Colon Adenocarcinoma Cell Line HT-29 Research Articles

Interferon-γ Decreases Cell Surface Expression of Galactosyl Ceramide, the Receptor for HIV-1 GP120 on Human Colonic Epithelial Cells

HT-29-A7, a CD4-negative clonal derivative of the human colonic adenocarcinoma cell line HT-29, is particularly sensitive to infection by several isolates of HIV-1 and, correspondingly, expresses high amounts of galactosylceramide (galactocerebroside, GalCer). GalCer is a neutral glycolipid which binds to the HIV-1 envelope glycoprotein gp120 and is present at abundant levels in normal human epithelial cells of the small and large intestine. Treatment of the HT-29-A7 cells with recombinant γ-interferon (rIFNγ) induced a dose-dependent inhibition of GelCar expression on the cell surface, as demonstrated by indirect immunofluorescence and by enzymatic labeling of cell surface glycoconjugates with oxidase-tritiated sodium borohydride. The rIFNγ effect was not associated with any toxicity and was specific for GalCer, since expression of carcinoembryonic antigen did not decrease following treatment. The decrease in GalCer expression was associated with resistance of the cells to HIV-1 infection. In contrast, rIFNγ did not alter cell surface expression of CD4, the classical HIV receptor, in HT-29-A7 cells that bad been transduced with a retroviral vector expressing full-length CD4, and there was no effect on their infection. These results strongly suggest that rIFNγ blocks HIV-1 infection of HT-29-A7 cells by decreasing GalCer synthesis and expression This effect on expression of a viral receptor is a novel antiviral property of rIFNγ which should be exploited for antiviral therapeutics.

Expression and function of CD59 on colonic adenocarcinoma cells.

The expression and function of CD59, a 19-25 kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement, was analyzed on normal and malignant human colonic epithelial cells. Analysis by immunofluorescence demonstrated a weak apical expression of CD59 on normal intestinal epithelium, with an increased expression on adenocarcinoma cells. The expression of CD59 was greatest on tumor cells with poor differentiation. The functional activity of CD59 on human adenocarcinoma cells was investigated using the colonic adenocarcinoma cell line HT29. CD59 on HT29 cells was glycosyl-phosphatidylinositol-linked, and had a molecular mass of 19-25 kDa. HT29 cells expressed approximately four times more CD59 than leukocytes, and showed a high resistance to antibody-dependent complement-mediated lysis. Blocking of CD59 with divalent antigen-binding F(ab')2 fragments of the anti-CD59 monoclonal antibody 1F5 resulted in a dose-dependent increase in complement-mediated lysis, suggesting that CD59 may be of importance in protecting colonic adenocarcinoma cells against complement-mediated cytolysis.

Growth inhibition of a colonic adenocarcinoma cell line (HT29) by T cells specific for mutant p21 ras

Mutations at codons 12, 13 or 61 of the ras proto-oncogenes are found in adenocarcinomas of the colon and rectum. Mutated ras encode tumor-specific proteins, and can elicit CD4+ HLA-class-II-restricted T cell responses both in mouse and man. The function of such T cells is, however, unclear. In a model system, we investigated whether HLA-class-II restricted CD4+ T cells, specific for a particular peptide derived from mutant p21 ras (Gln61-->Leu), might inhibit the growth of a colonic cancer cell line, when it was cultured in the presence of the corresponding peptide. We found in this case that the growth of the colonic adenocarcinoma cell line HT29, when also induced to express HLA class II molecules by interferon gamma treatment, was inhibited. The inhibition was peptide-specific and required the presence of HLA-DQ8 molecules on the target cell. However, HLA-DQ8-expressing HT29 cells functioned poorly as antigen-presenting cells and could only induce a weak proliferative T cell response in the presence of interleukin-2. The results suggest that colonic cancer cells expressing peptides derived from mutant p21 ras protein in a complex with HLA class II molecules may be a target for tumor-specific T cells. The results also indicate, however, that an initiation of the immune response will require "professional" antigen-presenting cells.

Induction by vasoactive intestinal peptide of interferon alpha/beta synthesis in glial cells but not in neurons.

Vasoactive intestinal peptide (VIP), a 28-amino acid peptide, plays a multifunctional neuromodulatory role in both peripheral and central nervous systems. We have recently reported that VIP induces interferon (IFN) alpha/beta synthesis in human colon adenocarcinoma cell line HT-29. It has been reported that VIP may counteract HIV-induced neuronal cell death; therefore, we postulated that the action of VIP may be mediated by a cascade regulation, involving the production of some cytokines such as IFN. Here we demonstrate that primary cultures of rat mesencephalic neurons and glial cells respond differently to VIP. Thus VIP enhanced 2'5' oligoadenylate (2'5' A) synthetase activity and inhibited vesicular stomatitis virus multiplication in glial cultures only. However, both cell cultures had functional adenylate cyclase coupled receptors for VIP. The increase in 2'5'A synthetase activity in glial cultures reached a maximum with 10(-6) M VIP and required cellular RNA and protein synthesis. Anti-IFN alpha/beta, but not anti-IFN gamma, antibodies abolished the induction of the antiviral and 2'5'A synthetase activities by VIP in rat glial-enriched cultures, suggesting that these inductions were mediated through IFN alpha/beta synthesis. Moreover, VIP or poly (i). poly (C12U) caused, in the glial cultures, the induction and secretion of an IFN of type alpha/beta with a titer value of 16 and 32 units/ml respectively. In contrast, neither of these two substances was able to induce IFN synthesis in neurons, which were, however, sensitive to IFN alpha/beta produced by VIP-treated glial cells. IFN produced by VIP in glial cells may therefore play an important role in defending the brain against viruses.

Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor.

A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR [Neuron, 4 (1990) 847-854]. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.

Interactions of cell-surface galactosyltransferase with immunoglobulins

Detection of the activity of β-1,4-galactosyltransferase (β-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of β-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of β-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface β-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of β-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-β-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn 2+ and UDP-[ 3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[ 3H]-galactose, β-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > α chain > μ chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, β-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the β-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, α-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37°C, the apparent affinity constants and association rate constants of interaction between cell surface β-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and α2 chain or monomeric IgA1 were in the range from 7.1 × 10 7 to 4.6 × 10 8 M −1 and from 1 × 10 5 to 3 × 10 6 M −1 s −1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 × 10 −2 to 5.0 × 10 −4 s −1 and from 33 to 1380 s, respectively. The number of α2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 × 10 5 to 1.3 × 10 6. The binding of IgA by the cell surface β-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.

The influence of type I collagen on the growth and differentiation of the human colonic adenocarcinoma cell line HT-29 in vitro

Abstract HT-29 Human colonic adenocarcinoma cells when grown on a plastic substratum were anaplastic in appearance and failed to express any morphological or biochemical features that were characteristic of intestinal differentiation. Growth of HT-29 cells subcutaneously in the flank of immune deprived mice gave rise to morphologically heterogeneous tumors which were poorly differentiated but contained approximately 11% of cells with an intestinal phenotype: these showed features typical of cell polarization with well-developed microvilli, tight junctional complexes and desmosomes between adjacent cells. The transfer of cells from plastic onto either a fixed (designated ‘non-released’) or floating (designated ‘released’) type I collagen gel induced some morphological features typical of intestinal differentiation; for example goblet-like cells were observed after 9 days, but biochemical markers of differentiation were expressed only modestly. The continued subculture of HT-29 cells on collagen type I gels, which were either attached to the plastic or floating in the medium, induced some morphological features of intestinal differentiation and changes in the activity of brush border-associated enzymes. Alkaline phosphatase activity was enhanced from 1.3×10 -3 μmoles/mg/min for cells cultured on plastic substrata to 2.1 × 10 -3 μmoles/mg/min when gels were non-released, and 2.9 × 10 -3 μmoles/mg/min when gels were released after 12 days of culture. This was confirmed by electron microscopical visualization of alkaline phosphatase activity. Elevated levels of aminopeptidase activity were also observed on day 12 (plastic = 26 milliunits/mg; non-released gel=41 milliunits/mg; released gel = 36 milliunits/mg). Similarly, changes occurred in the secretion of carcinoembryonic antigen from 0.96 × 10 -2 μg/mg/48 hours by cells cultured on plastic to 2.3 × 10 -2 μg/mg/48 hours by cells cultured on floating collagen gels. The effects of permitting HT-29 cells to undergo polarization were tested by culture on inert filter inserts: morphological features of intestinal differentiation were observed although this did not occur until after 21 days. These studies show that optimization of the growth conditions of anaplastic cells in vitro may provide cultures more representative of the tumor in vivo. This model system may be useful for cell biological and pharmacological studies of colon carcinoma.

Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines

Tight junctions (TJ) of the fascia occludens type can be induced in the human colon adenocarcinoma cell lines HT29 and Caco-2 by treatment with 320 mM cesium sulfate. This process can be completely inhibited by the protease inhibitors leupeptin and antipain. The concentration for 50% inhibition was 32 microM leupeptin and 270 microM antipain, respectively. In the polarized colon carcinoma cell line Caco-2, the spontaneous formation of histotypical TJ and the development of transepithelial electrical resistance do not occur when the cells are cultured in medium containing 400 microM leupeptin. Following the removal of leupeptin, zonula occludens type TJ and electrical resistance develop synchronously during a period of 4 h. Dihydroleupeptin, the alcohol analog of leupeptin, inhibits neither the spontaneous nor the induced assembly of TJ fibrils. Thus, the aldehyde group of leupeptin is essential for activity. These data suggest that the salt-induced as well as the spontaneous formation of TJ involve cellular proteases which are susceptible to protease inhibitors.

Cellular differentiation is required for cAMP but not Ca(2+)-dependent Cl- secretion in colonic epithelial cells expressing high levels of cystic fibrosis transmembrane conductance regulator.

The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of response in the unpolarized cells was not due to the inability of forskolin to raise cAMP levels. Neurotensin, a Ca(2+)-mobilizing agonist, stimulated 125I efflux from both cell lines. In the polarized cells, the magnitude of this response was attenuated at 8 days post-seeding. At this time, the undifferentiated line attained some cAMP responsiveness. This latter effect was paralleled by the appearance of monolayers within areas of the multicell layer. Cell-attached patch-clamp recording from apical membrane patches of polarized cells revealed the presence of a forskolin-stimulated 8-pS Cl- channel; no channel activity was observed in forskolin-stimulated unpolarized cells. Ca(2+)-activated Cl- channels were found in both cell lines. In agreement with the 125I efflux data, the single-channel activation response to [Ca2+]i was smaller in the polarized cell line. From these studies, we can conclude that CFTR expression, measured both at the mRNA and protein level, does not correlate with the colonocyte's ability to secrete chloride ions in response to a cAMP-generating agonist. Cyclic AMP-dependent Cl- secretion requires cellular polarization; specifically, the delineation of an apical membrane. Differences in the cellular location of CFTR during differentiation are likely to explain our results. In contrast, Ca(2+)-stimulated Cl- secretion occurred independently of cellular polarization but was reduced when the cells formed tight junctions.

Sorting of sphingolipids in the endocytic pathway of HT29 cells

The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3benzoxadiazol-4-yl)amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBDsphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37°C, both sphingolipid analogues were internalized by endocytosis, but their intracellular site of destination differed . After 30 min of internalization, C6-NBD-glucosylceramide was localized in the Golgi apparatus, as demonstrated by colocalization with fluorescently labeled ceramide, a Golgi complex marker, and by showing that monensininduced disruption of the Golgi structure was paralleled by a similar perturbation of the fluorescence distribution . By contrast, C6-NBD-sphingomyelin does SORTING and recycling are well-known phenomena in intracellular protein trafficking, occurring during biosynthesis and endocytosis (1, 11, 13) . The importance of these kind of processes in the concomitant intracellular flow of lipids is now also gradually emerging . Several sphingolipids have been shown to reappear at the plasma membrane by a recycling mechanism, after exogenous membrane insertion and subsequent internalization via the endocytic pathway (5, 6) . In polarized cells, sorting of two newly synthesized sphingolipids, derived from a common precursor, has been shown to occur, as reflected by lipid specific, outbound trafficking to apical, and basolateral membrane domains (14) . However, it remains to be resolved whether lipids are also subject to sorting during inbound cellular trafficking, i .e., in endocytic events . During endocytosis, an extensive flow ofmembranes takes place . To monitor the trafficking pathways of lipids during this process, advantage can be taken of the intracellular processing of certain ligand-receptor complexes . This processing involves delivery to endosomal compartments, sorting, and either recycling to the plasma membrane or further movement down the endocytic pathway towards the lysosomal system (1, 12) . In the present study, it is shown that two fluorescently laTeresa Babiâs present address is Autonomous University of Barcelona, Department of Biochemistry, Bellaterra, Spain . © The Rockefeller University Press, 0021-9525/91/07/231/9 $2 .00 The Journal of Cell Biology, Volume 114, Number 2, July 1991231-239 not colocalize with the tagged ceramide. Rather, a colocalization with ricin, which is internalized by endocytosis and predominantly reaches the lysosomes, was observed, indicating that the site of delivery of this lipid is restricted to endosomal/lysosomal compartments . Also, in monensin-treated cells no change in the distribution of fluorescence was observed . Thus, these results demonstrate that (sphingo)lipid sorting can occur in the endocytic pathway. Interestingly, the observed sorting phenomenon was specific for glucosylceramide, when compared to other glycolipids, while only undifferentiated HT29 cells displayed the different routing of the two lipids . In differentiated HT29 cells the internalization pathway of sphingomyelin and glucosylceramide was indistinguishable from that of transferrin . beled sphingolipids, C6-NBD-glucosylceramide and C6NBD-sphingomyelin, are sorted during internalization along the endocytic pathway in HT29 cells . Using transferrin (Tf)l and ricin (in conjunction with their receptors) as intracellular traffic markers, evidence is presented indicating that C6-NBD-glucosylceramide is transported to the Golgi apparatus, whereas C6-NBD-sphingomyelin follows the endocytic pathway, down to the endosomal/lysosomal system . Furthermore, when the trafficking ofthese sphingolipids was compared in an undifferentiated and a differentiated HT29 cell type, the latter being derived by clonal selection, it could be shown that this sorting phenomenon only occurred in the undifferentiated cell type . This distinct difference between the two cell types may imply that lipid transport routes and sorting phenomena are related to differentiation of cells . Materials and Methods

Sorting of sphingolipids in the endocytic pathway of HT29 cells.

The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBD-sphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37 degrees C, both sphingolipid analogues were internalized by endocytosis, but their intracellular site of destination differed. After 30 min of internalization, C6-NBD-glucosylceramide was localized in the Golgi apparatus, as demonstrated by colocalization with fluorescently labeled ceramide, a Golgi complex marker, and by showing that monensin-induced disruption of the Golgi structure was paralleled by a similar perturbation of the fluorescence distribution. By contrast, C6-NBD-sphingomyelin does not colocalize with the tagged ceramide. Rather, a colocalization with ricin, which is internalized by endocytosis and predominantly reaches the lysosomes, was observed, indicating that the site of delivery of this lipid is restricted to endosomal/lysosomal compartments. Also, in monensin-treated cells no change in the distribution of fluorescence was observed. Thus, these results demonstrate that (sphingo)lipid sorting can occur in the endocytic pathway. Interestingly, the observed sorting phenomenon was specific for glucosylceramide, when compared to other glycolipids, while only undifferentiated HT29 cells displayed the different routing of the two lipids. In differentiated HT29 cells the internalization pathway of sphingomyelin and glucosylceramide was indistinguishable from that of transferrin.

Production of MUC1 and MUC2 mucins by human tumor cell lines

A mucus secreting, clonal derivative (HT29-SB) of the human colonic adenocarcinoma cell line HT29, and the LS174T colon cancer cell line, secrete mucin into the culture medium as a viscoelastic gel. Mab BC2, which defines a peptide epitope present in the variable number of tandem repeats (VNTR) of the MUC1 core protein, reacted with this material after deglycosylation. Two high molecular weight bands were detected in TFMSA treated gel-formed mucin from HT29-SB and LS174T by western blotting (Mr 580 kDa and 420 kDa). A similar pattern of reactivity was seen with the culture supernatants from HT29-SB, the ovarian tumor cell line COLO-316, and the breast cancer cell line MCF-7. Mab CCP58 (anti-MUC2 VNTR) reacted with a 580 kDa band in gel-formed mucin produced by LS174T, but was not reactive with mucin produced by the other cell lines. The findings indicate that human colonic cell lines, in addition to breast and ovarian cell lines, may both express and secrete the MUC1 protein core, and that the LS174T cell line expresses and secretes both the MUC1 and MUC2 core proteins.

Effect of temperature on the assembly of tight junctions and on the mobility of lipids in membranes of ht29 cells

In the human colon adenocarcinoma cell line HT29, tight junctions can be induced by treatment with appropriate proteases or salt solutions. The temperature dependence of induced tight junction formation is characterized by a marked sigmoidal behavior. The different methods of induction used in this study were characterized by threshold temperatures ranging from 15 to 32 degrees C. Fluorescence photobleaching recovery measurements of the lateral diffusion of a fluorescent phospholipid probe in the cellular plasma membrane gave no evidence for a phase transition or for alteration in the organization of membrane lipids in lateral domains in the temperature range between 0 and 37 degrees C. Moreover, dynamic parameters of the probe in the plasma membrane did not change substantially on mild treatment with trypsin. Thus, the temperature dependence of tight junction formation is not dictated by the bulk properties of the cytoplasmic membrane lipids. The observed temperature dependence suggests that the assembly of tight junctions is a cooperative process, which may involve conformational rearrangement in a protein precursor subsequent to its proteolytic activation.

A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins.

A stably differentiated clonal derivative (Cl.16E) of the human colonic adenocarcinoma cell line HT29 secretes in culture high-Mr glycoproteins that were purified from the serum-free conditioned medium by preparative SDS/polyacrylamide-gel electrophoresis. Analysis of the oligosaccharides released from the [3H]glucosamine-labelled high-Mr glycoproteins by alkaline-borohydride treatment showed that this material consisted of O-linked oligosaccharides (without any detectable N-linked oligosaccharides) that were eluted as three fractions from Bio-Gel P-6 columns. The main oligosaccharide fraction obtained after such treatment and desialylation was eluted together with a six-unit glucose polymer from a Bio-Gel P-4 column. Polyclonal antibodies were raised against the high-Mr glycoproteins, and in immunoblot analysis they reacted specifically with the high-Mr glycoproteins present in the conditioned medium. Furthermore, immunohistochemical staining of sections in paraffin wax revealed that these antibodies labelled normal human gastrointestinal mucins. We conclude that (1) the high-Mr glycoproteins prepared by SDS/polyacrylamide-gel electrophoresis are pure mucus glycoproteins on the basis of sensitivity to alkaline-borohydride treatment, monosaccharide composition and immunochemical and immunohistological findings, and (2) these mucins have antigenic determinants in common with the normal human gastrointestinal mucins.

Effects of Gastrin, Proglumide, and Somatostatin on Growth of Human Colon Cancer

The effects of gastrin, proglumide (a gastrin receptor antagonist), and somatostatin on growth of human colon adenocarcinoma cell lines CX1, X56, and HT29 were examined in two experimental models. Nude mice bearing xenografts of colon cancer CX1 or X56 were treated for 14-25 days subcutaneously with saline, pentagastrin (0.5 or 1.0 mg/kg), proglumide (250 or 500 mg/kg), or somatostatin 14 (33, 100, or 300 micrograms/kg) twice daily. Tumor volume, weight, protein, and deoxyribonucleic acid were measured. HT29 cells were grown in vitro and the effects of gastrin 17, proglumide, and somatostatin on growth were evaluated by cell counts or [3H]thymidine incorporation. The larger dose of pentagastrin significantly increased tumor growth in the nude mouse (p less than 0.005) and gastrin induced a biphasic effect on deoxyribonucleic acid synthesis in tissue culture with significant increases of up to 39% (p less than 0.025). Somatostatin alone significantly inhibited tumor growth in two of the cell lines and also inhibited the gastrin-induced growth. Proglumide had no effect by itself but significantly inhibited gastrin-stimulated growth. These findings suggest that growth of some human colon cancers may be hormone-dependent.

Photoaffinity labelling of the vasoactive-intestinal-peptide-binding site on intact human colonic adenocarcinoma cell line HT29-D4. Synthesis and use of photosensitive vasoactive-intestinal-peptide derivatives.

N-Hydroxysuccinimidyl 4-azidobenzoate, a u.v.-sensitive heterobifunctional reagent, was used to synthesize photoreactive derivatives of the vasoactive intestinal peptide (VIP). Products of the reaction were purified by reverse-phase h.p.l.c. Three 4-azidobenzoyl-VIP (4-AB-VIP) derivatives were able to compete with monoiodinated 125I-VIP with an apparent KD of 2.5, 6.3 and 12.5 nM compared with 0.6 nM for native VIP. H.p.l.c.-purified mono[125I]iodinated VIP was used to synthesize 4-AB-125I-VIP derivatives. They were used to photoaffinity-label the VIP-binding site of HT29-D4 cells, a clone derived from the human colonic adenocarcinoma cell line HT29. Only one polypeptide, of Mr 70,000 +/- 5000 (mean +/- S.D.) was specifically labelled. The Mr of the component thus characterized was slightly higher than that of the major species (Mr 67,000) labelled after cross-linking experiments using 125I-VIP, conventional homobifunctional reagents and HT29 cells. Nevertheless, the specificity and extent of glycosylation of these two components were identical. These new photosensitive VIP derivatives should be useful tools with which to investigate further VIP-receptor structure and metabolism.

Cycloheximide induces accumulation of vasoactive intestinal peptide (VIP) binding sites at the cell surface of a human colonic adenocarcinoma cell line (HT29‐D4)

Incubation of monolayers of HT29-D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 microM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand. The increase in 125I-VIP-binding capacity was dose-dependent between 0.35 microM and 17.5 microM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5-100 microM) a plateau corresponding to a twofold increase in VIP-binding capacity was reached independently of the extent of protein synthesis inhibition. We found that VIP receptors of HT29-D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide). After inactivation of 90% of cell-surface VIP receptors by alpha-chymotrypsin, we observed a biphasic kinetic of reappearance of VIP-binding sites. 40% of VIP-binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide-treated cells. However, the slow recovery was inhibited in cycloheximide-treated cells probably because a pool of immature receptors was depleted by the drug before the alpha-chymotrypsin treatment. Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.

Determination of the HLA-DR profile of an HLA class II negative carcinoma cell line by restriction fragment length polymorphism (RFLP) analysis.

The human colonic adenocarcinoma cell line HT-29 does not normally express HLA class II molecules. By restriction fragment length polymorphism (RFLP) analysis of DNA with a DR beta-probe, we analysed the genomic DR beta polymorphism of this cell-line, and compared it with the RFLP patterns seen in DNA from a reference panel of different DR homozygous and heterozygous cells. The HT-29 cell-line expressed DR beta fragments similar to the sum of the fragments expressed by DR4 and DR7 homozygous cells. The DR4 and DR7 haplotypes of HT-29 was further confirmed by comparing the RFLP patterns of four DR4/7 heterozygotes with that of HT-29. Furthermore, the HT-29 cell line expressed a Hind III 9.3 kb fragment previously found to be strongly associated to DRw53. Following treatment with gamma-interferon, the HT-29 cells could be induced to express class II molecules. Serological typing revealed the presence of the DR4, DR7 and DRw53 antigenic determinants.

Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line HT29

Association of neurotensin to its receptor in HT29 cells increases the intracellular concentration of inositol phosphates. A rapid (20–30 s), transient stimulation of inositol trisphosphate (275% of the basal level) and inositol bisphosphate (420%) is first observed, followed by a slower, stable increase in inositol monophosphate (170%). Half-maximal stimulation of the three inositol phosphates was obtained with 50–100 nM neurotensin. These results indicate that neurotensin is able to regulate intracellular Ca 2+ levels in HT29 cells by using inositol trisphosphate as a second messenger.

Angiogenic activity of human tumor plasma membrane components

Plasma membranes from the human colon adenocarcinoma cell line HT-29 have been isolated and examined for the presence of angiogenic activity. Membrane-associated macromolecules extracted with Triton X-100 were fractionated on immobilized wheat germ agglutinin. The fraction which bound specifically (about 200 ng of protein/mL packed cells) was highly angiogenic when assayed on the chick embryo chorioallantoic membrane. As little as 0.2 ng of this human tumor derived material consistently induced neovascularization. Similarly, 1-2 ng of this material implanted into the rabbit cornea induced new vessel growth (5-8 mm) within 10 days. The plasma membranes of eight other human tumor lines were examined for angiogenic activity. For each, the wheat germ agglutinin bound material induced neovascularization at the low nanogram level. In contrast, the wheat germ agglutinin bound material derived from purified plasma membranes of two normal human diploid fibroblast cell lines failed to induce an angiogenic response on the chick chorioallantoic membrane, even at microgram levels.