The role of tumor suppressorp53 in nerve growth factor-mediated differentiation and apoptosis in PC12 cells.

摘要

The tumor suppressor protein p53 is a transcription factor that regulates the response to cellular insults such as DNA damage. Transcriptional activity of p53 requires post-translational modification by phosphorylation and acetylation. This study demonstrated that nerve growth factor (NGF) treatment results in p53 deacetylation, at Lys 382, using specific antibodies. NGF stimulation of p53 transactivation activity occurs through a MAP kinase pathway. Comparison of PC12 to PC12 [p53ts] cells showed that p53 deacetylation required functional p53. A deacetylase assay demonstrated that histone deacetylase (HDAC) activity is increased after NGF treatment and peaks before p53 deacetylation. Finally, inhibitors of MAPkinase that block p53 transactivation were also shown to abolish HDAC activation, suggesting that deacetylation of p53 is a NGF-dependent post-translational mechanism of p53 activation.; As PC12 cells differentiate, they are more poised to undergo apoptosis than their undifferentiated counterparts. XTT and TUNEL assays demonstrated that lack of p53 is initially protective against apoptosis. The window of protection seems to be about 20 hr for naïve and 36 hr for differentiated cells. Apoptosis involved caspase 2, 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53. When the expression of caspase 3 was silenced by RNAi, wild type PC12 cells revealed a morphology and biochemistry similar to PC12 [p53ts] cells, indicating that caspase 3 to p53 dysfunction accounts for the observed delay in apoptosis. Theses results suggest that p53 is a necessary, but not essential in factor-withdrawal mediated apoptosis. Other elements of caspase-mediated apoptosis are activated in the absence of functional p53.; To further analyze apoptosis, cDNA arrays for p53-related genes were used comparing PC12 cells either to PC12 [p53ts], to study the role of p53, or to PC12 cells after 12 hours of serum withdrawal, to study the components of apoptosis. The cDNA arrays revealed that both lack of p53 and induction of apoptosis after serum withdrawal alters the expression of about forty minimally overlapping, p53-related genes. The array study illustrates that both lack of functional p53 and induction of apoptosis changes the signal transduction transcripts in cell cycle, p53 modifiers, apoptotic genes, p53-stabilizing genes, and metastatic genes.