Integrins are transmembrane glycoproteins, composed of noncovalently associated ␣ and ␤ subunits, that are involved in cell-extracellular matrix (ECM) 1 and cell-cell interactions (1). Many integrin ␣ chains undergo a post-translational endoproteolytic cleavage. The ␣ 3 , ␣ 5 , ␣ 6 , ␣ 7 , ␣ 8 , ␣ 9 , ␣ v , and ␣ IIb subunits are cleaved in the membrane-proximal extracellular region, resulting in a heavy chain that is disulfide-linked to a membrane spanning light chain (2). The ␣ 4 and ␣ E subunits can also be cleaved, but at unusual positions, near the middle and in the N-terminal region of the molecule, respectively (3, 4). Endoproteolytic cleavage of integrin ␣ subunits occurs at specific sites comprising pairs of basic amino acids.Post-translational proteolysis is a common mechanism required for the synthesis of biologically active proteins in bacteria, fungi, yeast, invertebrates, and mammals (5). However, the role of endoproteolytic cleavage of integrin ␣ subunits is not clear. The cleavage is conserved, not only in different ␣ chains but also across species, suggesting that it might be of functional importance. It has been established, by site-directed mutagenesis of cleavage sites, that uncleaved ␣ IIb ␤ 3 and ␣ 4

The adhesion receptors of the integrin family play an essential role during tumour progression and thus represent interesting potential targets for the development of new therapeutic agents. The snake venom contains natural inhibitors of integrin-ligand interactions called disintegrins. It also contains C-type lectin proteins mainly known as modulators of platelet aggregation. In this study, we demonstrate that lebectin, a novel C-type lectin isolated from Macrovipera lebetina venom, displayed an anti-integrin activity. Lebectin inhibited the integrin-mediated attachment of various tumour cell lines to different adhesion substrata. The C-type lectin also completely blocked cell migration towards fibronectin in haptotaxis assays and prevented invasion of fibrin gels by tumour cells. In addition, lebectin proved to be a potent inhibitor of tumour cell proliferation. Although the specific integrins affected by lebectin are not identified in this study, the integrin alpha 5 beta 1 might be involved.

A completely defined medium has been designed to promote cell proliferation of 2 colonic adenocarcinoma cell lines of epithelial origin (HT 29 and HRT 18). The spreading of both cell types, especially of HT 29 cells, was not possible in a serum-free medium supplemented with growth factors. Spreading was obtained in a defined medium (a 1/1 mixture of DMEM and F12 media supplemented with 5 micrograms/ml transferrin, 5 ng/ml EGF, 10 ng/ml selenite and 15 mM HEPES pH 7.3) with an extracellular matrix-like material (ECM) secreted by the cells themselves. The properties of the ECM have been studied: ECM secreted by the 2 cell lines induced very quick spreading of HT 29 and HRT 18 cells (1 to 2 hr vs. 12 to 24 hr in serum-supplemented medium). ECM induced morphological differentiation of a rat pheochromocytoma cell line (PC 12). PC-12 cells grown under these conditions began to develop neurite extensions as early as 2 hr after seeding.

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Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days.

We have identified one class of IGF-I-binding sites and two classes of IGF-II-binding sites at the surface of the melanoma cell line IGR39. By means of affinity labeling with 125I-IGF-I, 290-300 kDa form was characterized. Using 125I-IGF-II, a 270 kDa polypeptide was labeled, corresponding to the type II IGF receptor. In the two serials of experiments, the order of potency in inhibiting 125I-IGF-I or 125I-IGF-II labeling of IGF-related peptides and alpha IR3, an antibody directed against type I receptor alpha subunit, was the same as in competition experiments. When IGR39 cells were cultured in a serum-free medium, the number of both high affinity IGF-II and IGF-I binding sites was increased, by 8- and 5-fold respectively, without any significant change in Kd values. In both culture conditions, we found IGFBP-2, -3 -4 and a 30 kDa form which Mr was consistent with IGFBP-5 or -6. Except for IGFBP-2, the amount of secreted IGFBPs was modified depending on culture conditions: in conditioned medium from cells cultured with 10% FCS, the amount of IGFBP-3 or -4 was higher, and the amount of the 30 kDa IGFBP was lower when compared to conditioned medium from cells cultured in serum-free medium.