[Effects of endocrine peptides on proliferation, migration and differentiation of human endothelial cells]. / Effets des peptides endocrines sur la prolifération, la migration et la différenciation des cellules endothéliales humaines.

AIMS: We aimed to evaluate the effects of several peptides (substance P, VIP, neuropeptide Y, bombesin, glucagon and somatostatin) on the proliferation, migration and differentiation of human endothelial cells and their modulation by an anti-angiogenic factor, endostatin. METHODS: Human endothelial cells (HUVEC) were isolated from umbilical veins. Their proliferation was measured by the incorporation of tritiated thymidine. Their migration was evaluated by using an haptotactic assay performed in Boyden chambers, after metabolic labeling of HUVEC through (35) S-methionin. Differentiation was evaluated as the capacity for HUVEC to form capillaries. RESULTS: Endothelial cell proliferation was increased by neuropeptide Y, bombesin and glucagon. Somatostatin induced a significant decrease in basal and stimulated endothelial cell proliferation. The migration of HUVEC increased in the presence of substance P, VIP, neuropeptide Y, bombesin, glucagon and somatostatin. The number of capillaries was increased by substance P and VIP and decreased by neuropeptide Y, bombesin and somatostatin. Endostatin induced a significant decrease in endothelial cell proliferation in the basal state and after stimulation by neuropeptide Y and bombesin. Endostatin had no additive effect on the anti-proliferative action of somatostatin. CONCLUSIONS: Our results suggest a role for endocrine peptides in the regulation of tumor angiogenesis. The potent anti-angiogenic effect of somatostatin may promote new therapeutic strategies.

Anti-beta4 integrin antibodies enhance migratory and invasive abilities of human colon adenocarcinoma cells and their MMP-2 expression.

Integrin-mediated adhesion of cells to extracellular matrix proteins has been shown to activate various intracellular signaling events. In the present study, we demonstrate that the addition of a monoclonal antibody raised against the beta4 integrin subunit in the culture medium of a clone derived from the colon adenocarcinoma cell line LoVo specifically results in stimulation of cell migration and invasion through reconstituted basement membrane matrices. Moreover, an increase in MMP-2 activity is observed. Conversely, monoclonal anti-alpha6 and anti-beta1 have no effect on MMP-2 expression. The s. c. co-injection of adenocarcinoma cells with antibodies raised against the beta4 integrin subunit to immunosuppressed newborn rats gives rise to tumors displaying altered and disorganized peri-tumoral basement membranes compared with tumors obtained when cells are injected with adenocarcinoma cells alone. Higher metastatic capacity of cells results when they are co-injected with antibodies to the beta4 integrin subunit. Our results suggest that the beta4 subunit of alpha6beta4 integrin, a laminin receptor in colon adenocarcinoma, may be responsible for the specific signals which stimulate cell motility, expression of MMP-2 and tumor invasion.

Site-specific epithelial-mesenchymal interactions in digestive neuroendocrine tumors. An experimental in vivo and in vitro study.

Little is known about the functional interactions between digestive neuroendocrine tumor cells and their stromal microenvironment. The focus of our study is whether mesenchymal cells modulate peptide expression, cell proliferation, and invasiveness in digestive neuroendocrine tumor cells. We designed an experimental in vivo and in vitro study using the mouse enteroendocrine cell line STC-1. In vivo, STC-1 cells were injected subcutaneously in 18 immunosuppressed newborn rats. At day 21, all animals presented poorly differentiated neuroendocrine tumors with lung metastases. Subcutaneous tumors were usually limited by a capsule containing basement membrane components and myofibroblasts that presented a low mitotic index. Lung tumors were devoid of capsule and poor in myofibroblasts, and their mitotic index was high. The profile of peptide expression in STC-1 tumors was different from that of cultured STC-1 cells. In vitro, STC-1 cells were cultured with fibroblasts of different origins, including dermis, lung, digestive tract, and liver. Based on their origin, myofibroblasts differentially modulated hormone synthesis, proliferation, spreading, and adhesion of STC-1 cells. In conclusion, our results show that site-specific functional interactions between mesenchymal and neuroendocrine cells may contribute to modulating the behavior of digestive neuroendocrine tumors, depending on their growth site.

[Integrins and metalloproteinases: an efficient collaboration in the invasive process]. / Intégrins et métalloprotéinases: une collaboration efficace dans le processus invasif.

During the invasive process, tumor cells must move through the extracellular matrix. They have to adhere to the extracellular matrix components, then proteolyse them and migrate on their fragments. This implicates integrins and proteinases, namely metalloproteinases. Numerous experiments which had been performed on various models, namely malignant melanomas proved that integrins have a major role in the transduction of signals from the outside to the inside of the cells, such signals enhancing the expression of the metalloproteinases or, in the contrary, inhibiting it. The modifications of this expression are dependent of extracellular matrix components and may be induced by the linking of specific antibodies to integrins. In some instances, the integrins localized on the tumor cell surface may act as receptors for extracellular matrix proteins and metalloproteinases at once, that may give to tumor cells an higher efficiency in the invasive process. Such mechanisms may result in interesting clinical perspectives for the control of metalloproteinases regulation in pathological processes.

Loss of epithelial differentiation markers and acquisition of vimentin expression after xenograft with laminin-1 enhance migratory and invasive abilities of human colon cancer cells LoVo C5.

Clone C5 of the human colon adenocarcinoma LoVo cell line was subcutaneously injected with or without exogenous laminin-1 (EHS laminin) into immunosuppressed newborn rats. Cultures were initiated from lung metastases obtained with or without laminin-1 and gave rise to the C5 sublines LM and M4, respectively. The LM subline was mainly composed of spreading cells whereas most C5 and M4 cells remained round and aggregated. The mesenchymal marker vimentin was expressed by very rare C5 and M4 cells (< 1%), and by many LM cells (about 35%). On the opposite, the epithelial markers villin and dipeptidylpeptidase IV were well expressed by C5 cells but not by LM cells. In in vitro migration and invasion assays, LM cells migrated and invaded basement membrane extract twice as much as the parental C5 clone and the M4 subline, probably in association with vimentin-expressing cells, because invasion of basement membrane extract Matrigel by LM cells gave rise to 100% vimentin-positive cells (sublines LM 22, LM 23 and LM 24). When subcutaneously injected, C5 cells induced tumors limited by an interrupted but well organized basement membrane, whereas LM cells induced tumor masses, occasionally limited by a very irregular basement membrane, as observed when C5 cells were injected with laminin-1. Gelatin zymographic analysis clearly showed an increased expression of matrix metalloproteinase-2 by LM cells. Our results suggest a specific role of laminin-1 on the in vivo proliferation of highly invasive vimentin-expressing colon carcinoma cells. This proliferation may result from the initial interaction of C5 cells with large amounts of laminin-1, leading to a selection of vimentin-expressing cells during the metastatic cascade.

Expression of the alpha 6, beta 1 and beta 4 integrin subunits, basement membrane organization and proteolytic capacities in low and high metastatic human colon carcinoma xenografts.

Malignant transformation is associated with alterations in both cell-cell and cell-matrix interactions. The E2 and C5 clones, derived from the human colon adenocarcinoma LoVo cell line, show, respectively, low and high metastatic capacity as experimental xenografts in vivo. In this study, we have assessed the adhesion and spreading of E2 and C5 cells on basement membrane laminin, expression of the laminin receptor integrins alpha 6 beta 1 and alpha 6 beta 4 and expression of gelatinolytic and plasminogen-dependent activities. On days 5 and 7 after subcutaneous grafting to immunosuppressed newborn rats, well-differentiated E2 tumors displayed a polarized expression of these integrin subunits, with the exception of the beta 1 subunit which remained pericellular. In contrast, C5 tumors were unorganized and the three integrin subunits remained nonpolarized and pericellular. Flow cytometry results showed that the expression of alpha 6 beta 1 and alpha 1 beta 4 integrins was weaker in the highly metastatic C5 clone than in the E2 clone whereas laminin expression was not significantly different. Under-expression and pericellular localization of these integrin receptors in C5 cells as compared to E2 cells may explain the difference in their binding and spreading capacity on laminin, organization of peritumoral basement membrane and maintenance of a differentiated phenotype. Whereas similar levels of gelatinolytic and plasminogen activator activities have been detected in the culture supernatant of the two clones, histozymograms showed that plasminogen-dependent caseinolysis appeared earlier in sections of C5 and parental tumors than in those of E2 xenografts. These results suggest that enhanced aggressiveness of C5 tumors in vivo may be linked to both an impairment of basement membrane setting due to integrin underexpression and distribution and of proteolytic activities modulated by tumor/host interactions.

Lentivirus-induced interstitial lung disease: pulmonary pathology in sheep naturally infected by the visna-maedi virus.

Visna-maedi virus is a lentivirus that causes a chronic disease in sheep affecting, among other organs, the lungs. Interstitial pneumonitis is similar to that in man associated with the infection by the human immunodeficiency virus type-1. We have compared the pathological features of lungs of sheep naturally infected with visna-maedi virus with the results obtained from bronchoalveolar lavage and virus isolation. Semi-quantitative grading of the lesions was performed on 147 sheep lungs obtained from the slaughterhouse. Seventy-seven were macroscopically and histologically normal, 39 had typical lesions of interstitial lung disease (maedi), and 13 had minor lesions of the same type. Eighteen of the affected lungs were heavily infested with parasites. Of these parasite-infected lungs, 9 showed typical maedi lesions and 4 showed minor lesions; parasite infection had no obvious effect on the development of maedi. In keeping with pathological findings, bronchoalveolar lavage disclosed an alveolitis process in the maedi lungs with increased macrophage, lymphocyte and neutrophil numbers. Cytopathic virus was detected from alveolar macrophage coculture with fibroblasts more often from maedi lungs (10/12) than from normal lungs (9/39). Electron microscopy of bronchoalveolar lavage cocultures revealed typical lentiviral particles. Animals with minor lesions may be at an early stage of the disease.

Comparative tumor morphogenesis of two human colon adenocarcinoma cell clones xenografted in the immunosuppressed newborn rat.

Two clones derived from the human adenocarcinoma cell line LoVo, E2 and C5 xenografted subcutaneously to immunosuppressed newborn rats, respectively produced well-differentiated and undifferentiated tumors. The comparative morphogenesis of these tumors was performed on xenografts explanted as early as 18 h and up to 21 days after grafting by studying the progressive setting of the enterocyte differentiation marker dipeptidylpeptidase IV, the basal lamina component laminin and the alpha 6 integrin subunit. E2 xenografts which were entirely undifferentiated 18 h after grafting, presented well-polarized acini-like tumoral islets 6 h later, i.e. only 1 day after injection. Basement membranes, which were not organized at this moment, may not be necessary for morphological polarization. The chronology of function antigens polarization was characterized by formation of a basement membrane 5 days after the graft with associated basal sorting of alpha 6 integrin. The polarization of alpha 6 integrin took, however, longer to be achieved while apical addressing of dipeptidylpeptidase IV was the last to be completed. In contrast, C5 tumors never differentiated. Even 21 days after grafting alpha 6 integrin remained pericellular, dipeptidylpeptidase IV was underexpressed and laminin was found as perilobular patches. Quantitative differences in laminin or alpha 6 integrin expression could not account for the differences in the polarization process observed in the two variants.

Characterization of the lymphocytic alveolitis in visna-maedi virus-induced interstitial lung disease of sheep.

In order to investigate the contribution of lymphocytes to interstitial lung disease in animals with visna-maedi infection, we studied in parallel bronchoalveolar cells and lung tissue from slaughter-house animals (n = 29) and from colostrum-deprived lambs transtracheally inoculated with field isolates of visna-maedi virus (n = 9) or saline (n = 6). Lymphocyte subpopulations were identified in bronchoalveolar lavage by immunofluorescence and flow cytometry analysis and in lung tissue using indirect immunohistochemistry. In infected animals a lymphocytic alveolitis containing CD4 and CD8 lymphocytes was observed. Peribronchovascular lymphoid nodules comprise mostly CD4 lymphocytes. Alveolar lymphocytes of both subsets displayed increased expression of MHC class II antigens in animals with naturally occurring maedi but not in experimentally infected ones. A sequential process of lymphocyte attraction and activation is likely to occur in vivo as part of the alveolitis.

Laminin expression by two clones isolated from the colon carcinoma cell line LoVo that differ in metastatic potential and basement-membrane organization.

In the present report we describe the characteristics of 2 clones, E2 and C5, isolated from the human colon adenocarcinoma cell line LoVo. When grafted to immunosuppressed newborn rats, these clones formed tumors that varied with regard to differentiation rate, basement-membrane organization and lung metastatic potential. Production and distribution of laminin by E2, C5 and related tumors was studied by immunohistochemistry with an anti-laminin monoclonal antibody 4C12 (MAb 4C12). In lowly metastatic E2-derived tumors, strong regular stainings were observed which were strictly peri-tumoral and corresponded to the basal lamina. Since the antibody interacted with human laminin (the graft) but not with rat laminin (the host), this result indicated that basement-membrane laminin was supplied mainly by tumor-cell synthesis. In highly metastatic C5-derived tumors, the staining obtained with MAb 4C12 was peri-cellular and unorganized. Laminin synthesis by E2 and C5 cells in sub-cultures or soon after dissociation from explanted tumors was studied by metabolic labelling with 35S-methionine under steady-state conditions followed by immunoprecipitation and SDS-PAGE. High-molecular-weight laminin comprised by disulfide-linked A and B chains, i.e., heterotrimeric laminin, was found in cell lysates and in the secretion medium of cell lines and tumor cells. In addition, B1B2 dimers and free B chains were observed in cell lysates. Quantitatively, laminin expression by E2 and C5 clones or tumor cells was not significantly different. These findings suggest that basement-membrane defects in invasive clone LoVo C5 were not due to laminin under-expression.

Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenocarcinoma cell line.

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.

[Basal lamina and metastatic potential of two tumorigenic clones from a human colonic adenocarcinoma cell line]. / Lame basale et potentialités métastatiques de deux clones tumorigènes issus d'une lignée d'adénocarcinome du colon humain.

We isolated from a human colonic adenocarcinoma cell line two clones with highly different metastatic abilities. One of them, which spreads rapidly in culture, produces, when injected in immunosuppressed newborn rats, well differentiated epithelial like tumors limited by a continuous basal lamina and never produces lung metastasis. The other clone, which spreads slowly in culture, produces undifferentiated tumors of irregular shape and with usually no basal lamina; tumor cells are often dispersed in the stroma and metastases are observed in the lungs. These two clones may hence constitute a model for the study of the link between the presence or absence of a basal lamina in human tumors and their ability to metastasize.

Antigenic analysis of African measles virus field isolates: identification and localisation of one conserved and two variable epitope sites on the NP protein.

Measles virus isolates from epidemics in the Cameroons (1983) and Gabon (1984) were analysed by a panel of monoclonal antibodies against four of the virion proteins. We observed no antigenic variation in the haemagglutinin, the fusion glycoprotein, or in the matrix protein. However, both inter- and intra-epidemic variation was observed in the nucleoprotein (NP). On the basis of strain reactivity and a competition binding assay, three epitopic sites were designated on the NP. One site was found on all the measles virus strains examined, whereas the other two were variable. Examination of proteolytic cleavage of the NP in situ (on the ribonucleoparticle) showed that the conserved site is located on a large fragment which remains bound to the viral genome. The peptide removed by proteolysis contained the two variable epitopes. The variability of the NP is discussed in relationship to its biological activity.

Different fates of hepatitis B virus markers during plasma fractionation: a clue to the infectivity of blood derivatives.

Hepatitis B surface antigen (HBs Ag) and associated particles, e antigen (e Ag) and DNA polymerase are unevenly distributed during Cohn's cold ethanol fractionation of plasmas positive for these markers of the hepatitis B virus (HBV). Most of the e Ag, Dane particles and DNA polymerase are retained in fraction III whereas the bulk of HBs Ag is recovered in fraction IV where only 22 nm spheres and short filaments are still identified. These results suggest that differences in quantitative distribution of HB virions together with alteration of infectious particles during the fractionation process may in addition to heat inactivation account for the relative hepatitis risk of the various plasma derivatives.