Ectopic p21(WAF1) expression induces differentiation-specific cell cycle changes in PC12 cells characteristic of nerve growth factor treatment.

Nerve growth factor treatment of PC12 cells results in neuronal differentiation, a process accompanied by induction of the Cdk inhibitor p21(WAF1). To determine the role of p21 in differentiation, PC12 clones containing an inducible p21 construct were utilized to induce growth arrest. Expression of p21 led to accumulation of cyclins D1 and E and to a decrease in cyclins A and B. Levels of Cdc2 and Cdk4 also decreased after p21 induction. Initially, thymidine incorporation into DNA was dramatically inhibited; however, low levels of incorporation were observed during prolonged p21 expression. Fluorescence-activated cell sorter analysis revealed that this low level of DNA synthesis resulted in the generation of polyploid cells. Results from Western blots were consistent with phosphorylation of p21 protein coincident with the resumption of DNA synthesis. Finally, treatment of p21-arrested populations with epidermal growth factor, a known PC12 mitogen, resulted in neurite extension, a key feature of neuronal differentiation. Overall, cell cycle changes following p21 overexpression in PC12 cells closely mimic distinctive events previously shown to occur during differentiation. These results suggest that the mechanism by which nerve growth factor induces the many cellular changes associated with growth arrest during differentiation is through p21(WAF1) induction.