Georges De Bruyne

Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers

Entamoeba (E.) histolytica trophozoites initiate amebiasis through invasion into the enteric mucosa. It was our aim to understand the molecular interactions between amebic trophozoites and en‐terocytes during the early steps of invasion. Tropho‐zoites of E. histolytica strain HM1:IMSS were seeded on the apical side of enteric T84 cell layers, which were established on filters in two‐compartment culture chambers. Cocultures were analyzed for para‐cellular permeability by measurement of transepithe‐lial electrical resistance (TER) and for the tight junction proteins ZO‐1, ZO‐2, occludin, and cingulin by immunocytochemistry and immunoprecipitation. On direct contact with the apical side of the enteric cells, trophozoites caused an increase in paracellular permeability as evidenced by a decrease of TER associated with an increase in [3H]mannitol flux. Immunoprecipitation of cocultures revealed dephos‐phorylation of ZO‐2, loss of ZO‐1 from ZO‐2, and degradation of ZO‐1 but less so of ZO‐2 and none of occludin or E‐cadherin. In conclusion, trophozoite‐associated increase in paracellular permeability of enteric cell layers is ascribed to disturbance of the molecular organization of tight junction proteins.—Leroy, A., Lauwaet, T., De Bruyne, G., Cornelissen, M., Mareel, M. Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers. FASEB J. 14, 1139–1146 (2000)

The flavonoid tangeretin inhibits invasion of MO4 mouse cells into embryonic chick heartin vitro

Tangeretin, a flavonoid from citrus plants, was found to inhibit the invasion of MO4 cells (Kirsten murine sarcoma virus transformed fetal mouse cells) into embryonic chick heart fragmentsin vitro. The flavonoid appeared to be chemically stable in tissue culture medium, and the anti-invasive effect was reversible on omission of the molecule from the medium. Unlike (+)-catechin, another anti-invasive flavonoid, tangeretin bound poorly to extracellular matrix. It did not alter fucosylated surface glycopeptides of MO4 cells. Tangeretin seemed not to act as a microtubule inhibitor, as immunocytochemistry revealed no disturbance of the cytoplasmic microtubule complex. However, at antiinvasive concentrations of tangeretin, cell proliferation and thymidine incorporation appeared to be inhibited. When cultured on an artificial substrate, treated MO4 cells were less elongated, covered a larger surface area and exhibited a slower directional migration than untreated cells. From the decrease in ATP content in MO4 cells after tangeretin treatment, we deduce that this flavonoid inhibits a number of intracellular processes, which leads to an inhibition of cell motility and hence of invasion.

Vinblastine, vincristine and vindesine: Anti-invasive effect on MO4 mouse fibrosarcoma cells in vitro ☆ ☆☆

Inhibition of the invasiveness of MO4 mouse fibrosarcoma cells by the vinca alkaloid, vinblastine (VLB), vincristine (VCR) and vindesine (VDS), has been examined in vitro. At doses between 0.006 microgram/ml (minimal effect) and 0.1 microgram/ml (complete inhibition) these drugs interfered with the invasion of MO4 cells from an aggregate confronting a fragment of embryonic chick heart in three-dimensional culture. We have also examined the effect of these drugs on the following activities of MO4 cells: growth, directional migration and assembly of the cytoplasmic microtubule complex. Growth and directional migration were affected by the same doses of vinca alkaloids as invasion. In contrast with the vinca alkaloids, 5-fluorouracil at 1 microgram/ml inhibited growth but allowed directional migration and invasion. At a dose of 0.3 microgram/ml VLB, VCR and VDS interfered with the assembly of cytoplasmic microtubules, as visible after immunocytochemical staining with tubulin antiserum. Ultrastructural analysis demonstrated that inhibition of invasion in three-dimensional culture corresponds with abolishment of the cytoplasmic microtubule complex. Anti-invasive concentrations of VLB, VCR and VDS represent clinically achievable plasma concentrations. We concluded that the anti-invasive effect of the vinca alkaloids may contribute to their antitumor activity.

β‐casein‐derived peptides, produced by bacteria, stimulate cancer cell invasion and motility

In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT‐8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility‐promoting factor, identified as a 13mer β‐casein‐derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin‐like metalloprotease, and a collagen‐associated trypsin‐like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a β‐casein source. The pro‐ invasive 13mer peptide‐signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21‐ activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3‐kinase. Delta opioid receptor (δOR) is a candidate receptor for the 13mer peptide since naloxone, an δOR antagonist, blocks both δOR serine phosphorylation and 13mer peptide‐mediated invasion.

Phagocytic capacity of invasive malignant cells in three-dimensional culture

SummaryTo study the phagocytic capacity of invasive malignant cells, fragments of the hypoblast from chick blastoderms were confronted in three-dimensional culture with spheroidal aggregates of 1) malignant virally transformed C3H mouse cells (MO4), 2) HeLa cells and 3) embryonic chick heart cells. The hypoblast was used because it contains yolk, a marker that is absent in the confronting cells and that can be identified histologically and ultrastructurally. The confronting tissues were incubated on semi-solid agar-agar medium or in fluid medium on a gyrotory shaker. Cultures were followed for 1 to 7 days by stereomicroscopy, cinemicrophotography, light and transmission electron microscopy.Confrontation with MO4 cells or HeLa cells, known to be invasive in vitro, led to complete disappearance of the hypoblast. The fragments of hypoblast were well conserved when cultured alone or confronted with aggregates of chick heart cells. Degeneration of the hypoblast is shown at the area of contact with MO4-cell or HeLa-cell aggregates, in contrast to heart cells. Filopodia-like extensions from the MO4 or HeLa cells penetrate intercellularly, transcellularly and intracellularly into the hypoblast. Phagosomes, containing yolk and unidentified debris are observed in MO4 cells and in HeLa cells, but not in heart cells. These observations demonstrate the phagocytic capacity of invasive malignant cells.

Morphotypic plasticity in vitro and in nude mice of epithelial mouse mammary cells (NMuMG) displaying an epithelioid (e) or a fibroblastic (f) morphotype in culture.

Transition from an epithelioid (e-) to a fibroblastic (f-) morphotype marks invasiveness in clinical and experimental cancer. To understand better the factors influencing such transitions, we have subcloned and manipulated mouse mammary gland (NMuMG) cell cultures and compared the invasive phenotype of multiple subclones in vitro and in vivo. Cell lines with an e-morphotype expressed E-cadherin homogeneously and were not invasive in vitro. Cells with an f-morphotype were E-cadherin-negative and became fully invasive in vitro upon expression of the ras oncogene. Invasive tumors were produced in nude mice after subcutaneous injection of e-type or f-type cells. These tumors showed cystic, glandular and undifferentiated structures. Tumors from f-type cells were E-cadherin-negative whereas e-type tumors stained heterogeneously in immunohistochemical preparations. Our observations demonstrate the impact of the micro-ecosystem on the invasive phenotype, with in vivo downregulation of E-cadherin and stimulation of the e- to f-morphotype transition.

Effect of temperature on invasion of MO4 mouse fibrosarcoma cells in organ culture

Invasion by MO4 mouse fibrosarcoma cells into fragments of embryonic chick heart or lung in organ culture was studied histologically and ultrastructurally at various temperatures between 12 and 40°C. Invasion was absent for at least 7 days at or below temperatures of 29°C. Invasion was invariably observed at or above 30·5°C. Differences in invasion between 29 and 30·5°C could not be ascribed to differences in growth, migration, or microtubule assembly/disassembly of MO4 cells. Neither could they be explained through differences in the attachment of MO4 cells to the heart fragments. Possible explanations for the absence of invasion at lower temperature are: altered resistance of the extracellular matrix in heart or lung fragments, and deficient expression of fucosylated glycoproteins at the surface of MO4 cells. A population of MO4 cells plated from the parent line and adapted to grow at 28°C (MO4 28 cell line) did not differ in invasiveness from the parent MO4 cells.We conclude that the temperature dependence of invasion in organ culture might indicate as yet unexplored aspects of the mechanisms of tumour invasion.

Immunohistochemistry of laminin in early chicken and quail blastoderms

SummaryWe have used immunohistochemical techniques to study laminin in quail blastoderms milked from the oviduct and the distribution of laminin in laid chicken and quail blastoderms. Laminin is a constituent of the basement membrane in both chicken and quail blastoderms. It is found at the ventral side of the upper layer cells. Laminin is first observed under individual upper layer cells in prelaid quail blastoderms 15 h post-ovulation, but is absent at the ingression site of endophyll cells. The presence of a continuous laminin layer coincides with the epithelialization of the epiblast after 5–10 h incubation. The laminin layer is discontinuous at the primitive streak and at Hensens node. It is thinner and partly discontinuous at the median part of the neural plate. By induction, either of an ectopic primitive streak or a neural plate, we have demonstrated, using the chicken-quail nucleolar marker technique, that at these sites the laminin layer is interrupted. A laminin layer might confer rigidity onto the epiblast, whereas disruption of a laminin layer seems to be correlated with ingression of cells or bending of the neural plate.

Specific anti-nuclear antibodies in systemic sclerosis patients with and without skin involvement: an extended methodological approach

OBJECTIVES To determine how sensitively SSc-associated ANAs are detected by a standard identification algorithm compared with an extensive panel of ANA identification assays, and to assess the distribution of SSc-associated ANAs and SSc organ system involvement in patients without skin involvement (limited SSc). METHODS Serum samples from 145 consecutive monocentric SSc patients fulfilling LeRoy and Medgsers criteria for early SSc were studied. ANAs were detected by IIF on HEp-2000 cells and identified by western blotting, protein radio-immunoprecipitation, RNA immunoprecipitation and line immunoassay (LIA). SSc organ involvement was assessed according to a modification of Medsgers disease severity scale. RESULTS At least one specific ANA reactivity was present in 88% of the patients. The standard algorithm (IIF and LIA) found at least one specific ANA in 74% of the patients. The main reactivities missed by this algorithm were anti-RNA polymerase III, anti-PM/Scl and anti-Th/To. Eighty-three percent of the patients with limited SSc had at least one ANA. ACAs and anti-Th/To antibodies were significantly associated with limited SSc, whereas anti-topoisomerase I and anti-RNA polymerase III were observed less frequently. SSc organ system involvement was present in 63% of the patients with limited SSc, most of whom had lung involvement. CONCLUSIONS Standard algorithms for ANA identification lack sensitivity for the detection of SSc-associated ANA and should be supplemented with additional assays, especially in a clinical environment that has particular interest in SSc. The spectrum of SSc-associated ANA differs according to the presence or absence of skin involvement.

Confrontation of an invasive (MO4) and a noninvasive (MDCK) cell line with embryonic chick heart fragments in serum-free culture media

SummaryConfronting cultures of precultured embryonic chick heart fragments (PHF) with aggregates of malignant cells in vitro have been shown to be relevant for a number of aspects of tumor invasion in vivo. Preculture of the heart fragments, formation of cell aggregates and subsequent culture of confronting pairs have so far been done only in serum-containing culture media. We describe here confronting cultures of PHF with invasive MO4 mouse cell aggregates or noninvasive MDCK dog kidney cell aggregates in serum-free media. Heart fragments precultured in the absence of serum seemed to be necrotic after confronting culture in serum-free media. However, preculturing in media supplemented with 10% fetal bovine serum allowed us to do subsequent confronting cultures in absence of serum. Cell aggregates were also prepared in serum-containing medium. MO4 cells occupied and replaced the heart tissue within 4 d, whereas MDCK cells remained at the periphery, of the PHF. This indicates that serum-free confronting cultures can discriminate between invasive and noninvasive cells. The viability of individual PHF and cell aggregates cultured in the same way as in confrontations was ascertained by histology and by explantation and postculturing on a solid tissue culture substrate. Growth of the cultures was smaller in serum-free media than in media supplemented with 10% fetal bovine serum. The main advantage of serum-free culture conditions in vitro is the elimination of the influence of serum components on invasion, and the ability to examine the effect on invasion of drugs that are, susceptible to inactivation by serum.