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Originally published In Press as doi:10.1074/jbc.M311500200 on January 22, 2004

J. Biol. Chem., Vol. 279, Issue 15, 15604-15614, April 9, 2004
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Nerve Growth Factor Withdrawal-mediated Apoptosis in Naïve and Differentiated PC12 Cells through p53/Caspase-3-dependent and -independent Pathways*

Houman Vaghefi{ddagger}, Allison L. Hughes{ddagger}§, and Kenneth E. Neet

From the Department of Biochemistry and Molecular Biology, The Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, Illinois 60064

Received for publication, October 20, 2003 , and in revised form, January 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Programmed cell death, or apoptosis, is a conserved mechanism that is strictly regulated and specifically activated in response to a wide variety of stimuli such as ultraviolet radiation (1), genotoxic agents (2, 3), and trophic factor withdrawal (4). Tumor suppressor protein p53 is one of the mediators of apoptosis, implicated in tumors of the central nervous system (5, 6), neurological disorders (7, 8), aging (9, 10), as well as the death of some developing neurons (11-16). Transcriptional products of an activated p53 pool lead to cell cycle arrest and programmed cell death (17-19). One such example is that p53-mediated transcriptional activation leads to up-regulation of p21/WAF1/CIP1 (20, 21), a mediator of cell cycle arrest, and Bax, a pro-apoptotic mitochondrial protein (22). In addition, up-regulation of p53 activates mediators of apoptosis, such as caspase 3 (23) and caspase 9 (24).

The neural crest-derived, rat pheochromocytoma cell line PC12 is a widely used model of the sympathetic and sensory nervous system (25-27) that responds to nerve growth factor (NGF)1 through high and low affinity binding sites (28). Although primary cell culture is a powerful tool to study signal transduction, PC12 cells contain a more homogeneous population, develop a faithful neuronal phenotype, and are available in large amounts for biochemical study. Moreover, our laboratory has produced a stably transfected PC12 cell line (29) carrying a retroviral, temperature-sensitive plasmid vector that overexpresses a conformationally inactive p53 pool that abrogates the normal function of p53 at the non-permissive temperature (30). Thus, comparison of wild type PC12 to the temperature-sensitive PC12 cell line, allows investigation of the importance of p53 in a variety of cell functions, including differentiation. PC12[p53ts] cells respond to NGF similarly to PC12 cells and differentiate into the neuronal phenotype; however, PC12[p53ts] cells fail to activate p21/WAF1 and lack cell cycle arrest (29).

PC12 cells, without exposure to NGF, are dependent on serum, and withdrawal of serum initiates apoptosis (31). 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). Thus trophic factor withdrawal allows a 2-fold approach toward studying different aspects of apoptosis: (a) serum withdrawal in naïve PC12 cells and (b) NGF withdrawal in terminally differentiated, neuronal-like PC12 cells. Trophic factor withdrawal-induced apoptosis is a relevant approach toward programmed cell death, because it mimics what happens, for example, in the dysfunctional nervous system as trophic factor production is compromised, contributing to neurodegenerative disorders such as Alzheimer's and Parkinson's disease (31-37).

The fundamental pathways of apoptosis have been delineated in several cell types. Trophic factor withdrawal serves as a stress factor on the mitochondria, which influences cytochrome c release, Bcl-2/Bax equilibrium (38), and the release of apoptosis-inducing factor (39). These events activate cysteine-dependent, aspartate-specific proteases (caspases) that act as effectors and executioners of apoptosis (40). The tumor suppressor protein p53 is upstream of Bax, a pro-apoptotic protein (41). Bax has an opposing effect on Bcl-2, an anti-apoptotic, mitochondrial protein (41). In apoptotic cells Bax overcomes the protective role of Bcl-2, leading to cytochrome c release from mitochondria (42). Procaspase 9, cytochrome c, and Apaf-1 (apoptotic protease activating factor-1) (42) form a complex called the apoptosome (43). In the presence of ATP, procaspase-9 undergoes autocatalysis to yield an active caspase 9 that activates caspase 3 (43). Activated caspase 3 cleaves cytoplasmic filaments, nuclear proteins, and enzymes responsible for DNA metabolism and repair, thus leading to programmed cell death (44).

Because programmed cell death follows an orchestrated sequence of events, temporal markers of apoptosis occur in stages (45, 46). Early apoptosis coincides with subtle membrane disruptions, leading to a change of topology in the bilayer, so that phosphatidylserines that mostly reside in the cytoplasmic leaflet can also be detected in the outer leaflet (47). The annexin V assay uses this concept to detect apoptotic cells in early stages. Caspase 3 activation is used as a middle marker of apoptosis downstream of caspase 9 activation (44). Late in the apoptotic pathway, DNA fragmentation is observed (48). All of these assays were used in this study.

The role of p53 in neuronal cell apoptosis remains controversial, because some groups have demonstrated that p53 participates in programmed cell death (49-54), whereas others argue that p53 is not significant in apoptosis (55-57). This study tested the hypothesis that p53 is involved in trophic factor withdrawal-mediated apoptosis in naïve and differentiated PC12 cells. We report here that p53 is needed for early apoptosis but that a delayed apoptosis occurs even in the absence of p53. Terminally differentiated PC12 cells underwent apoptosis more quickly and shared the same requirement for p53. RNAi silencing of caspase-3 mimicked the p53 temperature-sensitive cells in both naïve and terminally differentiated cells, thus implicating this pathway in early apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Cell Culture and Cell Lines—Exponentially dividing PC12 cells, PC12[vec], or PC12[p53ts] cells were grown in Dulbecco's modified Eagle's medium (DMEM) as described previously (25, 30) and supplemented with 15% serum (10% horse serum, 5% fetal bovine serum, Invitrogen), L-glutamine (final solution concentration: 4 mM, Sigma), 10 IU penicillin, 10 µg/ml streptomycin, and 0.025 µg/ml Amphotericin B (last three were added as 6 ml of antibiotic/antimycotic from Invitrogen, #15240-062, into 500 ml of final solution). PC12 cells were grown at 37 °C with an atmosphere of 10% CO2 and 90% air. The experiments were carried out at the non-permissive temperature of 38.5 °C. Under "Results" the PC12[p53ts] cells were compared with the PC12[vec] cells, and the results for the wild type PC12 cells were not shown for simplicity. Differentiation was achieved by placing PC12, PC12[vec], or PC12[p53ts] cells in a poly-L-ornithine-coated Petri dish in the presence of 15% serum and NGF (2 nM) for 7 days to obtain 7-day differentiated cells or for 14 days to obtain terminally differentiated cells. To ensure maximum bioavailability, NGF and serum were replaced every 2 days.

Induction of Apoptosis—In the case of naïve cells, serum was removed by washing the media four times in PBS (pH 7.4) and once in serum-free media (DMEM without serum but with L-glutamine and antibacterial/antimycotic). The cells were then placed in the incubator at 38.5 °C with 10% CO2. Apoptosis was induced in 7-day differentiated cells or terminally differentiated cells by washing the cells three times in PBS, pH 7.4, and once in serum-free media. Next the cells were placed in 1% serum without NGF and 10 µg/ml anti-NGF antibody (Santa Cruz Biotechnology) for 30 min (in the incubator at 38.5 °C with 10% CO2) and then placed in 1% serum without NGF. The cells were then placed in the incubator at 38.5 °C with 10% CO2 for various time points.

Western Blotting—Cells were washed three times with ice-cold PBS, harvested with SDS lysis buffer (5% SDS, 0.5 M Tris, pH 6.8, 10% glycerol), sonicated, and spun at 15,000 x g. Lysate containing 50 µg of protein per lane (BCA protein quantification, Pierce) was loaded on a 12% SDS-polyacrylamide gel. After electrophoresis, proteins were blotted onto nitrocellulose, and the membranes were blocked with 10% nonfat milk overnight at 4 °C. Primary antibodies used were rabbit anti-p53 polyclonal antibody (Cell Signaling Technology), rabbit polyclonal caspase 9 p35 (H-170, Santa Cruz Biotechnology) antibody, and rabbit polyclonal caspase 3 antibody (Cell Signaling Technology). Primary antibody incubation was performed in 5% bovine serum albumin in TBS-T (Tris base saline, pH 7.4, 0.1% Tween 20) and followed by staining with horseradish peroxidase-conjugated rabbit anti-mouse IgG secondary antibody (Bio-Rad), diluted at the recommended ratio, in TBS-T and 5% bovine serum albumin at room temperature. Washing with TBS-T was performed between all steps. All Western blots were visualized using the ECL enhanced chemiluminescence system (Amersham Biosciences) and Fuji XR film. All experiments were repeated at least twice with similar results. Lanes were scanned in a Bio-Rad Gel Doc 1000 to quantitate relative levels.

Trypan Blue Survival Assay—Cells were suspended in 0.4% trypan blue in PBS (pH 7.4), and 200 cells were counted. The cells that excluded the blue dye and had a well defined cellular outline were scored as live. Cells that did not exclude the dye were considered dead. The percentage of live cells in the total number of cells was plotted and averaged over at least three times.

XTT Survival Assay—XTT (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid), a yellow tetrazolium salt, is cleaved by the mitochondrial dehydrogenase in metabolically active cells to form an orange formazon dye. The formazon dye was measured in an optimal spectrophotometric range of 450 to 500 nm with a Tecan Genios plate reader. All assays were performed in 96-well tissue culture plates coated with 0.1 mg/ml poly-L-ornithine. Aliquots of 103-104 cells in 100 µl were plated per well with an Eppendorf repeat pipettor and allowed to adhere overnight. For survival assays, cells were washed four times with serum-free DMEM and then incubated in 100 µl of the serum-free DMEM with varying concentrations of NGF for varying time periods. All cells were kept at 38.5 °C with an atmosphere of 10% carbon dioxide. Then 50 µl of XTT was added to give a final concentration of 0.3 mg/ml, and absorbance was measured multiple times over a 4- to 20-h period thereafter. All experiments were repeated a minimum of three times with assays performed in triplicate.

Annexin V Staining—A fluorescein isothiocyanate-conjugated annexin V assay was used as a sensitive method of membrane disruption detection utilizing a fluorescent microscope according to a modified Oncogene Science protocol. Briefly, cells were plated on poly-L-ornithine-coated tissue culture plates, allowed to adhere overnight, and stained live in respective culture medium with 10 µl of Media Binding Reagent (Oncogene Science) followed by addition of 1.25 µl of annexin V-fluorescein isothiocyanate per 5 x 105 cells. The cells were incubated for 15 min at room temperature in the dark, washed five times with PBS, and submerged in 500 µl of cold PBS. Next, 10 µl of propidium iodide were added, and the sample was placed in the dark and promptly analyzed by fluorescent microscopy. Plates were photographed immediately at 20 times magnification with a Nikon F9 camera on a Nikon Diaphot TMD microscope. The propidium iodide is a cell-impermeable dye that is excluded from cells, unless they are necrotic.

TUNEL Assay—The TUNEL assay uses an enzyme (terminal deoxynucleotidyl transferase) to add biotinylated nucleotides to the strand breaks found in the DNA of apoptotic cells. Two types of TUNEL assay were used: a microscopic qualitative assay and a quantitative assay that utilized a plate reader (Trevigen, Inc., Gaithersburg, MD). Qualitative, microscopic TUNEL assay was performed by the manufacture's protocol. Briefly, naïve, 7-day differentiated, or terminally differentiated PC12 or PC12[p53ts] cells were grown on poly-L-ornithine-coated slides, fixed in paraformaldehyde, and washed with PBS. The cells were then covered with Cytonin (Trevigen), washed with PBS twice, treated with the quenching solution (methanol with 3% H2O2), washed in PBS, and placed in labeling buffer (Trevigen). Next, the sample was incubated in the labeling reaction mixture containing TdT deoxynucleotide triphosphate mix (Trevigen), cobalt, TdT enzyme, and labeling buffer. The reaction was quenched in Stop Buffer, washed in PBS, covered in strep-horseradish peroxidase solution, washed in PBS, and placed in diamidobenzidine solution. Finally, the sample was washed several times in PBS, counterstained in Methyl Green, and visualized by light microscopy. Quantitative TUNEL assay was performed on ~2 x 104 cells per well of a 96-well plate. Washed cells were fixed with paraformaldehyde in PBS, pH 7.4, washed, dried with 100% methanol, washed, treated with proteinase K, and washed with PBS. The remaining procedure was identical to the qualitative TUNEL up to the strep-horseradish peroxidase step, after which the samples were washed with 0.1% Tween 20 in PBS. Next, 100 µl/well of TACSTM-sapphire dye (Trevigen) were added. The reaction was stopped with 500 µl/well of 2 N HCl, and the absorbance was measured at 450 nm on a Genios Tecan plate reader.

Caspase Activity—Cell lysate (50 µg of protein, by BCA assay) was added to the colorimetric caspase substrate, containing the p-nitroaniline (pNA) moiety that upon cleavage by activated caspase absorbs at 405 nm (CaspACE assay, Promega), and was measured in a Genios Tecan multifunctional, multichannel fluorometric plate reader. A z-VAD-FMK inhibitor, which inhibits all caspases, was used to confirm that caspase was being measured. In some instances, a specific inhibitor was used to confirm the specificity of the reaction. Colorimetric caspase substrates and inhibitors were purchased from BIOMOL (Plymouth Meeting, PA) and included the following: caspase 2 (substrate: Ac-VDVAD-pNA and inhibitor: Ac-VDVAD-FMK), caspase 3 (inhibitor Ac-DEVD-FMK), caspase 6 (substrate: Ac-VEID-pNA and inhibitor: Ac-VEID-FMK), caspase 8 (substrate: Ac-LETD-pNA), and caspase 9 (substrate: Ac-LEHD-pNA and inhibitor: Ac-LEHD-FMK). A fluorometric caspase 3 assay was purchased from Promega, and experimental procedures are similar to the colorimetric caspase 3 substrate analysis.

Live cell caspase 3 activity was studied in situ with a fluorescently labeled, cell-permeable caspase substrate/inhibitor that binds irreversibly to active caspase 3 and produces a detectable fluorescence (Serological Corp., Norcross, GA; formerly Intergen). The company's protocol was followed, and the fluorescent level read at 535 nm (excitation 485 nm) in a Tecan Genios plate reader.

Preparation and Transfection of siRNA—The caspase 3 sequence (accession number NM_012922 [GenBank] ) was used in a BLAST 2 search to rule out sequence homologies to other peptides in the rat genome. Candidate sequences for siRNA were designed by the following rules, as recommended by the manufacturer (Xeragon, Germantown, MD): (a) the DNA target sequence was at least 100 bp from the 3' and from the 5' end of the DNA sequence; (b) the length of the siRNA was 21 nucleotides with a GC content between 45-55%; and (c) a dTdT was designed for the 3' overhang. Two non-overlapping sequences with these criteria were purchased from Xeragon with a 3'-fluorescein, 6-carboxyfluorescein. The first target DNA sense strand from the 5' position was designed against nucleotide 192 of the caspase 3 target DNA: 5'-UGGUACCGAUGUCGAUGCAdTdT-3'-fluorescein. The second target DNA sense strand from 5' position was designed against nucleotide 390 of the caspase 3 target DNA: 5'-CGGACCUGUGGACCUGAAAdTdT-3'-fluorescein. The transfection of the siRNA was performed when cells were undergoing serum deprivation or NGF withdrawal according to the recommended protocol using a TransMessenger system (Qiagen). Generally, transfection efficacy of about 50% was obtained in naïve cells and about 40% in differentiated cells, determined by fluorescent microscopy.

Statistics—Student's t test or ANOVA was done as indicated. Student's t test was calculated to determine significance (p < 0.05; 95% confidence level). When the same cell line (e.g. PC12[p53ts]) were treated and compared, a paired t test was calculated. When different cell lines were compared (e.g. PC12[vec] to PC12[p53ts]) after the same treatment and at the same time point, a Student's t test was carried out to establish a 95% confidence interval: p < 0.05). Statistical treatments were performed with GraphPad Prism (version 2.01), San Diego, CA. Differences between means were examined using one-way or two-way analysis of variance followed by a Tukey post-hoc comparison. Differences were considered significant if p < 0.05. All results are presented as mean ± S.D. Data were graphed using SigmaPlot 2000 or Microsoft Excel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
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 naïve PC12 cells against serum withdrawal-induced apoptosis. Trypan blue survival assays in naïve cells showed that, after serum withdrawal, 50% of naïve PC12 cells lost viability after 25 h, and 90% had died after about 84 h (Fig. 1A). In contrast, 50% of PC12[p53ts] cells survived after 70 h, and 90% of cell death was not reached until ~120 h after serum withdrawal. This result shows that a lack of a functional p53 pool makes the cells refractory to cell death.



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  		FIG. 1.
Survival assays of naïve and terminally differentiated PC12[vec] and PC12[p53ts] cells upon factor removal. A, trypan blue survival assay in naïve cells. Serum medium was removed from naïve cells and replaced with medium without serum at the times shown. Trypan blue exclusion was used to distinguish live cells from dead cells. B, XTT survival assay in naïve cells. Serum medium was removed from naïve cells and replaced with medium without serum and assayed for XTT cleavage (A450) at the times shown. C, XTT survival assay in 7-day NGF-differentiated cells. Cells were treated with 2 nM NGF in serum medium for 7 days and then replaced with medium with low serum and no NGF and assayed for XTT cleavage (A450) at the times shown. D, XTT survival assay in terminally differentiated cells. Cells were treated with 2 nM NGF in serum medium for 14 days. Serum medium and NGF were removed and replaced with medium with 1% serum and no NGF and assayed for XTT cleavage (A450) at the times shown. For all panels the experiments were carried out in triplicate, and the standard error is represented by bars. *, p < 0.05, significantly different from control at time zero; **, p < 0.01, significantly different form control at time zero; +, p < 0.05, significantly different from PC12 cells (ANOVA and Tukey's post hoc test).

 
A mitochondrially directed cell survival assay (XTT) was also used to assess the involvement of p53 in factor withdrawal-mediated cell death in PC12 cells at different stages of differentiation. The results with the XTT assay complement the trypan blue assays showing that a lack of functionally active p53 in naïve cells protects the cells against serum withdrawal-induced apoptosis. Naïve PC12 cells declined to half viability, by the XTT assay, after 30 h in contrast to PC12[p53ts] cells that reached half viability after 110 h (Fig. 1B). Interestingly, complete loss of viability was eventually reached after 120 h that was not mitigated by the absence of functional p53. To ascertain that waste product accumulation did not influence the survival data, experiments were carried out where media was changed after 2-3 days, and those results revealed similar survival results (data not shown).

PC12 cells that had been reversibly differentiated into a neuronal phenotype with 7 days of NGF treatment were also examined for p53 involvement in factor withdrawal-mediated cell death. As PC12 cells differentiate, NGF withdrawal is associated with a more rapid decline in cell survival. Fifty percent of differentiated PC12 cells lost viability after 12 h (Fig. 1C), which is about 24 h sooner than factor withdrawal-mediated apoptosis in naïve cells. Similar to naïve cells, differentiated PC12 cells were protected against cell death in the absence of p53, indicating the continued requirement for p53 in cell death these cells. In both terminally differentiated cells and 7-day differentiated cells; half of the PC12 cells lost viability in 12 h, whereas 50% of the PC12[p53ts] cells didn't perish until after 110 h of NGF withdrawal (Fig. 1D). However, the survival of both PC12 and PC12[p53ts] cells approached zero by 120 h after NGF withdrawal. Half of differentiated and naïve PC12[p53ts] cells lost viability between 100 and 110 h, demonstrating that cell death was clearly delayed in cells lacking a functional p53 pool.

Membrane Disruption after Factor Withdrawal-induced Apoptosis Is Delayed in the Absence of Functional p53—Because a delay was observed in the death of cells that do not have a functional p53 pool, we determined whether membrane disruption, an early event in programmed cell death, was also affected in PC12[p53ts] cells. The annexin V assay in PC12 cells showed that membrane disruption was detected after serum withdrawal in naïve cells after 24 h (Fig. 2A) but was not observed at 12 h (data not shown). In PC12[p53ts] cells membrane disruption was delayed, because annexin V reactivity was not strongly detected until 72 h after serum withdrawal. PC12[p53ts] failed to show annexin V staining 48 h after serum withdrawal (data not shown). Terminally differentiated PC12 cells after NGF withdrawal became annexin V reactive after 12 h of NGF withdrawal in presence of serum, consistent with a faster rate of dying than naïve PC12 cells. Lack of p53 initially protected the cells against membrane disruption, but after 72 h membrane disruption ensued (Fig. 2B). Moreover, cells that were annexin V-positive were not positive for propidium iodide, suggesting that cell death was mostly due to programmed cell death and not necrosis. In 7-day differentiated cells a similar pattern was observed with a delay in PC12[p53ts] cells (data not shown).



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  		FIG. 2.
Fluorescent annexin V labeling of naïve and terminally differentiated PC12[vec] and PC12 [p53ts] cells. Images A, C, E, G, and I are bright-field micrographs; B, D, F, H, and J are the same fields in micrographs that are labeled with fluorescein-annexin V. The time of withdrawal (hours) is shown above the panels. Cells are ~15-20 µm in diameter. Cells were counterstained with propidium iodide; cells that were annexin V-positive were not positive for propidium iodide (not shown). A, naïve cells in serum-free media. B, terminally differentiated cells in 1% serum media in the absence of NGF.

 
DNA Fragmentation by TUNEL Assays—Because annexin V staining indicated that initial apoptotic steps were present in PC12[p53ts] cells, chromosomal fragmentation, a late end point in apoptosis, was also examined. TUNEL staining showed, both quantitatively and qualitatively, that lack of p53 was initially protective toward DNA fragmentation in PC12 cells. Naïve PC12 cells showed TUNEL reactivity 24 h after serum withdrawal (Fig. 3, A and C). In contrast, naïve PC12[p53ts] cells that lack p53 were TUNEL-negative after 24 h of serum withdrawal and were positive only after 48-h serum withdrawal. In differentiated PC12 cells, a delay in apoptosis was also observed; 12 h after NGF withdrawal, PC12 cells showed TUNEL staining (Fig. 3, B and D). Terminally differentiated PC12[p53ts] cells lacked TUNEL reactivity and staining until 45 h of NGF withdrawal (Fig. 3, B and D). These results indicate that lack of p53 delays apoptosis in 7-day differentiated (not shown) or terminally differentiated PC12 cells (Fig. 3). However, after about 60 h, when most of the cells were dying (see XTT results Fig. 1), DNA fragmentation was also detected in PC12 cells lacking p53.



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  		FIG. 3.
TUNEL assay for DNA cleavage in PC12[vec] and PC12[p53ts] cells. A and C, naïve cells. Serum medium was removed from naïve cells and replaced with medium without serum at the times (hours) indicated. B and D, terminally differentiated cells. Cells were treated with 2 nM NGF in serum medium for 14 days and then replaced with medium with low serum and no NGF and assayed at the times shown. A and B, quantitative TUNEL. Absorbance at 405 nm was measured and the means ± S.E. of triplicate experiments is plotted as relative apoptotic cells, normalized to cell number. *, p < 0.05, significantly different from the untreated cells at time zero; **, p < 0.01, significantly different from the untreated cells at time zero; +, p < 0.05, significantly different from PC12 [p53ts] cells at the same time point (ANOVA and Tukey's post hoc test). C and D, TUNEL micrographs. The brown precipitate in the fixed cells (~15-20 µm in diameter) marks TUNEL positivity; cells were counterstained with methyl violet.

 
The qualitative TUNEL assays (Fig. 3, C and D) agreed with the quantitative (Fig. 3, A and B) results. In Naïve PC12 cells TUNEL staining showed evidence of programmed cell death 24 h after serum withdrawal (brown precipitate). In contrast, undifferentiated PC12[p53ts] cells displayed a delay in apoptosis with evidence of TUNEL staining only after 45 h of serum withdrawal (Fig. 3C). TUNEL reactivity was delayed in 7-day differentiated (not shown) and terminally differentiated PC12 (Fig. 3D) but occurred by 48 h as indicated by the brown precipitate in cells exhibiting neurites. These results agree with the quantitative TUNEL assay demonstrating a delay in apoptosis in the absence of p53.

Flow cytometric data indicated that both naïve PC12 and PC12[p53ts] cells showed increased hypodiploidicity after factor withdrawal (data not shown) as indicated by an increased percentage of cells with less than the normal diploid DNA content. However, PC12[p53ts] cells without functional p53 demonstrated a significantly reduced DNA fragmentation pattern after serum deprivation for 24 h. In both naïve PC12 and PC12[p53ts] cells, serum or NGF, respectively, was protective (data not shown), consistent with the TUNEL data above.

Caspase 3 Activation in PC12 Cells after Factor Withdrawal Is Absent in PC12[p53ts] Cells Extracts—Caspase 3 is an effector of apoptosis in NGF withdrawal-mediated apoptosis in PC12 cells (58, 59). Caspase 3 activity after serum withdrawal in naïve PC12 was activated after 2.5 h and peaked around 4 h (Fig. 4A), consistent with previous reports. Activation of caspase 3 decreased to a non-detectable level after 10 h. Furthermore, 7-day differentiated PC12 cells and terminally differentiated PC12 cells demonstrated an increasingly robust caspase 3 activation (Fig. 4C). Caspase 3 activation was shown to be inhibited with a general caspase inhibitor (z-VAD-FMK) and with a specific caspase 3 inhibitor (DEVD-aldehyde), confirming the identity of the activity assayed. Caspase activity was reduced to the initial level in naïve, 7-day differentiated, and terminally differentiated cells at all times tested with the inhibitors (data not shown). Surprisingly, PC12[p53ts] cells did not show any caspase 3 activation when subjected to the same conditions (Fig. 4, A and C). This finding suggests that the delay in cell death in PC12[p53ts] cells is due to the lack of caspase 3 activation and that the final dying process is due to caspase 3-independent pathways.



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  		FIG. 4.
Caspase 3 activation time course in PC12[vec] and PC12[p53ts] cells. A, serum withdrawal from naïve cells. Serum medium was removed from naïve cells and replaced with medium without serum. The cells were lysed, and 50 µg of cell lysate was incubated with the colorimetric caspase 3 substrate, Ac-DEVD-pNA, and quantitated at 405 nm (A405/4 h/50 µg of protein). Two right lanes (control): After 4 h of serum withdrawal, NGF was administered to the cells in serum-free medium for 60 min (NGF). After 4 h of serum withdrawal, z-VAD-FMK, a general caspase inhibitor was added to the assay (z-VAD). The specific caspase 3 inhibitor DEVD-aldehyde also reduced caspase 3 activation to the same levels as z-VAD (data not shown). Inset, caspase 3 is not cleaved in PC12[p53ts] cells. Naïve PC12 (left) and PC12[p53ts] (right) cells were lysed, and 100 µg of cell lysate were run on a 12% SDS-polyacrylamide gel, blotted, and incubated with anti-caspase 3 antibody. Lanes (left to right) are at 0, 2.5, 4, 10, 16, 24, 39, 48, and 72 h after factor withdrawal. Similar results were obtained with 7-day and terminally differentiated cells. The experiments were replicated at least three times. B, factor withdrawal in naïve and differentiated cells. Serum medium or NGF was removed from naïve, 7-day differentiated, or terminally (14 day) differentiated cells and replaced with medium containing no serum, 1% serum, or NGF for 4 h as indicated. The cell lysates were assayed for caspase 3 activity as in A. The experiments were carried out three times each, and the error bars indicate standard error. *, p < 0.05, significantly different from the untreated cells; **, p < 0.01, significantly different from the untreated cells; +, p < 0.01, significantly different from PC12 cells at the same time point (ANOVA and Tukey's post hoc test).

 
To ascertain that the caspase 3 activation signal obtained was specific, Western blot analysis was performed to detect cleaved caspase 3 peptide. Results showed a similar pattern of caspase 3 cleavage in PC12 cells, but not in PC12[p53ts] cells (Fig. 4A, inset), verifying the colorimetric assay. Unlike transient caspase 3 activation found with the colorimetric substrate, however, the caspase 3 cleaved activated peptide remained present for several hours after induction of programmed cell death. The presence of endogenous caspase inhibitors and subcellular sequestration has been proposed recently to explain why detection of cleaved product differs from caspase activity in vitro (60).

Caspase 3 activity was also measured in the live cell culture after serum withdrawal, utilizing a cell permeable caspase 3-specific peptide FAM-DEVD-FMK, which is cleaved by and irreversibly binds to the active caspase enzyme releasing a fluorophore (data not shown). A robust caspase 3 activity was detected in situ after 4 h in naïve, 7-day differentiated, and terminally differentiated PC12 cells. However, similar to the lack of activation of caspase 3 in cell lysates, PC12[p53ts] cells did not display activation of caspase 3 in situ. Thus, the results in live cells correlated well with the data obtained using a caspase 3-specific peptide in cell lysates.

Caspase 9 Activation Is Independent of p53 in PC12 Cells—Several groups have reported that caspase 9 activation, instead of caspase 3, is necessary for programmed cell death in some cells (15, 61, 62). Because, PC12 cells that lack p53 still undergo delayed apoptosis, but fail to activate caspase 3, we investigated whether caspase 9 was activated after factor withdrawal in PC12[p53ts] cells. An in vitro, assay specific for caspase 9, revealed that caspase 9 activation peaked 4 h after factor withdrawal in naïve, differentiated, and terminally differentiated PC12 cells (Fig. 5) (time data only shown for naïve cells). In addition, PC12[p53ts] cells also underwent caspase 9 activation after factor withdrawal in naïve, 7-day differentiated, and terminally differentiated cells (Fig. 5). Furthermore, as PC12 cells differentiated, a more robust activation of caspase 9 was observed, a pattern that mimics caspase 3 activation. This increasing pattern of caspase 9 activation was emulated in advancing stages of differentiated PC12[p53ts] cells. The caspase 9 activation was verified under all conditions by Western blot analysis of the cleaved form of 46-kDa procaspase 9 to active 35-kDa caspase 9 to rule out nonspecific signals from other caspase family members (Fig. 5A, inset). Thus, caspase 9 activation by factor withdrawal is independent of functional p53, in contrast to the p53-dependence of caspase 3 activation.



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  		FIG. 5.
Caspase 9 activation time course in naïve and terminally differentiated PC12[vec] and PC12[p53ts] cells. A, serum withdrawal from naïve cells. Serum medium was removed from naïve cells and replaced with medium without serum. The cells were lysed at the times shown, and 50 µg of cell lysate were incubated with the colorimetric caspase 9 substrate, Ac-LEHD-pNA, and quantitated at 405 nm (A405/4 h/50 µg of protein). Two right lanes (control): after 4 h of serum withdrawal, NGF was administered to the cells in serum-free medium for 60 min (NGF); after 4 h of serum withdrawal, z-VAD-FMK, a general caspase inhibitor, was added to the assay (inhib). The specific caspase 9 inhibitor LEHD-aldehyde also reduced caspase 9 activation to the same levels as z-VAD (data not shown). Inset, caspase 9 is cleaved in naïve PC12[vec] and PC12 [p53ts] cells. Serum was maintained (lanes 1 and 3) or removed for 4 h (lanes 2 and 4) from naïve PC12 cells (lanes 1 and 2) or PC12[p53ts] cells (lanes 3 and 4). Cells were then lysed, and 100 µg of cell lysate was run on a 12% polyacrylamide gel, blotted, and detected with anti-caspase 9 antibody. Similar results were obtained with 7-day and terminally differentiated cells. The experiments were replicated at least three times. B, factor withdrawal in naïve and differentiated cells. Serum medium or NGF was removed from naïve, 7-day differentiated, or terminally differentiated cells and replaced with medium containing no serum, 1% serum, or NGF as indicated. After 4 h, the cells were assayed for caspase 9 as in A. The experiments were carried out three times each, and the error bars indicate standard error. *, p < 0.05, significantly different from the untreated cells; **, p < 0.01, significantly different from the untreated cells; +, p < 0.01, significantly different from PC12 cells at the same time point (ANOVA and Tukey's post hoc test).

 
Caspase 3 Interference RNA and Cell-permeable Caspase 3 Inhibitor Reduce Cell Death in PC12 Cells—In contrast to PC12 cells, PC12[p53ts] cells failed to activate caspase 3, associated with a delay in programmed cell death. Therefore, ablation of caspase 3 in PC12 cells with interference RNA was used to determine if the delay in cell death observed in PC12[p53ts] cells could be mimicked. Caspase 3 was not detected after RNAi treatment, whereas tubulin levels remain constant (Fig. 6A), indicating the efficiency of the RNAi silencing. As expected, caspase 3 RNAi also suppressed caspase 3 activation in PC12 cells, as measured by the in vitro substrate assay (data not shown), similar to the activity level observed in PC12[p53ts] cells. Upon factor withdrawal, caspase 3 RNAi led to an increased early survival in naïve (Fig. 6B) and in terminally differentiated PC12 cells (Fig. 6C), whereas the negative control RNA had no effect. Indeed, the levels of survival in caspase 3 RNAi cells were similar to the control PC12[p53ts] cells, consistent with a common mechanism. Nevertheless, caspase 3-independent mechanisms ultimately led to cell death. Quantitative TUNEL studies after RNAi transfection supported the XTT experimental data (Fig. 7).



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  		FIG. 6.
Caspase 3 knockdown with interference RNA. A, caspase 3 and tubulin levels after RNAi treatment. Naïve (N), 7-day differentiated (7d), and terminally differentiated (TD) PC12[vec] cells and PC12 [p53ts] cells (not shown) were treated with RNAi or control RNA in the absence of serum (see "Materials and Methods"). Naïve cells were then placed in serum medium, and differentiated cells were re-placed in NGF and serum medium for 4 h. The cell lysate (50 µg) was electrophoresed on an SDS-polyacrylamide gel and immunoblotted with caspase 3 or tubulin antibodies. The experiment was done three times with similar results. Naïve (B) or terminally differentiated (C) PC12[vec] and PC12 [p53ts] cell survival after transfection with either non-silencing RNAi (neg ctrl) or caspase 3 silencing RNA (Cp3 RNAi) for 2 h. Subsequently, fresh 1% serum medium was added, and cells were allowed to incubate for 4 h. Next, serum was removed and replaced with serum-free medium for various times. Absorbance of cleaved XTT was measured at 450 nm. The experiment was replicated three times and plotted as means ± S.E. *, p < 0.05, significantly different from the untreated cells at time zero; **, p < 0.01, significantly different from the untreated cells at time zero; +, p < 0.05, significantly different from the PC12 cells (negative control) at the same time point (ANOVA and Tukey's post hoc test).

 



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  		FIG. 7.
Quantitative TUNEL assay in naïve (A) and terminally differentiated (B) cells transfected with either non-silencing RNAi (control) or caspase 3 RNAi for 2 h. Subsequently, fresh serum medium was added, cells were allowed to incubate for 4 h, serum or NGF was removed, and serum-free medium was added for various times. Absorbance at 405 nm was measured, and the means ± S.E. of triplicate experiments was plotted as relative apoptotic cells, normalized to cell number. *, p < 0.05, significantly different from the untreated cells at time zero; **, p < 0.01, significantly different from the untreated cells at time zero; +, p < 0.05, significantly different from the PC12 cells (control) at the same time point (ANOVA and Tukey's post hoc test).

 
To confirm these results, the cell-permeable caspase 3 inhibitor, FAM-DEVD-FMK, was used to suppress caspase 3 activity. Naïve and differentiated PC12 cells treated with the inhibitor demonstrated a prolonged survival, as determined by the XTT (Fig. 8) or TUNEL (data not shown) assay, but eventually exhibited cell death, similar to PC12[p53ts]. Thus, three different methods (p53 ablation, caspase 3 RNAi, and a specific caspase 3 inhibitor) demonstrate that the lack of caspase 3 activation correlates with a delayed onset of programmed cell death, following factor withdrawal, but with eventual completion of apoptosis.



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  		FIG. 8.
XTT survival of naïve (A) or terminally differentiated (B) PC12 [vec] and PC12 [p53ts] cells treated with the caspase 3 inhibitor (FAM-DEVD-FMK) for 1 h. Subsequently, fresh 1% serum medium was added, cells were allowed to incubate for 4 h, serum or NGF was removed, and serum-free medium was added for various times. Absorbance of cleaved XTT was measured at 450 nm, and the means ± S.E. of triplicate experiments is plotted as relative apoptotic cells. The caspase 8 inhibitor (FAM-LETD-FMK) was used as the control. *, p < 0.05, significantly different from the untreated cells at time zero; **, p < 0.01, significantly different from the untreated cells at time zero; +, p < 0.05, significantly different from the PC12 cells (control) at the same time point (ANOVA and Tukey's post hoc test).

 
Caspase 6 and Caspase 8 Activity after Factor Withdrawal in PC12 and PC12[p53ts] Naïve and Differentiated Cells—Caspases 6 and 8 were also assayed for thoroughness. A rapid (2-h) 2-fold caspase 6 activation was observed after serum withdrawal from naïve cells (Fig. 9A). Caspase 6 activity was reduced to undetectable amounts in lysates after addition a general caspase inhibitor (z-VAD) or specific caspase 6 inhibitor (Ac-VEID-aldehyde) (data not shown), indicating the specificity of the assay. In terminally differentiated PC12 cells (Fig. 9B) and 7-day differentiated cells (not shown), activation of caspase 6 in both PC12 and PC12[p53ts] cells was observed 2 h after NGF withdrawal, sustained for several hours, and declined around 16 h (Fig. 9B). The process of serum withdrawal-mediated caspase 6 activation appears to be independent of p53 as PC12[p53ts] cells have a similar pattern of caspase 6 activation.



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  		FIG. 9.
Caspase 6 activation time course after serum withdrawal in naïve (A) and terminally differentiated (B) cells. Serum medium was removed from naïve cells and replaced with medium without serum and analyzed at various times. Cells lysate (50 µg) was incubated with the colorimetric caspase 6 substrate Ac-VEID-pNA and quantitated at 405 nm (A405/4 h/50 µg of protein). Ac-VEID-aldehyde, a caspase 6 inhibitor, reduced caspase 6 activation at 8 h in both PC12 and PC12 [p53ts] cell lines (data not shown). The experiment was carried out three times, and error bars indicate standard error. *, p < 0.05, significantly different from the untreated cells at time zero; **, p < 0.01, significantly different from the untreated cells at time zero (ANOVA and Tukey's post hoc test).

 
Caspase 8 activity was measured in cell lysates using a peptide specific to caspase 8 after serum withdrawal in naïve cells and NGF withdrawal in terminally differentiated PC12 Cells (data not shown). Caspase 8 activation was not detected in naïve nor differentiated cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
This study demonstrated that factor withdrawal-mediated apoptosis is delayed in PC12 cells lacking functional p53 throughout varying stages of differentiation. After the delay, however, apoptosis is achieved by both mitochondrial-mediated factors and through parallel pathways such as caspase 6 and caspase 2-mediated programmed cell death.2 This apoptosis network is vast, involving caspases 2, 3, 6, and 9, but selective, because caspase 8 activation was not detected after serum withdrawal. Thus, both sides of the controversy about the role of p53 in apoptosis are probably correct. During initial stages of apoptosis, particularly early in programmed cell death, the presence of functional p53 seems to be important in the fate of PC12 cells after factor withdrawal. However, after such a delay, the absence of p53 fails to prevent the outcome of apoptotic machinery, and death becomes inescapable in the PC12 cells.

Lack of p53 Resulted in a Delay in Cell Death, But Not the Ability to Terminally Differentiate—Results from both the trypan blue survival assay and the XTT assay showed that a lack of p53 was protective against programmed cell death. However, the point where 50% of PC12 cells die as measured by trypan blue exclusion was 20 h, whereas the XTT assay showed 50% viability at 35 h. One might expect trypan blue positivity to occur after mitochondrial damage, measured by XTT; however, these two assays measure different phenomena. Trypan blue reflects cellular plasma membrane integrity, whereas XTT detects mitochondrial damage, distinct phenomena of dying, but not necessarily programmed cell death. Necrotic cells also stain with trypan blue and the XTT method. XTT is also used as a proliferation assay, because it measures the ability of the mitochondria to maintain electron transport chain reaction. Mitochondria are central in the endogenous apoptotic pathway believed to be important in PC12 programmed cell death; however, more definitive assays are needed to establish apoptosis.

To ascertain that the cell death detected was truly programmed cell death, more direct assays of apoptotic death were used. Annexin V binding, an early stage marker of apoptosis, showed that membrane disruption occurred as early as 8 h after serum withdrawal in naïve PC12 cells (29) and was clearly detected 24 h after factor withdrawal. Membrane disruption occurred later in PC12[p53ts] cells, consistent with the general delay in apoptosis in these cells. In addition, the delay in annexin V reactivity was verified in PC12[p53ts] cells with flow cytometry (data not shown). One-dimensional flow cytometry for DNA content and hypodiploidicity measured this hallmark of late apoptosis (63). Flow cytometric and TUNEL experiments confirmed that apoptosis in terminally differentiated cells occurred and was not observed until 24 h. Qualitative and quantitative TUNEL assays also showed that lack of p53 in PC12[p53ts] did not prevent apoptosis but delayed the apoptotic process in terminally differentiated cells by 36 h. Thus, annexin V reactivity was observed before cell death and apoptosis in PC12 cells, as detected by trypan blue assay and TUNEL assay, respectively. Once PC12 cells have differentiated, the onset of cell death rapidly accelerated, suggesting that differentiated cells become more poised to undergo apoptosis. Our results agree with studies in which oligodendrocytes were shown to be more susceptible to apoptosis as they mature, due to a change in balance of protective and proapoptotic mechanisms (64).

These experiments also provided evidence that lack of functional p53 does not prevent terminal differentiation of PC12 cells as determined by factor dependence. Thus, PC12 cells without functional p53 continued to replicate DNA (29), extended neuritic processes, and became dependent on NGF for survival (Figs. 1 and 2) even in the presence of serum. Although PC12 cells require p53 to exit the cell cycle (29), the lack of functional p53 does not eliminate the ability of either both naïve and terminally differentiated PC12 cells retain the ability to undergo cell death in the absence of functional p53. Cell cycle arrest depends upon the p53-dependent induction of p21/WAF1, whereas p53-mediated Bax activation leads to programmed cell death. Our results suggest that apoptotic processes may be more redundant than steps in the cell cycle. With respect to evolution, it is intriguing to wonder, is achieving death more crucial than differentiating or exiting the cell cycle? Understanding which steps are more unique than others offers the potential for specific therapeutic strategies.

In animal studies, p53 has been shown to be dispensable for normal survival and development, because p53 null mice develop normally, albeit with a greater susceptibility to tumorigenesis (65, 66). Moreover, sensory and sympathetic neurons from the p53 knockout mice, as with normal mice, can survive in culture with the addition of neurotrophins (67). Importantly, however, neuronal precursors from transgenic mice lacking functional p53 demonstrate enhanced proliferation, suggesting that p53 mediates anti-proliferation rather than differentiation signals in neurons (68). Although p53 is pro-apoptotic for neurons, the closely related family member p73, whose levels are also regulated by NGF, appears to be pro-survival in both peripheral and central nervous system neurons, probably through the truncated form of p73 acting in a dominant negative manner on p53 (69). In homozygous p73-/- mice, the numbers of both sympathetic (70) and cortical (71) neurons are markedly decreased. The balance between truncated p73 and p53 appears to be quite important in both neuronal development and neurodegeneration during aging and disease (69). PC12 cells, studied in this communication, appear to express endogenous p73, although the isoform has not been determined (72).

Parallel Caspase Pathways Are Activated after Factor Withdrawal in PC12 Cells—Because programmed cell death eventually occurs in naïve and differentiated cells regardless of the presence of p53, we determined which pathways were important in early and late apoptosis after factor withdrawal. The tumor suppressor p53 is a transcription factor for Bax that directly affects mitochondria to release cytochrome c for assembly of the apoptosome complex. The apoptosome is composed of autocleaved caspase 9, cytochrome c, ATP, and apaf-1 and is important in activating the intrinsic apoptotic pathway (43). Therefore, we studied the role of caspases involved in the endogenous pathway.

Caspase activation is accepted to be involved toward the middle to late timeline of apoptotic events; changes in the plasma membrane, detected by annexin V is considered to be an earlier event (for a review, see Ref. 73). In this study, caspases 3 and 9 were both detected 4 h after factor withdrawal, preceding annexin V staining, trypan blue uptake, and flow cytometric changes that occurred 12-24 h after withdrawal. In addition, caspase 6 was detected only 2 h after withdrawal, suggesting that caspases are activated earlier in the chronological order of apoptosis. Recent studies also suggest that caspases may be activated before changes in the plasma membrane (60, 74, 75), agreeing with the data obtained in our study. Both caspase 3 and caspase 9 activity increased in magnitude as cells differentiated. This increase in magnitude may explain the XTT and the TUNEL results that indicate that apoptosis occurred more rapidly as PC12 cells became differentiated and primed for cell death in the absence of growth factors. Caspase 6 is known to be involved in neuronal cell death (76, 77) and is activated fairly rapidly (3-5 h) in hippocampal neurons following trophic factor withdrawal (78). Our data show that caspase 6 activation was detected as early as 2 h after serum withdrawal, independent of p53, because PC12[p53ts] cells also showed caspase 6 activation with the same magnitude and same kinetics. Although this result may explain the observation that both PC12 and PC12[p53ts] eventually undergo apoptosis through a caspase 6 pathway, the delay is less easily understood. We have not yet demonstrated definitively that caspase 6 is required for the later phase of apoptosis. One possible explanation is that caspase 6 represents a back-up mechanism that is activated early and poised to execute apoptosis in the event of failure of other, redundant pathways.

None of the other caspase inhibitors completely blocked apoptosis, detected by TUNEL assay. When caspase 6 inhibitor and caspase 9 inhibitor were co-incubated with cells after serum withdrawal, apoptosis still occurred (data not shown), possibly through a caspase-independent programmed cell death mechanism (79, 80). Also, the pan-caspase inhibitor (z-VAD-FMK) was more effective than specific caspase 3 inhibitor (data not shown), suggesting that inhibiting more caspase pathways delays programmed cell death more effectively than blocking one pathway. Other investigators have provided evidence that caspase 9 activation seems to bypass caspase 3 (15), which agrees with the findings in this study. Redundancy in other pathways, such as caspase 6 and caspase 2 (81), appear to push the cell to die by apoptosis to maintain cellular homeostasis. A model showing some of these aspects of the parallel pathways is shown in Fig. 10.



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  		FIG. 10.
A schematic of parallel apoptotic pathways in PC12 cells. The diagram shows how a stress factor such as serum or neurotrophic factor withdrawal can activate caspase 9 and result either in apoptosis through caspase 3 mediation or independently of caspase 3. Caspase 6 also seems to play a role in factor withdrawal-mediated apoptosis, independently of p53, which seems to be in parallel to caspase 9- and caspase 3-mediated programmed cell death. The solid arrows illustrate the "favored" pathways, and the dashed arrows suggest parallel pathways.

 

   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
Caspase 3 RNAi and a specific caspase 3 inhibitor led to a delay in apoptotic kinetics identical to that observed in PC12[p53ts] cells. Therefore, we conclude that p53 and caspase 3 are essential in the initiation phase of cell death after factor withdrawal and utilize the endogenous apoptotic pathways. The exact role of p53 in programmed cell death remains controversial. Some researchers believe that p53 is not important in apoptotic death (55-57, 81), whereas others maintain that p53 is essential in programmed cell death (49-54). In this study, we have demonstrated that tumor suppressor p53 is involved, but not essential in factor-mediated programmed cell death. In addition, our study supports the notion that, after a delay, programmed cell death resumes even in the absence of functional p53. Thus, p53 probably participates in the early phases of programmed cell death in PC12 cells through caspase 3 activation, but the absence of functional p53 results in a delay. With no p53 the PC12 cell still dies through other mechanisms (82), including caspase 6. One report indicates that half of cisplatin-induced apoptosis is mediated by p53 activation of caspase 3 independently of either caspase 8 or 9 or mitochondrial dysfunction (83). These studies, taken together, suggest that both sides of the controversy around p53 are probably right, depending upon which apoptotic initiators, which stage of apoptosis, and which cell type are being studied. In short, we conclude that death must go on.


   FOOTNOTES
 
* This work was supported in part by a grant from the USPHS, National Institutes of Health Grant NS24380 (to K. E. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy, Finch University of Health Sciences, The Chicago Medical School, 2002. Back

§ Current address: Dept. of Dermatology, Wayne State University, Detroit, MI 48201. Back

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, The Rosalind Franklin University of Medicine and Science, 3333 Green Bay Rd., North Chicago, IL 60064. Tel.: 847-578-3220; Fax: 847-578-3240; E-mail: Kenneth.Neet{at}rosalindfranklin.edu.

1 The abbreviations used are: NGF, nerve growth factor; Ac, acetyl; BCA, bicinchoninic acid; DMEM, Dulbecco's modified Eagle's medium; FMK, fluoromethyl ketone; p73, p53 family member p73; PBS, phosphate buffered saline; PC12, pheocytochromatoma 12 cell line; PC12[p53ts], pheocytochromatoma 12 cells with temperature-sensitive p53; PC12[vec], pheocytochromatoma cells with an empty vector; pNA, p-nitroaniline; RNAi, RNA interference; ts, temperature-sensitive; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; XTT, sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid; z-VAD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; PBS, phosphate-buffered saline; siRNA, small interference RNA; ANOVA, analysis of variance. Back

2 H. Vaghefi and K. E. Neet, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Barbara Vertel, Daniel Peterson, and Tung Ling Chen for assistance with the microscopy; Roopa Bhat, Laurie Dvorak, Sang B. Woo, and Nandan Lad for useful discussions; and Debbie Messineo-Jones for technical assistance.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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