12-O-Tetradecanoylphorbol-13-acetate promotion of carcinogen-treated rat liver endothelial cells

Established RLC-1 adult rat liver endothelial cells were utilized to assess promotional requirements of hepatic endothelial elements to malignant transformation following in vitro exposure to carcinogens requiring metabolic activation. Diethylnitrosamine failed to transform RLC-1 cells with or without tetradecanoylphorbol acetate (TPA) promotion. Benzo (a) pyrene was previously shown to initiate certain growth changes in the RLC-1 line; these cells, however, required TPA promotion prior to expression of the fully transformed phenotype.     

The toxic response of preneoplastic rat tracheal epithelial cells to 12-O-tetradecanoylphorbol-13-acetate

The purpose of our studies was to examine the effect of 12-O-tetradecanoylphorbol-13-acetate(TPA) on survival, proliferation, and differentiation of normaland preneoplastic rat tracheal epithelial (RTE) cells. NormalRTE cells obtained from 8-week-old rats were plated into mediumcontaining TPA at concentrations ranging from 0.1 to 100 ng/ml.A dose-related increase in colony-forming efficiency (CFE) wasobserved at concentrations above 0.1 ng/ml. At 100 ng/ml theCFE was increased 5-fold above that observed in controls. Similarstudies were carried out with immortal, preneoplastic RTE cellsderived from carcinogen-exposed primary cultures. Upon exposureto 10 ng/ml of TPA, 9 out of 11 cell lines showed a marked decreasein CFE ranging from a 70 to a >99% reduction. Two out of11 cell lines showed only a minor reduction in CFE at this TPAconcentration. Teleocidin B was as potent as TPA in causinginhibition of CFE in preneoplastic RTE cells; the potency of4-O-methyl TPA was approximately 1/100 that of TPA and phorbolwas virtually inactive. Further studies suggested that inhibitionof clonal growth of the preneoplastic tracheal cells by TPAwas due to rapid cell death. As early as 2 h after additionof TPA to the cultures, a loss of viable cells from the culturesas well as a reduction in CFE was observed. Morphological studiessupport the interpretation that a large number of cells wasseverely damaged within 2–4 h after TPA exposure. Theformation of cross-linked envelopes (CLE), a measure of keratinocytedifferentiation, was determined in cultures of preneoplasticRTE cells. TPA caused only minor increases in CLE formationindicating that the cells were not dying from TPA-induced terminalkeratinocyte differentiation, but rather from TPA toxicity.Thus, preneoplastic RTE cells respond differently to TPA fromnormal RTE cells and also from normal and transformed keratinocytesof various origins. The role of TPA toxicity in promoting preneoplasticRTE cells to the neoplastic state is being investigated.     

Differential effects of 12-O-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on 10 human lung carcinoma cell lines were compared to those seen on normal human bronchial epithelial (NHBE) cells. TPA (0.1 to 100 nM) did not enhance the clonal growth rate for any of the cell lines. As little as 3 nM TPA induced the NHBE cells to undergo terminal squamous differentiation and thus completely inhibited their proliferation; in contrast, none of the carcinoma cell lines was significantly inhibited at this concentration, and they all continued to proliferate in as much as 100 nM TPA. To determine if this lack of TPA inhibition of clonal growth reflected resistance to TPA induction of terminal squamous differentiation, we measured the ability of TPA to induce cross-linked envelope formation and to increase plasminogen activator activity in four carcinoma cell lines. Cross-linked envelopes were not induced in two lines, and only a small number were induced in the other two lines relative to NHBE cells; plasminogen activator activity was induced in NHBE cells but not in any of the cell lines.     

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells

As a prelude to study the promotion with TPA of in vitro transformationof human urothelial cells (HUC) in culture, we characterizedtumor promoter TPA receptors in primary cultures of HUC. [³H]TPAbound specifically to intact living HUC; maximum specific bindingwas attained in 30 min at 37°C. [³H]TPA bound to HUC ina saturable and competitive manner. Scatchard analysis of specificbinding to intact cells displayed a single slope correspondingto an equilibrium dissociation constant (Kd) of 0.56 nM; atsaturation TPA-binding capacity was 2.37 pmol/10⁶ HUC (1.43x 10⁶ sites per cell). [³H]TPA bound specifically and with highaffinity to the particulate fractions of HUC; binding was bothsaturable and reversible. Saturation of the specific bindingof [3H]TPA occurred at 1 nM at 4°C. Scatchard analysis ofspecific binding to the particulate fraction displayed a singleslope corresponding to a Kd of 1.08 nM; at saturation TPA-bindingcapacity was 2.05 pmol/mg protein (750 000 molecules per HUC).[³H]TPA binding was inhibited by the biologically active phorbolester, phorbol didecanoate, whereas inactive phorbol did notcompete for TPA binding. Binding was not affected by sodiumsaccharin, epidermal growth factor, retinoic acid or dexamethasone.[³H]TPA bound specifically to the HUC cytosolic fraction butonly in the presence of calcium and phosphatidylserine. Calcium-activatedand phospholipid-sensitive protein kinase activity was detectedin HUC fractions. These results indicate the presence of high-affinityspecific receptors for TPA in HUC.     

Effects of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate on normal and "preneoplastic" mouse submandibular gland epithelial cells in vitro.

The effect of the promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied on mixed primary cultures of C57BL/Icrf-at mouse submandibular gland and on slowly proliferating preneoplastic epithelial foci derived from such cultures. No cytologic alterations were noted in the TPA-treated epithelium. The development of three-dimensional epithelial ducts occurring between 30 and 60 days in vitro in untreated cultures was markedly inhibited in TPA-treated cultures. Epithelial proliferation, as shown by the [H]thymidine labelling index and mitotic index, seemded to increase during treatment with TPA and fall between the weekly treatments. Long-term TPA treatment increased the incidence of slow-growing foci in cultures previously treated with 7,12-dimethylbenz[a]anthracene but not in cultures treated with dimethyl sulfoxide. TPA had no effect on the emergence of tumorigenic cell lines at a late stage in culture or on the development of such lines from preexisting foci derived from non-TPA-treated cultures.     

12-O-Tetradecanoylphorbol-13-acetate releases human diploid fibroblasts from contact-dependent inhibition of growth

Treatment of human diploid fibroblasts at varying cell densitieswith 12-O-tetradecanoylphorbol-13-acetate (TPA) (10^–7M) resulted in a bimodal response of proliferation rate: whileat low cell densities (3 x 10³-1.5 x 10⁴ cells/cm²) TPA inhibitedthe proliferation by up to 50%, at high cell densities (1–1.6x 10⁵ cells/cm²) a 2-fold higher proliferation rate as in untreatedcultures was observed. When sparsely seeded normal diploid fibroblastswere grown in the presence of immobilized plasma membrane giycoproteins,as in confluent cell cultures strongly decreased proliferationand enhanced collagen type III synthesis is found. Using thistest system, it emerged that the addition of plasma membraneproteins from untreated as well as from TPA-treated fibroblaststo untreated fibroblasts resulted in a strong inhibition ofproliferation rate. In contrast, the addition of either untreatedor TPA-treated plasma membrane proteins to cells cultured inthe presence of TPA had no effect on growth. It is suggestedthat TPA treatment of normal diploid cells in culture resultsin a loss of responsiveness against cell-cell contacts, leadingto an escape from the contact-dependent inhibition of growth.     

Acute effects of 12-Otetradecanoylphorbol-13-acetate, teleocidin B, or 2,3,7,8-tetrachlorodibenzo-p-dioxin on cultured normal human bronchial epithelial cells

Effects of teleoddin B, 12-O-tetradecanoylphorboi-13-acetate(TPA), phorbol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and2,7-dichlorodibenzo-p-dioxin (DCDD) on normal human bronchialepithelial cell cultures were assessed by quantitation of cellularmorphology, clonal growth (population doublings per day), cross-linkedenvelope (CLE) formation and the enzymatic activities of arylhydrocarbon hydroxylase (AHH), ornithine decarboxylase (ODC)and plasminogen activator (PA). Toxicity was assessed by clonalgrowth assays. Teleocidin B and TPA had similar effects on growth,morphology and enzyme activities. When the cells were incubatedwith TPA or teleocidin B at concentrations of 1–100 nMfor 6 h, RNA synthesis was unaffected, but DNA synthesis decreasedand squamous differentiation, marked by an increase in cellsurface area and cross-linked envelope formation, was increased.TPA and teleocidin B also increased ODC activity in LHC-0 medium(a maintenance medium without epidermal growth factor) but causeda decrease of ODC activity in LHC-4 (a growth medium containingepidermal growth factor). Finally, TPA and teleocidin B eachcaused an increase of PA and a decrease of AHH activities inboth media. Phorbol, a non-promoting analogue of TPA, had noeffect on growth, morphology or biochemical assays. TCDD (100nM) caused a 15% decrease in cell growth when cells were incubatedin LHC-4, and this was accompanied by an increase in cell surfacearea, PA activity, and CLE formation. TCDD caused an increasein AHH and ODC activities when the cells were incubated in eitherLHC-0 or LHC-4 medium. DCDD did not alter cell growth, and itsmorphological and biochemical effects were similar to thoseof TCDD although less marked. In conclusion, results reportedhere are consistent with the hypothesis that an important propertyof some tumor promoters is their ability to induce terminaldifferentiation in normal, non-initiated epithelial cells.     

Enhanced synthesis of a specific protein by 12-O-tetradecanoylphorbol-13-acetate in cultured chick embryo muscle cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces in cultured postmitotic myotubes specific alterations of synthesized proteins as revealed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pulse labeling with [35S]methionine. Synthesis of myosin heavy chain is remarkably inhibited after exposure to 1.6 X 10(-7) M TPA for periods of 9 hr or longer. During shorter periods of TPA treatment (2 hr), an enhanced synthesis of a Mr 31,000 polypeptide is observed, which is associated with the particulate fraction of cultured myotubes. "Pulse chase" experiments show that this polypeptide is not a degradation product induced by TPA. The stimulation of Mr 31,000 polypeptide requires simultaneous RNA synthesis, since actinomycin D completely and selectively abolishes [35S]methionine incorporation into this polypeptide. The stimulation of Mr 31,000 polypeptide is a transient biosynthetic event not detectable after prolonged incubation (24 hr) of myotubes with the tumor promoter. However, TPA-containing medium preincubated with cultures for up to 24 hr induces stimulation of Mr 31,000 polypeptide when administered to untreated cultures. The early stimulatory effect on Mr 31,000 polypeptide synthesis and the late inhibitory effects on contractile protein synthesis are also observed when postmitotic, unfused myoblasts, rather than myotubes, are treated with TPA.     

Dynamics of glycoprotein metabolism in familial polyposis coli-derived colonic epithelial tissue

Familial polyposis coli is a disease state that offers an opportunity to obtain colon tissue in a predictable manner in terms of identifying putative premalignant markers. Our results support the hypothesis that a sequential series of alterations occur in the dynamics of glycoprotein metabolism as normal colonic mucosa progresses to colon adenocarcinoma. Specifically, both nonmalignant polyps and colon adenocarcinoma tissue expressed diminished glycosylation of proteins, whereas an elevated level of activity for membrane-associated glycosyltransferases does not appear until colon cancer itself develops.     

Differentiation of normal and cultured preneoplastic tracheal epithelial cells in rats: importance of epithelial mesenchymal interactions

Changes in the dependence on mesenchymal tissues for survival and differentiation in inbred F344 female rats were investigated in tracheal epithelial cells exposed to 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol 13-acetate (TPA). Fresh suspensions of normal tracheal epithelium or cultured preneoplastic cells were inoculated into isolated organ segments (trachea, esophagus, bladder, or small intestine) or into Dacron containers that were then implanted subdermally into isogenic recipients. At various times after cell inoculation and implantation, tissues were removed for histologic evaluation. Normal cells inoculated into frozen-thawed trachea, esophagus, bladder, and intestine yielded a regular mucociliary epithelium. Normal cell inocula did not, however, survive in tracheae previously heated (100 degrees C), fixed in ethanol, or digested with collagenase; nor did normal cells survive in Dacron containers unless tracheal fibroblasts plus epithelial cells were inoculated together. DMBA- and TPA-exposed cell populations with increased growth capacity in vitro survived and differentiated on all of the above substrates. Our observations were consistent with those of other investigators in that normal cell survival and differentiation depend to some extent on interaction with extracellular material(s) present in various organs. The essential elements were not supplied by subdermal fibroblasts alone. For survival and differentiation in vivo, preneoplastic cells appeared to have less stringent substrate requirements than did normal cells. Application of the described techniques to the study of changes occurring early in the development of neoplastic disease is discussed.     

A keratin epitope that is exposed in a subpopulation of preneoplastic and neoplastic mouse mammary epithelial cells but not in normal cells

Three monoclonal anti-keratin antibodies, AE1, AE3, and AE4, were used to compare the expression of keratins in normal, preneoplastic, and malignant mouse mammary epithelial cells growing in primary culture. In indirect immunofluorescence, AE1 did not stain normal cells but did stain a minority of preneoplastic and carcinoma cells. AE3 reacted with a subpopulation of epithelial cells in both the normal and abnormal cultures, except for certain cultures from one type of tumor wherein all of the epithelial cells were reactive. AE4 decorated an elaborate keratin filament network in all cultured mammary epithelial cells, regardless of neoplastic state. In double-label immunofluorescence, a guinea pig anti-keratin antiserum, which reacts preferentially with myoepithelial cells, exhibited coincident staining with AE1 in the tumor cultures and AE3 in the normal and most tumor cultures, indicating that the cells recognized by the antibodies in these populations were myoepithelial. Immunoblot experiments with cytoskeletal polypeptides extracted from the normal and tumor cells demonstrated that the set of keratins recognized by each monoclonal antibody was essentially the same in all of the cells except for a Mr 40,000 component that was present in normal cells but either absent or diminished in the cancer cells. Thus, while normal cells had Mr 40,000 and 50,000 keratins recognized by AE1, the epitope detected by this antibody was apparently concealed or "masked" in situ. AE3 reacted in immunoblots with a major keratin group (Mr 54,000-55,000) and a minor keratin (Mr 57,000), while AE4 reacted only with the Mr 54,000-55,000 keratin species. Because immunofluorescence with AE4 showed that the Mr 54,000-55,000 keratin group was present in all mammary epithelial cells, the AE3-reactive epitope must be masked in the majority of normal and tumor cells. The data therefore showed that epitopes on three major keratins, the Mr 40,000, 50,000, and 54,000-55,000 group, were "masked" in normal cells, whereas in tumor cells "masking" involved primarily the Mr 54,000-55,000 keratin. Attempts to "unmask" the epitopes recognized by AE1 in normal cells or to increase the number of cells reactive with AE3 in the normal and tumor cultures failed. Thus, certain cultured preneoplastic and neoplastic mammary cells with a myoepithelial phenotype have an altered organization of keratins that is manifested by a keratin antigenic determinant which is visible by immunocytochemistry in the abnormal cells but not in normal mouse mammary cells. This is the first demonstration that the immunoreactivity of keratins can be modified during neoplastic progression of epithelial cells.     

Establishment of epithelial cell lines following exposure of cultured tracheal epithelium to 12-O-tetradecanoyl-phorbol-13-acetate

In vitro exposure of rat tracheal epithelium to the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate results in a marked increase in growth capacity. The growth changes are manifested in an increased rate of cell division and growth in primary cultures and in the establishment of permanent epithelial cell lines. Such changes did not occur in control cultures. Fourteen of the cell lines have been inoculated into immunosuppressed recipients, and all are nontumorigenic.     

Ornithine decarboxylase activity and DNA synthesis after treatment of cells in culture with 12-O-tetradecanoylphorbol-13-acetate.

The ability of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce the enzyme ornithine decarboxylase (ODC) and to stimulate DNA synthesis was studied in four different cell types in vitro. The effects of this agent on each cell type were different: (a) in hamster embryo cells, TPA induced ODC but had no effect on DNA synthesis; (b) TPA induced ODC and stimulated DNA synthesis in BALB/c 3T3 mouse cells; (c) it did not induce ODC in human fibroblasts but did stimulate DNA synthesis; and (d) it induced neither ODC nor DNA synthesis in rat embryo fibroblasts. In contrast to the effects of TPA, ODC was induced and DNA synthesis was stimulated in all cell types by fresh serum-containing medium. Treatment of the cells with a combination of fresh medium and TPA resulted in an approximate summation of the effects of treatment with each agent alone. These results emphasize the differences in the responses of various cells to TPA. They also show that in some cells, at least, the induction of ODC and stimulation of DNA synthesis following TPA treatment can be regulated independently.     

Transient protection of cultured human cells against antitumor agents by 12-O-tetradecanoylphorbol-13-acetate

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown in cell cultures to enhance the frequency of resistance to methotrexate. However, we found that TPA could also partially protect human KB cells over a short time (72 h) from the cytotoxicity of several antitumor agents, including 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene)-beta-D-glucopyranoside (VP-16), vincristine, mitoxantrone, and methotrexate, but not 1-beta-D-arabinofuranosylcytosine or 5-fluorouracil. The modes of protection were different for methotrexate and VP-16. Protection by TPA was concentration dependent up to 40 nM and could be accomplished by a 2-h incubation of cells with TPA alone prior to drug treatment. This protection disappeared after a 24-h drug-free incubation. TPA-induced protection could not be mimicked by treatment of cells with 1-oleoyl-2-acetyl-glycerol (a stimulator of protein kinase C) or phospholipase C, which increased the cellular content of diacylglycerols. Thus the action of TPA on protein kinase C may not be sufficient to exert protection. Verapamil, a calcium-channel blocker which has been found to circumvent resistance of multiple drug-resistant cells, also circumvented the protective effect of TPA when used with VP-16. The presence of TPA during a 24-h exposure to radiolabeled VP-16 reduced the cellular drug content by about 30%, whereas verapamil enhanced drug content by at least 50% in TPA-treated and untreated cultures. Since substances with some TPA-like activity have been found in foods and in human circulation, the observation of clinical resistance to some compounds may partly be due to a related mechanism.     

Role of oxygen radicals in 12-O-tetradecanoylphorbol-13-acetate-induced squamous differentiation of cultured normal human bronchial epithelial cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits growth and induces terminal squamous differentiation of normal human bronchial cells when added to the culture media [J. C. Willey, A. J. Saladino, C. Ozanne, J. F. Lechner, and C. C. Harris, Carcinogenesis (lond.), 5: 209-215, 1984]. We have investigated the possibility of oxygen free radicals being involved as intermediates in this process. Electron paramagnetic resonance measurements using the spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide failed to detect oxygen free radicals in bronchial epithelial cells exposed to TPA, although oxy radicals were detected in bronchial epithelial cells after a nontoxic exposure to menadione, and in human neutrophils after exposure to TPA. Addition to the culture media of free radical scavenger, i.e., reduced glutathione, N-acetylcysteine, D-alpha-tocopherol, copper (II) (3,5-diisopropylsalicyclic acid)2, or the combination of superoxide dismutase and catalase did not affect the dose-dependent growth inhibition of TPA on the bronchial epithelial cells. Moreover, exposure of the bronchial epithelial cells to TPA did not result in increased DNA single strand breaks measured by alkaline elution, as would be expected with a free radical mediated mechanism. Thus, our results argue against the importance of oxygen free radicals in the inhibition of growth and the induction of squamous differentiation by TPA in normal human bronchial epithelial cells.     

12-O-Tetradecanoylphorbol-13-acetate and the induction of prostaglandin E2 generation by human keratinocytes: a re-evaluation

12-O-Tetradecanoylphorbol-13-acetate (TPA) induces prostaglandinE₂ (PGE₂ synthesis in mouse keratinocytes and is associatedwith the induction of keratinocyte proliferation as well asaccelerated differentiation. In human keratinocytes, TPA hasbeen reported not to induce the release of either ³H-labeledarachidonic acid or ³H-labeled prostaglandins, even though celldifferentiation is stimulated. Because PGE₂ has been associatedwith the modulation of cell differentiation and because of technicalproblems inherent in evaluating arachidonic acid metabolismusing only radlolabeled substrates, we evaluated the abilityof TPA to induce endogenous PGE₂ generation by cultured humankeratinocytes using a specific and sensitive enzyme immunoassay.With this technique, TPA was found to induce a dose-dependent(1.6x10^–12–1.6x10^–8 M) increase in PGE₂ generation.These results are consistent with observations made not onlyin mouse keratinocytes but in other mammalian and human celltypes. Documenting the ability of TPA to stimulate PGE₂ productionin human keratinocytes is very relevant to current theoriesregarding the role of PGE₂ In keratinocyte differentiation aswell as to establishing parallels between the murine and humanskin models.     

12-O-tetradecanoylphorbol-13-acetate activates the synthesis of phosphatidylethanol in animal cells exposed to ethanol

Tumor-promoting phorbol esters acutely activate a pathway in lymphocytes leading to the synthesis and accumulation of phosphatidylethanol, using exogenous ethanol as a precursor. This product is a representative of a unique class of acidic glycerophospholipids in which the head group is a primary alcohol. The formation of this lipid, in response to different phorbol ester derivatives, correlates with their activity as tumor promoters and inducers of growth changes in a variety of animal cells. Since phosphatidylethanol represents an unusual metabolite of ethanol, it is proposed that studies of its synthesis and biological functions may also provide new perspectives on the biology of alcohol addiction as well as the role of this biological pathway in tumor promotion.     

Calcium ionophore A-23187 and 12-O-tetradecanoyl-phorbol-13-acetate stimulation of prostaglandin synthesis in herpes simplex virus type 2-transformed rat cells

The purpose of this investigation was to determine whether cells transformed by herpes simplex virus type 2 (HSV-2) can be stimulated to synthesize prostaglandins (PG). Stimulation was determined by measuring the release of PG into overlay fluids from cell monolayers prelabeled with [3H]arachidonic acid. Results showed that Ca2+ ionophore A-23187 markedly stimulated arachidonic acid release starting 30 min after treatment of HSV-2-transformed and nontransformed rat embryo fibroblast cells. However, only HSV-2-transformed cells were stimulated in production of PG. HSV-2-transformed, nontumorigenic, rat embryo fibroblast, line G, clone 2.0 cells synthesize nearly equal amounts of prostaglandin E2 (PGE2) and prostaglandin F2 alpha, while tumor (rat fibrosarcoma) cells synthesize primarily PGE2. Stimulation of PGE2 synthesis by Ca2+ ionophore A-23187 or 12-O-tetradecanoyl-phorbol-13-acetate decreased as rat fibrosarcoma cells were serially passaged in tissue culture. At low passage of parental rat fibrosarcoma cells, four distinct morphological clonal cell lines were isolated, which varied markedly in their capacity to be stimulated in PG synthesis by 12-O-tetradecanoyl-phorbol-13-acetate. There was correlation between the capacity of clone 1 cells to be stimulated in PGE2 synthesis by serum alone and capacity of the tumors produced by the clone 1 cells to metastasize to the lungs of syngeneic tumor-bearing rats. In summary, cell transformation by HSV-2 appears to be essential for stimulation of PG synthesis in cells. The capacity to be stimulated in arachidonic acid metabolism and PG synthesis may be important in the process of carcinogenesis by a putative human cancer virus.     

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 JH-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. © 1994 Wiley-Liss, Inc.