(423h) Dose-Dependent p21(Cip1/Waf1) Cell Cycle Regulation

p21(Cip1/Waf1) is a member of the Cyclin-dependent kinase inhibitor (CKI) class of proteins involved in arresting the cell cycle in response to DNA damage.  Previous studies have shown that the N-terminal domain of p21 is involved in binding to and inhibiting the activity of Cyclin-CDK complexes, and the C-terminal domain of p21 binds to and inhibits proliferating cell nuclear antigen (PCNA), a DNA polymerase auxiliary factor involved in DNA synthesis and repair. 

Our lab has developed genetic tools and methods to evaluate cellular responses over varying levels of gene expression.  Recently, we have developed a library of expression vectors that control the level of protein translation.  We have used this vector library to express exogenous p21 levels in an Hct116 human colon cancer cell line in which the endogenous p21 has been inactivated by gene-targeting.  We were able to generate a full range of p21 expression levels, including those greater than the p21 levels induced by DNA damaging agents and those below levels observed in undamaged wild-type Hct116 cells. This wide range of expression allows us to monitor the behavior of cells exposed to varying amounts of p21, including levels that are physiologically relevant. 

Our results show that cells expressing exogenous p21 are not actively cycling through S phase.  To  explore the underlying mechanism of p21 expression strength affecting cell cycle arrest, we made two mutant versions of the p21 gene—one that does not bind to Cyclin/CDK complexes (p21 CDK-) and one that does not bind to PCNA (p21 PCNA-).  While each of these mutant versions of p21 alters the cell cycle, neither has been able to fully eliminate S phase and arrest the cell cycle in the same manner as non-mutated p21.