Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling.

Rho-like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T-lymphoma cells into a fibroblast monolayer is induced by Tiam1, an activator of the Rho-like GTPase Rac, and by constitutively active V12Rac1. Here we show that activated V12Cdc42 can also induce invasion of T-lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho-inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T-lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum-borne lipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) (10(-7)-10(-6) M), which act on distinct G protein-linked receptors to activate RhoA and phospholipase C (PLC)-Ca2+ signaling. LPA- and S1P-induced invasion is preceded by Rho-dependent F-actin redistribution and pseudopodia formation. However, expression of both V14RhoA and V12Rac1 does not bypass the LPA/S1P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U-73122, but not the inactive analog U-73343, abolishes invasion. Our results indicate that T-lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation, and is dependent on LPA/S1P receptor-mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.     

Motility and invasive potency of murine T-lymphoma cells: effect of microtubule inhibitors.

ESb and BW-O-Li1 are T-lymphoma cell lines that form metastases in various organs after injection into syngeneic mice. In vitro, both cell lines invade through a fibroblastic monolayer, but ESb cells do so much slower than BW-O-Li1. By the use of Fourier analysis of cell outlines, we can relate this difference in invasiveness to a difference in cell motility: ESb cells do not perform any conspicuous shape change, whereas BW-O-Li1 cells are actively protruding and retracting large pseudopodia. However, the low-motile ESb cells become as motile and deformable as BW-O-Li1 cells when they have eventually invaded under a fibroblastic monolayer. This indicates that ESb cells do have inherent capability for shape change. Treatment of ESb cells with the microtubule disrupting agent nocodazole concomitantly increases their shape change intensity, and their invasion rate through fibroblast monolayers. On the contrary, the microtubule stabilizing drug taxol inhibits both motility and invasion of BW-O-Li1 cells. Our observations suggest that the microtubule network can repress invasion-bound motility of lymphoid cells.     

Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.

Neisseria gonorrhoeae is a facultative intracellular bacterium capable of penetrating into certain human epithelial cell types. In order to identify gonococcal factors essential for invading Chang human conjunctiva cells, a gentamicin selection assay for the quantification of viable intracellular bacteria was used in conjunction with microscopy. The results demonstrate a correlation between the invasive behaviour of gonococci and the expression of Opa proteins, a family of variable outer membrane proteins present in all pathogenic Neisseria species. However, only particular Opa proteins supported invasion into Chang cells as indicated by the use of two unrelated gonococcal strains. Invasion was sensitive to cytochalasin D, and strong adherence mediated by the Opa proteins appeared to be essential for the internalization of gonococci. In contrast pili, which also conferred binding to Chang conjunctiva cells, did not support cellular invasion but rather were inhibitory.     

Cyclic AMP content and invasive capacity of metastatic variants of the BW-5147 murine T-cell lymphoma

The invasive behaviour of 8 lymphoma cell lines were tested by an in vitro monolayer invasion assay. The metastatic cell lines (TAM 4D1.2, DCH10Sp, TAM 4D6.2, E4 and BWLi) were more invasive than their non-metastatic counterparts (TAS 5C4, BWO and DCH 10). There was a positive correlation between their invasiveness and the PGE1- and forskolin stimulated cellular cAMP levels. Invasiveness and basal cAMP levels could not be correlated. Pretreatment with pertussis toxin (50 ng/ml) for 24 hours provoked did not significantly affect the basal and PGE1-stimulated cAMP levels in all cells. Yet, the toxin catalysed the ADP-ribosylation of 40 kDa components in all cells and provoked a significant increase in the invasiveness of non-metastatic cell lines and a decrease in the invasiveness of metastatic cell lines. These data suggest that the invasiveness of T-lymphoma cell lines might be controlled by a complex interplay between different signal transducing pathways in the membrane, rather than by the intracellular level of cAMP.     

Edwardsiella ictaluri invasion of IEC-6, Henle 407, fathead minnow and channel catfish enteric epithelial cells

Invasion of Edwardsiella ictaluri into cultured mammalian, fish and enzymatically harvested catfish enteric epithelial cells is described. Gentamicin survival assays were used to demonstrate the ability of this catfish pathogen to invade IEC-6 (origin: rat small intestinal epithelium), Henle 407 (origin: human embryonic intestinal epithelium), fathead minnow (FHM, minnow epithelial cells) and trypsin/pepsin-harvested channel catfish enteric epithelial cells. Invasion of all cell types occurred within 2 h of contact at 26 degrees C, in contrast to Escherichia coli DH5 alpha, which did not invade cells tested. Eight Edwardsiella ictaluri isolates from diseased catfish and the ATCC (American Type Culture Collection) strain were evaluated for invasion efficiency using FHM cells. All isolates were invasive, but at differing efficiencies. Invasion blocking assays using chemical blocking agents were performed on a single isolate (LA 89-9) using IEC-6 epithelial cells. Preincubation of IEC-6 cells with cytochalasin D (microfilament depolymerizer) and monodansylcadaverine (blocks receptor-mediated endocytosis) significantly reduced invasion by E. ictaluri, whereas exposure to colchicine (microtubule depolymerizer) had no effect on bacterial internalization. Results indicate that actin polymerization and receptor-mediated endocytosis are involved in uptake of E. ictaluri by IEC-6 epithelial cells. Invasion trials using freshly harvested cells from the intestine of the natural host, Ictalurus punctatus, show that invasion occurs, but at a low efficiency. This is possibly due to loss of outer membrane receptors during enzymatic cell harvest. This study provides the first documentation of the invasion of cultured mammalian and fish cells by E. ictaluri, and identifies possible mechanisms used for intracellular access. Additionally, the study describes several functional in vitro invasion models using commercially available cell lines as well as cells from the natural host (channel catfish, I. punctatus).     

A mathematical model for pattern formation of glioma cells outside the tumor spheroid core

Glioblastoma is the most common and the most aggressive type of brain cancer. The median survival time from the time of diagnosis is approximately one year. Invasion of glioma cells from the core tumor into the surrounding brain tissue is a major reason for treatment failure: these migrating cells are not eliminated in surgical resection and cause tumor recurrence. Variations are seen in number of invading cells, and in the extent and patterns of migration. Cells can migrate diffusely and can also be seen as clusters of cells distinct from the main tumor mass. This kind of clustering is also evident in vitro using 3D spheroid models of glioma invasion. This has been reported for U87 cells stably expressing the constitutively active EGFRVIII mutant receptor, often seen expressed in glioblastoma. In this case the cells migrate as clusters rather than as single cells migrating in a radial pattern seen in control wild type U87 cells. Several models have been suggested to explain the different modes of migration, but none of them, so far, has explored the important role of cell–cell adhesion. The present paper develops a mathematical model which includes the role of adhesion and provides an explanation for the various patterns of cell migration. It is shown that, depending on adhesion, haptotactic, and chemotactic parameters, the migration patterns exhibit a gradual shift from branching to dispersion, as has been reported experimentally.     

Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion.

The role of the Campylobacter jejuni flagella in adhesion to, and penetration into, eukaryotic cells was investigated. We used homologous recombination to inactivate the two flagellin genes flaA and flaB of C. jejuni, respectively. Mutants in which flaB but not flaA is inactivated remain motile. In contrast a defective flaA gene leads to immotile bacteria. Invasion studies showed that mutants without motile flagella have lost their potential to adhere to, and penetrate into, human intestinal cells in vitro. Invasive properties could be partially restored by centrifugation of the mutants onto the tissue culture cells, indicating that motility is a major, but not the only, factor involved in invasion.     

Non-encapsulated strains reveal novel insights in invasion and survival of Streptococcus suis in epithelial cells

Streptococcus suis is a porcine and human pathogen causing invasive diseases, such as meningitis or septicaemia. Host cell interactions of S. suis have been studied mainly with serotype 2 strains, but multiple capsular serotypes as well as non-typeable strains exist with diverse virulence features. At present, S. suis is considered an extracellular pathogen. However, whether or not it can also invade host cells is a matter of controversial discussions. We have assessed adherence and invasion of S. suis for HEp-2 epithelial cells by comparing 10 serotype 2 strains and four non-typeable (NT) strains. Only the NT strains and a non-encapsulated serotype 2 mutant strain, but none of the serotype 2 strains, adhered strongly and were invasive. Invasion seemed to be affected by environmental signals, as suggested from comparison of strains grown in different media. Further phenotypic and genotypic characterization revealed a high diversity among the different strains. Electron microscopic analysis of invasion of selected invasive NT strains indicated different uptake mechanisms. One strain induced large invaginations comparable to those seen in 'caveolae' mediated uptake, whereas invasion of the other strains was accompanied by formation of filipodia-like membrane protrusions. Invasion of all strains, however, was similarly susceptible to hypertonic sucrose, which inhibits receptor-mediated endocytosis. Irrespective of the uptake pathway, streptococci resided in acidified phago-lysosome like vacuoles. All strains, except one, survived intracellularly as well as extracellular acidic conditions. Survival seemed to be associated with the AdiS protein, an environmentally regulated arginine deiminase of S. suis. Concluding, invasion and survival of NT strains of S. suis in epithelial cells revealed novel evidence that S. suis exhibits a broad variety of virulence-associated features depending on genetic variation and regulation.     

Fibroblasts-dependent invasion of podoplanin-positive cancer stem cells in squamous cell carcinoma

To clear whether podoplanin-positive cancer stem cells in squamous cell carcinoma have higher invasion activity during a fibroblasts-dependent invasion. A collagen gel invasion assay was performed using fluorescent ubiquitination-based cell cycle indicator-labeled A431 cells. The total number and number of invading cells in S/G2/M phase were counted using time-lapse imaging cocultured with fibroblasts. There was no significant difference between the number of invading podoplanin-positive and negative A431 cells when fibroblasts did not exist. On the contrary, the number of invading podoplanin-positive cells was significantly higher when fibroblasts existed. The frequency of cells in S/G2/M phase among invasion was no difference. Knockdown of podoplanin decreased the number of invaded A431 cells significantly when fibroblasts existed. Podoplanin-positive A431 cells display higher invasion activity when fibroblasts exist, suggesting that some biological functions of cancer stem cells might become evident only within the fibrous tumor microenvironment.     

Antisense Tiam1 down-regulates the invasiveness of 95D cells in vitro

As a specific guanine nucleotide exchange factor of Rac 1, Tiam 1 (T-lymphoma invasion and metastasis inducing protein 1) is involved in a number of cellular events, such as cytoskeleton reorganization, cell adhesion, and cell migration. Since Tiaml was implicated in the invasion and metastasis of T-lymphoma cells and breast tumor cells, we compared the expression level of Tiaml in two human giant-cell lung carcinoma cell strains with high or low metastasis potential, and found that Tiaml expression level in high-metastatic 95D cells was higher than that in low-metastatic 95C cells. To further confirm the role of Tiam I in invasion and metastasis, we constructed the antisense Tiaml expression plasmid (pcDNA3-anti-Tiaml), which was transfected into 95D cells. A stable transfected clone with decreased Tiaml expression was screened and selected for further research. Transwell assay showed that down-regulation of endogenous Tiam1 by anti-Tiam1 can reduce the in vitro invasiveness of 95D cells. Our results suggested that Tiam1 signaling contributed to the invasion and metastasis of the human giant-cell lung carcinoma cells.     

Plasmodium falciparum: protease inhibitors and inhibition of erythrocyte invasion

Invasion of human red blood cells by Plasmodium falciparum is inhibited by the protease inhibitors, leupeptin and chymostatin. The efficacy of chymostatin was reduced if the cells were first treated with chymotrypsin. On the other hand, exposure of fresh cells to the supernatant from a synchronous culture at the reinvasion stage showed no such effect. This suggests that a proteolytic step occurs in the course of invasion and may be confined to the region of contact between the invading parasite and the erythrocyte. To test this, leupeptin or chymostatin was introduced into lysed cells, which were then resealed. The intracellular inhibitor strongly reduced invasion. Leupeptin also caused a striking effect on the development of the trophozoite stage of the parasites: a massive vacuole, apparently containing undigested haemoglobin, developed within the parasite. This did not totally stop development and the vacuolated parasites could be recovered in relatively pure form by lysis of the parasitised host cells with saponin.     

Basic fibroblast growth factor and transforming growth factor beta-1: Synergistic mediators of angiogenesis in vitro

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC₅₀ of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10–20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley-Liss, Inc.     

The destruction of allogeneic tumour cells by antibody and adherent cells from peritoneal cavities of mice.

Antisera to a DBA2 lymphoma (SL2) were raised in C57 black mice. The sera contained cell-dependent antibodies which lysed SL2 cells in conjunction with a monolayer of adherent peritoneal cells from unimmunised mice. The strongest lytic reaction was observed when the three components of the system, monolayer, target, and antiserum, were incubated together. The free antibody was not cytophilic for macrophages. It combined specifically with the target cell but precoated SL2 cells were not lysed effectively, probably because cells in the monolayer also accelerated the inactivation of antibody on the surface of the target cell.     

How do extracellular pathogens cross the blood-brain barrier?

Bacterial invasion of the meninges can occur as a consequence of bloodstream invasion by some bacterial pathogens. Bacteria enter the central nervous system following a direct interaction with the luminal side of the cerebral endothelium, which constitutes the blood-brain barrier. To breach the barriers protecting the brain, extracellular pathogens must cross a monolayer of tight junction-expressing endothelial or epithelial cells. The limited number of pathogens capable of crossing these tight barriers and invading the meninges suggests that they display very specific attributes. For Neisseria meningitidis, type IV pili have been identified as being essential for meningeal invasion and it is believed other, as-yet-unidentified factors are also involved.     

Effects of Helicobacter pylori on biological characteristics of gastric epithelial cells

Infection with Helicobacter pylori (H. pylori) strains is linked to an increased risk of inflammation and gastric cancer. To investigate the effects of H. pylori on biological characteristics of gastric epithelial cells SGC-7901, derived from human adenocarcinoma, morphological appearances of both the pathogen and these cells, as well as features of attachment and internalization were observed by using transmission electron microscopy (TEM). We also investigated cell junctions and invasion by TEM and Transwell Invasion Assay. Cell proliferation and apoptosis were assessed by using chromogenic methylthiazol tetrazolium bromide (MTT) dye and flow cytometry. Three types of H. pylori were observed around, attaching to, or invading tumor cells. Cellular damage was characterized by vacuolar degeneration, dilated endoplasmic reticulum (ER), and reduction of organelles. Cell junctions and cell microvilli reduced or disappeared. H. pylori inhibited cell proliferation, whereas it had no effect on apoptosis. It also promoted gastric carcinoma cell invasion. H. pylori damages cell construction, destroys cell junctions, inhibits cell proliferation, promotes cell invasive ability, and, therefore, might accelerate the malignant progress and metastasis of gastric cancer.     

Membrane and membrane-associated proteins involved in the aggressive phenotype displayed by highly invasive cancer cells

Invasion, the penetration of tumour cells into adjacent tissues, is a fundamental characteristic of malignant carcinomas and a first step in the metastatic process. The molecular mechanisms involved in tumour cell invasion are complex, but over the last couple of decades the knowledge base has grown quite considerably and many proteins with important roles in invasion have been identified and characterised. Benign tumours typically are encapsulated, which inhibits their ability to behave in a malignant manner, meaning these tumours do not grow in a location-limited less aggressive manner, do not invade surrounding tissues and do not metastasise. The ability of malignant tumours to invade and metastasise is the major cause of death for cancer patients. A greater insight into the molecular basis of cancer invasion and metastasis will lead to the development of novel therapies and specific panels of biomarkers for use in the treatment and diagnosis/monitoring in many types of metastatic cancer.     

IFN-gamma amplifies NFkappaB-dependent Neisseria meningitidis invasion of epithelial cells via specific upregulation of CEA-related cell adhesion molecule 1

Temporal relationship between viral and bacterial infections has been observed, and may arise via the action of virus-induced inflammatory cytokines. These, by upregulating epithelial receptors targeted by bacteria, may encourage greater bacterial infiltration. In this study, human epithelial cells exposed to interferon-gamma but not tumour necrosis factor-alpha or interleukin 1-beta supported increased meningococcal adhesion and invasion. The increase was related to Opa but not Opc or pili adhesin expression. De novo synthesis of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a major Opa receptor, occurred in epithelial cells exposed to the cytokine, or when infected with Opa-expressing bacteria. Cell line-dependent differences in invasion that were observed could be correlated with CEACAM expression levels. There was also evidence for Opa/pili synergism leading to high levels of monolayer infiltration by capsulate bacteria. The use of nuclear factor-kappa B (NFkappaB) inhibitors, diferuloylmethane (curcumin) and SN50, abrogated bacterial infiltration of both untreated and interferon-gamma-treated cells. The studies demonstrate the importance of CEACAMs as mediators of increased cellular invasion under conditions of inflammation and bring to light the potential role of NFkappaB pathway in Opa-mediated invasion by meningococci. The data imply that cell-surface remodelling by virally induced cytokines could be one factor that increases host susceptibility to bacterial infection.