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J Fantini

Publications and source records attributed to J Fantini.

At least 73 records · Page 4Linked to original sources
PubMed1992

Insulin like growth factor I is an autocrine regulator of human colon cancer cell differentiation and growth.

The polyanionic compound suramin triggers enterocyte-like differentiation of the human colic adenocarcinoma cell clone HT29-D4. We now demonstrate that suramin interferes with the binding of IGF-I to its receptor at the surface of HT29-D4 cells. Half-maximum inhibition of 125I-IGF-I binding was obtained in the presence of 25 micrograms/ml suramin. Moreover, the drug was able to dissociate 125I-IGF previously bound to its cell surface receptor. Affinity labeling HT29-D4 cells were cultured in the presence of 10 micrograms/ml of alpha-IR3, a monoclonal antibody directed against the binding site of IGF-I, an inhibition of cell proliferation and a stimulation of cell differentiation was observed. After 10 days of treatment with alpha-IR3, HT29-D4 cells formed a regular monolayer of enterocyte-like cells exhibiting an apical brush border and tight junctions delimiting two domains of the plasma membrane (apical and basolateral). Furthermore, we show that IGF-I significantly increased the initial rate of glucose uptake by HT29-D4 cells, while we have previously shown that suramin decreased glucose consumption. From these data we conclude that IGF-I secreted by the cells themselves, stimulates proliferation of HT29-D4 cells via an autocrine mechanism. Blockade of this stimulation by suramin or by a specific monoclonal antibody inhibits cell growth, glucose uptake and triggers the process of enterocytic differentiation.

Biological Transport↗
PubMed1992

Inhibition of human immunodeficiency virus infection in human colon epithelial cells by recombinant interferon-gamma.

Pretreatment of human colon epithelial cells HT29 by recombinant gamma interferon (IFN)-gamma was found to protect the cells from infection with various isolates of human immunodeficiency virus (HIV)-1 and HIV-2, as assessed by co-cultivation with human T lymphoblastoid cells and gene amplification by polymerase chain reaction technique. Additionally, IFN-gamma induced a dose-dependent inhibition of HIV-1 and HIV-2 production in chronically infected HT29 cells. In situ hybridization studies demonstrated that IFN-treated cells were still able to synthesize viral messenger ribonucleic acid. However, the expression of the p24 product of the gag gene was markedly decreased after IFN treatment as demonstrated by radio-immunoprecipitation assay. Taken together, these data suggested that the cytokine acted at the post-translational level by inhibiting the processing of structural viral proteins. It is concluded from this study that IFN-gamma has a potent anti-HIV effect on epithelial gastrointestinal cells.

Cells, Cultured↗
PubMed1992

Short-term suramin treatment followed by the removal of the drug induces terminal differentiation of HT29-D4 cells.

Suramin is an anti-cancer drug which induces the differentiation of the human colon cancer clone HT29-D4. Yet chronic suramin treatment of these cells eventually leads to a marked disturbance of the lysosomal system, which consists in an accumulation of hypertrophied autophagic vacuoles and the occurrence of lamellated inclusion bodies. We report here the effect of a prime treatment of HT29-D4 cells with suramin during various periods of time, followed by the removal of the drug and a subsequent culture in suramin-free medium. A prime treatment of cells in the presence of the drug for 2 days or 4 days was found ineffective to induce the organization of cells into polarized monolayers. On the contrary, a prime treatment of cells for 5 days is sufficient to allow the cellular organization to proceed normally toward a fully polarized monolayer, without any lysosomal damage. The cells did not require the continuous presence of suramin to develop an electrical resistance and a transepithelial potential difference. Moreover the basolateral localization of HLA class I molecules was achieved 9 days after the removal of the drug from the culture medium. Finally prime treatment of cells in the presence of suramin for times longer than 5 days induced the morphological, biochemical, and electrophysiological differentiation of HT29-D4 cells. However, in this case, severe lysosomal disturbances were constantly observed. These data demonstrate that the impaired lysosomal system is a post-differentiation event due to prolonged exposure of the cells to suramin. A metabolic analysis of HT29-D4 cells primed for various times with the drug showed that differentiated cells have a reduced glycolytic activity and this suggests an action of suramin at the level of autocrine growth factors which are known to regulate glucose uptake and degradation.

Cell Count↗
PubMed1992

Structural variability of env and gag gene products from a highly cytopathic strain of HIV-1.

The glycoprotein precursor of the highly cytopathic Zairian virus HIV1-NDK synthesized in CEM leukemic cells displayed a molecular mass of 140 kDa (gp140) as compared to the 160 kDa of gp160 of HIV1-LAV prototype strain. This precursor was cleaved to produce a smaller than prototype extra-cellular envelope glycoprotein (gp100) and a transmembrane component with a usual size (gp41). Immunoprecipitates from tunicamycin-treated infected cells demonstrated the presence of a non-glycosylated precursor of 100 kDa for HIV1-LAV prototype strain and 90 kDa for HIV1-NDK. Digestion of labeled precipitates with a mixture of endoglycosidase F and glycopeptidase F reduced the size of HIV1-LAV gp160 and gp120 to 100 and 60 kDa, respectively, while HIV1-NDK gp140 and gp100, after treatment with the same enzymes, displayed an apparent molecular mass of 90 kDa and 55 kDa, respectively. From these data we conclude that HIV1-LAV gp120 and HIV1-NDK gp100 differ both in their proteic moiety (60 kDa and 55 kDa, respectively) and in their carbohydrate moiety (60 kDa and 45 kDa, respectively). These differences could not be deduced from the available gene sequences of the two viruses. A chimeric virus containing the first 124 amino acid residues of the envelope glycoprotein coded by HIV1-LAV sequence and the rest by HIV1-NDK displayed normal size envelope glycoproteins, demonstrating the involvement of this N-terminal sequence in the alteration of the molecular mass characteristic of HIV1-NDK gp140 and gp100. Finally, characterization of the gag gene products from both strains demonstrated that HIV1-NDK p18 and p15 have a slower electrophoretic mobility as compared to its HIV1-LAV counterparts. Therefore, structural properties of HIV1-NDK env and gag products, reflected by their unusual electrophoretic mobilities, may be responsible for HIV1-NDK biological properties.

Amidohydrolases↗
PubMed1992

Tumor necrosis factor-alpha stimulates both apical and basal production of HIV in polarized human intestinal HT29 cells.

The human colon epithelial cell line HT29 can be infected by selected strains of the human immunodeficiency virus (HIV) [9]. In the present study, it is shown that tumor necrosis factor-alpha (TNF-alpha) is a potent stimulator of HIV replication in chronically infected differentiated HT29 cells, but not in undifferentiated cells. The polarity of HIV production upon TNF-alpha stimulation has been studied in polarized monolayers of differentiated HT29 cells grown on porous-bottomed dishes. It is shown that the cytokine induced a dramatic increase of HIV production through the two opposite sides of the monolayer, i.e. the apical and basolateral plasma membrane domains. The effect of TNF-alpha was mainly localized at the level of viral mRNA synthesis as demonstrated by in situ hybridization. These data support the concept that cytokines released as a result of intestinal inflammatory responses could promote HIV replication and contribute to the gastrointestinal disease in HIV-infected patients.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Human colon epithelial cells productively infected with human immunodeficiency virus show impaired differentiation and altered secretion.

Selected strains of the human immunodeficiency virus (HIV) types 1 and 2 are able to infect human colon epithelial cells in vitro, suggesting a mechanism for the anal route of HIV transmission. In some cases, HIV is not produced by infected colon cells but can be rescued after coculture with T-lymphoid cells. One of the HIV strains (HIV1-NDK) replicated well in colonic cells. A transmission electron microscope study demonstrated two major structural perturbations in producer colon cells: an unusual number of secretion bodies and the appearance of intracellular lumina with disorganized microvilli, indicating a defect in brush border assembly and differentiation. Either abnormality could account for HIV-induced enteropathy consisting of chronic diarrhea and malabsorption in the absence of enteric pathogens. Moreover, HT29 cells infected with HIV provide a unique model for selection of enterotropic HIV strains.

Cell Differentiation↗
PubMed1992

Galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type 1 on human colon epithelial HT29 cells.

The gastrointestinal tract is considered to be a major route of infection for human immunodeficiency virus (HIV). Infection of human colon epithelial cells by HIV is not blocked by anti-CD4 antibodies known to block infection of lymphoid cells (J. Fantini, N. Yahi, and J. C. Chermann, Proc. Natl. Acad. Sci. USA 88:9297-9301, 1991), suggesting the presence of an alternate receptor for HIV on these cells. In this report, we show that (i) a monoclonal antibody specifically directed against galactosyl ceramide inhibited the infection of HT29 cells by two markedly different strains of HIV-1, as assessed by polymerase chain reaction amplification and reverse transcriptase assay; (ii) this antibody strongly labeled the surface of HT29 cells by immunofluorescence and electron microscopic immunolocalization; (iii) the labeling was preferentially but not totally restricted to the basolateral membrane domain of differentiated colonic cells, in agreement with the ability of HIV to infect both the apical and basolateral surfaces of these epithelial cells; and (iv) in thin-layer chromatography-immunostaining experiments with neutral glycolipids prepared from HT29 cells, the antibody specifically reacted with a ceramide monoglycoside fraction corresponding to galactosyl ceramide. We did not detect this glycolipid in lymphoid cells, and anti-galactosyl ceramide antibodies consistently failed to inhibit HIV infection of these cells. These data suggest that galactosyl ceramide (or a derivative) is an essential component of the receptor for HIV on the surface of HT29 cells.

Antibodies, Monoclonal↗
PubMed1992

Replication and apical budding of HIV-1 in mucous-secreting colonic epithelial cells.

The human colonic adenocarcinoma cell line HT29 can be infected with various isolates of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). In some cases, the virus was able to perform its complete cycle of replication as demonstrated by the persistent production of mature viral particles in the cell-free culture supernatant. We have cultured HT29 cells chronically infected with the replicative strain HIV1-NDK in a chemically defined serum-free medium. Under these conditions, the cells were able to maintain a high level of viral replication, as demonstrated by reverse transcriptase activities and in situ hybridization studies. By indirect immunofluorescence labeling and electron microscopy, we observed that serum starvation was associated with the differentiation of HIV-1-infected HT29 cells into mucous-secreting cells resembling epithelial goblet cells of the colonic mucosa. These mucous-secreting cells, which accounted for 50% of the overall population, produced mature particles of HIV through their apical membrane in the vicinity of mucous granules. These data suggest that HIV-infected goblet cells in the colonic mucosa may produce the virus in the colorectal lumen; this could explain the route of transmission of HIV in the case of anal intercourse.

Adenocarcinoma↗
PubMed1992

[Suramin induces lysosomal storage disorder in HT29-D4 cells during a mechanism of polarized endocytosis].

During the course of suramin-induced differentiation, a marked lysosomal storage disorder is observed in HT29-D4. This impairment could account for the toxic side effects of the drug during clinical trials in humans. It is shown that the perturbation is caused by a process of endocytosis of suramin-serum albumin complexes by the apical membrane of differentiated HT29-D4 cells.

Adenocarcinoma↗
PubMed1991

Double screening of suramin derivatives on human colon cancer cells and on neural cells provides new therapeutic agents with reduced toxicity.

Suramin is a polyanionic compound currently used under evaluation for antineoplastic activity. One of the main problems encountered during clinical trials was an adverse neurotoxic effect, probably due to a direct cytotoxic effect on neural cells. Suramin is also known to trigger differentiation of human colon cancer cells, yet a chronic treatment induces a lysosomal storage disorder. The aim of this study was to evaluate suramin analogs for their effect: (i) on the lysosomal system of the human colon cancer cell clone HT29-D4; and (ii) on C6 glioma cell growth and morphology. One of the derivatives tested, NF036, induced terminal differentiation of HT29-D4 cells without any impairment of the lysosomal system. Furthermore, in contrast to suramin, NF036 did not alter C6 cell growth and morphology. We conclude that there is a relationship between the ability of a suramin derivative to induce a lysosomal storage disorder in human colon cancer cells and its neurotoxic effect. A double screening of suramin analogs on HT29-D4 and C6 cells allowed us to identify a new candidate antineoplastic drug: NF036.

Antineoplastic Agents↗
PubMed1991

Kinetics of biochemical, electrophysiological and morphological events (including lysosomal disorder) during the course of suramin-induced differentiation of the human colon-cancer cell clone HT29-D4.

Suramin, a polysulfonated naphtylurea, has been shown to bind to a wide variety of tumor growth factors, and to exhibit anti-proliferative effects on several cell lines. We have followed the suramin-induced (100 micrograms/ml) evolution of morphological, biochemical and electrophysiological changes in HT29-D4 human colonic adenocarcinoma cell clone as a function of culture time. After 5 days of culture in the presence of the drug the cells were polarized and exhibited apical brush border and tight junctions. The polarization process of carcino-embryonic antigen (CEA) in the apical membrane domain was achieved after 8 days of treatment, while the correct localization of HLA class-I molecules in the basolateral membrane domain occurred after 14 days of culture in the presence of suramin. Spontaneous potential difference (PD) and transepithelial resistance (Rt) were recorded from the 9th day of treatment and reached maximum values at day 15 (PD = 3 mV; RT = 450 omega cm2), giving evidence that the differentiation process triggered by suramin concerned virtually all cells in the monolayer. Untreated cells were consistently found to be electrically inactive. Finally, from day 10 of suramin treatment, the lysosomal system was perturbed including accumulation of large autophagic vacuoles and, later, typical lamellar inclusion bodies. These structures were never seen when cells were induced to differentiate in suramin-containing serum-free medium. Moreover, similar perturbations of the lysosomal system could be obtained by adding BSA in the suramin-containing defined medium, suggesting that the lysosomal storage disorder occurring upon suramin treatment was due to endocytosis of suramin-BSA complexes. We conclude that lysosomal impairment due to the presence of BSA in the culture medium did not prevent HT29-D4 cells from differentiating and that it was not an early event which could be involved in the mechanism of action of suramin. However, this perturbation might account for some of the toxic effects occurring during chronic suramin treatment in humans.

Cell Differentiation↗
PubMed1991

Human immunodeficiency virus can infect the apical and basolateral surfaces of human colonic epithelial cells.

The gastrointestinal tract is considered to be a major route of infection for the human immunodeficiency virus (HIV). To understand the interaction of HIV with epithelial cells of the intestinal mucosa, we have studied the infection of a human colon cancer cell clone HT-29-D4. The enterocyte-like differentiation of this clone can be modulated in vitro according to the concentration of glucose. We show that: (i) undifferentiated HT-29-D4 cells can be infected by HIV types 1 and 2 (HIV-1 and HIV-2) strains with no subsequent effect on cell growth; (ii) undifferentiated HT-29-D4 cells express a CD4-related antigen bearing epitopes of the immunoglobulin-like variable (V) region domains V1 and V2 of CD4 but lacking the epitope known to be involved in HIV envelope recognition; (iii) differentiated HT-29-D4 cells can be infected by HIV after an interaction with either the apical brush border membrane (luminal side) or the basolateral side (serosal side); (iv) the CD4-like molecule is restricted to the basolateral domain of differentiated cells; and (v) the infection is not inhibited by anti-CD4 monoclonal antibodies (mAbs) OKT4, OKT4A, Leu-3a, Bl4, 13-B-8-2, S-T4 or S-T40. We conclude that epithelial intestinal cells may represent a major site of entry for HIV. Infection of these epithelial cells may occur via the basolateral membrane by HIV-bearing lymphocytes or macrophages of the lamina propria and via the apical membrane by HIV present in the bowel lumen. This infection may remain silent for up to 9 months, and the virus can be rescued by cocultivation with lymphoid cells. These data may give an explanation for the long latent seronegative state that may occur in a HIV-infected individual.

Adenocarcinoma↗
PubMed1991

Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells. Obtention of clones exhibiting different patterns of enterocytic differentiation.

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.

Cell Differentiation↗
PubMed1991

Selected human immunodeficiency virus replicates preferentially through the basolateral surface of differentiated human colon epithelial cells.

We have used HIV1-NDK-infected HT29 cells grown on permeable substratum to study the polarity of virus maturation in human intestinal cells. When cultured in glucose-containing medium, these cells are mostly undifferentiated. The removal of glucose from the medium allowed the emergence of a selected differentiated subpopulation which continued to produce viral particles in the culture supernatant. The polarity of viral production was evaluated by harvesting virus from the two sides of the monolayer. Seventy-five percent of released HIV was found on the basolateral side of the monolayer. Mature viral particles were observed by electron microscopy near the apical (luminal) and the basolateral (serosal) membrane. These data suggest that epithelial cells of the colon productively infected by a selected strain of HIV are able to produce the virus through both sides of the epithelium but mainly through the serosal side.

Cell Differentiation↗
PubMed1991

Vectorial release of carcinoembryonic antigen induced by IFN-gamma 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-gamma) added only in the basal chamber, suggesting a basolateral restriction for IFN-gamma receptors.

Blotting, Western↗
PubMed1991

Suramin inhibits vasoactive intestinal peptide (VIP) binding and VIP-induced cAMP accumulation into two human cancerous cell lines.

The antihelminthic drug suramin inhibits the binding of monoradioiodinated VIP (125I-VIP) to two human cancerous cell lines, namely HT 29-D4 and IGR 39 derived from a colic adenocarcinoma and a superficial melanoma respectively, with an IC50 of 280 micrograms/ml. The drug is not able to remove 125I-VIP previously bound to either types of cells even with concentration as high as 1500 micrograms/ml. Neither 125I-VIP binding nor VIP binding sites molecular weight are affected by pretreatment of the cells by the drug. Suramin at 1000 micrograms/ml inhibits by 56% to 99% the cAMP accumulation induced by VIP, depending on the VIP concentrations and the cell lines used for the experiments. On the contrary the drug does not have any effects on the cAMP accumulation induced by the beta receptor agonist isoproterenol. Also suramin does not affect the basal accumulation of cAMP in both types of cells either in acute or chronic treatment with the drug. We speculate that these observations may account, at least in part, for the in vivo and in vitro effects of VIP and suramin on cell proliferation and survival.

Cross-Linking Reagents↗