for the nonclonal HT29 cells [Pinto, M., (1982) Biol. Cell, 44:193-196]. An essential property of the HT29-D4 cell line is the fact that no cell loss occurs after the medium change, so that the differentiated cells can be considered as the true counterpart of the undifferentiated one. This model is particularly suitable to study morphological and biochemical events associated with the progressive establishment of the differentiation state. We report here that carcinoembryonic antigen (CEA), a 180 kDa glycoprotein originally described as a colon tumor associated antigen, is faintly expressed at the surface of undifferentiated HT29-D4 cells. These cells release a small amount of CEA (2.5 ng/10(6) cells/24 hr) in the culture medium. Fourty-eight hours after glucose substitution by galactose, both CEA cell surface expression and release are strongly enhanced as demonstrated by immunofluorescence and immunoprecipitation studies. Ten days after the medium change, the amount of CEA released reaches a maximum value of 130 ng/10(6) cells/24 hr, which remains stable for differentiated HT29-D4 cells cultured in glucose-free, galactose-containing medium (Gal-medium) for several months. HT29-D4 cells grown in Gal-medium in porous-bottom culture dishes generate leakproof epithelial monolayers. We have successfully performed an independent radioiodination of the apical and basolateral domains of these cells, followed by immunoprecipitation. We demonstrate that CEA is expressed exclusively at the apical surface of differentiated HT29-D4 cells, since the 180 kDa polypeptide was immunoprecipitated only when the radioiodination was performed at the apical side of the monolayer. Leakproof HT29-D4 monolayers cultured in permeable chambers were also used to demonstrate that CEA was exclusively released in the medium bathing the apical side of the cells. In conclusion, this study of cell surface CEA expression and CEA release during the process of differentiation of HT29-D4 cells demonstrated that 1) CEA cell surface expression and CEA release are correlated with cell differentiation; 2) CEA is expressed in the apical brush border membrane of differentiated HT29-D4 cells; and 3) CEA release is exclusively oriented toward the apical side of the polarized monolayer.