Jean-Baptiste Rognoni

α2β1-integrin signaling by itself controls G1/S transition in a human adenocarcinoma cell line (Caco-2): implication of NADPH oxidase-dependent production of ROS

In this work, we report that type IV collagen, mainly via alpha2beta1-integrin ligation, was able to induce cyclin expression and G1/S transition in a colic adenocarcinoma cell line (Caco-2) cultured without soluble growth factors or fetal bovine serum. This process involved Erk 1/2 activation and the production of reactive oxygen species (ROS) by a membrane-bound NADPH oxidase. Data presented here show that NADPH oxidase-dependent production of ROS increased following alpha2beta1-integrin ligation with type IV collagen or with a specific monoclonal antibody (Gi9 mAb). NADPH oxidase activation and, therefore, the production of ROS were shown to be involved in the increase of alpha2beta1-integrin plasma membrane expression, p38 MAPK phosphorylation, cyclin expression, and G1/S transition. We thus identified in this work a new integrin-signaling pathway in colon tumor cells involved in cell cycle regulation by the extracellular matrix.

Up‐regulation of α2β1 integrin cell‐surface expresssion protects A431 cells from epidermal growth factor–induced apoptosis

High epidermal growth factor (EGF) concentration (10–8 M) induces inhibition of A431 cell proliferation, resulting in part from an apoptotic process. For some cells escaping this process, proliferation was associated with a decrease in apoptosis. Moreover, these surviving cells displayed marked morphological changes consisting of filopodia formation and cell aggregation. Disrupting cell–cell contacts by lowering extracellular calcium concentration reversed the resistance process, suggesting that apoptosis protection by aggregation may involve intercellular adhesion and cell–cell survival signals probably mediated by calcium‐requiring molecules such as integrins. From a panel of integrins tested, only α2β1 integrin cell‐surface expression was up‐regulated after high apoptotic EGF treatment, and this up‐regulation was not observed under a growth‐stimulatory EGF concentration (10–11 M). Double‐labeling analysis (α2β1/DNA) implicated α2β1 integrin in the resistance process since 99% of cells that up‐regulated α2β1 integrin survived a high dose of EGF. Moreover, the involvement of α2β1 integrin up‐regulation in the survival of A431 cells that escape EGF‐induced apoptosis was verified using the blocking anti‐α2β1 integrin antibody, which was shown to decrease the survival of EGF‐stimulated cells. Furthermore, under our culture conditions, α2β1 integrin–dependent cell–cell adhesion can be inhibited without affecting other cell‐adhesive interactions, suggesting that α2β1 integrin is involved more directly in cell–cell interaction than in cell–substrate adhesion. Our results provide evidence that EGF‐induced up‐regulation of α2β1 integrin contributes to the enhancement of cell–cell adhesion, leading to cell aggregate formation, which permits the escape of A431 cells to EGF‐induced death by α2β1 integrin signaling. Int. J. Cancer 87:360–367, 2000.

Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.