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J Fantini

Publications and source records attributed to J Fantini.

At least 55 records · Page 3Linked to original sources
PubMed1996

Intracellular localisation of suramin, an anticancer drug, in human colon adenocarcinoma cells: a study by quantitative autoradiography.

Suramin is a polysulphonated naphthylurea currently investigated for the treatment of advanced malignancy. In the present study, we have analysed the uptake and the intracellular localisation of tritiated suramin in human colon adenocarcinoma cells (HT-29-D4), using quantitative autoradiographic techniques at the optical and electron microscopy levels. Our results show that the drug is able to enter both undifferentiated and differentiated HT-29-D4 cells. The process of suramin uptake is time-dependent, and significantly inhibited by the presence of the suramin-binding protein serum albumin in the culture medium of HT-29-D4 cells. Autoradiographic analysis revealed two distinct patterns of intracellular localisation of tritiated suramin labelling, according to the presence or absence of serum albumin. Indeed, in the absence of serum albumin, the labelling of free suramin was distributed over the nucleus, the Golgi apparatus and the mitochondria, while it was restricted to the lysosomal system when suramin was complexed with albumin. These data show that a serum factor, i.e. albumin, influences the biological activity of suramin by determining its intracellular localisation. The presence of suramin in a given compartment may account for specific effects of the drug including mitochondrial hypertrophy, altered gene expression and lysosomal perturbation.

Adenocarcinoma↗
PubMed1995

The development of Na(+)-dependent glucose transport during differentiation of an intestinal epithelial cell clone is regulated by protein kinase C.

The sodium-dependent glucose transporter SGLT1 is expressed on the apical plasma membrane of fully differentiated enterocytes. Recently, we have found that the cotransport function appears gradually during the process of differentiation of the human intestinal epithelial cell clone HT-29-D4. However, the SGLT1 protein was detected in both undifferentiated and differentiated HT-29-D4 cells suggesting that sodium-glucose cotransport was dependent on post-translational events controlling the efficient targeting of the protein in the plasma membrane. In the present study, we have analyzed the molecular mechanisms controlling the functional expression of the SGLT1 protein during the course of HT-29-D4 differentiation. We show that the appearance of the cotransport function in the apical membrane is blocked by 1-5-isoquinolinesulfonyl)-2-methylpiperazine-HCl (H-7), a potent inhibitor of protein kinase C activity. Moreover, H-7 treatment was associated with an inability of HT-29-D4 cells to organize into a polarized monolayer of differentiated cells. Reciprocally, short term treatment (15 min) of undifferentiated cells by 0.1 microM phorbol myristyl acetate resulted in the appearance of the cotransport function. In contrast, inhibition of cAMP and cGMP-dependent protein kinases by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl did not prevent the development of sodium-glucose cotransport during the differentiation of HT-29-D4 cells. In addition, stimulation of cAMP-dependent protein kinases by 8-Cl-cAMP did not induce the cotransport function in undifferentiated HT-29-D4 cells. By using immunogold labeling at the electron microscopy level, we demonstrated that phorbol myristyl acetate induced the redistribution of SGLT1 protein from intracellular sites to the plasma membrane. In conclusion, our data show that the appearance of a functional sodium-glucose cotransporter in HT-29-D4 cells is controlled, at least in part, by intracellular pathways regulated by the activity of protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Antigens, CD↗
PubMed1995

Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line.

In this study, we have characterized the Na/glucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT-29-D4. Isotopic tracer flux measurements show that differentiated HT-29-D4 cells possess a sodium-dependent alpha-methyl-D-glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D-glucose, D-galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium-dependent AMG transporter with a Km of 1.2 +/- 0.12 mM and a Vmax of 3.24 +/- 0.25 nmol/mg of protein per min. Hill analysis reveals a coefficient of 1.9 +/- 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402-Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT-29-D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT-29-D4 cells express the sodium/glucose cotransporter SGLT1 at their apical membrane and that this transporter generates the basal transepithelial potential difference.

Blotting, Western↗
PubMed1995

Production of a highly cytopathic HIV-1 isolate from a human mucosal epithelial cell line cultured on microcarrier beads in serum-free medium.

The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins.

Adenocarcinoma↗
PubMed1995

Intracellular calcium release induced by human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein in human intestinal epithelial cells: a putative mechanism for HIV-1 enteropathy.

Intracellular Ca2+ ([Ca2+]i) was measured in single human epithelial intestinal HT-29-D4 cells with the Ca2+ probe Fura-2 and digital imaging microscopy. Treatment of these cells with HIV-1 surface envelope glycoprotein gp120 (or a soluble form of its precursor gp160) induced an important increase of [Ca2+]i. This effect was abolished by preincubation of the viral glycoprotein with neutralizing antibodies specific for the V3 domain of gp120. These antibodies inhibited the binding of both gp120 and gp160 to galactosylceramide (GalCer), the alternative HIV-1 receptor in HT-29-D4 cells. Moreover, treatment of HT-29-D4 cells with an anti-GalCer mAb induced an increase in [Ca2+]i and rendered the cells insensitive to HIV-1 glycoprotein stimulation. The calcium response resulted from release of Ca2+ from caffeine-sensitive intracellular stores. Finally, the viral glycoprotein specifically abrogated the calcium response to the neuropeptide agonist neurotensin, a stimulator of chloride secretion via inositol trisphosphate-mediated calcium mobilization. Reciprocally, after neurotensin stimulation, the cells did not respond to gp120, showing that neurotensin and gp120 stimulate a common pathway of [Ca2+]i mobilization. These results suggest that HIV-1 may directly alter ion secretion in the intestine and thus be the causative agent of the watery diarrhea associated with HIV-1 infection.

Calcium↗
PubMed1995

Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4-negative mucosal epithelial cell line.

The glycosphingolipid galactosylceramide (GalCer), which binds gp120 with high affinity and specificity, is a potential alternative receptor for human immunodeficiency virus type 1 (HIV-1) in some CD4-negative neural and epithelial human cells, including the human colonic epithelial cell line HT-29. In the present study, we demonstrate that synthetic multibranched peptides derived from the consensus sequence of the HIV-1 V3 loop block HIV-1 infection in HT-29 cells. The most active peptide was an eight-branched multimer of the motif Gly-Pro-Gly-Arg-Ala-Phe which at a concentration of 1.8 microM induced a 50% inhibition of HIV-1 infection in competition experiments. This peptide was not toxic to HT-29 cells, and preincubation with HIV-1 did not affect viral infectivity, indicating that the antiviral activity was not due to a nonspecific virucidal effect. Using a high-performance thin-layer chromatography binding assay, we found that multibranched V3 peptides recognized GalCer and inhibited binding of recombinant gp120 to the glycosphingolipid. In addition, these peptides abolished the binding of an anti-GalCer monoclonal antibody to GalCer on the surface of live HT-29 cells. These data provide additional evidence that the V3 loop is involved in the binding of gp120 to the GalCer receptor and show that multibranched V3 peptides are potent inhibitors of the GalCer-dependent pathway of HIV-1 infection in CD4-negative mucosal epithelial cells.

Amino Acid Sequence↗
PubMed1994

Interferon-gamma 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 gamma-interferon (rlFN gamma) induced a dose-dependent inhibition of GalCer expression on the cell surface, as demonstrated by indirect immunofluorescence and by enzymatic labeling of cell surface glycoconjugates with oxidase-tritiated sodium borohydride. The rIFN gamma 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, rIFN gamma did not alter cell surface expression of CD4, the classical HIV receptor, in HT-29-A7 cells that had been transduced with a retroviral vector expressing full-length CD4, and there was no effect on their infection. These results strongly suggest that rIFN gamma 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 rIFN gamma which should be exploited for antiviral therapeutics.

Colon↗
PubMed1994

Suramin inhibits binding of the V3 region of HIV-1 envelope glycoprotein gp120 to galactosylceramide, the receptor for HIV-1 gp120 on human colon epithelial cells.

The infection of human colonic epithelial cells HT-29 by human immunodeficiency virus type 1 (HIV-1) occurs independently of CD4, the main HIV-1 receptor expressed on lymphocytes and macrophages. Recent studies from our group have shown that HT-29 cells express the glycosphingolipid galactosylceramide (GalCer), a potential alternative receptor for the HIV-1 envelope glycoprotein gp120. The binding of recombinant gp120 to GalCer was blocked by monoclonal antibodies directed against the third variable region (V3) of gp120, suggesting that the V3 domain was implicated in GalCer recognition. In the present report, we show that suramin, a polysulfonyl naphtylurea known to inhibit retroviral reverse transcriptases in vitro, blocks HIV-1 infection in HT-29 cells. The effect is dose dependent, with a half-maximal inhibition (IC50) achieved for a suramin concentration of 54 micrograms/ml. Since [3H]suramin was not significantly internalized into HT-29 cells during our infection assay (i.e. 2 h), we have considered the possibility that the drug could act at an extracellular step of the HIV-1 cycle. Using a high performance thin layer chromatography binding assay, we show that suramin inhibits binding of HIV-1 gp120 to purified GalCer with an IC50 of 25 micrograms/ml. Suramin does not bind to GalCer, since preincubation of GalCer with suramin did not prevent the subsequent attachment of gp120. Using a solid-phase assay, we show that [3H]suramin specifically binds to recombinant gp120 and that this binding could be blocked by a monoclonal antibody specific for the conserved GPGRAF motif of the V3 domain of gp120. We also demonstrate that [3H]suramin binds to multibranched synthetic GPGRAF peptides that block HIV-1 infection in HT-29 cells. Binding of [3H]suramin to V3 peptides is specific and inhibited by unlabeled suramin (IC50 of 28 micrograms/ml). In contrast, the suramin derivative NF036, that is unable to block HIV-1 infection in HT-29 cells, does not inhibit the binding of [3H]suramin to V3 peptides. Taken together, these results suggest that suramin blocks HIV-1 infection in HT-29 cells because it binds to the V3 domain of gp120 and hence prevents the interaction between gp120 and the GalCer receptor.

Amino Acid Sequence↗
PubMed1994

Binding of human immunodeficiency virus type I (HIV-1) gp120 to galactosylceramide (GalCer): relationship to the V3 loop.

The primary receptor for the human immunodeficiency virus (HIV) is the CD4 molecule. However, a large body of evidence has demonstrated that some cells that do not express the CD4 receptor can be infected by HIV-1 and HIV-2, indicating that an alternative mechanism of infection must exist for some cell types. Recently it was reported that antibodies against the glycosphingolipid, galactosylceramide (Gal beta 1-1'Cer;GalCer), blocked infection of several CD4 negative cell lines derived from the brain and colon. The hypothesis that GalCer might be involved in the process of HIV entry into these cells was further supported by the finding that recombinant gp120 bound GalCer with high affinity in a high performance thin layer chromatography (HPTLC) binding assay. We have examined the interactions between GalCer and gp120, and found that the oligosaccharides that constitute a large proportion of the molecular mass of this glycoprotein are not involved in binding to this glycolipid. Furthermore, using a panel of monoclonal and monospecific antibodies we have determined that gp120 binds GalCer, and the related molecule 3' sulfo galactosylceramide (sulfatide), at a site that is conformationally close to the its principal neutralizing domain (V3 loop) or at the V3 loop itself.

Animals↗
PubMed1994

Physical contact with lymphocytes is required for reactivation of dormant HIV-1 in colonic epithelial cells: involvement of the HIV-1 LTR.

HIV-1 transmission from mucosal epithelial cells to lymphocytes is a potential mechanism of HIV-1 contamination during sexual intercourse. The human colon epithelial cell line HT-29, that is infectable by various HIV-1 strains, is a useful model for studying the molecular mechanisms involved in this process. In the present study, we show that HT-29 cells, when exposed to either HIV-1(LAI) or HIV-1(NDK) at a low multiplicity of infection, became infected but did not produce infectious virions. Using two-compartment cell culture chambers separated by a porous membrane, we showed that PBL were able to rescue infectious HIV-1 from latently infected HT-29 cells following a physical interaction between the two cell populations. In contrast, HT-29 cells, infected with the same viruses at a high multiplicity of infection, were able to produce mature viral particles that were infectious to PBL in absence of cellular contacts. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that cell-to-cell contact induced an activation of the HIV-1 promoter. These observations provide a putative molecular mechanism for transmission of HIV-1 from mucosal epithelial cells to lymphocytes.

Cell Communication↗
PubMed1994

Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages.

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.

Amino Acid Sequence↗
PubMed1993

Autoradiographic localization of tritiated suramin in polarized human colon adenocarcinoma cells.

In this report we demonstrated that [3H]suramin enters polarized human colon adenocarcinoma cells when added to the apical side of the monolayer. Using light microscopic quantitative autoradiography, we showed that suramin was accumulated in the apical cytoplasm and in the nucleus. In contrast, a weak labeling was noted in other compartments such as the basolateral cytoplasm and the intercellular space. The accumulation of suramin in the apical region of the cells is consistent with previous data showing that suramin elicited a lysosomal storage disorder in HT29-D4 cells by a mechanism of polarized endocytosis.

Adenocarcinoma↗
PubMed1993

Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor.

The gastrointestinal tract plays a major role in the pathogenesis and pathophysiology of infection by the type 1 human immunodeficiency virus (HIV-1). It is a potential route for viral entry and it is the site of a number of complications, including both opportunistic infections and a primary HIV-induced enteropathy. Correspondingly, both in vivo and in vitro studies have demonstrated HIV infection of gastrointestinal cells of lymphoid and epithelial origin. HT-29, a human colonic epithelial cell line that is infectable with many HIV-1 strains, does not express CD4 protein or mRNA. Recent studies showed that antibodies recognizing a neutral glycolipid related to galactosylceramide (GalCer) in HT-29 cells inhibited HIV-1 infection of this cell line, extending previous findings in neural cells. In the current studies, we further analyzed the neutral glycolipids of HT-29 cells and showed that they contained authentic GalCer and that recombinant gp120 bound to this glycolipid. Moreover, by analyzing GalCer expression in clones derived from HT-29 and Caco-2 (another human colonic cell line), we observed that the level of expression of this glycolipid was associated with the sensitivity to HIV-1 infection. Subclones of Caco-2 did not express GalCer and were not infectable with any of three HIV-1 strains. These results strengthen the possibility that GalCer is an alternative receptor in CD4- cell lines. Furthermore, since GalCer is a major glycolipid in epithelial cells of the small intestine and colon, these results provide a structural basis for the binding of HIV-1 by gastrointestinal epithelial cells and the entry of the virus into those cells.

Adenocarcinoma↗
PubMed1993

Polarized distribution of gamma interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells).

The characteristics of a polarized epithelial cell line and dynamics of an endogenous polarized plasma membrane constituent were studied by use of an subclone, FRT-9, from the Fisher rat thyroid cell line, FRT. Transmission electron microscopy (conventional, freeze-fracture), determination of transepithelial electrical parameters and immuno-fluorescence study, were used to establish polarity and demonstrated the basolateral distribution of transferrin receptors and the major histocompatibility complex antigens (constitutive Class I or gamma interferon-induced Class II).

Animals↗
PubMed1993

Multi-branched peptides based on the HIV-1 V3 loop consensus motif inhibit HIV-1 and HIV-2 infection in CD4+ and CD4- cells.

The V3 loop is an hypervariable region of the HIV-1 surface envelope glycoprotein gp120 that is able to generate neutralizing antibodies. These antibodies are generally type-specific. They inhibit the interaction between the V3 loop and the membrane molecules involved in virus-cell and cell-cell fusion. Synthetic peptides based on the V3 loop sequence have been tentatively used to block the fusion process, but the results were unsuccessful. In this report, we confirm that monomeric V3 peptides are devoid of any anti-HIV activity. However, we found that, when assembled in a synthetic polymeric construction (SPC), these peptides have been able to inhibit the infection of human CD4+ lymphocytes and macrophages as well as CD4- epithelial intestinal cells by distantly related isolates of HIV-1 and HIV-2. V3 SPCs, based on the consensus HIV-1 sequence, may therefore represent a new class of therapeutically useful anti-HIV agents able to neutralize a wide range of viral isolates in both CD4+ and CD4- susceptible cells.

CD4 Antigens↗