Caco-2 intestinal cell differentiation is associated with G₁ arrest and suppression of CDK2 and CDK4

The cellular mechanisms regulating intestinal proliferation and differentiation remain largely undefined. Previously, we showed an early induction of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1) in Caco-2 cells, a human colon cancer line that spontaneously differentiates into a small bowel phenotype. The purpose of our present study was to assess the timing of cell cycle arrest in relation to differentiation in Caco-2 cells and to examine the mechanisms responsible for CDK inactivation. Caco-2 cells undergo a relative G₁/S block and cease to proliferate at day 3 postconfluency; an increase in the activity of terminally differentiated brush-border enzymes (sucrase and alkaline phosphatase) was noted at day 6 postconfluency. Cell cycle block was associated with suppression of both CDK2 and CDK4 activities, which are important for G₁/S progression. Treatment of the CDK immune complexes with the detergent deoxycholate (DOC) resulted in restoration of CDK2, but not CDK4, activity at day 3 postconfluency, suggesting the presence of inhibitory protein(s) binding to the cyclin/CDK2 complex at this time point. An increased binding of p21(Waf1/Cip1) to CDK2 complexes at day 3 postconfluency was noted, suggesting a potential role for p21(Waf1/Cip1) in CDK2 inactivation; however, immunodepletion of p21(Waf1/Cip1) from Caco-2 protein extracts demonstrated that p21(Waf1/Cip1) is only partially responsible for CDK2 suppression at day 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears to contribute to the CDK2 inactivation noted at days 6 and 12 postconfluency. Taken together, our results suggest that multiple mechanisms contribute to CDK suppression during Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leads to G₁ arrest and inhibition of proliferation that precede Caco-2 cell differentiation.