Introduction

The tumor suppressor protein p53 is a multipotent transcriptional factor involved in apoptosis (Wu and Levine, 1994), cell cycle arrest (Wu and Levine, 1994), and differentiation (Chandrasekaran et al., 1982; Oren et al., 1982; Ben-Dori et al., 1983; Vousden and Lu, 2002; Morrison et al., 2003). Inactivation of p53 occurs in many tumor forms and lack of function of this protein leads to loss of cell cycle check point and resistance to antitumor therapy (Makris et al., 1995). Activation of p53 is mediated through post-translational modification (for a review see Ito et al., 2001), leading to increased stability (Pise-Masison et al., 1998) and translocation of p53 into the nucleus (Boulikas, 1995; Higashimoto et al., 2000). Transcriptional products of an activated p53 pool lead to cell cycle arrest and programmed cell death (Selter and Montenarh, 1994; Blaydes et al., 2001). The mechanism of these cellular effects is generally accepted to occur, at least partly, through p53-mediated transcriptional upregulation of p21WAF/CIP (Liu and Pelling, 1995; Parker et al., 1995), a mediator of cell cycle arrest, and Bax (Zhan et al., 1994), a proapoptotic mitochondrial protein (for a review see Budihardjo et al., 1999).

The p53 protein is post-translationally modified in as many as 18 amino-acid positions, depending on the nature of the stress factor and cell type (Milczarek et al., 1997), as demonstrated with poly- and monoclonal antibodies raised to specific phosphorylated or acetylated amino acids and often confirmed by mass spectroscopy (Meek, 1994; Maxwell et al., 1996; Merrick et al., 2001). For example, phosphorylation of Ser 20 in p53 has been demonstrated to be associated with p53-mediated apoptosis after 1–2 h of UV treatment (Chehab et al., 1999; Craig et al., 1999a, 1999b; Cregan et al., 1999; Jabbur et al., 2001). In addition, acetylation of p53 at lysine (Lys) 320 is believed to be important in transactivation of p53 after stress factors (Liu and Pelling, 1995; Mauser et al., 2002). Moreover, dephosphorylation at Ser 376 is associated with an increased p53 transcriptional activity (Liu and Pelling, 1995; Mauser et al., 2002). Recent studies have shown that deacetylation of p53 regulates its influences on apoptosis and cell cycle arrest (Luo et al., 2001; Vaziri et al., 2001; Zeng et al., 2003).

Acetylation of p53 was widely believed to be synonymous with the activation of the tumor suppressor (Bayle and Crabtree, 1997; Lakin and Jackson, 1999; Grossman, 2001; Woods and Vousden, 2001). Espinosa and Emerson (2001) challenged this notion that acetylation was not necessarily associated with activation. Langley et al. (2002) have provided evidence that, in an embryonic fibroblast model, deacetylation of p53 by Sir 1 may be important for p53-mediated cell growth.

Nerve growth factor (NGF) is a member of the neurotrophin family that mediates differentiation, survival, and apoptosis of neurons through TrkA and p75 receptors (for reviews see (Neet and Campenot, 2001; Lad et al., 2003). Our laboratory (Hughes et al., 2000) and others (Scotto et al., 1999) have demonstrated that NGF treatment leads to p53 nuclear translocation and activation. The TrkA NGF receptor, and not the p75 receptor, appears to mediate this effect for the following reasons: (a) In a PC12nnr cell line that does not express TrkA receptor, p53 is not activated (Gollapudi and Neet, 1997). (b) A mutant NGF (9/13), which binds with more affinity to p75 than TrkA (Woo et al., 1995), does not activate the transcriptional activity of p53 (Hughes et al., 2000). (c) The TrkA inhibitor, K252a, blocks the NGF-mediated p53 activation (Gollapudi and Neet, 1997). This line of evidence indicates that TrkA mediates p53 activation in the PC12 cell model system. Similar to UV or genotoxic stress-mediated activation of p53, NGF activates p53 to cause cell cycle arrest, but NGF-mediated p53 activation does not lead to apoptosis (Hughes et al., 2000).

The neural crest-derived, rat pheochromocytoma cell line (PC12 cell line) has been widely used as an established neuronal model to investigate cell signaling and apoptosis (Greene and Tischler, 1976; Lee et al., 1977). PC12 cells contain a more homogeneous population, are available in large amounts, and have most characteristics of differentiated neurons (Greene and Tischler, 1976; Lee et al., 1977; Luckenbill-Edds et al., 1979; McGuire and Greene, 1979). In addition, a low basal level of p53 expression in PC12 cell line makes it a practical choice in this study.

In this study, we have utilized a stably transfected PC12 cell line, produced with retroviral, temperature-sensitive plasmid vector, that overexpresses a conformationally inactive p53 pool that abrogates the normal function of p53 at a nonpermissive temperature (Hughes et al., 2000). Wild-type PC12 cells have been compared to the temperature-sensitive PC12 cell line to investigate the post-translational modification state of p53 that is an important mediator of stability and function in the tumor suppressor p53. We present evidence that NGF induces a MAP kinase-mediated p53 deacetylation in PC12 cells concomitant with differentiation.

Results

NGF treatment in PC12 cells leads to a dose-dependent, time-dependent, and reversible deacetylation of p53

Preliminary experiments using a pan-acetylated p53 antibody suggested that NGF caused deacetylation of p53. Subsequent experiments confirmed that NGF leads to a dose-dependent p53 deacetylation (Figure 1a). The concentration, at which half of this effect is observed, 50 pM, agrees with NGF-mediated stimulation of PC12 cells. In addition, the time dependence of NGF-mediated p53 deacetylation showed a fairly rapid response (Figure 1b). The time at which p53 is half deacetylated is about 40 min. At 2 nM NGF for 60 min, the acetylated p53 is virtually absent. Finally, after a period of NGF treatment, the factor was removed and the acetylated p53 band reappeared indicating that the deacetylation process is reversible (Figure 1c). Washing the cells with the media with no NGF but containing anti-NGF antibody assured complete NGF withdrawal. However, the reacetylation was not completely achieved, as the acetylated band was not fully recovered within the 12 h time tested. In these experiments, the blot was first probed for total p53 in the lysate and then reprobed for acetylated p53. This procedure provides a control for equal loading and the proper internal control.

Figure 1.

Deacetylation of p53 after NGF treatment. PC12 cells in 1% serum were pretreated with 0.6 nM TSA for 12 h and then placed in fresh serum media with NGF in the presence of 0.6 nM TSA. Cells were lysed and 75 g of total protein were electrophoresed on a 12% polyacrylamide gel, and immunoblotted with a pan-monoacetylated antibody. A separate gel was run to detect total p53 amounts in the PC12 cell after NGF treatment. Blots were scanned for quantitation (lower panel). The gels are a single run representative of triplicate experiments with means.d. for the three experiments shown in the lower panel. (a) Dose–response curve. NGF treatment was for 1 h at 1 pM to 20 nM (lanes from left to right) as indicated in the lower panel. (b) Time course. NGF treatment (2 nM) was for times from 0 to 100 min as indicated in the lower panel. (c) Reversibility of deacetylation upon NGF withdrawal. After 120 min NGF treatment (see arrow), NGF was removed and replaced with fresh serum media in the presence of 0.6 nM TSA and anti-NGF antibody (10 g/ml) for complete removal of NGF. Cell lysates were analysed at different times for another 600 min (lower panel)

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The antibody used in Figure 1 recognizes any acetylated Lys on p53. To determine which p53 site is deacetylated, the blots in Figure 1a were stripped and reprobed with specific acetylated p53 antibodies, one for Lys 373 and another antibody for Lys 320 (Figure 2a). Both of these antibodies showed no change in band intensity, indicating that the site on p53 is not Lys 373 or Lys 320.

Figure 2.

Lys residues 382 is deacetylated after NGF treatment. PC12 or PC12[p53ts] cells were pretreated with 0.6 nM TSA for 12 h and then placed in fresh media in the presence of 0.6 nM TSA in 1% serum and 2 nM NGF for the times shown. Cells were lysed and 75 g (a) or 100 g (b) of total protein were electrophoresed on a 12% polyacryamide gel and immunoblotted with anti-acetyl-Lys 320 p53 antibody (a), with anti-acetyl-Lys 373 p53 antibody (a), or with an anti-acetyl-p53 (Lys 373- and Lys 382-specific) antibody (b). A separate gel was run to detect total p53 amounts loaded onto the gel. The gels are representative of triplicate experiments. No significant differences were detected in levels of acetylated p53 at Lys 320 and Lys 373 (a) but acetylated Lys 320 plus Lys 382 was reduced (b). PC12[p53ts] were also tested with the anti-acetyl-p53 (Lys 373- and Lys 382-specific) antibody (b, three right lanes)

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A newly available specific antibody directed to both Lys 373 and Lys 382 was used and demonstrated that NGF-mediated p53 deacetylation occurs at Lys 373 or Lys 382 in PC12 cells since the intensity of staining with this dual-specific antibody decreased with time of treatment (Figure 2b). Since the site-specific anti-Lys 373 p53 antibody did not show deacetylation after NGF treatment (Figure 2a), Lys 382 must be the major contributor to NGF-mediated p53 deacetylation. Figure 2b also demonstrates that PC12[p53ts] cells with a nonfunctional p53 do not exhibit NGF-mediated p53 deacetylation at Lys 373/382, suggesting that a wild-type p53 protein conformation may be necessary for p53 deacetylation.

Deacetylation pattern in p53 is specific to NGF

PC12 cells were treated with other growth factors and cytokines to investigate the specificity of the trophic factor deacetylation pattern in p53. Epidermal growth factor (EGF) activates a similar cascade of events as NGF in PC12 cells, but with only a transient (rather than sustained) activation of the MAP kinase pathway (Traverse et al., 1992), and consequently weakly stimulates proliferation with no differentiation (Boonstra et al., 1985). Fibroblast growth factor (FGF) also activates similar pathways in PC12 cells that lead to differentiation (Togari et al., 1983). Tumor necrosis factor-alpha (TNF-) leads to apoptotic cell death in PC12 cells (Heneka et al., 1998). EGF or TNF- did not lead to p53 deacetylation (Figure 3). Surprisingly, FGF also did not stimulate p53 deacetylation even though it is generally believed to activate most of the same pathways as NGF (see Discussion). The data show that only NGF in wild-type PC12 cells leads to p53 deacetylation detected by Western blot analysis with acetyl-Lys-specific antibodies. Serum withdrawal also leads to apoptosis and p53 is known to be involved in this process (Zhong et al., 1993; Gartenhaus et al., 1996; Park et al., 1996; Stefanis et al., 1996; Simpson et al., 2001; Vaghefi et al., 2004). However, serum withdrawal did not lead to p53 deacetylation (data not shown). The PC12[p53ts] cells did not promote NGF-, or any other growth factor-–, stimulated deacetylation of p53 (Figure 3).

Figure 3.

NGF-specific p53 deacetylation. The p53 deacetylation pattern was determined using a pan-acetylated p53 antibody after NGF (2 nM), EGF (10 ng/ml), TNF (5 ng/ml), or bFGF (5 ng/ml) treatment at various times in the presence of 0.6 nM TSA. Similar experimental procedures were carried out with wild-type PC12 (rows 1 and 3) and PC12[p53ts] (rows 2 and 4) cells. Samples were processed and analysed as in Figure 1. The gels are a single run representative of triplicate experiments

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Deacetylation of p53 after NGF treatment occurs through the MAP kinase pathway

PC12 cells were treated under conditions that result in p53 deacetylation and the cells were cotreated with various inhibitors of signaling pathways initiated after NGF treatment in PC12 cells. Western blot analysis was performed to determine the acetylated state of p53 (Figure 4). The deacetylation process was inhibited by the MAP kinase or ERK kinase kinase (MEK) 1/2 inhibitor, PD98059, in a dose-dependent manner (data not shown). Such inhibition of deacetylation was not detected with SB 203580 (p38 MAPK inhibitor), Wortmannin (PI3 kinase inhibitor), or LY 294002 (PI3 kinase inhibitor) that are known to inhibit other pathways activated in PC12 cells after NGF treatment (Kimura et al., 1994; Yao and Cooper, 1995; Iwasaki et al., 1999). Since PD98059 has been shown to be a rather specific inhibitor of MEK1/2, thereby preventing activation of MAPK (Alessi et al., 1995; Dudley et al., 1995), the deacetylation pattern associated with NGF treatment appears to be activated through a MAPK-dependent pathway. Moreover, K252a, a receptor tyrosine kinase inhibitor (Ohmichi et al., 1992; Tapley et al., 1992), was also able to abolish NGF-mediated p53 deacetylation, indicating that the MAP kinase pathway to deacetylation is initiated through TrkA. This result is consistent with previous data that links TrkA and p53 activation (Gollapudi and Neet, 1997; Hughes et al., 2000). These inhibition results confirm the specific effect of the main NGF signaling pathway to stimulate the p53 deacetylation process.

Figure 4.

Effects of inhibitors on NGF-mediated p53 deacetylation. The first lane is a control lane indicating the basal amounts of acetylated p53 at 60 min. PD98059 (MEK1/2 inhibitor, PD), K252a (tyrosine kinase inhibitor), Wortmannin (PI3 kinase inhibitor, W) at 1 M concentration or TSA at high concentrations (0.6 M) was tested with NGF (2 nM) at 45 min or 60 min as shown. The last lane is a positive control with NGF-mediated deacetylation. All cells were also pretreated with low TSA (0.6 nM). Gels were immunoblotted with the pan-acetylated p53 antibody. For cell treatment see Figure 1. The gels are a single run representative of triplicate experiments

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NGF-mediated p53 deacetylation is Trichostatin A (TSA) sensitive

Deacetylation is carried out by two major types of deacetylases (de Ruijter et al., 2003). Class I deacetylases, such as histone deacetylases (HDAC), are categorized by TSA sensitivity; M concentrations of TSA inhibit HDAC activity. Another category of deacetylases is the TSA-insensitive (Sir class) class that is insensitive to TSA (Gasser and Cockell, 2001) and needs NAD⁺ for full activation. Cell incubations for Western blot experiments were performed using low, nanomolar concentrations of TSA prior to NGF addition to minimize deacetylation during cell processing (see Materials and methods) (Furumai et al., 2001). NGF treatment without nanomolar concentrations of TSA did not lead to reproducible demonstration of deacetylation and was subject to variability due to random deacetylation during processing (data not shown). When the concentration of TSA was increased to the micromolar range (0.6 M TSA) during incubation, the deacetylation was abolished as shown by Western blots (Figure 4). These results suggest that the deacetylase involved in NGF-mediated post-translational modification of p53 belongs to a TSA-sensitive class of deacetylase and probably an HDAC family member.

NGF stimulates HDAC activity in a TSA-sensitive manner, which is mediated by a MAP kinase cascade

HDAC assay was performed using a fluorometric reagent fluor-de lys™ that fluoresces when active HDAC cleaves the reagent. Lysate from NGF-treated PC12 cells or PC12 cells cotreated with NGF and inhibitor was incubated with the fluorogenic substrate. These results with a direct assay indicate that NGF treatment leads to an activation of HDAC (Figure 5a). However, the NGF-mediated HDAC activation, observed in the HDAC studies, is transient and peaks at 15 min after NGF treatment. In contrast, deacetylation observed by Western blot after NGF treatment results in a sustained level of p53 deacetylation. This result suggests that no rapid reacetylation occurs in the presence of NGF. The pattern of HDAC activation is also TSA sensitive (right most set of columns), identical to the deacetylation pattern of p53. NGF-mediated HDAC activation is dependent on the dose of NGF (data not shown), similar to p53 deacetylation. HDAC activation is also blocked by PD98059 (Figure 5a). Thus, NGF-mediated p53 deacetylation is consistent with an HDAC-mediated event, promulgated by a MAP kinase pathway. Neither FGF nor EGF stimulates HDAC activity (Figure 5a), which is consistent with the trophic factors' specificity of p53 deacetylation (Figure 3).

Figure 5.

Stimulation of HDAC activity. PC12 or PC12[p53ts] cells were treated with growth factors and/or inhibitors for various times. Cells were then harvested, lysed, and incubated with the Fluor de Lys™ substrate for 30 min at room temperature. The experiment was performed in triplicate and the error bars represent standard error. (a) HDAC activity with various growth factors. PC12 cells were treated with NGF (2 nM), EGF (2 nM), or bFGF (2 nM) for 2 min to 5 h. LY 294002 (PI3 kinase inhibitor, LY), PD98059 (MEK1/2 inhibitor, PD), K252a (tyrosine kinase inhibitor), SB 203580 (p38 MAPK inhibitor, SB), U0185 (MEK1/2 inhibitor, U), Wortmannin (PI3 kinase inhibitor, Wtm) at 1 M concentration or TSA at 0.6 nM (as indicated) was preincubated for 60 min before growth factor addition for 15 min. The three open bars on left were controls with no growth factor. 'Control' refers to cells with no growth factor or TSA added. 'Hi TSA' (second column) indicates cells to which 0.6 M TSA was added without growth factor. 'Lo TSA' indicates the addition of 0.6 nM TSA to the cells but with no growth factor. ^*Significantly different from the untreated and significantly different from EGF and FGF treatment (P<0.05). ^#Significantly different from 15-min NGF treatment P<0.05 (ANOVA and Tukey's post hoc test). (b) HDAC activity in PC12 cells and PC12[p53ts] cells. PC12 cells and PC12[p53ts] cells were treated with 2 nM NGF for the times indicated. ^*Significantly different from PC12 cells at t=0 (P<0.05). ⁺Significantly different from the PC12[p53ts] cells at t=15 min (P<0.05) (ANOVA and Tukey's post hoc test)

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A PC12 cell line lacking transactivation activity for p53[p53ts] fails to deacetylate after NGF treatment

Cell extract from NGF-treated PC12[p53ts] cells was analysed for the detection of HDAC activation. Interestingly, HDAC activation was deficient in these PC12[p53ts] cells. In agreement with the experiments noted above, we did not detect any p53 deacetylation by Western blot when we treated the PC12[p53ts] cells with NGF, suggesting that deacetylation requires wild-type p53 (Figures 3 and 4). In addition, PC12[p53ts] cells did not show HDAC activation, indicating that p53 function may be important in the deacetylation process (Figure 5b). The tumor suppressor p53 is also important in serum withdrawal-mediated apoptosis. Deacetylation activity was not observed using the HDAC assay in either PC12 or PC12[p53ts] cells upon serum withdrawal (data not shown).

MAP kinase pathway is involved in NGF-mediated transcriptional activity of p53

NGF treatment of PC12 cells has previously been demonstrated to lead to an increased transcriptional activity of p53, measured by the luciferase/-galactosidase reporter assay (Hughes et al., 2000). Temperature-sensitive PC12[p53ts] cells have a lower transcriptional activity in comparison to the PC12 cell line counterparts. We determined whether the MAP kinase pathway and HDAC activation is also involved in p53 transactivation. When NGF was used with the MAP kinase inhibitor PD89059, transactivation by p53 was decreased, measured by the luciferase assay (Figure 6). SB 203580 (p38 MAPK inhibitor) did not decrease the luciferase reporter assay activity, suggesting that p38 kinase is not involved in NGF-mediated p53 activation (Figure 6). Wortmannin (PI3 kinase inhibitor) and LY 294002 (PI3 kinase inhibitor) did not decrease luciferase activity (data not shown). However, K252a did decrease luciferase activity (data not shown) comparable to PD 89059, indicating that the observed p53 transcriptional activation is initiated through a TrkA tyrosine receptor pathway. Finally, TSA (0.6 nM) was effective at blocking the reporter response in this 1-day assay compared to its inefficacy in the 1 h deacetylation assay (Figures 1, 2 and 3). This result suggests that MAP kinase pathway, as well as HDAC, is involved in NGF-mediated p53 activation.

Figure 6.

Inhibitors of deacetylation or MAPK block p53 transcriptional activity with a luciferase reporter assay. PC12 cells were transiently cotransfected with -galactosidase reporter for normalization of transfection efficacy and either a p53 response element (+, response element) or a reporter, which lacks p53 response element (-, response element). After transfection, cells were incubated with either 1% serum only media or with the inhibitors PD 98059 (PD), SB203580 (SB), or TSA (0.6 nM) plus 1% serum medium for 1 h prior to NGF addition. Cells were then harvested at 1 day, assayed, and a normalized luciferase unit was calculated and plotted. The error bars indicate standard error of triplicate determinations. ^*Significantly different from control (-, p53 response element) NGF-treated cells with no inhibitor P<0.05. ^**Significantly different from NGF-treated cells transfected with (+) p53 response element with no inhibitor P<0.05 (ANOVA and Tukey's post hoc test)

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Discussion

NGF is a trophic factor that has been demonstrated to activate p53 in the PC12 cell line and other cells. This study outlines the first evidence that NGF treatment in PC12 cells leads to deacetylation of p53. In accord with the p53 deacetylation experiments, NGF stimulation was also shown to activate HDAC, a general deacetylase in the cell, since TSA (Figures 3 and 4), suberoylanilide hydroxamic acid (SAHA), or butyrate (data not shown) blocked NGF-mediated p53 deacetylation. However, the HDAC inhibitor TSA alone (no NGF) does not alter the acetylation of p53 in PC12 or PC12[p53ts]. Since a specific MAP kinase inhibitor, PD98059, blocked p53 deacetylation, HDAC activation, and p53 transcriptional activity on a reporter gene, it appears that the p53 deacetylation is regulated through the MAP kinase pathway, concomitant with cell cycle arrest.

Recently, p53-mediated deacetylation has been linked to the p21WAF1 pathway with evidence that p53 is deacetylated at Lys 382, as well as Lys 320 and Lys 373, after increased laminar flow in the vein (Zeng et al., 2003). NGF-mediated deacetylation, however, seems to be site specific. Deacetylation at Lys 320 or Lys 373 was ruled out by the lack of change with acetyl-Lys-specific antibodies for those sites (Figure 2a). However, an antibody that is specific to both acetylated Lys 373 and Lys 382 detected deacetylation after NGF treatment (Figure 2b), indicating that the site of deacetylation is Lys 382. In vitro studies showed that when exposed to the same conditions, Lys 382 is a more reactive site than the Lys 381 (Gu and Roeder, 1997).

In PC12[p53ts] cells where p53 is conformationally inactive, p53 is not deacetylated and HDAC is not activated, as occurs in the wild-type PC12. This result suggests that deacetylation might require a conformationally active pool of p53 in the cell. Further, PC12[p53ts] cells differ from PC12 in two aspects. One, PC12[p53ts] are more resistant to programmed cell death after serum or NGF withdrawal (Vaghefi et al., 2004). However, we did not observe any deacetylation in PC12[p53ts] or PC12 cells after serum or NGF withdrawal (data not shown). In addition, we did not detect any HDAC activation after serum withdrawal or NGF withdrawal in PC12 or PC12[p53ts] cells. Therefore, we reason that factor withdrawal-mediated apoptosis is not correlated with deacetylation of p53. The second difference between PC12 and PC12[p53ts] is that, although both PC12 and PC12[p53ts] cells differentiate, PC12[p53ts] cells do not undergo complete cell cycle arrest since they incorporate BrdU after differentiation (Hughes et al., 2000). Thus, deacetylation could be involved in the transactivation ability of p53 to cause cell cycle arrest. Indeed, the inhibitors of MAPK that block NGF-mediated HDAC activity also block p53 transcriptional activity (Figures 5 and 6), suggesting that NGF-mediated HDAC activity could be linked to cell cycle arrest. We propose the deacetylation-mediated p53 activation pathway in PC12 cells as shown in Figure 7. However, more investigation is needed to fully determine this point.

Figure 7.

A schematic model of NGF-mediated p53 deacetylation and distinct cell response. NGF-induced deacetylation of p53 is mediated through TrkA, MAPK, and HDAC activation. The post-translational modification state of p53 may favor assembly of a particular set of coactivators to associate with p53. Deacetylation of p53 on Lys 382 may lead to a different set of coactivators to interact with p53, leading to selective gene transactivation, and cell cycle arrest rather than apoptosis

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The kinetics of the events starting from HDAC activation to p53 deacetylation to luciferase activation are consistent with a causal series of events. HDAC is activated after 15 min, deacetylation of p53 reaches half-activation at about 40 min and p53 luciferase reporter activity is observed within a day after transient transfection. Luciferase activity may peak well before 1 day observed in our study, but a time course cannot be established before 1 day because that time is required for effective transfection.

In this study, FGF and NGF led to divergent end points. While similar pathways have been documented to be activated with both FGF and NGF, a considerable difference was observed here with p53 deacetylation (Figures 3 and 5). Although NGF results in p53 deacetylation, FGF fails to do so. This result is consistent with the observation that FGF does not activate deacetylases in the PC12 cells according to the HDAC assay. Both NGF and FGF lead to comparable levels of differentiation and protect against apoptosis after serum withdrawal. Although FGF and NGF are very similar in signaling through the MAPK pathway, subtle differences exist, such as NGF-mediated Akt activation that is not detected after FGF treatment in PC12 cells (Wert and Palfrey, 2000). Parallel events and or neurotrophin-specific actions may influence why only NGF leads to p53 deacetylation and not FGF. Furthermore, since EGF does not activate HDAC or p53 deacetylation, a transient activation of the MAP kinase pathway is not sufficient for deacetylation activity.

Considerable evidence suggests that alteration in p53 acetylation status can modulate how p53 interacts with cofactors such as acetylase (p300), thereby modulating the ability of p53 to function as a transcription factor (Shiseki et al., 2003). In addition, acetylation can alter the properties of the C-terminus in the p53 molecule so that the domain would interact differently with the other domains, altering how p53 interacts with its target sequence (Gu et al., 1997; Gu and Roeder, 1997). Upon activation, the tumor suppressor p53 is acetylated in some systems, preceding transcriptional activation of factors such as Bax that are needed for apoptosis (Juan et al., 2000; Vaziri et al., 2001; Langley et al., 2002). Several studies, including ours, lead to a more diverse regulatory perspective, suggesting that the effect on p53 is more subtle.

NGF-induced cell cycle arrest of PC12 cells is a p53-mediated (Hughes et al., 2000) and p300-mediated (Billon et al., 1996) process that leads to p21WAF1 transcriptional activation, probably involving a multiprotein complex (Billon et al., 1996). Our data show that trophic factor activates p53, correlated with p53 deacetylation. Thus, we speculate that the post-translational status of p53 may favor interaction with a specific set of cofactors, thereby supporting transactivation of specific transcripts. In support of this position, HDAC has been shown to interact in a complex with transcription suppressors such as REST to favor the neuronal phenotype (Grimes et al., 2000; Ballas et al., 2001). Also, a different post-translational modification status may alter p53 binding ability required for transcription (Hupp and Lane, 1995; Dornan et al., 2003). Other reports have shown that different modes of acetylation and interaction with cofactors may be responsible for various p53-dependent transcriptional activation (Kobet et al., 2000; Webley et al., 2000; Ito et al., 2001). Thus, p53 deacetylation strongly suppresses the p53 transcription activity needed for apoptosis, thereby leading to survival (Luo et al., 2000). The two deacetylases, Sir-2 and HDAC, also appear to form a complex to alter p53 function, probably negative regulation of p53 (Nagy et al., 1997). Another study indicates that HDAC activation leads to the activation of p53 targets such as the transcription factor SP-1 that interacts with coactivators such as p300, leading to the formation of an active transcription factor complex (Choi et al., 2002).

Conversely, deacetylation does not equate to inactivation. For example, deacetylation is necessary for p53 cell cycle arrest in vascular endothelial cells (Zeng et al., 2003). A fine balance between acetylation, executed by p300, CREB binding protein (CBP), and p300 and CREB binding protein-associated factor (PCAF), and deacetylation, catalysed by HDAC and Sir proteins are important in gene regulation (Roeder, 1998) and seem to be essential for normal cell growth (Lehrmann et al., 2002). Such post-translational modifications will slightly modify the proteins in the transcriptional complex and lead to differential transcription by the same transcription factor. Depending on the acetylation state of p53, different cofactors may form complexes with the transcription factor that could then determine whether p53 transactivates cell cycle arrest or apoptosis-related genes (Figure 7). This mechanism explains how p53 may be involved in various pathways in the cell. A similar model has been proposed that outlines how phosphorylation–dephosphorylation may be involved in determining the p53 gene targets (Vousden and Lu, 2002).

Materials and methods

Cell culture and cell lines

Exponentially dividing PC12 cells, PC12[p53ts] cells (stably transfected PC12 cell line carrying a retroviral, temperature-sensitive plasmid vector that over expresses a conformationally inactive p53 pool that abrogates the normal function of p53 at the nonpermissive temperature), or PC12[vec] (carrying the empty plasmid as a control cell line) were grown in Dulbecco's modified Eagle's medium as described (Greene and Tischler, 1976; Sladek and Jacobberger, 1992) supplemented with 15% serum (10% horse serum and 5% fetal bovine serum; Gibco), L-glutamine (final concentration: 4 mM; Sigma), and PEN-STREP antibiotic–antimycotic (6 ml into 500 ml of final solution; Gibco). PC12 cells were grown at 37°C with an atmosphere of 10% CO₂ and 90% air. All experiments were carried out at the nonpermissive temperature of 38.5°C. In the Results section, the PC12[p53ts] were compared with the PC12[vec] cells and the results for the wild-type PC12 cells were not shown for simplicity.

Western blotting

Cells were washed three times with ice-cold phosphate-buffered saline (PBS), harvested with sodium dodecyl sulfate (SDS) lysis buffer (5% SDS, 0.5 M Tris pH 6.8, 10% glycerol), sonicated, and spun at 15 000g. All cells for Western blots were pretreated with TSA (0.6 nM) to suppress random deacetylation of Lys residues, as suggested by the protocol from Cell Signaling Technology. Lysate containing 50–100 g of protein (BCA protein quantification; Pierce) per lane was loaded onto 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 mouse monoclonal monoacetylated Lys (Ac-K-103) antibody (catalog number 9681; Cell Signaling), rabbit anti-p53 polyclonal antibody (catalog number 9282; Cell Signaling Technology), anti-acetyl-p53 (Lys 320) polyclonal antibody (catalog number 06–915; Upstate), and anti-acetyl-p53 (Lys 373) polyclonal antibody (catalog number 06–916; Upstate). The rabbit polyclonal anti-acetyl-p53 (Lys 373 and 382) was purchased from Upstate (catalog number 06–758). The rabbit polyclonal p53 antibody (Cell signaling, catalog number 9282) was used as the primary antibody to detect total p53 in this study. Primary antibody incubation was performed in 5% bovine serum albumin (BSA) 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) in TBS-T and 5% BSA at room temperature (diluted at the recommended ratio). Washing with TBS-T was performed between all subsequent steps. All Western blots were visualized using the ECL chemiluminescence system (Amersham) 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 estimate relative levels.

HDAC assay

The cells were treated and subsequently lysed with Triton buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 g/ml leupeptin, 1 mM PMSF, and 1 mM sodium orthovanadate). Then 50 g of the lysate were incubated for 30 min at room temperature with the proprietary HDAC-specific substrate compound Fluor de lys™ from Biomol (Plymouth Meeting, PA, USA). In the presence of a deacetylase, the compound hydrolyses the substrate, creating a fluorophore with excitation at 360 nm and emission at 460 nm. The assay was read in a multifunctional plate reader (Tecan Genios). Proper flat-bottom, black 96-well plates suited for fluorescence were used (Whatman, Clifton, NJ, USA).

Reagents and inhibitors

PD98059 (MEK1/2 inhibitor) (Dudley et al., 1995), LY294002 (PI3 kinase inhibitor), and Wortmanin (PI3 kinase Inhibitor) were purchased from Upstate Biotechnologies. SB 203580 (p38 MAPK inhibitor) was purchased from Sigma. K252a (receptor tyrosine kinase inhibitor) was purchased from Calbiochem. TSA, sodium butyrate, and SAHA were purchased from Sigma. EGF, bFGF, and TNF- were purchased from Research Diagnostic Inc. NGF was purified from male Webster mice salivary gland (Luo and Neet, 1992).

Transient transfections and luciferase -galactosidase assays

Effects on endogenous wild-type p53 activity were monitored with a p53 response element/luciferase reporter construct that was cotransfected with a -galactosidase reporter for normalization of transfection efficiency (Hughes et al., 2000). The p53 reporter plasmid contained a strong hsp70 promoter element upstream of the luciferase gene. The DNA binding sites for p53 (p53RE) are located upstream of the hsp70 element with the p53 consensus binding elements from a vector named 'p53CON-Frag.A', (Martinou et al., 1995; Hughes et al., 2000). PC12 cells were plated on poly-L-ornithine-coated tissue culture plates at 70% confluency the day before transfection. Plasmids were packaged in Lipofectamine-plus (Gibco-BRL) liposome vehicles. The transfection protocol was performed according to Gibco-BRL's specific instructions for transfection of PC12 cells. Cells were then placed in medium containing serum with or without NGF. Cells were harvested 1 day later and lysed in RLB buffer (Promega proprietary). Total cell lysates were assayed colorimetrically with a -galactosidase assay kit (Promega) and for luminescence with a luciferase assay kit (Promega). Luciferase data were normalized relative to overall transfection efficiency as determined by -galactosidase expression and then data were presented as normalized luciferase units. All transfection experiments were performed three times with the assays in duplicate for each experiment.

Induction of apoptosis

In the case of naive cells, serum was removed by washing the media four times in PBS/pH 7.4 and once in serum-free media (Dulbecco's modified Eagle's medium, without serum and with L-glutamine and PEN-STREP antibacterial–antimycotic). The cells were then placed in the incubator at 38.5°C/10% CO₂ and 90% air. Apoptosis was induced in 7-day 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 g/ml anti-NGF antibody (Santa Cruz, catalog number sc-485) for 30 min (in the incubator at 38.5°C/10% CO₂ and 90% air) and then placed in 1% serum without NGF. Finally, the cells were placed in the incubator at 38.5°C/10% CO₂ and 90% air for various time points.

Statistics

Student's t-test or ANOVA were carried out as indicated. Student's t-test was calculated to determine significance (P<0.05; 95% confidence level). When the exact cell lines (i.e. PC12[p53ts]) were treated and comparison was made, a paired t-test was calculated. When different cell lines were compared (i.e. PC12[vec] to PC12[p53ts]) after the same treatment and at the same time point, 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, USA. Differences between means were examined using one- or two-way analysis of variance followed by a Tukey's post hoc comparison. Differences were considered significant if P<0.05. All results are presented as meanstandard deviation (s.d.). Data were graphed using SigmaPlot 2000 or Microsoft Excel.

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Acknowledgements

This work was supported by a grant from the USPHS, National Institutes of Health, NINDS, NS24380 to KEN. We thank Laurie Dvorak, Sang B Woo, and Nandan Lad for useful discussions and Debbie Messineo-Jones for technical assistance.