Patterns of expression of lineage-specific markers during the in vitro- induced differentiation of HT29 colon carcinoma cells

The four different cell types present in the mature colon, absorptive enterocytes, mucus-secreting goblet cells, Paneth cells, and enteroendocrine cells, are believed to derive from a common precursor, the stem cell, anchored near the base of the crypt. Stem cell descendants undergo several rounds of cell division, creating a pool of transit cells that are committed to differentiation. The mechanisms by which committed cells are allocated to the different cell lineages of the intestine are poorly understood. We have used the colon carcinoma cell line HT29 and Cl.16E cells, a clonal derivative of HT29 cells, to investigate the regulation and pattern of expression of several markers (MUC2, MUC3, carcinoembryonic antigen, and alkaline phosphatase) that are associated with a more differentiated phenotype and that, in the mature cells, are lineage restricted. HT29 cells can express, upon exposure to the appropriate inducers, distinct intestinal specific markers; they are, therefore, considered multipotent, similar to the stem cells of the crypt. Conversely, Cl.16E cells are lineage restricted and respond to cell contact inhibition by expressing a fully differentiated goblet cell phenotype. We show that, in HT29 cells, different inducers (12- O-tetradecanoylphorbol-13-acetate, forskolin, and sodium butyrate) modulate specific sets of markers. Forskolin induces the expression of both MUC2 and MUC3, whereas 12-O-tetradecanoylphorbol-13-acetate is capable of inducing only MUC2, and sodium butyrate, only MUC3 gene expression. Carcinoembryonic antigen, a marker common to enterocytes and goblet cells, can be induced by all the agents, whereas the alkaline phosphatase gene, the expression of which is characteristic of enterocytes, is responsive solely to sodium butyrate treatment. Furthermore, in Cl.16E cells, we show that the expression of MUC2, MUC3, and carcinoembryonic antigen depends upon the cells reaching confluence, yet each marker analyzed displays distinct kinetics of induction. The complexity of the intestinal differentiation process is further emphasized by our data showing that the induction of MUC2 mRNA and protein in HT29 cells is not associated with the production of glycosylated, fully mature mucin. Thus, our data indicate that colonic differentiation programs result from the integration of numerous pathways. Furthermore, in the intestine, there may be transit cells that coexpress markers restricted to different lineages upon maturation.