Biomedical subjects

H Verschueren

Publications and source records attributed to H Verschueren.

At least 37 records · Page 2Linked to original sources

PubMed1994

The role of the spleen in the organ-specific metastasis of murine BW 5147 T lymphomas.

Organ-specific metastasis of tumour cells may result from selective invasion and growth or from selective host cell responses. The present study demonstrates how selective interactions with the host affect the metastatic pattern of two murine T cell hybridoma lines, derived from the BW 5147 thymoma. Upon intravenous inoculation into syngeneic mice BW-14 cells preferentially colonize the kidneys, whereas BW-19 cells metastasize mainly to the spleen and the liver. The organ-specific behaviour of the two cell lines appears to be determined by a differential interaction with the spleen microenvironment. Inoculation of BW-14 cells into splenectomized mice results in increased liver colonization, indicating a negative effect of the spleen on BW-14 tumour development in the liver. Macrophages are likely to be involved in this inhibition, since inoculation of BW-14 cells into macrophage-depleted mice also leads to increased liver and spleen metastasis. In contrast, inoculation of BW-19 cells into splenectomized mice results in decreased liver metastasis, which indicates that the spleen exerts a stimulating effect on BW-19 cells. Macrophages also appear to be involved in this stimulation, since macrophage depletion causes a similar decrease in liver and spleen colonization. Hence components of the splenic microenvironment, probably macrophages, exert inhibiting or stimulating activities on BW-14 or BW-19 cells respectively, thereby determining the subsequent liver or kidney colonization.

Animals↗

PubMed1994

Tumorigenicity of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Kk antigens.

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2Kk antigen expression. This suggested that H-2Kk antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2Kk expression by gene transfection. Transfected cells expressing a high level of H-2Kk antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selection in vivo for cells expressing a reduced level of H-2Kk antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2Kk expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2Kk transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2Kk transfectants is due to an immune rejection mechanism, mediated by CD8+ immune effector cells, as revealed by in vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2Kk antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity.

Animals↗

PubMed1993

Methods for computer assisted analysis of lymphoid cell shape and motility, including Fourier analysis of cell outlines.

Locomotion of lymphocytes and other leukocytes is an essential feature of the immune system, and therefore the evaluation of the locomotor behaviour of a lymphocyte population is part of its functional analysis. Paradoxically, the locomotor status of leukocytes is usually assessed on the basis of static information, by counting the number of spherical versus non-spherical cells. In this paper we describe two methods for the measurement of shape changes in microscopic images of lymphoid cells. First we computed a simple shape change factor, coined incongruence factor, based on the degree of non-overlap of the contours of the cell at the beginning and at the end of a 1 min time interval. Second we have used Fourier analysis of the cell outline: a function describing the undulations of the cell outline is broken down into sinusoidal 'waves' of increasing frequency, each with its corresponding amplitude. The amplitude values for the first ten frequencies produced a satisfactory mathematical description of lymphoid cell shapes, and the change of these amplitudes over a 1 minute time interval produced a quantitative description of the shape alterations of the cells. We have used five approaches to evaluate the shape and shape changes in the following populations of mouse lymphoma cells: a constitutively low-motile T lymphoma cell line (BW5147), a high-motile hybridoma (BW-O-Li1) either on plastic or on a precultured fibroblast-like monolayer, BW-O-Li1 cells after penetration through the monolayer, and BW-O-Li1 cells after treatment with cytochalasin B. We compare the results from direct visual evaluation of cell shape, from computer assisted assessment of sphericity and from Fourier analysis of cell shape at one moment, with the two methods for quantitative shape change analysis. All approaches revealed a clear distinction between spherical low-motile populations, and non-spherical high-motile cells. Moreover, the incongruence factor proved to be a reliable single parameter of active cell deformation. In addition, the Fourier analysis of cell outlines produced useful measures of static shape and of dynamic shape change, at any user-defined level of accuracy.

Animals↗

PubMed1993

Expression of L1 cell adhesion molecule is associated with lymphoma growth and metastasis.

The cell adhesion molecule (CAM) L1 is involved in homotypic and heterotypic adhesion between neural cells. It has recently also been identified on leucocytes. We have investigated the expression of L1 on hematopoietic tumor cell lines and found that several tumors including the ESb-MP lymphoma are positive for L1. A potential role for L1 in spontaneous metastasis formation was examined using these cells. From wild-type (wt) L1high lymphoma cells we selected by a fluorescence-activated cell sorter (FACS) stable L1low expression variants. Syngeneic DBA/2 mice injected subcutaneously with L1low clones showed faster primary tumor growth, developed visceral metastases significantly faster and died earlier than animals carrying L1high wt cells. L1 high revertants from the L1low variants showed again a reduced metastatic capacity and a malignancy similar to the wt cells. Expression of L1 on the tumor variants and revertants correlated directly with their homotypic aggregation behaviour in vitro. L1 expression correlated negatively with metastatic capacity. These results suggest that L1 molecules may contribute to the overall malignant potential of the lymphoma cells, presumably by interfering with cell-cell interactions critical for tumor growth and dissemination.

Animals↗

PubMed1991

The lymphocytosis promoting action of pertussis toxin can be mimicked in vitro. Holotoxin but not the B subunit inhibits invasion of human T lymphoma cells through fibroblast monolayers.

Pertussis toxin is known to elicit lymphocytosis in whooping cough patients and experimental animals, by blocking the extravasation of lymphocytes and stimulating their release from lymphoid organs such as the thymus. The mechanisms responsible for these unique effects of PT are not fully understood. The effect of pertussis toxin (PT) on the invasive behavior of human CCRF-CEM T lymphoma cells has been investigated with the use of a monolayer invasion assay (MIA). We had previously found that invasion of murine T lymphoma cells in this model system was correlated with their ability to extravasate and form metastases after i.v. injection in syngeneic animals. We now show that human CEM cells can also penetrate through a precultured confluent monolayer of murine 10T1/2 fibroblast-like cells within a few hours. In a quantitative MIA run over 24 h, PT at concentrations above 10(-14) M inhibited invasion of the CEM cells. In addition, PT stimulated the release ('evasion') of CEM cells that had invaded under the monolayer before the toxin was added. The A subunit of PT was totally inactive, the B subunit had a small residual effect, and reconstitution of the AB complex partially restored the activity. The invasion-inhibiting activity of two different holotoxin preparations and of the subunits perfectly matched their activity in the Chinese hamster ovary cell clustering assay, which is known to depend on a functional AB complex. We suggest that inhibition of monolayer invasion by PT can be used as an in vitro model system to investigate the cellular and molecular mechanisms underlying the lymphocytosis-promoting action of the toxin. Furthermore, the method is sufficiently sensitive to be used for titration of toxin activity. Our data indicate that the ADP-ribosylating activity of the A subunit is indeed required, and that the promotion of lymphocytosis is not elicited by the binding of the B subunit alone.

Cells, Cultured↗

PubMed1991

Dynamic morphology of metastatic mouse T-lymphoma cells invading through monolayers of 10T1/2 cells.

We have used an in vitro model system to analyze cytomechanical aspects of tissue infiltration by T-lymphocytes. The interaction of metastatic T-lymphoma cells with a precultured monolayer of 10T1/2 fibroblast-like cells was recorded in time-lapse video with alternating phase contrast and reflection interference contrast microscopy. Sectioning of embedded specimens as well as cytoskeletal stainings have been performed on matching cocultures. The lymphoma cells did not strongly attach or spread on the dorsal surface of the monolayer cells. Invasion started with the protrusion of a pseudopodium through a narrow gap, and conspicuous constriction of the invading cell's body and nucleus was a consistent feature during the later steps. Overt retraction of the target cells was not seen, but the invading lymphoma cells elevated the fibroblasts over relatively large areas, thereby creating dome-shaped open spaces, allowing for further migration under the monolayer with minimal resistance. Invasion was not unidirectional but was readily reversible at any stage. Due to this wavering character, an invasion event could take more than 1 hour, although the shape alterations involved were fast. Even after the invasion process had been completed, the lymphoma cells could come out from below the monolayer again. Therefore we propose that invasion in this model should be considered as a dynamic equilibrium. Invading T-lymphoma cells displayed diffuse F-actin staining and a well-organized microtubular complex with the centrosomes behind the nucleus in the uropod, which also contained most vesicular organelles.

Actins↗

PubMed1990

Identification of T-cell antigens expressed by metastatic T-cell hybridomas and lymphomas.

Using the murine BW5147 tumor model system, we have identified 3 MAbs that discriminate between metastatic and non-metastatic BW5147-derived T-cell hybridomas and BW5147-unrelated T-lymphomas. The 3 rat MAbs appear to recognize an identical membrane-associated sialoglycoprotein with an approximate molecular weight of 95-100 kDa. We thus defined "metastatic T-cell hybridoma" antigens (MTH-Ags) that are also expressed on normal T-lymphocytes. No correlation was found between the expression of the MTH Ags and in vitro invasive behavior of normal and malignant cells. Neither did we find any relationship between organ specificity of i.v. inoculated tumor cells and their MTH-Ags expression. It thus remains unclear whether our MTH-Ags are functionally involved in the metastatic process, or whether their expression is only incidentally related to the metastatic potential.

Animals↗

PubMed1990

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.

Alprostadil↗

PubMed1990

Association between MHC class I antigen expression and malignancy of murine T lymphoma variants.

Tumor cell variants were derived from an AKR T-cell lymphoma cell line (BW5147, H-2k haplotype). These variants differed in their malignant potential and in their membrane expression of class I MHC antigens. High tumorigenic and spontaneous metastatic capacity was found to be predominantly associated with a decrease of H-2Kk class I major histocompatibility complex (MHC) antigen expression. In contrast, high experimental metastatic capacity correlated strongly with an increased H-2Dk antigen expression. The in vitro invasive potential and the LFA-1 expression of the BW variants showed no correlation with the differential MHC antigen expression and the differential metastatic and tumorigenic capacity of the BW variants. Furthermore, the susceptibility of the BW 5147 variants to TNF and NK-mediated cytotoxicity was not related to the differential metastatic potential and the expression of the class I MHC antigens.

Animals↗

PubMed1989

Suggestive evidence that genes controlling invasion and metastasis of T-cell lymphomas are located on mouse chromosome 3.

Cell lines differing in their malignant potential have been derived from the murine BW5147 T-cell lymphosarcoma. To evaluate the involvement of chromosomal aberrations in tumor progression within this model, we have analyzed the karyotypes and the in vitro invasiveness of 13 related nonmetastatic and metastatic variants. Giemsa banding revealed the presence of several marker chromosomes, one of which was of particular importance. Depending on the cell line, four variants of this marker I were found: Marker Ia corresponds to two translocated chromosomes 3, marker Ib is a deleted Ia marker, marker Ic is a Ib translocated to small unidentified chromosome fragment, and marker Id is a further deleted Ib marker. The Ia and Id markers were characteristic for the noninvasive, nonmetastatic lines, whereas the Ib and Ic markers predominated in the invasive, metastatic variants. The results suggest that metastasis-enhancing genes are located between the D and FI band of mouse chromosome 3 and that metastasis-suppressing genes are located between the FI and H band of the same chromosome.

Animals↗

PubMed1989

The in vitro invasive behavior of human myelomonocytic cell lines is modulated by HuIFN-gamma and pertussis toxin.

Using an in vitro monolayer invasion assay (MIA), we analyzed the interaction of human myeloid cell lines representing different maturation stages with murine fibroblastic monolayers. After 24 h of coculture, only promonocytic U937 cells invaded the monolayer to an appreciable extent in contrast to less-differentiated KG1a, KG1, and HL-60 cells and more mature THP-1 cells. Human interferon-gamma (HuIFN-gamma) treatment was found to induce maturation in the U937 line and resulted in a considerable reduction of interaction with the monolayer. Thus, the capacity of myeloid cells to interact with a fibroblastic monolayer is restricted to a specific maturation stage. Interaction of U937 cells was also abolished when they were treated with pertussis toxin (PT), an agent known to induce monocytosis in vivo, indicating that the MIA may serve as an in vitro simulation of the extravasation of blood borne cells. Finally, although both HuIFN-gamma and PT are able to block cell spreading in the MIA, no effect could be seen on the capacity of U937 cells to phagocytose.

Humans↗

PubMed1988

Syngeneic in vivo passage of the murine BW 5147 lymphoma results in the expression of a stable metastatic phenotype.

BW 5147 lymphoma cells are non-invasive tumor cells which do not generate experimental metastases following i.v. inoculation. In contrast, s.c. and intra-splenic (i.s.) growth of BW cells resulted in widespread colonization of liver and spleen. Cells derived from either s.c. tumors or metastatic lesions did generate metastases after i.v. administration. The capacity of these tumor-derived BW cells to disseminate via blood-borne cells was irreversible and stable, indicating that one in vivo passage of BW cells results in the generation of new, metastatic BW variants. Concomitantly, these variants exhibited an inherent invasive potential as manifested by their capacity to infiltrate in vitro monolayers of hepatocytes and fibroblasts. The BW variants expressed new membrane markers such as H-2 antigens, the Lyt 1.2 T-cell differentiation antigen and the MTH antigen (a newly defined membrane antigen expressed predominantly on murine metastatic T-cell lymphomas and mature T lymphocytes). This phenomenon was observed with both cloned and uncloned BW populations, suggesting that an inductive rather than a selective mechanism accounts for the transition of BW cells towards a more malignant phenotype. These observations confirm the concept that local factors at the growth site of a tumor might influence the metastatic behavior of that tumor, possibly via induction of silent differentiation programs.

Animals↗

PubMed1988

Difference in motile behavior between lymphoma variants with different invasive and metastatic capabilities.

The motile behavior of two tumor cell variants of the murine BW 5147 lymphosarcoma line, displaying different metastatic capabilities, was analyzed. When placed on top of a confluent monocellular layer of fibroblastic cells, the nonmetastatic lymphoma cells did not carry out any appreciable translocation or shape modification, whereas the metastatic cells displayed intense pseudopodal activity and performed positional shifts. Both these aspects of cell motility were approached through quantitative assays, demonstrating a highly significant difference between the two variant lines. In addition, the metastatic cells were shown to penetrate underneath the fibroblastic monolayer, whereas the nonmetastatic cells were unable to invade. We suggest that the difference in motile behavior is at the basis of the different invasive potencies of the variants. Since in vitro monolayer invasion assays mimic the extravasation of blood-borne cells, we further speculate that in this particular model system, cell motility is the discriminating property that determines whether disseminated tumor formation will occur after intravenous injection of either cell line.

Animals↗

PubMed1987

Development of a monolayer invasion assay for the discrimination and isolation of metastatic lymphoma cells.

A new in vitro invasion model system, based on monolayers of 10T1/2 fibroblastic mouse embryo cells, is presented. When inoculated onto such a confluent monolayer, cells from a nonmetastatic T-cell lymphoma line remained on top, whereas cells from metastatic derivative lines penetrated through the monolayer. Combined differential interference contrast and interference reflection microscopy were used to follow the relative vertical cell positions in the co-cultures. The number of underlying lymphoma cells per unit area can be used as a quantitative invasion index, allowing a systematic comparison between cell lines. Moreover, since the invasive cells could be recovered separately, this invasion system allowed the isolation of minor subpopulation of invasive cells from a 1000-fold excess of noninvasive cells.

Animals↗

PubMed1985

The effect of tumor promoters and antagonists on in vitro directional migration of 10T1/2 mouse embryo cells.

In vitro directional migration of 10T1/2 fibroblasts is partially inhibited by TPA but not by its non-promoting analogues. Other tumor promoters, e.g., phenobarbital, saccharin, and benzoylperoxide had no measurable effect when added in concentrations known to affect in vitro two-step transformation or intercellular communication. Inhibitors of in vitro transformation do not affect migration, except for dexamethasone, which inhibited it. Hence, there is no evidence for a general correlation between tumor promoting potential and inhibition of in vitro directional migration.

Animals↗

PubMed1985

Interference reflection microscopy in cell biology: methodology and applications.

Since its introduction into cell biology by Curtis in 1964, interference reflection microscopy (IRM) has been used by an increasing number of researchers to study cell-substrate interactions in living cells in culture. With the use of antiflex objectives, high-contrast IRM images can now be readily obtained. From the different theories on image formation in IRM that have been put forward, it can be seen that a zero-order interference pattern is generated at high illuminating numerical aperture. This yields information on the closeness of contact between cell and substrate, with only minor perturbation by reflections from the dorsal cell surface. Therefore, the proper use of illuminating apertures is crucial. Nevertheless, IRM images have to be interpreted with caution, especially under thin cytoplasmic sheets. Quantitative IRM is possible only with a mathematical model for finite illuminating aperture interferometry and with an independent measurement of cell thickness for values up to 1 micron. IRM has been applied qualitatively to a large number of cell types, and it seems that there are two universal types of adhesion. Focal contacts are small regions of closest cell-substrate apposition, possibly of immediate contact, that are associated with the distal end of actin filament bundles. They are firm attachment structures that hold the cell in place and in its spread shape. Close contacts are broad areas of reduced cell-to-substrate distance. They are weaker but highly dynamic adhesions that sustain rapid movements of cells or cell parts over the substrate. Although a number of independent observations suggest that adhesion patterns of malignantly transformed cells differ from those of their normal counterparts, there is no simple correlation between malignancy in vivo and altered contact formation in vitro. The adhesion pattern seems to be determined by the locomotory state of the cells rather than by their tissue of origin. Finally, IRM can also be used to enhance contrast in images of fixed preparations.

Animals↗

PubMed1984

A new model for the quantitative analysis of cell movements in vitro: definition of a shape change factor.

Changes in shape, in addition to translocations, are an important aspect of cell motility. We propose a simple geometrical model for the quantitative analysis of shape changes undergone by cultured cells. The extent to which images of a given cell do not overlap at the beginning and end of a time interval is used as a measure of motility, and a translation step included to eliminate translocation effects. Initial findings suggest that the method is widely applicable.

Animals↗

PubMed1984

Dipyridamole reduces the motility of 10T1/2 cells and disturbs their stress fibre pattern.

Dipyridamole (DPD), a widely used anti-thrombotic agent, inhibits in vitro directional migration of 10T1/2 mouse embryo cells in a dose dependent way. The inhibition is detectable at DPD concentrations comparable to those in human plasma after oral intake of the drug. Using a model for quantitative shape change analysis, it is demonstrated that DPD inhibits pseudopodal activity and stabilizes the outlines of 10T1/2 cells. The inhibition of pseudopodal activity is thought to be involved in the reduced migratory capacity and the decreased area occupied by DPD treated cells. Coomassie blue staining of detergent-extracted cells shows that stress fibre patterns in 10T1/2 cells are altered by DPD. In particular, highly regular, polygonal networks, reminiscent of those described in spreading cells, appear after DPD treatment. It is suggested that interference of DPD with the normal organization of the actin microfilament system accounts for its effects on spreading, motility, and growth of cultured cells, and possibly for part of its in vivo anti-thrombotic activity as well.

Animals↗