Frans Van Roy

Cloning and characterization of the human invasion suppressor gene E-cadherin (CDH1)

E-cadherin is a Ca(2+)-dependent epithelial cell-cell adhesion molecule. Downregulation of E-cadherin expression often correlates with strong invasive potential and poor prognosis of human carcinomas. By using recombinant lambda phage, cosmid, and P1 phage clones, we isolated the full-length human E-cadherin gene (CDH1). The gene spans a region of approximately 100 kb, and its location on chromosome 16q22.1 was confirmed by FISH analysis. Detailed restriction mapping and partial sequence analysis of the gene allowed us to identify 16 exons and a 65-kb-long intron 2. The intron-exon boundaries are highly conserved in comparison with other classical cadherins. In intron 1 we identified a 5 high-density CpG island that may be implicated in transcription regulation during embryogenesis and malignancy.

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.

The invasive phenotypes.

The expression of the invasive (I+ or I-) phenotypes determines cancer metastasis (M+ or M- phenotype). The invasive (I+ or I-) phenotypes can be divided according to time and site of expression into subphenotypes, which can be assessed separately. At various sites along the metastatic pathway the expression of the I phenotypes can be accompanined by the presence of uncontrolled growth (G+ phenotype) or its absence (G- phenotype). Various combinations of the I and G phenotypes determine the behaviour of metazoan or parasitic cells under normal, pathological non-neoplastic and neoplastic conditions. Although the G+I+M+ combination correlates with full malignancy, the sequence of events leading to the acquisition of these phenotypes during tumor development is not clear. Conditional invasion in experimental systems indicates that a tumor may be invasive and metastatic when part of its population temporarily expresses the I+ phenotype. These experiments further stress the importance of the tumor-host ecosystem for the regulation of the I phenotypes. As distinct from some parasites, the invasive morphotype of vertebrate cells cannot be simply identified. Nevertheless, within the tumor-host ecosystem morphological correlates of the activities of invasive cells may be recognized. They reflect one or more of the I+ functions, namely: motility; loss of homotypic cell-cell adhesion; establishment of alternative cell-substrate and heterotypic cell-cell adhesion; breakdown of extracellular matrices. These functions are not exclusive for I+ tumor cells, and neither are the molecular markers investigated so far. Oncogene activation leads mainly to G+ expression, and in this way serves as a signal amplifier for the I and M phenotypes. Attractive candidate molecular markers of I phenotypes are: regulators of hydrolase activities; cell-cell adhesion molecules; cell surface receptors. From data presently available, we hypothesize that invasion depends upon the balance between an I+ and an I- pathway, with both pathways being sensitive to stimulation and inhibition.

Tumour invasion: effects of cell adhesion and motility

Metastasis is the major cause of failure in cancer therapy. Recent studies of the molecular cell biology of the metastatic process have provided new insights into the mechanisms of cell-cell adhesion, cell-substrate adhesion and cell motility that underly invasion by tumour cells. In this review, Van Roy and Mareel discuss the role of proteins with invasion-promoting and invasion-suppressing functions in metastasis.

Tumor necrosis factor and interleukin 1 activate phospholipase in rat chondrocytes

Tumor necrosis factor (TNF) and interleukin 1 (IL‐1) are both cytokines of macrophage origin with similar activity on several cell types. We investigated whether TNF can, analogously to IL‐1, stimulate phospholipase activity of chondrocytes. Addition of each of these cytokines to cells, isolated from the xiphisternum of adult rats, resulted in a time‐ and dose‐dependent increase in phospholipase activity in both secreted and membrane‐associated form. Moreover, TNF and IL‐1 both induce a transformation of chondrocyte morphology. In conclusion, TNF stimulates chondrocyte phospholipase activity and extends the long list of actions shared by IL‐1 and TNF in a diversity of cellular systems.

Inhibition by glucocorticoids of tumor necrosis factor-mediated cytotoxicity: Evidence against lipocortin involvement

The role of the phospholipase inhibitor proteins, lipocortin‐I and ‐II, in tumor necrosis factor (TNF)‐mediated cytotoxicity against L929 fibrosarcoma cells was investigated. We previously reported that TNF‐mediated cytotoxicity was inhibited by dexamethasone (DEX), suggesting an involvement of lipocortins [1]. Now we show that, despite inhibition by DEX of TNF‐induced arachidonic acid release, DEX has no effect on the synthesis of these lipocortins. Moreover, TNF itself has no effect on the synthesis and phosphorylation of lipocortin‐I and ‐II. Also there was no difference in expression levels of lipocortin‐I and ‐II between TNF‐sensitive and ‐resistant cells. These data strongly suggest that the protective effect of DEX and other glucocorticoids is not mediated by lipocortins.The role of the phospholipase inhibitor proteins, lipocortin-I and -II, in tumor necrosis factor (TNF)-mediated cytotoxicity against L929 fibrosarcoma cells was investigated. We previously reported that TNF-mediated cytotoxicity was inhibited by dexamethasone (DEX), suggesting an involvement of lipocortins [1]. Now we show that, despite inhibition by DEX of TNF-induced arachidonic acid release, DEX has no effect on the synthesis of these lipocortins. Moreover, TNF itself has no effect on the synthesis and phosphorylation of lipocortin-I and -II. Also there was no difference in expression levels of lipocortin-I and -II between TNF-sensitive and -resistant cells. These data strongly suggest that the protective effect of DEX and other glucocorticoids is not mediated by lipocortins.

Lithium chloride potentiates tumor necrosis factor-induced and interleukin 1-induced cytokine and cytokine receptor expression

The antimalignant cell activity of tumor necrosis factor (TNF) in many cell types can be enhanced by lithium chloride (LiCl). This study shows the in vitro effect of LiCl on the TNF-induced or interleukin 1 (IL-1)-induced expression of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-2, and the IL-2 receptor-alpha (IL-2R alpha). The levels of IL-6 and GM-CSF in the medium of TNF-treated L929 fibrosarcoma cells were increased by cotreatment with LiCl. In contrast, enhancement of IL-6 production by dibutyryl cyclic AMP or cycloheximide was not affected by LiCl. The production of IL-6 and GM-CSF was not correlated with sensitivity to TNF-mediated cell killing. IL-1 by itself had no measurable effects on L929 cells. However, LiCl potentiated the IL-1-induced synthesis of IL-6, GM-CSF, IL-3, and IL-2 in PC60 murine T-cell hybridoma cells. TNF alone induced only GM-CSF production in these cells, but in the presence of LiCl, increased amounts of GM-CSF as well as small amounts of IL-2 and IL-6 could be detected. It is also shown that in these PC60 cells the expression of the IL-2R alpha was induced by TNF + LiCl treatment but not by TNF alone. IL-2R alpha expression was likewise considerably enhanced by IL-1 + LiCl treatment, as compared with treatment with IL-1 alone. The effects of LiCl on the TNF-induced and the IL-1-induced gene expression seem to be independent of the protein kinase A and C pathways. These results show that LiCl can modulate both TNF-mediated cytotoxicity and TNF-induced and IL-1-induced cytokine expression, suggesting that Li+ acts early in the TNF-signaling pathway, but at a step shared with the IL-1-signaling pathway.

ISOLATION AND CHARACTERIZATION OF A HUMAN PSEUDOGENE (CTNNAP1) FOR ALPHA-E-CATENIN (CTNNA1) - ASSIGNMENT OF THE PSEUDOGENE TO 5Q22 AND THE ALPHA-E-CATENIN GENE TO 5Q31.

A pseudogene (CTNNAP1) for the human alpha E-catenin gene was isolated from a human genomic phage library. The pseudogene sequence shows 90% similarity to the alpha E-catenin mRNA at the nucleotide level. Thirty-eight stop codons in all three reading frames and multiple other mutations were found, indicating that the pseudogene does not encode a functional protein. No introns were found in the region corresponding to the open reading frame of the alpha E-catenin cDNA, and two direct repeats flank this same region. Hence, the pseudogene can be classified as a processed pseudogene. Polymerase chain reaction with pseudogene-specific primers on genomic DNA and cDNA from human cell lines and healthy blood donors demonstrated the general occurrence of the pseudogene and the lack of its transcription. By fluorescence in situ hybridization the pseudogene was mapped to human chromosome 5q22 and the alpha E-catenin gene to the formerly disputed locus 5q31. This is the first report of a pseudogene for a member of the cadherin-catenin cell-cell adhesion complex.

Qualitative and quantitative analysis of tumour invasion in vivo and in vitro.

SUMMARY Qualitative and quantitative methods for the analysis of invasion in ‘natural’ and in experimental tumours in vivo and in vitro are reviewed. In human tumours the functional consequences of invasion were evaluated histologically through staging on the basis of depths of invasion and through the presence of tumour cells inside vessels. Antibodies against components of the basement membrane have facilitated the definition of minimal invasion. With new probes derived from oncogene research the search for molecular differences between invasive and non-invasive parts of the tumour has begun. Since the same methods as those used for analysis of natural tumours also apply to experimental tumours in vivo, the major advantage of the latter is the possibility of manipulation. We have described a new mesenterium assay that may permit the selection of invasive cells from non-invasive ones in transfection experiments. Invasion relative to growth as a function of time was quantified in the kidney invasion test. In three-dimensional confrontations between embryonic chick heart fragments and invasive cells, we have used both a subjective grading and a qualitative computer-assisted image analysis of serial histological sections to score invasion. In two-dimensional confrontations supplementary methods could be applied, since such confrontations permitted direct observations on living cultures. In a variety of natural and experimental tumours, ultrastructural analysis, transmigration in two-compartment chambers, and release of metabolic label have demonstrated the role of motility and of lytic activity in tumour invasion.

Modulation of cellular susceptibility to the cytotoxic/cytostatic action of tumor necrosis factor by adenovirus E1 gene expression is cell type-dependent

Primary baby rat kidney cells, primary human embryonic retinoblast cells, established NIH3T3 and established normal rat kidney (NRK) cells, expressing E1A and/or E1B gene regions of adenovirus 5 (Ad5) or Ad12, were investigated for susceptibility to the cytotoxic/cytostatic action of Tumor Necrosis Factor (TNF). In the primary cells and in the NRK cells, there was no correlation between TNF sensitivity and E1 gene expression; neither did sensitivity to TNF correlate with the oncogenicity of the Ad serotype. In contrast, the expression of Ad E1 gene regions in NIH3T3 cells was found to enhance TNF sensitivity of this cell line. Differences in E1A expression levels between cell types cannot explain this discrepancy regarding modulation of TNF sensitivity by E1A.