Biomedical subjects

J Luis

Publications and source records attributed to J Luis.

At least 19 recordsLinked to original sources

PubMed2002

Prevention of cytokine-induced apoptosis by insulin-like growth factor-I is independent of cell adhesion molecules in HT29-D4 colon carcinoma cells-evidence for a NF-kappaB-dependent survival mechanism.

We have previously established that insulin-like growth factor (IGF)-I, -II and insulin exert a strong protective effect against tumor necrosis factor-alpha (TNF)-induced apoptosis in interferon-gamma (IFN)-sensitized HT29-D4 human colon carcinoma cells. In this study, we report that this effect was still operative when cells were cultured in the absence of integrin- and E-cadherin-mediated cell-extracellular matrix and cell-cell interactions. In this model, IGF-I did not activate the focal adhesion kinase, whereas it induced tyrosine phosphorylation of the insulin receptor substrate-1 and activation of the extracellular signal-related kinase 1 and 2, p38, phosphatidylinositol 3'-kinase and protein kinase B/Akt. However, the use of specific inhibitors indicated that these pathways did not play a role in the adhesion-independent IGF-I anti-apoptotic signal. In contrast, inhibition of the NF-kappaB activation induced a complete reversal of the IGF-I anchorage-independent protective effect. Correspondingly, IGF-I markedly enhanced the TNF- and IFN/TNF-induced NF-kappaB-dependent interleukin-8 production. Our results provide evidence that IGF-I induces resistance against cytokine-induced cell death even in the absence of cell adhesion-mediated signaling. NF-kappaB appears to be a key mediator of this anti-apoptotic effect that should contribute to the resistance of colon cancer cells to immune-destruction during metastasis.

Apoptosis↗

PubMed2001

Inhibition of proprotein convertases is associated with loss of growth and tumorigenicity of HT-29 human colon carcinoma cells: importance of insulin-like growth factor-1 (IGF-1) receptor processing in IGF-1-mediated functions.

Proprotein convertases (PCs) of the subtilisin/kexin family are responsible for the activation of prohormones, protrophic factors, and their receptors. We sought to determine whether loss of PC-mediated activities might affect the malignant phenotypes of cancer cells. Stable transfectants of alpha(1)-antitrypsin Portland (alpha(1)-PDX) cDNA, coding for a potent PC inhibitor, were analyzed in model HT-29 cells (HT-29/PDX) and in other cell lines. Expression of alpha(1)-PDX resulted in a proinsulin-like growth factor-1 receptor (pro-IGF-1R) processing blockade, hence inhibiting the ability of exogenous IGF-1 to induce tyrosine phosphorylation of its beta-subunit and insulin-related substrate-1. Coexpression of IGF-1R with four different PCs or the novel convertase SKI-1 in the furin-defective LoVo-C5 cells demonstrated that pro-IGF-1R ( approximately 200 kDa) cleavage into IGF-1R (beta-subunit, approximately 105 kDa) can be achieved by furin and PC5A, but not by PACE4, PC7, or SKI-1. Expression of alpha(1)-PDX resulted in reduction of DNA synthesis and in anchorage-independent growth. Following serum deprivation, the alpha(1)-PDX transfectants exhibited an enhanced apoptotic phenotype and were insensitive to IGF-1-mediated [(3)H]thymidine incorporation and protection against apoptosis. These cells showed reduced invasiveness that paralleled decreased mRNA levels of urokinase-type plasminogen activator and its receptor, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. Comparative subcutaneous inoculation of cells in nude mice revealed that animals injected with HT-29/PDX cells exhibited delayed and lower incidence of tumor development as well as reduced tumor size. Immunohistochemical analysis of CD31 antigen expression, a marker of endothelial cells, revealed reduced HT-29/PDX tumor vascularization. These findings indicate that PCs actively contribute to the growth and malignant phenotypes of HT-29 tumors, suggesting that PC inhibition strategies may be a useful adduct to the arsenal of colorectal anticancer gene therapies.

Animals↗

PubMed2000

Role of endoproteolytic processing in the adhesive and signaling functions of alphavbeta5 integrin.

Some integrin alpha subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation belong to the subtilisin-like endoprotease family (convertases). To understand the role of the alpha subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing alpha(1)-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that alpha(3), alpha(6) and alpha(v) integrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the alpha(v)beta(5) integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by alpha(v) integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of alpha(v) integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion.

Animals↗

PubMed2000

Endoproteolytic processing of integrin pro-alpha subunits involves the redundant function of furin and proprotein convertase (PC) 5A, but not paired basic amino acid converting enzyme (PACE) 4, PC5B or PC7.

Several integrin alpha subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-alpha5, alpha6 and alphav by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-alpha subunit substrates in vitro; the defective pro-alpha chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-alpha subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin alpha-subunit precursors. In conclusion, these results indicate that integrin alpha-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.

Amino Acid Motifs↗

PubMed2000

Increased prevalence of polycystic kidney disease type 2 among elderly polycystic patients.

Autosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous, with at least three chromosomal loci (PKD1, PKD2, and PKD3) accounting for the disease. Mutations in the PKD2 gene, on the long arm of chromosome 4, are estimated to be responsible for 15% of the cases of ADPKD, based on linkage studies. PKD2 is a milder form of the disease, with a mean age of end-stage renal disease (ESRD) approximately 20 years later than PKD1. The object of this study is to determine the proportion of elderly patients with ADPKD with ESRD who harbor mutations in the PKD2 gene. We analyzed all exons and intron-exon boundaries of the PKD2 gene by single-strand conformation polymorphism analysis and silver staining technique in 46 patients with ADPKD who reached ESRD after the age of 63 years or were not yet undergoing renal replacement therapy (RRT) by that age. We performed exactly the same studies in a control group of 40 patients with ADPKD with unknown gene status aged younger than 63 years. In 22 patients, a mutation in the PKD2 gene was defined: 18 of 46 patients from the elderly group and 4 of 40 patients from the control group. We identified 14 different mutations: 4 nonsense mutations, 1 missense mutation, 5 small deletions, 2 insertions, 1 deletion of the whole PKD2 gene, and 1 splicing mutation. Five of these mutations previously were described by our group. Three of the mutations reported in the present study are recurrent. The prevalence of PKD2 disease among elderly patients with ADPKD undergoing RRT is 39.1%, almost three times the prevalence of the disease in the general ADPKD population.

Adult↗

PubMed1999

Integrins and E-cadherin cooperate with IGF-I to induce migration of epithelial colonic cells.

Although the detailed mechanisms of cell migration remain largely unknown, it is now clear that growth factors and cell adhesion molecules are crucial for this process. We have shown that type I insulin-like growth factor (IGF-I) promotes migration of human colonic tumour cells. Since morphological analysis suggested an involvement of adhesion molecules, we have now examined the role of integrins (cell-matrix adhesion molecules) and E-cadherin/catenins complex (cell-cell adhesion molecules) in the IGF-I-induced migration. Using a monolayer wounding assay, we have determined that, except for alpha2beta1, all of the integrins expressed in HT29-D4 cells are involved in the induced cell migration. Immunofluorescence studies revealed that upon IGF-I stimulation the integrins reorganized at the leading edge of migrating cells. We also demonstrate that E-cadherin is involved in cell migration. A rapid tyrosine phosphorylation of E-cadherin and beta-catenin was detected upon IGF-I stimulation. Tyrosine phosphorylation was associated with reduced membranous expression of E-cadherin and promotion of cell motility, suggesting a regulation of the E-cadherin/catenins complex. This effect can be reversed by incubating cells with tyrosine kinase inhibitors. Taken together, our results suggest that IGF-I promotes colonic cell migration through reorganization of integrin receptors and through modulation of E-cadherin/catenins complex function.

Antibodies, Monoclonal↗

PubMed1999

The RGD motif and the C-terminal segment of proprotein convertase 1 are critical for its cellular trafficking but not for its intracellular binding to integrin alpha5beta1.

Cellular trafficking of subtilisin/kexin-like precursor convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin alpha5beta1 associates primarily with the zymogens proPC1, proPC1-DeltaC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as alpha5beta1.

Amino Acid Sequence↗

PubMed1999

Protein kinases C-gamma and -delta are involved in insulin-like growth factor I-induced migration of colonic epithelial cells.

BACKGROUND & AIMS: The mechanisms by which epithelial cells migrate during the repair of damaged colonic mucosa are poorly understood. This study investigated the insulin-like growth factor I (IGF-I) signaling pathway leading to HT29-D4 human colonic epithelial cell line migration. METHODS: IGF-stimulated cell migration was determined using a wound model in the presence or absence of kinase inhibitors. Activation of protein kinase C (PKC) was determined by immunodetection. RESULTS: IGF-I and insulin induce cell migration without affecting cell proliferation through their cognate receptors. Des(1-3)-IGF-I, a truncated analogue of IGF-I, was more potent than IGF-I, suggesting that IGF-binding proteins secreted in the medium modulated IGF-I-induced cell migration. Inhibition of phosphatidylinositol 3-kinase, PKC, and mitogen-activated protein kinases eliminated cell restitution. Long-term exposure of cells to phorbol myristate acetate caused the depletion of PKC-delta and -gamma and prevented also IGF-I-induced cell motility. IGF-I also induced activation of PKC-delta and -gamma only. CONCLUSIONS: IGF-I stimulates colonic restitution through the activation of multiple signaling pathways including activation of phosphatidylinositol 3-kinase, PKC-delta and -gamma, and mitogen-activated protein kinases.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Biogenesis of alpha6beta4 integrin in a human colonic adenocarcinoma cell line involvement of calnexin.

The heterodimer alpha6beta4 is a major integrin and the main laminin receptor in epithelia. The alpha6 integrin subunit is proteolytically cleaved, probably by furin, and glycosylated during its biosynthesis. In the present work, we have investigated the kinetics of the assembly process of alpha6beta4 heterodimers in the colonic adenocarcinoma cell line HT29-D4. We demonstrate that the association of alpha6 and beta4 precursors occurs within the ER, while the endoproteolytic cleavage of pro-alpha6 occurs later, probably in the trans-Golgi network. When pro-alpha6 was blocked within the ER by treatment with brefeldin A, its maturation processing was completely prevented without any consequence on its association with beta4 subunit. Low temperature (20 degrees C) also blocked pro-alpha6 maturation, like brefeldin A, but in addition impaired the integrin assembly. Calnexin, an ER resident protein chaperone, was found to be associated with both the alpha6 and beta4 subunit precursors. Our data suggest that calnexin might be responsible for the prolonged retention of pro-alpha6 within the ER compartment and for the defect of integrin subunit association observed at low temperature.

Adenocarcinoma↗

PubMed1998

Functional reconstitution of membrane glycoproteins into lipid vesicles using lectin precipitation. Application to the VIP receptor.

We studied the interaction of n-octyl-beta-d-glucopyranoside-solubilized VIP receptors (VIPR) with wheat germ agglutinin and found that the addition of the lectin to the detergent extract led to the formation of aggregates that could be pelleted by high speed centrifugation. Resuspension of the pellet in the presence of the competing trisaccharide, N,N', N"-triacetylchitotriose (TAC), dissociated the lectin from the complex without altering the precipitability of VIPR. The final pellet (referred to as TAC pellet) contained an average of 12% of total protein and 96% of total VIP binding activity with a typical rank order of potency for VIP-related peptides. Lipid analysis and electron microscopic examination indicated that the precipitated material was composed of lipid vesicles. VIPR molecules were shown to be integrally inserted in the liposomes because they could not be dissociated from the vesicles at pH 11 or with high salt concentration. By comparing the liposome-associated VIP binding activity in the presence and absence of detergent and by showing accessibility of VIPR to PNGase F, it was concluded that VIP binding sites were not simply trapped within the reconstituted vesicles but likely exposed at the external surface of the liposomes.

Amidohydrolases↗

PubMed1998

Effect of microtubule disruption on cell adhesion and spreading.

Microtubules have been involved in a variety of cellular processes. In this study, we examined the role of the microtubular system in the adhesion and spreading of the adenocarcinoma cell line HT29-D4. Disruption of microtubules by nocodazole or navelbine resulted in an increase in cell adhesion to purified ECM proteins. This enhanced cell adhesion is mediated by integrins, but is not attributable to quantitative changes in the number of integrin receptors at the cell surface, as determined by flow cytometric analysis. In contrast to attachment, spreading of HT29-D4 cells was reduced by nocodazole treatment in a dose-dependent manner. Thus, microtubule depolymerization appears to increase initial attachment of cells to extracellular matrix, while impeding subsequent cell spreading.

Adenocarcinoma↗

PubMed1998

Convergent effects of growth factors, hormones, and fibronectin are necessary for the enterocyte differentiation of a colon adenocarcinoma cell line (HT29-D4).

The aim of this work was to show in serum-free medium a convergent effect of physiological factors and extracellular matrix proteins on the differentiation process of enterocytes by taking as a model the HT29-D4 clone that has the feature of differentiating when subcultured in fetal bovine serum glucose-free medium. We show that triiodothyronine (T3) as well as insulin promotes limited cell growth and differentiation, whereas fibronectin or bovine serum albumin (BSA) induces cell growth and a low level of differentiation. However, insulin, T3, fibronectin, and BSA together with epidermal growth factor and transferrin promoted satisfactory growth and enterocyte morphology with epithelial electrophysiological properties in HT29-D4 cells. With these factors adequate protein targeting was achieved since cells apically expressed the carcinoembryonic antigen, and basolaterally transferrin and insulin receptors, beta 1 and alpha v beta 6 integrins, talin, vinculin, and focal adhesion kinase (FAK). Talin, vinculin, FAK, and alpha v beta 6 integrin, the fibronectin receptor, were clustered in focal contacts, which agrees with a possible role of fibronectin in final cell growth, the latter process mediating the final phase of differentiation. This level of differentiation can be maintained for a long time. Thus HT29-D4 cells appear to be a suitable model to study the implication of integrins in the differentiation process of human enterocytes.

Adenocarcinoma↗

PubMed1998

Integrin ligation and PKC activation are required for migration of colon carcinoma cells.

The activation of protein kinases C (PKCs) is an essential step in integrin-dependent cell adhesion and spreading. In this report we examined the effect of the phorbol ester PMA, a PKC activator, on adhesion, spreading and migration of a colon carcinoma cell line, HT29-D4. Treatment with PMA increased the rate of cell spreading and induced the migration of these cells towards purified matrix proteins in haptotaxis assays on Boyden chambers. PMA-induced effects were the result of PKCs activation, as shown by using the inactive isomer 4alpha-PMA and PKCs inhibitors. The involvement of integrins in the phorbol ester-induced cell migration was demonstrated both by the absence of migration of cells plated on membranes coated with poly-L-lysine and by the use of function blocking antibodies. Thus, interactions between alpha 2beta1, alpha3beta1, alpha6beta4, alpha vbeta5, alphavbeta6 integrins and their specific ligands are necessary for the PKC-mediated migration. However, adhesion, immunoprecipitation and immunocytofluorometry experiments clearly showed that HT29-D4 cell haptotaxis induced by PKC activation is not a consequence of quantitative or qualitative changes in the cell surface integrins. We also demonstrated that PKCs were able to activate the MAP kinase pathway and that the impediment of MAP kinase activation resulted in the loss of cell migration. Moreover, stimulation of the insulin-like growth factor I signalling pathway led to MAP kinase activation and to the induction of cell migration. In addition, the growth factor-induced motility of HT29-D4 cells was affected both by PKC and MAP kinase cascade inhibitors. It thus appears that both integrin ligation and MAP kinase activation by PKCs are required to promote the migration of HT29-D4 cells.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Activation of transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat by the vasoactive intestinal peptide (VIP).

The ability of the human immunodeficiency virus type 1 (HIV-1) to persist and replicate in human CD4+ cells is under the control of both virally encoded proteins and a variety of host-related factors. Transcription of human immunodeficienty virus type 1 (HIV-1) is regulated by multiple cis-acting regulatory elements located in the viral long terminal repeats (LTRs). Here we report that the human vasoactive intestinal peptide (VIP) can activate the HIV-1-LTR. The activity of VIP is dose-dependent and specific. The effective concentration of VIP is within the physiological range suggesting that VIP release may modulate the activity of HIV-1 in vivo. A sequence in U3 region could be involved in VIP and in various VIP-related peptides' activation of HIV-1-LTR. This cis-acting element conferring VIP responsiveness is indistinguishable from HIV NF-kappa B tandem repeat binding sites (HIV-1 kappa B).

Enhancer Elements, Genetic↗

PubMed1996

[Aggressive behavior of the male volcano mouse Neotomodon alstoni (Rodentia: Cricetidae)].

A study was carried out on the agonistic behavior of the male volcano mouse, Neotomodon alstoni, with 50 pairs of males which were classified as possible dominants (D) and subordinates (S), utilizing Melzack-Thompson's Method. The aggressiveness levels exhibited by this mouse were recording in the combinations: D vs. D and S vs. S. Two groups were formed: Group I with 12 pairs of D males and 13 of S males, and Group II with 11 pairs of D males and 14 of S males. In Group I the aggressiveness level was quantified after one week of mating and after another week of isolation, and in Group II the sequence of observation was inverted. The aggressiveness level was measured by the number of attacks per hour, an attack being defined as the aggressive physical contac of an animal (aggressor) with another (attacked). The kinds of behavior registered, including offense, defense, and submission patterns, revealed hierarchic relationships. Dominance was correlated significatively (p < 0.05) with a higher level of aggressiveness.

Agonistic Behavior↗

PubMed1994

A receptor for VIP-related peptides with an unusual selectivity profile on the melanoma cell line IGR37.

A receptor capable of recognizing VIP-related peptides with unusual functional characteristics and selectivity profile was characterized on human IGR37 melanoma cells. When using either [125I]VIP or [125I]N-AcPACAP27 as a tracer, PACAP38 had the highest affinity, while PACAP27, VIP, helodermin, GRF and the VIP fragment VIP10-28 showed the same low affinity. Moreover, this receptor did not recognize PHM, PHV, helospectin, secretin, GIP, glucagon and glucagon-like peptide-1(7-36). Surprisingly, none of the peptides significantly stimulated the cAMP production. By covalent crosslinking, the receptor was shown to have a M(r) = 60,500.

Binding, Competitive↗

PubMed1994

Surface distribution of the EGF receptor during differentiation of the human colon carcinoma cell line HT29-D4.

The clone HT29-D4 can be induced to differentiate into enterocyte-like cells, by simply removing glucose from culture medium. In this report, we used the HT29-D4 model to study the membrane segregation of the EGF receptor on epithelial intestinal cells. Differentiated and undifferentiated cells displayed a single class of EGF binding sites with similar dissociation constants. However, differentiation of HT29-D4 led to a 3-fold decrease in the total number of EGF binding sites, while the number of IGF-I binding sites was unchanged. Fifteen percent of EGF receptors present on differentiated HT29-D4 cells were localized in the apical surface, whereas 98% of IGF-I receptors were segregated to the basolateral domain. By covalent cross-linking experiments using 125I-EGF and by immunoprecipitation with an anti-EGF receptor antibody, we have characterized the HT29-D4 EGF receptor as a Mr = 165,000 protein in both differentiated and undifferentiated cells. Apical EGF receptors were functional, as evidenced by their ability to be internalized in response to EGF binding. Thus, intact and functional EGF receptors are present at the apical surface of differentiated HT29-D4 cells, suggesting the presence of EGF receptors on the apical domain of enterocytes.

Adenocarcinoma↗

PubMed1993

Structural and functional analysis of the human vasoactive intestinal peptide receptor glycosylation. Alteration of receptor function by wheat germ agglutinin.

The vasoactive intestinal peptide (VIP) receptor from the human melanoma cell line IGR39 has been shown to be a 60-kDa glycoprotein. Using serial lectin affinity chromatography, as well as specific glycosidases, we demonstrate that VIP receptor-linked carbohydrates are predominantly tri- or tetraantennary sialylated N-linked oligosaccharides, 27% of which are fucosylated, and some may have terminal galactose residues. Treatment of 125I-VIP receptor complexes with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase revealed the presence of at least three N-linked carbohydrate chains/receptor polypeptide. To investigate the functional role of the carbohydrate moiety, 125I-VIP binding to IGR39 cell membranes was tested in the presence of soluble lectins. Among the lectins tested, only wheat germ agglutinin (WGA) was found to markedly inhibit VIP binding in a dose-dependent manner. Binding data indicated that the presence of the lectin led to a 3-fold increase in Kd value, from 0.15 to 0.44 nM, without any change in the number of available binding sites. The potent inhibitor of WGA binding, N,N',N"-triacetylchitotriose, completely reversed the effect of the lectin. On the other hand, VIP binding inhibition persisted even after neuraminidase treatment, suggesting that sialic acids were not directly involved. Furthermore, WGA inhibition was not abolished although most, if not all, VIP receptor oligosaccharides were converted to high mannose type structures by treating IGR39 cells with deoxymannojirimycin. Finally, whereas the pharmacological profile of VIP receptor was virtually identical, the presence of WGA greatly reduced the VIP-stimulated cAMP in IGR39 cells, indicating that the lectin alters the ability of the receptor to interact with the adenylate cyclase system.

Binding, Competitive↗