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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.

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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.

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Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3':5'-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3 X 10-12 M. Maximum VIP-induced cAMP levels were observed with 10-9 M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3 X 10-1°M VIP. 125I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10-10 and 10-7 M. Half-maximum inhibition of binding was observed with 2 X 10-9 M VIP. For experimental studies, 3-4 X 106 cells were transfered into 150-cm2 flasks in 30 ml of culture medium. The medium was changed on day 4, and 3 days later, cells were collected with the buffer described above, without trypsin. Under such conditions, the cells were in exponential growth phase. The average yield was 30 X 106 cells per flask. The cells were washed three times in Krebs-Ringer phosphate buffer, pH 7.4, containing 2%

The complete sequence of the 6 kb cDNA and the 5' genomic structure are reported for the gene coding for the human intestinal brush border hydrolase sucrase-isomaltase. The human sucrase-isomaltase cDNA shows a high level of identity (83%) with that of the rabbit enzyme, indicating that the protein shares the same structural domains in both species. In addition to the previously reported homology with lysosomal alpha-glucosidase, the sucrase and isomaltase subunits also appear to be homologous to a yeast glucoamylase. A 14 kb human genomic clone has been isolated which includes the first three exons and the first two introns of the gene, as well as 9.5 kb 5' to the major start site of transcription. The first exon comprises 62 bp of untranslated sequence and the second starts exactly at the initiation ATG codon. Typical CAAT and TATA boxes are seen upstream of the first exon. A genetic polymorphism is described which involves a PstI site in the second intron. Southern blotting, sequencing and mRNA studies indicate that the structures of the sucrase-isomaltase gene and its mRNA are unaltered in the two human colon cancer cell lines Caco-2 and HT-29 in comparison with normal human small intestine.