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.