Human colonic carcinoma Caco-2 cells grown in vitro undergo epithelial differentiation. Electrical measurements showed that they form resistant monolayers of polarized cells. On millipore filters, transepithelial electrical resistance (154 +/- 6.5 omega X cm2) was accompanied by a small potential difference (0.29 +/- 0.02 mV, serosal side positive) and by short-circuit current (1.9 +/- 0.14 microA X cm-2), both of which were ouabain sensitive. Micropuncture of domes formed on plastic supports under standard culture conditions revealed electrical polarity similar to that of filter-grown cells (0.8 +/- 0.2 mV, serosal side positive) combined with a highly negative cytoplasm (-57 +/- 1 mV) and very marked cell asymmetry (76% of total electrical cell resistance was located in the mucosal membrane). These parameters were not affected by the diuretic amiloride nor the hormone aldosterone, suggesting that sodium conductance is very limited in the mucosal membrane. Addition to the mucosal side of the ionophore nystatin or amphotericin B unmasked the possibility of high electrical transport activity. Electrical measurements made it possible to define the epithelial properties of Caco-2 cells, which may resemble those of colonic crypt or fetal cells. These measurements also confirmed that functional differentiation is homogeneous in Caco-2 cells. It is suggested that dome cell micropuncture may be useful in investigating the functional properties of other dome-forming cell lines.

In order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT-29 cells was selected for its capacity to grow in the total absence of sugar. These cells (Glc-cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+ cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase-isomaltase. The differentiation of Glc- cells is reversible: the addition of glucose to postconfluent cultures of Glc- cells results in an inhibiting effect on the expression of sucrase-isomaltase; switching growing cultures of Glc- cells to the Glc+ medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage-related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+ cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+ medium for 20 passages, are switched back to the Glc- medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc- cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of differentiation of HT-29 cells.

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.