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Ectopic p21(WAF1) expression induces differentiation-specific cell cycle changes in PC12 cells characteristic of
1Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. erhardt@pharm.med.upenn.edu
Abstract:
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.
Insights
Nerve growth factor induces neuronal differentiation in PC12 cells by upregulating p21(WAF1), a cell cycle inhibitor. This study reveals p21
Area of Science:
- Cell Biology
- Molecular Biology
- Neuroscience
Background:
- Nerve growth factor (NGF) induces neuronal differentiation in PC12 cells.
- This differentiation process involves the induction of the cyclin-dependent kinase (Cdk) inhibitor p21(WAF1).
Purpose of the Study:
- To investigate the role of p21(WAF1) in NGF-induced neuronal differentiation of PC12 cells.
- To elucidate the cell cycle mechanisms underlying p21(WAF1) mediated differentiation.
Main Methods:
- Utilized PC12 cell clones with an inducible p21(WAF1) construct to induce growth arrest.
- Analyzed cell cycle progression using Western blots for cyclin and Cdk levels, thymidine incorporation assays, and fluorescence-activated cell sorter (FACS) analysis.
- Assessed the effect of epidermal growth factor (EGF) on p21-arrested cells.
Main Results:
- p21(WAF1) induction caused cell cycle arrest with altered cyclin and Cdk levels (increased D1/E, decreased A/B/Cdc2/Cdk4).
- Initial DNA synthesis inhibition was followed by polyploid cell generation during prolonged p21 expression, with evidence of p21 phosphorylation.
- Epidermal growth factor treatment of p21-arrested cells induced neurite extension, a hallmark of differentiation.
Conclusions:
- Overexpression of p21(WAF1) in PC12 cells recapitulates key cell cycle events observed during NGF-induced neuronal differentiation.
- These findings suggest that p21(WAF1) induction is a critical mechanism by which NGF promotes neuronal differentiation and associated cellular changes.