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Nerve growth factor withdrawal-mediated apoptosis in naive and differentiated PC12 cells through
Houman Vaghefi1, Allison L Hughes, Kenneth E Neet
1Department of Biochemistry and Molecular Biology, The Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, Illinois 60064, USA. Kenneth.Neet@rosalindfranklin.edu
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.
Insights
The tumor suppressor protein p53 plays a role in programmed cell death, but is not essential. Lack of p53 initially protects cells from apoptosis, delaying caspase activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Neuroscience
Background:
- Programmed cell death (apoptosis) is a critical cellular process regulated by various stimuli.
- The tumor suppressor protein p53 is known to mediate cell cycle arrest and apoptosis.
- The role of p53 in neuronal cell apoptosis is debated, with some studies suggesting requirement and others independence.
Purpose of the Study:
- To investigate the role of p53 in trophic factor withdrawal-mediated apoptosis in PC12 cells.
- To compare the apoptotic response in wild-type PC12 cells versus a temperature-sensitive PC12 cell line with impaired p53 function.
Main Methods:
- Utilized PC12 cell lines (wild-type and temperature-sensitive for p53).
- Assessed apoptosis using XTT and TUNEL assays.
- Investigated caspase activation (caspases 3, 6, 9) and employed RNA interference to silence caspase 3 expression.
Main Results:
- Differentiated PC12 cells are more susceptible to apoptosis than undifferentiated cells.
- Absence of functional p53 initially protected cells from apoptosis for a defined period (20h naïve, 36h differentiated).
- Caspase 3 activation was absent in p53-deficient cells, correlating with delayed apoptosis; silencing caspase 3 mimicked this delay in wild-type cells.
Conclusions:
- p53 is important but not essential for trophic factor withdrawal-mediated apoptosis.
- Caspase 3 activation is a key event in p53-mediated apoptosis, and its absence leads to delayed cell death.
- Parallel apoptotic pathways exist and are activated later in the absence of functional p53.
