P53 Transcription-Dependent and -Independent Regulation of Cerebellar Neural Precursor Cell Apoptosis

By Geng, Ying; Akhtar, Rizwan S; Shacka, John J; Klocke, Barbara J; Et al

Abstract

Regulation of cerebellar neural precursor cell (NPC) death is important for both normal brain development and prevention of brain tumor formation. The tumor suppressor p53 is an important regulator of NPC apoptosis, but the precise mechanism of p53-regulated cerebellar NPC death remains largely unknown. Here, by using primary cerebellar NPCs and a mouse cerebellar NPC line, we compared the molecular regulation of cerebellar NPC death produced by staurosporine (STS), a broad-spectrum kinase inhibitor, with that caused by genotoxic agents. We found that both STSand genotoxin- induced cerebellar NPC death were markedly inhibited by p53 or Bax deficiency. Genotoxin-induced cerebellar NPC death required new protein synthesis and PUMA, a p53 transcriptionally regulated BH3- only molecule. In contrast, STS caused cerebellar NPC death without requiring new protein synthesis or PUMA expression. In addition, genotoxic agents increased nuclear p53 immunoreactivity, whereas STS produced rapid cytoplasmic p53 accumulation. Interestingly, STS- induced death of cerebellar granule neurons was p53-independent, indicating a differentiation-dependent feature of neuronal apoptotic regulation. These results suggest that STS-induced cerebellar NPC death requires a direct effect of p53 on cytoplasmic apoptotic mediators, whereas genotoxin-induced death requires p53-dependent gene transcription of PUMA. Thus, p53 has multiple death promoting mechanisms in cerebellar NPCs.

Key Words: Bax, Cell death, Genotoxic injury, PUMA, Staurosporine.

INTRODUCTION

Neural precursor cells (NPCs) consist of multipotent neural stem cells and lineage-restricted neural progenitor cells (1). During development, cerebellar NPCs reside in the external granule layer (EGL) of the cerebellum and migrate inward to differentiate into granule neurons (2). Cerebellar NPCs are believed to be the cells of origin for medulloblastoma, the most common malignant brain tumor of childhood (3).

NPC fate is regulated by proliferation, differentiation, and death. Therefore, it is essential to define the molecular regulation of cerebellar NPC apoptosis. On receipt of an intrinsic death stimulus, proapoptotic multidomain Bc1-2 family members Bax and/or Bak are activated, increase the permeability of mitochondria, and promote the release of mitochondrial cytochrome c into the cytosol (4). BH3domain only (BH3-only) Bc1-2 subfamily molecules such as Noxa and PUMA directly or indirectly activate proapoptotic multidomain Bc1-2 subfamily molecules Bax or Bak (5). Cytochrome c, together with Apaf-1 and procaspase-9, forms the apoptosome, which in an energy-dependent fashion activates caspase-9 and triggers the activation of downstream effector caspases (4). Our previous studies have shown that genotoxic injury induces embryonic telencephalic NPC death through the intrinsic pathway and requires Bax/Bak, Apaf-1, and caspase-9 (6, 7).

Another important regulator of genotoxin-induced NPC death is the tumor suppressor p53. The p53 gene is the most commonly mutated gene in human cancers and has been described as the “guardian of the genome” (8). As a transcription factor, p53 is implicated in both cell-cycle arrest and apoptosis regulation in different cell types or under different stress stimuli (9, 10). p53 may act to induce expression of genes mediating cell cycle arrest (e.g. p21) or to activate transcription of target genes (e.g. noxa, puma) to promote apoptosis (11). Recently, p53 has also been found to regulate transcription-independent induction of apoptosis by directly interacting with Bc1-2 family members (12). In NPCs, p53 has been shown to mediate ionizing radiation-induced cerebellar NPC death in vivo (13) and cytosine arabinoside (AraC)induced telencephalic NPC death both in vivo and in vitro (14, 15). However, whether p53 is required for staurosporine (STS)-induced cerebellar NPC death is unknown. STS is a broad-spectrum protein kinase inhibitor and its analog, UCN01, is under clinical trials for antitumor activity (16). In the present study, we compared the molecular regulation of cerebellar NPC death produced by genotoxic agents versus that caused by STS. We report here that both Bax and p53 are critical regulators of both genotoxin- and STS-induced cerebellar NPC death. However, unlike genotoxin-induced death, STS-induced cerebellar NPC death is independent of p53-dependent gene transcription.

MATERIALS AND METHODS

Chemicals

AraC, bleomycin, STS, and cycloheximide (CHX), were all purchased from Sigma (St. Louis, MO). BOC-aspartyl(Ome)-fluoromethyl ketone (BAF) was purchased from MP Biomedicals (Aurora, OH).

Mice

Generation of box^sup -/-^ (17), bak^sup -/-^ (18), noxa^sup -/- ^, and puma^sup -/-^ (19) mice have been described previously. p53^sup +/-^ mice were purchased from Taconic (Germantown, NY). Endogenous and disrupted genes were detected by polymerase chain reaction analysis of tail DNA extracts as described previously (20). The day the pups were born was counted as day 0. Mice were cared for in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals. All animal protocols were approved by Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.

Cell Cultures

Cerebellar NPCs were harvested from the cerebellum of postnatal day 6 to 7 mice. The isolation process was similar to the isolation of telencephalic NPCs, as we previously published (15). Cells were placed in uncoated flasks (Corning, Inc., Coming, NY) at 37C in humidified 5% CO2/95% air atmosphere and glial cells, postmitotic neurons, and other adherent cell populations were allowed to attach to the bottom of the flask. Twenty-four hours later, floating cells, consisting almost exclusively of NPCs, were transferred to poly-L- lysine (Sigma)/laminin (BD Bioscience, Bedford, MA)-coated flasks. Cerebellar NPCs formed adherent monolayer cultures and were allowed to grow. Fresh media was added to cultures every 3 days. Cell suspensions were prepared by trypsinization as stated previously and plated to poly-L-lysine/laminin-coated 48-well tissue culture plates (Corning, Inc.). A small aliquot of cells was stained with Trypan Blue (Sigma) and counted and 30,000 cells were plated per well. Cultures were then incubated for another 2 to 4 days in medium containing fibroblast growth factor-2 (FGF-2) before they were used for experiments. Genotoxic agents and STS were dissolved in medium containing FGF-2 and added to the cells for various length of time as indicated.

Cerebellar granule neurons (CGNs) were isolated from postnatal day 7 mice as described previously (21, 22). Briefly, the cerebella were dissociated in dissection media containing 0.02% trypsin and further trituration. After allowing debris to settle briefly, the cell suspension was centrifuged through a column of 4% BSA in dissection media. The resulting pellet was then resuspended in Neurobasal media (Gibco, Grand Island, NY) supplemented with 1% Pen Strep (Gibco), 0.5 mM glutamine, 25 mM KCl, B-27 Supplement (Gibco), and β-mercaptoethanol. Cells were plated in poly-L-lysine- coated plates at a density of 1250 cells/mm^sup 2^. Forty-eight- well plates were used for viability assays; CGNs were treated on day 4.

The C17.2 cell line was cultured in high-modified DMEM (Gibco) containing 1% penicillin/streptomycin, 1% L-glutamine (all from Sigma), 5% horse serum, and 10% fetal calf serum (FCS) (all from Gibco). Fresh media was added to cultures every 2 days. Cell suspensions were prepared by trypsinization and plated onto uncoated 48-well plates at a density of 30,000/well. Cultures were then incubated overnight before being used in experiments. During drug treatment, cell culture medium was switched to high modified DMEM without horse serum or FCS.

Cell Viability and In Vitro Caspase Cleavage Assays

As previously described (21, 22), cells were washed once with Locke’s buffer and then incubated at 37C for 30 minutes in Locke’s buffer containing 5 M calcein AM (Molecular Probes, Eugene, OR). Calcein-AM conversion was read using a fluorescence plate reader (excitation 488 nM, emission 530 nm). Cells used for in vitro caspase-3 cleavage assays were lysed in the 48-well plate followed by the addition of buffer containing 10 M DEVD-7-amino-4- methylcoumarin (AMC) (Biomol, Plymouth Meeting, PA). The plate was incubated for 30 minutes at 37C in the dark and the generation of the fluorescent AMC cleavage product was measured at 360 nm excitation, 460 nm emissions, using a fluorescence plate reader. Both calcein-AM conversion and DEVD-AMC cleavage was expressed relative to untreated controls.

Western Blot

Cells were washed and collected from plates in phosphate- buffered saline (PBS), resuspended with 2 sample buffer, and boiled for 5 minutes. Proteins were then resolved in a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. After transfer, blots were used for detection of p21 (Santa Cruz Biotechnology, Santa Cruz, CA) or PUMA (Prosci Inc., San Diego, CA). Total protein levels were normalized to actin (Sigma).

Immunocytochemistry and Immunohistochemistry

For immunocytochemical detection, cells were fixed in 4% paraformaldehyde for 20 minutes at 4C, washed with PBS 3 times and then incubated for 3\0 minutes in PBS-Blocking Buffer (PBS-BB; PBS containing 1% BSA, 0.2% powdered milk, and 0.3% Triton X-100). Primary antibodies were diluted in PBS-BB (without Triton X-100) at the indicated dilutions and applied overnight at 4C. Primary antibodies used were antinestin (1:20,000; Rat-401 mouse monoclonal antibody; Developmental Studies Hydridoma Bank, University of Iowa, Iowa City, IA), antiglial fibrillary acidic protein (GFAP) (1:10,000; Z0334 rabbit polyclonal antiserum; Dako Corp., Carpinteria, CA), antineuron-specific nuclear protein (NeuN) (1:10,000; MAB377; Chemicon, Temecula, CA), and anti-p53 (1:5000; Ncl-p53-Cm5P rabbit polyclonal antiserum; Novocastra Laboratories, Newcastle upon Tyne, UK). After washes with PBS, plates were incubated with a horseradish peroxidase-conjugated donkey anti- rabbit or anti-mouse secondary antibodies (Jackson Immunoresearch, West Grove, PA) diluted to 1:2000 in PBS-BB (without Triton X-100) for 1 hour at room temperature. After washes with PBS, immunoreactivity was detected using a tyramide signal amplification system (Perkin-Elmer Life Science Products, Boston, MA) according to the manufacturer’s instructions. Cultures were counterstained with bisbenzimide (2 g/mL; Hoechst 33258; Sigma) and examined with a Zeiss-Axiovert fluorescence microscope. To quantitate immunocytochemistry, the number of positive cells was counted as a fraction of total cells in 4 randomly selected fields for each well. At least 3 independent wells from multiple preparations were immunostained and quantitated for each experiment.

Immunohistochemistry was performed as previously described (6). Mouse brains were isolated 6 hours after injection of either AraC (25 mg/kg subcutaneously) or vehicle fixed in Bouin’s fixative at 4C overnight and then embedded in paraffin. AraC was selected as prototypical genotoxic agent for the in vivo analysis because unlike bleomycin, which has poor blood-brain barrier penetration (23), AraC is a potent activator of neural precursor cell apoptosis in vivo (15). Sections of cerebellar EGL were analyzed for cleaved-caspase- 3 (1:500; Cell Signaling, Danvers, MA) and p53 (1:1000; Ncl-p53- Cm5P rabbit polyclonal antiserum; Novocastra Laboratories) immunoreactivity.

Statistics

All data points represent mean standard error of the mean (n = 6 wells for all experiments except those stated otherwise). All experiments were repeated at least 3 times except stated otherwise. Representative data are shown. Significant effects of treatment were analyzed by one-way analysis of variance. Genotype-specific effects of treatment were analyzed for significance by 2-way analysis of variance. Post hoc analysis was conducted using the Bonferroni test. A level of p < 0.05 was considered significant.

RESULTS

Both Genotoxic Injury and Staurosporine Induce Concentration- Dependent Increases in Cell Death and Caspase-3 Enzymatic Activity in Cerebellar Neural Precursor Cells

To study the molecular regulation of cerebellar NPC apoptosis, we first established FGF-2 expanded primary cerebellar NPC cultures from mice at postnatal day 6 to 7 in which more than 90% of the cells express nestin, a neural precursor marker (data not shown). The cultures were almost completely negative (<1% of cells) for NeuN immunoreactivity, a marker of mature neurons, and showed only a low level of GFAP expression (<5% of cells), a marker of astrocytes (data not shown). These results indicate that FGF-2 expands and maintains cerebellar NPCs in an undifferentiated state in vitro.

FGF-2 expanded cerebellar NPCs were treated with genotoxic agents, including AraC, a nucleoside analog or bleomycin sulfate, an inducer of double-strand DNA breaks or with STS. AraC (Fig. 1A), bleomycin (Fig. 1B), and STS (Fig. 1C) all induced cerebellar NPC death in a concentration-dependent manner. AraC (Fig. 1D), bleomycin (Fig. 1E), and STS (Fig. 1F) also caused concentration-dependent caspase-3 activation measured by in vitro enzymatic cleavage of DEVD- 7-amino-4-methylcoumarin, a fluorogenic caspase-3 like substrate. In comparison with genotoxic injury, STS produced more rapid caspase-3 activation and cell death (Fig. 1 and data not shown). These results indicate that both STS and genotoxic injury induce cerebellar NPC apoptosis.

Both Genotoxin- and Staurosporine-Induced Cerebellar Neural Precursor Cell Apoptosis Require Bax

We further determined whether genotoxic injury and STS induced cerebellar NPC apoptosis through the intrinsic death pathway, which requires Bax and/or Bak, 2 multi-BH domain, proapoptotic Bcl-2 family members. Cerebellar NPCs were prepared from Bax-deficient, Bax heterozygous, and wild-type littermate mice. Compared with cerebellar NPCs prepared from wild-type or heterozygous mice, Bax- deficient cerebellar NPCs exhibited significantly less death induced by bleomycin (Fig. 2A) or STS (Fig. 2B). In contrast, cerebellar NPC death was not attenuated by the targeted deletion of Bak (Fig. 2C, D). Overall, these data indicate that Bax, not Bak, is the major proapoptotic, multidomain Bcl-2 molecule regulating cerebellar NPC death and that both genotoxic injury and STS produce cerebellar NPC death through the Bax-dependent intrinsic pathway.

p53 Mediates Both Genotoxin- and Staurosporine-Induced Cerebellar Neural Precursor Cell Death

To study whether p53 is required for both genotoxin- and STS- induced cerebellar NPC death, we compared p53-deficient, p53 heterozygous, and wild-type cerebellar NPCs after exposure to different concentrations of either bleomycin (48 hours) or STS (6 hours). p53 deficiency decreased both bleomycin- (Fig. 3A) and STS- (Fig. 3B) -induced cerebellar NPC death. These findings indicate that p53 is required for both genotoxin- and STS-induced cerebellar NPC death.

To further determine whether p53-mediated, STS-induced cerebellar NPC death is cell differentiation-dependent, CGNs, the postmitotic neurons derived from cerebellar NPCs, were treated with 0.1 M STS for 24 hours. p53 deficiency failed to protect CGNs from STS- induced death (Fig. 3C), suggesting that p53-dependent, STS-induced death is a differentiation-dependent feature of cerebellar NPCs.

PUMA is Required for Genotoxin- but not Staurosporine-Induced Cerebellar Neural Precursor Cell Death

After genotoxic injury, p53 transcriptionally upregulates expression of the downstream proapoptotic BH3 only molecules Noxa and PUMA (11). We previously reported that genotoxin-induced telencephalic NPC death is regulated by PUMA both in vivo and in vitro (24). To test the contribution of PUMA to genotoxin- and STS- induced cerebellar NPC death, we examined cerebellar NPCs in vitro. Similar to the previous experiments, PUMA-deficient or wild-type cerebellar NPCs were exposed to 0.03 U/mL bleomycin for 48 hours or 1 M STS for 6 hours. PUMA deficiency significantly decreased bleomycin-induced cerebellar NPC death but not STS-induced cerebellar NPC death (Fig. 4A). To determine if a separate p53 regulated BH3-only molecule, Noxa, is required for STS-induced cerebellar NPC death, Noxa-deficient cerebellar NPCs and wild-type cells were exposed to bleomycin or STS. Noxa deficiency failed to inhibit either bleomycin- or STS-induced cerebellar NPC death (data not shown). Overall, our results indicate that genotoxin-induced cerebellar NPC death requires p53 transcriptionally regulated PUMA expression, whereas STS-induced cerebellar NPC death does not require either PUMA or Noxa expression.

FIGURE 1. Exposure to genotoxic agents or staurosporine (STS) induces cell death and caspase-3 activation in primary cerebellar neural precursor cells (NPCs). Exposure for 48 hours to cytosine arabinoside (AraC) (A), bleomycin (B), or 6 hours to STS (C) induces a significant decrease in cell viability. Similarly, exposure for 24 hours to AraC (D), bleomycin (E), or 6 hours to STS (F) significantly decreases caspase-3-like enzymatic activity. Cell viability and caspase-3 activation are normalized to the untreated group. Data points represent mean standard error of the mean with n = 6 (*, p < 0.001 by one-way analysis of variance/Bonferroni posttest compared to untreated group).

To confirm the neuroprotective effects of PUMA deficiency in vivo, postnatal day 7 mice were injected with AraC. In wild-type cerebellum, AraC produced a marked increase in cleaved caspase-3 immunoreactivity in the EGL. AraC-treated PUMA-deficient mice showed only limited activated caspase-3 immunoreactivity in the EGL (Fig. 4B). Compared with wild-type littermates, PUMA deficiency decreased cleaved caspase-3 immunoreactivity after AraC injection. In addition, markedly increased nuclear p53 immunoreactivity, detected by a p53-specific antibody was also found in the EGL of the PUMA- deficient mice after AraC injection. These results confirm that PUMA is a key regulator of genotoxin-induced cerebellar NPC death in vivo and that after genotoxic injury, deficiency of PUMA leads to p53 nuclear accumulation in NPCs located in the EGL as a result of downstream inhibition of cerebellar NPC apoptosis.

Genotoxic Injury Causes p53 Transcription-Dependent Death, Whereas Staurosporine Produces p53 Transcription-Independent Death

Our data showed that the p53 transcriptionally regulated molecule, PUMA, is required for genotoxin-induced, but not STS- induced, cerebellar NPC death. Thus, we hypothesized that p53 causes transcriptional-dependent death upon genotoxic injury, whereas p53 causes transcriptionindependent death after STS treatment. CHX, an inhibitor of protein synthesis (25), was used to determine whether new macromolecular synthesis is required for genotoxininduced or STS- induced activation of caspase-3 in cerebellar NPCs. The effect of CHX was compared with treatment with BAF, a broad-spectrum caspase inhibitor. CHX decreased bleomycin-induced caspase-3 activation (Fig. 5A), but not STS-induced caspase-3 activation (Fig. 5B). As expected, BAF inhibited caspase-3 activity pro\duced by both stimuli. These results indicate that genotoxin-induced caspase-3 activation requires new protein synthesis, whereas STS-induced caspase-3 activation does not.

To further test the hypothesis that genotoxic injury induces p53 transcription-dependent death and STS-induced death depends on p53 transcription-independent function, p53-regulated genes were analyzed as indicators of p53 transcriptional function. p21^sup Wafl/ CiP1/Sdi1^ is a p53 transcriptionally regulated gene and inhibits different phases of the cell cycle by inhibiting cyclin/cyclin- dependent kinase complex (26). The BH3-only molecule PUMA is also upregulated by p53 and can induce apoptosis upon genotoxic injury (19, 27). Therefore, p21 and PUMA were chosen as markers of p53 transcriptional function. Primary cerebellar NPCs were treated with AraC (3 M; 6 or 12 hours), bleomycin (0.03 U/mL; 6 or 12 hours), or with STS (0.3 M; 3 or 6 hours), and levels of p21 and PUMA proteins were measured by Western blot. Compared with untreated control, levels of p21 and PUMA did not change after exposure to STS, whereas levels of p21 and PUMA increased significantly after genotoxic injury (Fig. 5C and data not shown).

C17.2 Cells Recapitulate Cerebellar Neural Precursor Cell Apoptotic Death Pathways

To further characterize the molecular regulation of p53 in cerebellar NPC death, we used a cerebellar precursor cell line, C17.2, which was generated from the mouse cerebellar EGL by retroviral vector-mediated transduction of avian myc oncogene (28). C17.2 cells transplanted into the developing mouse cerebellum integrate in a nontumorigenic fashion and differentiate into neurons and glia (29). Our immunocytochemical analysis of C17.2 cells in vitro showed that more than 99% of the cells express the NPC marker nestin and were almost completely negative for NeuN and GFAP immunoreactivity (data not shown).

FIGURE 2. Bax deficiency, but not Bak deficiency, decreases both genotoxin- and staurosporine (STS) -induced cell death. Bax deficiency attenuates both bleomycin (48 hours) (A) and STS (6 hours) (B) induced neural precursor cell (NPC) death. Bak deficiency shows no protection from bleomycin (C) or STS (D) treatment. Data points represent mean standard error of the mean with n = 6 (*, p < 0.001 by 2-way analysis of variance/Bonferroni posttest compared to wild-type treated group).

C17.2 cells undergo concentration-dependent caspase-3 activation and cell death after AraC, bleomycin, or STS treatment, similar to that of cerebellar NPCs (data not shown). Also similar to primary cerebellar NPCs, CHX decreased caspase-3 activation induced by bleomycin (Fig. 6A) but not STS (Fig. 6B) in C17.2 cells. Exposure of C17.2 cells to 3 M AraC or 0.03 U/mL bleomycin for 6 hours increased p21 and PUMA protein expression levels, whereas levels of p21 and PUMA were unaffected by exposure to STS (Fig. 6C). These results suggest that, like primary cerebellar NPCs, genotoxic injury causes p53 transcription-dependent and STS induces p53 transcription- independent caspase-3 activation and death in C17.2 cells.

Genotoxic Injury Causes Nuclear p53 Accumulation, Whereas Staurosporine Produces Cytosolic p53 Accumulation

In response to genotoxic injury and other stresses, posttranslational modification of p53 leads to its nuclear accumulation and p53 binds to DNA to induce downstream gene transcription (30). The subcellular localization of p53 in C17.2 cells after genotoxic injury or STS was determined by immunocytochemical analysis. Untreated C17.2 cells showed relatively weak nuclear p53 immunoreactivity (5.0 2.5% of nucleated cells, n = 3 wells) (Fig. 7A). After exposure to AraC (3 M) or bleomycin (0.03 U/mL) for 6 hours, there was a marked increase in the percentage of cells displaying nuclear p53 immunoreactivity (13.3 5.7%, n = 3 wells; 15.5 2.9%, n = 3 wells, respectively; Fig. 7B, C). In contrast, p53 cytosolic accumulation occurred after treatment with STS (0.3 M) for 3 hours (Fig. 7D) and no increase in the percentage of cells with p53 nuclear immunoreactivity was observed (1.1 1.1%, n = 3 wells).

FIGURE 3. p53 deficiency decreases both genotoxin- and staurosporine (STS) -induced cerebellar neural precursor cell (NPC) death. p53 deficiency attenuates both bleomycin- (48 hours) (A) and STS- (6 hours) (B) induced NPC death. Data points represent mean standard error of mean with n = 6 (*, #, p < 0.001 by 2-way analysis of variance/Bonferroni posttest compared to wild-type treated group). (C) p53 deficiency does not protect STS-induced cerebellar granule neuron (CGN) death. Data points represent mean standard error of the mean with n = 28 (*, p < 0.001 by 2-way analysis of variance/Bonferroni posttest compared to wild-type treated group).

DISCUSSION

In this study, we found that p53 has dual death-promoting actions in cerebellar NPCs that are differentially activated by specific death stimuli. Both genotoxin- and STS- induced cerebellar NPC death require p53. Genotoxic injury, but not STS, upregulates expression of PUMA, which is required for genotoxin-induced, but not STS- induced, cerebellar NPC death. In addition, caspase-3 activation caused by genotoxic injury requires new protein synthesis, whereas caspase-3 activation produced by STS does not. Finally, STS promoted p53 cytosolic accumulation, whereas genotoxic injury increased nuclear p53 immunoreactivity. Taken together, our experiments indicate that genotoxin-induced cerebellar NPC death requires p53- dependent gene transcription, whereas STS-induced cerebellar NPC death requires p53 but is independent of p53-dependent gene transcription.

Proapoptotic p53 activity in the absence of gene transcription or protein translation has been known for over 10 years (31, 32). However, the molecular mechanism for this effect has only recently been investigated and is incompletely understood. Several reports indicate that after genotoxic injury, a small fraction of p53 translocates to mitochondria in addition to its nuclear accumulation (33, 34). Targeting p53 to mitochondria in p53-deficient cells has been shown to directly trigger apoptosis (34). Other groups have reported that after genotoxic injury, p53 may directly trigger Bax activation (35), and this process involves PUMA (36). In our study, STS triggered cytoplasmic p53 accumulation and p53 transcription- independent cell death in which PUMA expression was not required. Thus, we clearly define a p53 transcription-independent death pathway in cerebellar NPCs that can be activated by STS.

FIGURE 4. PUMA deficiency decreases genotoxin- but not staurosporine (STS) -induced cerebellar neural precursor cell (NPC) death. PUMA deficiency attenuates bleomycin- (0.03 U/mL) (48 hours), but not STS- (1 M) (6 hours) (A) induced cerebellar NPC death. Data points represent mean standard error of the mean with n = 6 (*, p < 0.001 by 2-way analysis of variance/Bonferroni posttest compared with wild-type treated group). (B) Untreated wild-type mice exhibits minimal cleaved caspase-3 immunoreactivity at baseline. After wild- type mice are exposed to cytosine arabinoside (AraC), there is a significant increase of cleaved caspase-3 immunoreactivity in the external granule layer (EGL). PUMA deficiency dramatically decreases caspase-3 activation after AraC treatment. Compared with untreated wild-type mice, AraC-treated wild-type mice shows only a slight increase of p53 immunoreactivity in the EGL. AraC-treated PUMA- deficient mice shows a marked increase of p53 immunoreactivity in the EGL.

FIGURE 5. Inhibition of new macromolecular synthesis attenuates genotoxin-induced caspase-3 activation in cerebellar neural precursor cells (NPCs) but not staurosporine (STS) – induced caspase- 3 activation. (A) Cycloheximide (CHX) (0.1 g/mL) significantly attenuates caspase-3-like enzymatic activity in cerebellar NPCs after 12-hour exposure to bleomycin. BOC-aspartyl(Ome)-fluoromethyl ketone (BAF) (150 M), a broad-spectrum caspase inhibitor, also prevented caspase activation after genotoxic injury. (B) CHX (0.1 g/ mL) does not affect STS-induced caspase-3-like enzymatic activity. However, BAF (150 M) significantly decreases caspase-3 activation. Data points represent mean standard error of the mean with n = 6 (*, p < 0.001 by analysis of variance/ Bonferroni posttest compared with bleomycin or STS alone treated group). Caspase-3 activity is normalized to untreated control cerebellar NPCs. (C) Compared with untreated (U) cerebellar NPCs, 3 M AraC (A)- or 0.03 U/mL bleomycin (B)-treated cells shows increased protein expression of PUMA and p21. 0.3 M STS (S) has much less effect on either PUMA or p21 protein levels. Actin protein expression serves as an internal control.

FIGURE 6. C17.2 cells recapitulate cerebellar neural precursor cell (NPC) apoptotic death pathways. (A) Cycloheximide (CHX) (0.1 g/ mL) significantly attenuates caspase-3-like enzymatic activity in C17.2 cells after 6-hour exposure to bleomycin. BOC-aspartyl(Ome)- fluoromethyl ketone (BAF) (150 M) also prevents caspase activation after genotoxic injury. (B) CHX (0.1 g/mL) does not affect staurosporine (STS)-induced caspase-3-like enzymatic activity. However, BAF significantly decreases caspase-3 activation. Data points represent mean standard error of the mean (*, p < 0.001 by analysis of variance/Bonferroni posttest compared with bleomycin or STS alone treated group). Caspase-3 activity is normalized to untreated control C17.2 cells. (C) Compared with untreated (U) C17.2 cells, 3 M AraC (A)- or 0.03 U/mL bleomycin (B)-treated cells shows increased protein expression of PUMA and p21. 0.3 M STS (S) has no effect on either PUMA or p21 protein levels. Actin protein expression serves as an internal control.

p53-induced apoptotic cell death is cell-type and stimulus specific. In the nervous system, p53 mediates neuronal cell death on a variety of injuries, including genotoxic stress, hy\poxia, tropic factor insufficiency, and excitotoxicity (37, 38). STS-induced hippocampal neuron death has been reported to be p53-independent (39). We found that p53 deficiency protected cerebellar NPCs but not CGNs from STS-induced death. This differential dependence of NPCs and postmitotic neurons on p53 may be explained by the higher basal expression of p53 in NPCs relative to postmitotic neurons (40), which can result in a rapid cytosolic accumulation of p53 after STS treatment. In contrast, postmitotic neurons may have insufficient levels of p53 to induce p53 transcription-independent cell death after STS treatment. Alternatively, differential expression of Bcl- 2 family members in NPCs versus postmitotic neurons may affect the ability of cytosolic p53 to trigger apoptosis. For example, levels of the important antiapoptotic protein Bcl-XL are low in NPCs relative to postmitotic neurons and this may predispose NPCs to the death-promoting effects of cytosolic p53 (41).

FIGURE 7. Genotoxins and staurosporine (STS) produce different patterns of p53 immunoreactivity in C17.2 cells, Immunodetection of p53 in untreated C17.2 cells shows only weak immunoreactivity (A). Treatment with either 3 M cytosine arabinoside (AraC) (B) or 0.03 U/ mL bleomycin (C) for 6 hours produces extensive nuclear p53 immunoreactivity (red) as evidenced by colocalization with the nuclear label Hoechst 33,258 (blue). In contrast, 0.3 M STS for 3 hours produces marked cytoplasmic p53 immunoreactivity (D).

Proapoptotic, multidomain Bcl-2 molecules Bax and Bak have been found to critically regulate the intrinsic apoptotic death pathway. In unstressed cells, Bax and Bak exist in inactive conformations (42). After apoptotic stimuli, proapoptotic BH3-only molecules such as Noxa and PUMA directly or indirectly activate Bax or Bak (5). Upon activation, the cytosolic, monomeric Bax translocates to the mitochondria where it homodimerizes and inserts into mitochondrial membranes (43). This process correlates with exposure of the aminoterminal domain of Bax, which can also be found in Bak activation (44, 45). We previously reported that both Bax and Bak are involved in genotoxin-induced embryonic telencephalic NPC death. Single deficiency of Bax or Bak did not completely protect such cells from genotoxic injury (6). In contrast, other studies have shown that both naturally occurring and ionizing irradiation- induced cerebellar NPC death is Bax-dependent (46, 47). In this study, we found that Bax alone is the key regulator of both genotoxin- and STS-induced cerebellar NPC death. Therefore, different subsets of NPCs may use different multidomain, proapoptotic Bcl-2 molecules to regulate apoptosis.

p53 mutations are found in approximately 10% of human medulloblastomas (48), and several clinical-pathologic studies have shown that intense p53 immunoreactivity in tumor cells correlates with a poor prognosis for medulloblastoma patients (49, 50). In mouse models of medulloblastomas, p53 deficiency significantly accelerates and increases the incidence of tumor formation (51, 52). Interestingly, p53-dependent, transcription-independent apoptotic function has been reported to mediate tumor suppression in primary lymphomas both in vitro and in vivo (53). These results suggest that inhibition of p53 apoptotic function might also be involved in human medulloblastoma formation and progression. In this study, we have shown that STS induces p53 transcription-independent cell death in cerebellar NPCs, the cell origin of medulloblastomas. Therefore, activation of p53 transcription-independent cell death by STS or its analog, UCN-01, might have therapeutic potential for human medulloblastomas, especially those with high levels of p53 and a poor prognosis.

ACKNOWLEDGMENTS

The authors thank Dr. Evan Snyder (Burnham Institute) for generously providing the C17.2 cell line; Dr. Andreas Strasser (Walter and Eliza Hall Institute of Medical Research) for the Noxa and PUMA-deficient mice; and Ms. Cecelia B. Latham, Mr. Enfu Bi, and the UAB Neuroscience Core Facilities (NS4746 and NS57098) for technical assistance. The nestin antibody was developed by Susan Hockfield and was obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa.

REFERENCES

1. Svendsen CN, Smith AG. New prospects for human stem-cell therapy in the nervous system. Trends Neurosci 1999;22:357-64

2. ten Donkelaar HJ, Lammens M, Wesseling P, et al. Development and developmental disorders of the human cerebellum. J Neurol 2003;250: 1025-36

3. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain rumour initiating cells. Nature 2004;432:396-401

4. Hengartner MO. The biochemistry of apoptosis. Nature 2000;407: 770-76

5. Willis SN, Adams JM. Life in the balance: How BH3-only proteins induce apoptosis. Curr Opin Cell Biol 2005; 17:617-25

6. D’Sa C, Klocke BJ, Cecconi F, et al. Caspase regulation of genotoxin-induced neural precursor cell death. J Neurosci Res 2003;74:435-45

7. D’Sa-Eipper C, Roth KA. Caspase regulation of neuronal progenitor cell apoptosis. Dev Neurosci 2000;22:116-24

8. Whitesell L, Sutphin P, An WG, et al. Geldanamycin-stimulated destabilization of mutated p53 is mediated by the proteasome in vivo. Oncogene 1997;14:2809-16

9. Fei P, El-Deiry WS. p53 and radiation responses. Oncogene 2003;22:5774-83

10. Vousden KH, Lu X. Live or let die: The cell’s response to p53. Nat Rev Cancer 2002;2:594-604

11. Yu J, Zhang L. The transcriptional targets of p53 in apoptosis control. Biochem Biophys Res Commun 2005;331:851-58

12. Schuler M, Green DR. Transcription, apoptosis and p53: Catch- 22. Trends Genet 2005;21:182-87

13. Herzog KH, Chong M, Kapsetaki M, et al. Loss of Atm and p53 is radioprotective in the central nervous system. Eur J Neurosci 1998;10:12

14. Akhtar RS, Geng Y, Klocke BJ, et al. Neural precursor cells possess multiple p53-dependent apoptotic pathways. Cell Death Differ 2006;13:1727-39

15. D’Sa-Eipper C, Leonard JR, Putcha G, et al. DNA damage- induced neural precursor cell apoptosis requires p53 and caspase 9 but neither Bax nor caspase 3. Development 2001;128:137-46

16. Senderowicz AM. Small-molecule cyclin-dependent kinase modulators. Oncogene 2003;22:6609-20

17. Knudson CM, Tung KSK, Tourtellotte WG, et al. Bax-deficient mice with lymphoid hyperplasia and male germ-cell death. Science 1995;270:96-99

18. Lindsten T, Ross AJ, King A, et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 2000;6:1389-99

19. Villunger A, Michalak EM, Coultas L, et al. p53- and drug- induced apoptotic responses mediated by BH3-only proteins Puma and Noxa. Science 2003;302:1036-38

20. Kuida K, Haydar TF, Kuan CY, et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 1998;94:325-37

21. Nowoslawski L, Klocke BJ, Roth KA. Molecular regulation of acute ethanol-induced neuron apoptosis. J Neuropathol Exp Neurol 2005;64:490-97

22. Shacka JJ, Klocke BJ, Shibata M, et al. Bafilomycin A1 inhibits chloroquine-induced death of cerebellar granule neurons. Mol Pharmacol 2006;69:1125-36

23. Neuwelt EA, Barnett PA, Glasberg M, et al. Pharmacology and neurotoxicity of cis-diamminedichloroplatinum, bleomycin, 5- fluorouracil, and cyclophosphamide administration following osmotic blood-brain barrier modification. Cancer Res 1983;43:5278-85

24. Akhtar RS, Geng Y, Klocke BJ, et al. BH3-only proapoptotic Bcl-2 family members Noxa and Puma mediate neural precursor cell death. J Neurosci 2006;26:7257-64

25. Setkov NA, Kazakov VN, Rosenwald IB, et al. Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: A radioautographic and cell fusion study. Cell Prolif 1992;25:181-91

26. Roninson IB. Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): Association with cell senescence and tumour- promoting activities of stromal fibroblasts. Cancer Lett 2002;179:1- 14

27. Yu J, Zhang L, Hwang PM, et al. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673-82

28. Ryder EF, Snyder EY, Cepko CL. Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer. J Neurobiol 1990;21:356-75

29. Snyder EY, Deitcher DL, Walsh C, et al. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 1992;68:33-51

30. Xu Y. Regulation of p53 responses by post-translational modifications. Cell Death Differ 2003;10:400-03

31. Caelles C, Helmberg A, Karin M. p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature 1994;370:220-23

32. Haupt Y, Rowan S, Shaulian E, et al. Induction of apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev 1995;9:2170-83

33. Erster S, Mihara M, Kim RH, et al. In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation. Mol Cell Biol 2004;24:6728-41

34. Marchenko ND, Zaika A, Moll UM. Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem 2000;275:16202-12

35. Chipuk JE, Kuwana T, Bouchier-Hayes L, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 2004;303:1010-14

36. Chipuk JE, Bouchier-Hayes L, Kuwana T, et al. PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Science 2005;309:1732-35

37. Morrison RS, Kinoshita Y. The role of p53 in neuronal cell death. Cell Death Differ 2000;7:868-79

38. Wood KA, Youle RJ. The role of free radicals and p53 in neuron apoptosis in vivo. J Neurosci 1995;15:5851-57

39. Johnson MD, Xiang H, London S, et al. Evidence for involvement \of Bax and p53, but not caspases, in radiation-induced cell death of cultured postnatal hippocampal neurons. J Neurosci Res 1998;54:721-33

40. van Lookeren CM, Gill R. Tumor-suppressor p53 is expressed in proliferating and newly formed neurons of the embryonic and postnatal rat brain: comparison with expression of the cell cycle regulators p21Waf1/Cip1, p27Kip1, p57Kip2, p16Ink4a, cyclin G1, and the proto-oncogene Bax. J Comp Neurol 1998;397:181-98

41. Motoyama N, Wang F, Roth KA, et al. Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science 1995;267:1506-10

42. Scorrano L, Korsmeyer SJ. Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochem Biophys Res Commun 2003;304:437-44

43. Korsmeyer SJ, Wei MC, Saito M, et al. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 2000;7:1166-73

44. Goping IS, Gross A, Lavoie JN, et al. Regulated targeting of BAX to mitochondria. J Cell Biol 1998;143:207-15

45. Griffiths GJ, Dubrez L, Morgan CP, et al. Cell damage- induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 1999;144:903-14

46. White FA, Keller-Peck CR, Knudson CM, et al. Widespread elimination of naturally occurring neuronal death in Bax-deficient mice. J Neurosci 1998;18:1428-39

47. Chong MJ, Murray MR, Gosink EC, et al. Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system. Proc Natl Acad Sci U S A 2000;97:889-94

48. Adesina AM, Nalbantoglu J, Cavenee WK. p53 gene mutation and mdm2 gene amplification are uncommon in medulloblastoma. Cancer Res 1994;54:5649-51

49. Eberhart CG, Chaudhry A, Daniel RW, et al. Increased p53 immunopositivity in anaplastic medulloblastoma and supratentorial PNET is not caused by JC virus. BMC Cancer 2005;5:19

50. Woodburn RT, Azzarelli B, Montebello JF, et al. Intense p53 staining is a valuable prognostic indicator for poor prognosis in medulloblastoma/central nervous system primitive neuroectodermal tumors. J Neurooncol 2001;52:57-62

51. Tong WM, Ohgaki H, Huang H, et al. Null mutation of DNA strand break-binding molecule poly(ADP-ribose) polymerase causes medulloblastomas in p53(-/-) mice. Am J Pathol 2003;162:343-52

52. Wetmore C, Eberhart DE, Curran T. Loss of p53 but not ARF accelerates medulloblastoma in mice heterozygous for patched. Cancer Res 2001;61:513-16

53. Talos F, Petrenko O, Mena P, et al. Mitochondrially targeted p53 has tumor suppressor activities in vivo. Cancer Res 2005;65:9971- 81

Ying Geng, MS, Rizwan S. Akhtar, PhD, John J. Shacka, PhD, Barbara J. Klocke, MS, Jin Zhang, PhD, Xinbin Chen, PhD, and Kevin A. Roth, MD, PhD

From the Division of Neuropathology, Department of Pathology (YG, RSA, JJS, BJK, KAR), Department of Neurobiology (RSA), and Department of Cell Biology (JZ, XC), University of Alabama at Birmingham, Birmingham, Alabama.

Send correspondence and reprint requests to: Kevin A. Roth, MD, PhD, Division of Neuropathology, Department of Pathology, University of Alabama at Birmingham, SC 961, 1530 3rd Avenue South, Birmingham, Alabama 35294-0017; E-mail: kroth@path.uab.edu

This work was supported by grants from the National Institutes of Health (NS35107 and NS41962).

Copyright Lippincott Williams & Wilkins Jan 2007

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