Postmitotic differentiation of colon carcinoma caco-2 cells does not prevent reentry in the cell cycle and tumorigenicity

Our purpose was to analyze whether postmitotic Caco-2 colon cancer cells, although they express most of the differentiation characteristics of terminally differentiated intestinal epithelial cells, still maintain, unlike normal cells, a proliferation potential. Experiments were performed with clone TC7 of the Caco-2 cell line. Dividing TC7 cells are undifferentiated and express detectable levels of thymidylate synthase (TS) and cytochrome P450 1A1 (CYP1A1) mRNAs. When reaching confluence TS and CYP1A1 are downregulated, mitosis is no longer detectable, and differentiation takes place, as demonstrated by appearance and increasing levels of differentiation-associated marker mRNAs (e.g., sucrase-isomaltase (SI), dipeptidylpeptidase-IV (DPP-IV) or GLUT5), increasing activities of sucrase and DPP-IV, and increasing expression, on immunofluorescence analysis, of SI on the surface of the cell layer. Trypsinization and seeding of late postconfluent cells (day 30) expressing complete differentiation results within 24 h in upregulation of TS and CYP1A1, a concomitant and dramatic disappearance of differentiation marker mRNAs associated with a decrease in sucrase and DPP-IV activities, and delayed resumption of cell division. This is followed, after the cells have reached confluence again, by downregulation of TS and CYP1A1 and resumption of cell differentiation. The ability of differentiated cells to dedifferentiate was further confirmed by wounding the cell layer of late postconfluent differentiated cultures: within 24 h following the wound, cells migrate from the wound edge and dedifferentiate, as demonstrated by transmission electron microscopy and disappearance of SI from the cell surface of migrating cells. Late postconfluent differentiated cells were tumorigenic in nude mice. These results raise the question of the validity of the concept of differentiation therapy when applied to colon cancer cells.