INTESTINAL-CELL CYCLE REGULATION - INTERACTIONS OF CYCLIN-D1, CDK4, AND P21(CIP1)

Objective The p21(Cip1) protein is a potent stoichiometric inhibitor o f cyclin-dependent kinase activity, and p21(Cip1) mRNA expression is l ocalized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (IEC s) underwent reversible cell cycle arrest by contact inhibition, and d etermined whether increases in the relative amount of p21 associated w ith Cyclin D/Cdk4 protein complexes were associated with cell growth a rrest. Methods Density arrest was achieved by prolonged culture of IEC -6 in confluent conditions (5 or mote days). Release from density arre st was achieved by detaching the cells from the culture plate and rese eding them at a 1:4 ratio. The DNA synthesis was estimated by [H-3]-th ymidine incorporation and expressed as mean plus or minus standard err or of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein leve ls were determined by standard Northern and Western blot analyses, res pectively. Cyclin D1, Cdk4, and p21 protein complex formation was anal yzed by immunoprecipitating the complexes from cell lysates with an an tibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitat ed complexes using antibodies to the other proteins, The kinase activi ty of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. Results The IEC-6 [H-3]-thymidine incorporation was decr eased 7.5-fold from day 1 of confluence to day 7 of confluence. Twenty -four hours after release from density arrest, there was a 43-fold inc rease in [H-3]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immun oblotting showed that the levels of cyclin D1 and Cdk4 proteins decrea sed by 70.9% and 68.7%, respectively, comparing day 3 with day 9 durin g density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-ar rested cultures, coincident with the increase in DNA synthesis. The am ount of p21 associated with the cyclin D1 and Cdk4 complex in the dens ity-arrested cells was 170% of that observed in the reseeded, prolifer ating cells. More important, the p21::Cdk4 ratio was 6.4-foId higher i n the density-arrested (quiescent) cells as compared with rapidly prol iferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release f rom growth arrest, coincident with decreased binding of p21 to the com plex. Conclusions Intestinal epithelial cells in culture can undergo d ensity-dependent growth arrest. This process involves downregulation o f cyclin D1 and Cdk4 at the level of protein expression, whereas the m RNA levels remain relatively unchanged. Further, during contact inhibi tion, there is more p21 associated with cyclin D1/Cdk4, which further contributes to the inhibition of the kinase complex. The authors also have shown that the process of contact inhibition is reversible, which may explain partly the ability of the intestinal epithelium to increa se proliferative activity in response to injury.