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Papers In Press, published online ahead of print January 22, 2004
J. Biol. Chem, 10.1074/jbc.M311500200
Submitted on October 20, 2003
Revised on January 22, 2004
Accepted on January 22, 2004

Nerve growth factor withdrawal-mediated apoptosis in naive and differentiated PC12 cells through p53/caspase-3 dependent and independent pathways

Houman Vaghefi, Allison L. Hughes, and Kenneth E. Neet

Department of Biochemistry and Molecular Biology, FUHS/Chicago Medical School, North Chicago, IL 60064

Corresponding Author: neetk{at}finchcms.edu

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53 that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53 while other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53, has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal mediated apoptosis in both naïve and differentiated PC12 cells. Our data show that as PC12 cells differentiate, they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT and TUNEL demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 hr for naïve and 36 hr for differentiated cells. Apoptosis involved caspase 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12 [p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor-withdrawal mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.


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