INTRODUCTION Colon cancer is a consequence of the accumulation of genetic alterations that lead to an abnormal balance in the events that regulate intestinal epithelial cell growth and differentiation (Fearon and Vogelstein, 1990). In vitro, the study of these processes has been hampered by the lack of reproducible methods for the culture of normal intestinal epithelium and the undifferentiated phenotype of most col- orectal cancer cell lines (Neutra and Louvard, 1991; Zweibaum et al., 1991). Recently, intestinal epithelial cell lines that are able to differentiate in culture into goblet or absorptive cells have been described (Neutra and Louvard, 1991; Zweibaum et al., 1991). HT-29 M6 is a subpopula- tion of the HT-29 cell line that is isolated by its ability to grow in medium supplemented with 10 -6 methotrexate (Lesuffleur et al., 1990)*. After confluence, HT-29 M6 cells develop a polarized phenotype, with a moderately well defined apical brush border and a cytoplasm essentially full of mucous droplets (Lesuffleur et al., 1990, 1991). Prolif- erating HT-29 M6 cells resemble pre-differentiated colon crypt cells that are committed to differentiate into goblet cells (Lesuffleur et al., 1990, 1991). Thus, although HT-29 M6 cells are derived from a tumour, the characteristics described above make them a suitable model with which to identify the molecular mechanisms controlling epithelial cell proliferation and differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent activator of protein kinase C (PK-C), its specific cellular receptor (Blumberg, 1988; Jaken, 1990). Great interest in this phorbol ester has evolved from its use in carcinogenesis models, in particular in skin and in the gastrointestinal tract (Boutwell, 1974). In vivo carcinogen- esis models have shown that tumour development proceeds in two distinct stages: initiation, which involves a genetic change caused by a mutagen; and promotion, an amplifi- cation of the mutated cell population as the result of the stimulation of cell proliferation by a ‘promoter’. TPA falls in this second category of molecules (Goerttler et al., 1979). Moreover, a role for other activators of PK-C in the devel- opment and/or progression of colon cancer has been suggested (Weinstein, 1991). To characterize further the role of PK-C in intestinal epithelial cells, we have investi- 513 Journal of Cell Science 106, 513-522 (1993) Printed in Great Britain © The Company of Biologists Limited 1993 The effects of tumor promoter 12-O-tetradecanoylphor- bol-13-acetate (TPA) on the growth characteristics of the colon cancer cell line HT-29 M6 were studied. TPA induced the scattering of proliferative HT-29 M6 cells: in the presence of the phorbol ester, HT-29 M6 colonies scattered and the cells acquired a flatter aspect with diminished cell-cell contacts. This effect of TPA required a persistent activation of PK-C and was acom- panied by a slight decrease (30%) in the growth rate. Modifications by TPA of two scattering associated prop- erties of these cells were also detected: TPA decreased cell-to-cell aggregation and enhanced the cellular attachment to matrix substrata (collagen, laminin). The decrease in cell-to-cell adhesion was correlated with a loss of cellular E-cadherin as evidenced by immunoflu- orescence or immunoblotting with a specific monoclonal antibody. Cell scattering was dependent on the extra- cellular concentration of Ca 2+ ; an increase from 1.6 to 10 mM in the concentration of this ion completely blocked the morphological effects of TPA as well as its action on cell aggregation. This high concentration of Ca 2+ also prevented the down modulation of E-cadherin as determined by immunofluorescence. However, the TPA-induced increase in cell attachment to the matrix was not affected by high calcium. These findings sup- port the importance of altered cell-cell adhesion in the process of scattering and provide a good system for the study of down modulation of E-cadherin, a protein involved in the control of cell growth, differentiation and invasion of epithelial cells. Key words: HT-29 cells, TPA tumor promoter, E-cadherin, cell scattering, calcium SUMMARY Phorbol ester-induced scattering of HT-29 human intestinal cancer cells is associated with down-modulation of E-cadherin Myriam Fabre and Antonio García de Herreros* Departament d’Immunologia, Institut Municipal d’Investigació Mèdica, Universitat Autònoma de Barcelona, Spain *Author for correspondence *For the sake of brevity we have given the name of HT-29 M6 to the cell line previously named HT-29 (10-6 MTX) (Lesuffleur et al., 1990)