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The role of calcium and extracellular materials in the in vitro growth and contractility of carcinoma cells Lee, Hong-Chao


Epithelial morphogenesis i n many organs depends on coordinated, microfilament-mediated cellular contractions. Effects of ionophores and Ion transport inhibitors have shown indirectly that these contractions are calcium dependent. To determine if extracellular calcium is required for contraction and to determine by a direct method whether epithelial cell contraction is associated with an increase in intracellular calcium contraction, contractions were induced in vitro in monolayers of an epithelial cell line, C-4II cells, in the absence of heterologous tissues. Cytoplasmic microfilaments have been implicated as the contractile organelles responsible for the convolution of C-4II cell sheets into structures resembling epithelia. The "Lanthanum method", used to differentiate between extracellular and intracellular calcium, developed originally for studies of muscle contraction, was adapted to the study of cultured epithelial cells. The method is based on removal of extracellular calcium by lanthanum and inhibition of calcium flux by lanthanum and cold. Prior to contraction, intracellular calcium concentrations ranged from 0.22 to 0.68 μg/mg protein. These values were influenced by the physiological state of the cells, by dissociation procedures used for subculture and by the concentration of calcium in the medium. Upon removal from the substratum, cell sheets contracted gradually and, concomitantly, there was a highly significant increase in intracellular calcium (1.02 to 4.33 μg/mg protein). Ionophore A23187 did not increase the rate of contraction. In Ca⁺⁺ -free medium the cells did not contract but released calcium to the medium. Contraction was inhibited by 10⁻⁵ M LaCl₃. These results show directly that contraction of epithelial cells depends on influx of extracellular calcium. These observations support the theory that contraction of non-muscle cells may be controlled by the calcium content of the cells, as is contraction in muscle cells. In vivo, the intracellular polarity of epithelia, i.e. the asymmetric distribution of organelles along the apical-basal axis, may be induced by interactions of the developing epithelia with heterologous tissues and specialized components of extracellular materials (e.g. basal lamina) at the basal surface. The behavior of the C-4II cultures shows that such polarity can also be established autonomously by isolated epithelial cell populations, in vitro. To determine whether this polarity was associated with an asymmetric distribution of extracellular glycosaminoglycans, glucosamine incorporation into extracellular materials by C-4II cells was examined autoradiographically. In addition, basal lamina formation was examined histochemically and electron microscopically. Although C-4II cells form basal lamina in vivo, they did not form any in vitro under the conditions tested. Autoradiographic studies showed that there was an asymmetric distribution of glycosaminoglycans between the apical and basal sides of the cells, in vitro. It follows, that the establishment of polarity in cytofilament distribution and contractility in these cells may depend on a gradient in amounts or concentration of glycosaminoglycan-containing materials, but not on the presence of a structured basal lamina.

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