Nerve Growth Factor Withdrawal-mediated Apoptosis in Naïve and Differentiated PC12 Cells through p53/Caspase-3-dependent and -independent Pathways

Houman Vaghefi,Allison L Hughes,Kenneth E Neet

doi:10.1074/jbc.m311500200

Abstract

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, whereas 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 (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for naïve and 36 h for differentiated cells. Apoptosis involved caspases 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.

Highlights

Programmed cell death, or apoptosis, is a conserved mechanism that is strictly regulated and activated in response to a wide variety of stimuli such as ultraviolet radia
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
Lack of Functional p53 Protects against Serum or Factor Withdrawal-induced Apoptosis in PC12 Cells—Control PC12 cells were compared with the PC12[p53ts] cells that lack a functional p53 pool to determine whether lack of functional p53 protects the naıve PC12 cells against serum withdrawal-induced apoptosis

Summary

Introduction

Programmed cell death, or apoptosis, is a conserved mechanism that is strictly regulated and activated in response to a wide variety of stimuli such as ultraviolet radia-. Transcriptional products of an activated p53 pool lead to cell cycle arrest and programmed cell death [17,18,19]. PC12[p53ts] cells respond to NGF to PC12 cells and differentiate into the neuronal phenotype; PC12[p53ts] cells fail to activate p21/WAF1 and lack cell cycle arrest [29]. After about 7–10 days of NGF treatment, PC12 cells terminally differentiate into a neuronal phenotype, become dependent on NGF, and undergo apoptosis after NGF withdrawal even in the presence of serum [31]. Trophic factor withdrawal allows a 2-fold approach toward studying different aspects of apopto-

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