Background and Aims: We have previously shown that autocrine insulin- like growth factor (IGF)-II synthesis through IGF-I receptor stimulates proliferation and inhibits differentiation of Caco-2 cells. To demonstrate whether differentiation of Caco-2 cells is dependent on cell growth status, we analyzed the effect of cell cycle arrest on differentiation of wild-type and IGF-II-overexpressing cells. Methods: Cells were treated with drugs that inhibit the progression either to S phase (I-b-D-arabinofuranosylcytosine or M phase (nocodazole). Cell differentiation was analyzed by assessing apolipoprotein A-1 and sucrase-isomaltase expression. Cell proliferation and DNA content were assessed by thyroidine incorporation and fluorescence- activated cell sorter analysis, respectively. Cell cycle regulatory molecules were analyzed by assessing p21 and retinoplasma protein (pRb) expression and pRb phosphorylation. Results: Cell cycle block at G1-S phase was associated with increased expression of differentiation markers in both parental and IGF-II-transfected cells. On the contrary, cell cycle arrest at G2-M phase correlated with the expression of differentiation markers in parental but not in IGF-II-transfected cells. Constitutive IGF-II-expressing cells actively incorporated thymidine and showed an increase in the proportion of cells with >4N DNA ploidy in the presence of nocodazole. Nocodazole treatment of constitutive IGF-II-expressing cells stimulated p21 expression in the presence of hyperphosphorylated pRb. Conclusions: The data show that cell cycle arrest increases differentiation of Caco-2 cells. IGF-II-mediated proliferation may prevent cell differentiation through effects on control cell checkpoint proteins.
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