Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine Charlesene McNeill-Blue a , Barbara A. Wetmore a , Joseph F. Sanchez b , William J. Freed b , B. Alex Merrick a,⁎ a

Proteomics Group, National Center for Toxicogenomics, National Institute of Environmental Health Sciences, National Institute of Health, Department of Health and Human Services, D2-04, P.O. Box 12233, Research Triangle Park, NC 27709, USA b Cellular Neurobiology Research Branch, National Institute on Drug Abuse, National Institute of Health, Department of Health and Human Services, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

AF5 neural cells derived from fetal rat mesencephalic tissue were immortalized with a

Accepted 4 July 2006

truncated SV40 LT vector lacking the p53-inactivating domain to maintain long-term

Available online 9 August 2006

cultures with a p53-responsive phenotype. This study examined p53 function in producing programmed cell death in propagating AF5 neural cells after exposure to hydrogen peroxide

Keywords:

(H2O2) and the kinase inhibitor staurosporine (STSP). Concentration-dependent exposure of

Apoptosis

AF5 cells to 0–800 mM H2O2 and STSP at 0–1000 nM revealed increasing cytotoxicity from

Hydrogen peroxide

MTS cell viability assays. Apoptosis occurred at 400 mM H2O2 as evidenced by subG1 DNA

Neural cells

and Annexin V flow cytometry analyses and cellular immunofluorescence staining with

p53

propidium iodide, anti-Annexin V and DAPI. DNA fragmentation, caspase-3/7 activity and

Rat

cytochrome c release into cytosol also confirmed H2O2-mediated apoptotic events. p53

Staurosporine

protein levels were increased over 24 h by H2O2 in a coordinated fashion with mdm2

Neurodegeneration

expression. p53 activation by H2O2 was evidenced by elevated Ser15 phosphorylation, increased luciferase p53 reporter activity and upregulation of the downstream p53 targets p21waf1 and apoptotic proteins, bax, Noxa and PUMA. STSP exposure produced apoptosis demonstrated by DNA fragmentation, caspase-3/7 activity, cytochrome c release and over 24 h was accompanied by sustained increase in p53 and Ser15 phosphorylation, rise in p21waf1 and bax and a transient increase in p53 reporter activity but without Annexin V binding. These findings demonstrate that AF5 cells undergo apoptosis in response to H2O2mediated oxidative stress and signal pathway disruption by STSP that therefore would be useful in studies related to p53-dependent neuronal cell death and neurodegeneration. Published by Elsevier B.V.

⁎ Corresponding author. Fax: +1 919 541 4704. E-mail address: [email protected] (B.A. Merrick). 0006-8993/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.brainres.2006.07.024

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Abbreviations: bFGF, basic fibroblast growth factor CNS, central nervous system CO2, carbon dioxide DAPI, 4,6-diamidino-2-phenylindole DAT, dopamine transporter ddH2O, distilled, deionized water DMEM, Dulbecco's modified Eagle's medium EDTA, ethylenediaminetetraacetic acid G1, gap1 G2, gap2 GFAP, glial fibrillary acidic protein H2O2, hydrogen peroxide MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine MTS, [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium O−2, superoxide radical OH−, hydroxyl radical PC12, pheochromocytoma cells PD, Parkinson's disease PMSF, phenylmethylsulfonylfluoride P-Ser15-p53, phosphorylation of p53 at Ser15 PI, propidium iodide PMSF, phenylmethylsulfonylfluoride PUMA, p53 upregulated modulator of apoptosis ROS, reactive oxygen species SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis STSP, staurosporine TH, tyrosine hydroxylase

1.

Introduction

Millions of people worldwide suffer from neurodegenerative disorders, but the pathogenic mechanisms leading to neuronal cell death are not well understood. Neural cell lines are valuable research tools for understanding the effectors and biochemical pathways underlying the neurodegenerative process. However, many oncogenically derived cell models often possess abnormal p53 function, altered cellular pathways and functions, and an inability to undergo apoptosis in a manner that limits their usefulness in the study of neuronal cell death. Tumor cell lines such as rat PC12 (Greene and Tischler, 1976), human SH-SY5Y (Pahlman et al., 1981) and mouse MN9D (a dopaminergic–neuroblastoma hybrid) (Choi et al., 1992) routinely used for neuronal studies face similar limitations in growth control, differentiation and apoptosis (Truckenmiller et al., 2002; Whittemore and Snyder, 1996). The AF5 cell line was derived from the central nervous system (CNS) of 14-day-old rat mesencephalic tissue (Truckenmiller et al., 1998). Primary cultures were immortalized with

an N-terminal fragment of SV40 large T antigen (T155 g) lacking the p53 inactivating domain while retaining wt p53 genotype. AF5 cells displayed genomic stability and growth factor responsiveness after months of continuous culture while retaining considerable plasticity in culture (Truckenmiller et al., 2002). These cells can be maintained in a propagating state in which nestin, a marker for immature neural precursors, is expressed or they can be differentiated upon confluency and exhibit differentiated morphologies, growth arrest and expression of mature neuronal cell markers such as βIII-tubulin. Approximately 1% of the cells in confluent cultures were immunopositive for tyrosine hydroxylase (TH), a marker of dopaminergic neurons and important for study of Parkinson's disease (Chen et al., 2005a,b,c). Microarray analysis showed gene expression or altered transcription consistent with neural specification when comparing undifferentiated (with and without bFGF) and differentiated AF5 cells (Truckenmiller et al., 2002). More recently, additional fetal rat mesencephalic and cerebral cortical lines have been derived using a similar construct T155c (Freed et al., 2005). While both necrosis (Leist and Nicotera, 1998; Martin et al., 1998) and apoptosis (Anglade et al., 1997; Hirsch et al.,

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1999; Mochizuki et al., 1996) are primary causes of neuronal cell death, apoptosis predominates in chronic neurodegenerative diseases (Emerit et al., 2004). The tumor suppressor protein p53 plays a pivotal role in apoptosis or neuronal programmed cell death (Bonini et al., 2004) by a multitude of molecular pathways after activation by phosphorylation (Wu, 2004). Activated p53 functions by upregulating target genes such as bax, PUMA (p53 upregulated modulator of apoptosis) and Noxa (Miyashita and Reed, 1995; Morrison et al., 2003; Xiang et al., 1998), which causes the release of mitochondrial protein cytochrome c (Schuler et al., 2000) and the activation of caspases (Martinou et al., 1998). Although initial studies of AF5 cells using a luciferase reporter construct showed p53 activation in response to adriamycin (Truckenmiller et al., 2002), a more thorough investigation of AF5 cellular response to other apoptotic agents has not yet been reported. Oxidative stress from reactive oxygen species (ROS) has been widely implicated for many chronic neurodegenerative disorders (Alexi et al., 2000; Behl, 1999; Zhang et al., 2000), and neuronal cell exposure to hydrogen peroxide (H2O2) has often been used to simulate ROS effects experimentally (Datta et al., 2002). The protein kinase inhibitor staurosporine also frequently induces apoptosis in rodent cell cultures including mouse and rat neural stem cells (Sleeper et al., 2002; Tamm et al., 2004), rat PC12 cells (De Simone et al., 2003), rat olfactory bulb neurons (Farso et al., 2006), and many others. This study was undertaken to characterize potential p53 activation in the AF5 cell line in response to the oxidative stressor H2O2 and the broad kinase inhibitor staurosporine (STSP) as a signaling pathway dysregulator. Cell cycle analyses, DNA fragmentation, and expression of proapoptotic proteins bax, PUMA, and Noxa were found to be altered consistent with p53-mediated apoptosis. Results suggest that the AF5 cell line can provide insights into p53-mediated neuronal cell death and neurodegeneration.

2.

Results

2.1. AF5 cell viability assay after H2O2 and STSP exposures Several apoptotic agents known for p53-dependent cytotoxicity were screened to determine their effects on AF5 cells using the MTS viability assay. Preliminary experiments showed that H2O2 and STSP reduced AF5 cell viability much more effectively than other agents such as cycloheximide, dexamethasone, etoposide, actinomyocin D, and camptothecin (data not shown). Concentration–response time course studies were then conducted to determine an effective range for H2O2 and STSP upon AF5 cell viability. At 24 h after exposure to H2O2, only the 800 μM concentration showed a significant loss of cell viability as seen in Fig. 1A. After 48 h of exposure to 400 μM H2O2, only a slight reduction in cell viability occurred. AF5 cells treated with 0–1000 nM concentrations of STSP over 24 h underwent a significant loss in cell viability when exposed to concentrations ≥250 nM that were slightly augmented with increasing concentration as shown in Fig. 1B. These results demonstrated an effective concentration

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Fig. 1 – Hydrogen peroxide (H2O2) and staurosporine (STSP) induce dose-dependent cell death in neural AF5 cells. (A) AF5 cells seeded at 4 × 104 cells/cm2 were treated with H2O2 at concentrations ranging from 0, 100, 200, 400 and 800 μM and harvested 24 and 48 h later. The MTS assay was used to determine the number of viable cells after treatment. Results are expressed as a percentage of viable cells with untreated control cells set at 100%. Cell viability is shown as mean ± SEM derived from at least three separate experiments done in triplicate. ANOVA and Fishers Exact Test were performed to determine statistical differences from control (*P < .0001, **P < .005). (B) AF5 cells treated with 0, 250, 500 and 1000 nM of STSP for 24 h showed a significant increase in cytotoxicity for all treatments. range to examine cell death from treatment of AF5 cells with H2O2 and STSP.

2.2.

Cell cycle analysis and subG1 population assessment

FACS analyses of PI stained AF5 cells revealed possible G1 to G2 growth arrest with lower concentrations of H2O2 at 100–200 μM at 24 to 48 h. A measurable subG1 population at 15.93% indicated that some cell death occurred with 400 μM H2O2 beginning at 24 h and became more prominent at a value of 65.99% at 48 h (Fig. 2A). AF5 cells treated with higher concentrations of H2O2 (800 μM) yielded considerably greater cytotoxicity as noted by an increasing proportion of a subG1 cell fraction proceeding from 20.88% to 92.63% at 24 h and 48 h, respectively. For STSP, the cell cycle distribution in vehicle (DMSO)treated cells is comparable to control cells for H2O2 and the flow cytometry profile is not dramatically altered up to 500 nM. The subG1 fraction does not progress greatly at 2.40– 7.55% from 0 to 500 nM. However, 1000 nM STSP produces a sizeable increase in the G1 population to 39.01% at 24 h. By 48 h, STSP-treated cell cultures generally had recovered and were growing normally.

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Fig. 2 – H2O2 and STSP increase sub G1 population in AF5 cells in a Dose-Dependent Manner. (A) AF5 cells treated with 0–800 μM concentrations of H2O2 for 48 h revealed possible G1 to G2 growth arrest with lower (100–200 μM) H2O2 concentrations. Cytotoxicity, determined by a measurable subG1 population, was observed at 400 μM concentration. Data analysis by the Cell Quest™ software showed 65.99% cell death in treated as compared to 1.06% of baseline cell death in untreated control. AF5 cells treated with a cytotoxic concentration of H2O2 at 800 μM reflected a large amount (92.63%) of cytotoxicity. (B) AF5 cells treated with 0–1000 nM concentrations of STSP over a 24 h period showed increasing cell death with gradual increases in the subG1 population with a maximum (39.01%) at 1000 nM.

2.3.

Annexin V binding and PI staining by flow cytometry

The Annexin V staining measures the presence of an externalized phosphatidylserine, a phospholipid normally found in

the inner leaflet of the plasma membrane and externalized during the apoptotic process. Concurrent staining with impermeable dye PI was used as an indicator of plasma membrane integrity which is compromised during necrosis.

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We noted that a consistent observation in control AF5 preparations was a small proportion of strong PI staining cells in quadrant IV (about 5%; range 1–9%) that was characteristic of AF5 cells. Cytotoxicity observed in H2O2-treated AF5 cells during PI flow cytometric analysis (Fig. 2A) was also reflected in Annexin V staining data as shown in Fig. 3A. Lower concentrations (100– 200 μM) of H2O2 showed minimal Annexin V signal (2–17%) in quadrant II (Annexin V+/PI−). By comparison, 400 μM H2O2 exposure produced the highest Annexin V positive staining (63%) in quadrant II. At this concentration, no substantial increase in the PI-positive cells appeared in quadrant III (Annexin V+/PI+). This observation suggests that 400 μM H2O2 was most effective in producing apoptosis in AF5 cells under these conditions. An increase (24%) in proportion of quadrant

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III cells (Annexin V+/PI+) occurred at 800 μM H2O2 which suggests the emergence of late stage apoptosis and early necrosis. These data show the progression of apoptosis from lower concentrations of H2O2 (100–400 μM) to apoptosis and necrosis at higher levels of H2O2 at 800 μM. STSP-treated cells at 0–1000 nM were also evaluated by flow cytometry analysis for Annexin V and PI. Vehicle control cells treated with DMSO showed 90% viable cells with small distribution (<5%) in other quadrants. Generally, the cytotoxicity observed in Fig. 1B with STSP was similarly reflected in flow cytometry analysis in Fig. 3B. After 24 h exposure to the lowest concentration at 250 nM, STSP reduced the population in quadrant I to 72% from 90% in control. The proportion of cells was evenly distributed (8–11%) among quadrants II, III, and IV at 250 nM STSP. Interestingly, at higher STSP concentrations of

Fig. 3 – Increase in positive annexin V and PI staining following H2O2 and STSP exposures of AF5 cells (A) AF5 cells treated with 0–800 μM H2O2 for 48 h. Lower concentrations (100–200 μM) of H2O2 showed minimal to increasing Annexin V positive binding (0–17%). In comparison, treatment with 400 μM H2O2 produced the highest Annexin V positive percentage (63%) in quadrant II in which plasma membranes were intact (PI negative). Increases (24%) in the PI-positive and Annexin V negative, quadrant lll, indicates late apoptosis/early necrosis as seen with 800 μM H2O2. (B) AF5 cells treated with 0–1000 nM of STSP for 24 h showed minimal Annexin V positive binding in quandrant II with STSP exposure.

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500 and 1000 nM, proportions of viable cells in quadrant I remained about the same at 75% and 72%, respectively. Unlike H2O2, the distribution of Annexin V staining cells in quadrant II did not rise with increasing STSP achieving only 14% at 1000 nM. Late apoptotic and necrotic cells (quadrants III and IV) combined for all STSP treatments ranged from 19% to 24% after 24 h. Thus, cell viability was higher and total cell death (25–28%) was lower in STSP cells treated after 24 h STSP compared to maximum effects of H2O2 at 48 h with 25% to 10% remaining viable cells at 400 and 800 μM, respectively. Cell cycle distribution and dual labeling flow cytometry analysis in Figs. 2A and 3A support concentrations of 200 to 400 H2O2 μM range for apoptosis. MTS data in Fig. 1 for H2O2 did not show changes until 800 μM which may reflect differences between incubation conditions and mitochondrial reduction capacity of MTS versus PI/Annexin V staining. For STSP, data in Figs. 1B, 2B, and 3B indicate substantial effects upon cell viability, cell cycle distribution and PI/Annexin V staining that begin at 250 nM and gradually increase with rising concentration.

2.4. Triple immunofluorescence staining after H2O2 and STSP exposures Immunofluorescence analyses were performed to better describe cell death compared to the collective population analyses represented in cell viability and flow cytometry analyses. Triple immunofluorescent staining of AF5 cells was performed with anti-Annexin V (phosphatidylserine membrane binding for apoptosis), PI (plasma membrane integrity), and DAPI (nuclear staining) as shown in Fig. 4. Preceding experiments in Figs. 1–3 suggested a useful range for observing apoptosis up to 400 μM. We found for immunofluorescence staining under coverslips that optimal adherence, viewing of individual cells, and cell response occurred at 50% of the cell density (2 × 104/cm2) of biochemical studies and that H2O2 concentrations were downward adjusted accordingly. Immunofluorescence staining is shown after 4 h of treatment with 0, 100, 200, and 300 μM H2O2. Control healthy cells receiving only aqueous vehicle in Fig. 4A,a show DAPI bluestained nuclei without PI nuclear staining or Annexin V cellular staining. For comparison, a characteristic necrotic cell that was occasionally observed at the high concentration of H2O2 (see inset) is shown for reference and contains merged signals for all three stains. Here, the nuclei stain pink (DAPI blue and PI red stain) and the membrane stains heavily for Annexin V (green). At 100 μM H2O2 exposure, only a few cells (i.e. cell at lower right in Fig. 4A, b) whose presence is indicated by DAPI nuclear staining show slight Annexin V stain. Annexin V staining is noticeably increased at 200 μM H2O2 to show the faint outline of the cell membrane. At 300 μM H2O2 in Fig. 4A, d, Annexin V staining is prominent throughout membranes of affected cells indicating phosphatidylserine exposure and increasing apoptosis. Note that plasma cell membranes are intact in Fig. 4A, a–d since no PI staining was noted. Immunofluorescence studies were also carried out with STSP using 250 nM as the highest concentration. A range of 0, 62.5, 125, and 250 nM STSP showed almost no Annexin V staining with increasing of STSP concentration up to 6 h in Fig. 4B (a–d). However, nuclear shrinkage (DAPI staining) became

more prominent with higher STSP concentrations as well as increasing cytoplasmic PI staining (Fig. 4B, c, d). Note inset in Fig. 4B, d showing enlargement of adjacent cell to left in which PI staining occurs throughout the cytoplasm without concurrent nuclear staining. Cytoplasmic PI staining likely indicates PI binding to RNA early in the cell death process after loss of plasma membrane integrity (Degen et al., 2000).

2.5.

DNA fragmentation analysis

Activation of endonucleases produces a characteristic set of DNA fragments (DNA ladders) that are a hallmark of apoptosis. Genomic DNA was extracted from cells treated for 48 h with 100 to 800 μM H2O2 and separated by electrophoresis on agarose gels (Fig. 5A). Results showed that DNA fragmentation was visible at 200 μM and greatest at 400 μM of H2O2. Reduced laddering at 800 μM likely reflects mixed populations of apoptotic and necrotic cells. DNA fragments were detectable beginning at 250 nM after 24 h, with the most intense staining at 500 nM concentration that was somewhat reduced in intensity at 1000 nM which may also reflect a shift from apoptosis to necrosis. Therefore, both H2O2 and STSP were capable of producing DNA ladders, indicating detectable apoptosis in a sizeable population of AF5 cells.

2.6.

Assessment of caspase activity in AF5 cells

Activation of cytoplasmic proenzyme caspase-3 produces the active proteolytic form as a potent effector of apoptosis (Cohen, 1997; Granville et al., 1998; Patel et al., 1996). A concentration of 400 μM H2O2 had been shown in prior analyses to be effective in producing apoptosis. After 24 h of exposure to 400 μM H2O2, cell lysates were incubated with substrate for 2–4 h and fluorescence was measured. Results indicated a 2- to 4-fold increase above control (gray bars) over time in H2O2-treated AF5 cells (solid bars as shown in Fig. 6A). This increase in fluorescence was blocked in the presence of inhibitor (open bars). Caspase3/7 activation occurred much earlier in STSP-treated AF5 cells (Fig. 6B) compared to H2O2. After 6 h of incubation with 500 nM STSP, fluorescence measurements increased to about 4-fold above control caspase-3/7 activity after 4 h of incubation with substrate. The AC-DEVD-CHO inhibitor alone did alter relative fluorescence values compared to control (data not shown). These data show that both H2O2 and STSP were capable of caspase-3/7 activation, although the times of enzyme activity were earlier for STSP compared to H2O2.

2.7. Upregulation of p53 and transcription-dependent targets in AF5 cells following initiation of apoptosis Western blot analysis of p53 and its downstream gene targets was conducted in AF5 cells treated over time with H2O2 and STSP in Fig. 7A. As in the prior assessment of caspase-3/7 activity, a concentration of 400 μM H2O2 was examined over a 24 h period. Total p53 briefly increased at 2–4 h after 400 μM H2O2 treatment of AF5 cells returning to control after 6 h. Similarly, a marked increase in p53 phosphorylation at Ser15, an indicator of p53 activation, occurred at 2–6 h after H2O2 over the same period as for total p53. The increase in p53 protein levels was accompanied by subsequent induction of a

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Fig. 4 – Increase in positive annexin V immunofluorescence staining following H2O2 but not STSP treatments of AF5 cells. (A) H2O2 exposure at 0–300 μM at 4 h in panel a–d showed enhanced Annexin V staining (green) with increasing H2O2 concentration. Inset in Panel A,a is from a necrotic cell (occasional at 400 μM, 6 h). Absence of PI (red) staining indicated intact plasma membranes. DAPI stained nuclei (blue) became smaller and more condensed at higher H2O2 concentrations. (B) STSP treatment did not show Annexin V staining (green) from 62.5 nm to 250 nM after 4 h treatment (Panel B, a–d). Increasing amounts of cytoplasmic PI staining were observed around DAPI stained nuclei (blue) with rising levels in STSP. Insert in panel d, is an enlargement of PI cytoplasmic staining at 250 nM STSP without concurrent nuclear staining at this time.

downstream target p21waf1 at 4 h after H2O2 treatment which was sustained up to 24 h. Mdm2, a downstream target and negative transcriptional regulator of p53 expression, was also upregulated from 4 to 12 h after H2O2 which coincided with

observed downregulation of p53 after 6 h. Bax appeared to increase only slightly after H2O2 exposure, while recently discovered proapoptotic proteins, PUMA and Noxa, also began a mild, sustained increase after 4 h. There was a baseline level

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experiments compared to panel A (ddH2O vehicle). Increases in p21waf1 and bax expression compared to control were also evident from 2 to 12 h treatment with STSP (Fig. 7B) and returned to control levels by 24 h. Levels for proapoptotic

Fig. 5 – H2O2 and STSP treatments cause DNA fragmentation in AF5 cells (A) Genomic DNA isolation of AF5 cells treated with 0–800 μM H2O2 for 48 h revealed increased DNA fragmentation at increasing concentrations with the highest population occurring at 400 μM. Jurkat cells exposed to actinomyocin D served as a positive control (+) for DNA fragmentation. (B) Genomic DNA isolation of AF5 cells treated with 0–1000 nM STSP for 24 h also showed increasing DNA laddering with increasing treatment with the maximal occurring at 500 nM. Positive control (+) was Jurkat cells exposed to actinomyocin D.

of protein expression detectable at 0 h before treatment for p53, p21waf1, bax, PUMA, and Noxa. Enriched cytosolic fractions of H2O2-treated AF5 cells showed a transient increase of cytochrome c above control that was detectable in the cytosol at 2–4 h after H2O2 (Fig. 7C). Increased p53, phosphorylated p53, and downstream target gene products suggest involvement of a p53-mediated process of apoptosis in AF5 cells after H2O2 treatment. A level of 500 nM STSP produced the most intense DNA fragmentation after STSP treatment (Fig. 5B) forming the rationale for selecting this STSP concentration to examine its effect upon p53 and downstream target protein levels over a 24 h period (Fig. 7B). Treatment with 500 nM STSP produced a sustained increase in total p53 protein levels from control from 2 to 24 h. This observation was paralleled by a sustained phosphorylation of p53 at Ser15 (P-Ser15-p53) from 4 to 24 h after STSP treatment, suggesting activation of p53. In response to increasing p53 levels, mdm2 was transiently upregulated at 2 and 4 h after STSP treatment returning to control levels at 6 h. We also noted mild expression of mdm2 at 0 h in panel B which may represent a slight vehicle effect (DMSO) in STSP

Fig. 6 – Caspase-3, 7 activity increases in AF5 cells following H2O2 and STSP treatments. Caspase-3/7 activity was measured using the specific pro-fluorogenic peptide substrate Z-DEVD R-110 versus control. Specific inhibition of Caspase-3/7 was achieved by the addition of caspase inhibitor AC-DEVD-CHO. ANOVA and Fishers Exact Test were performed to determine significance from control (*P < .0001, **P < .005). (A) Exposure of AF5 cells to 400 μM H2O2 showed significant caspase-3/7-like activity 26–28 h after exposure. (RFU is relative fluorescence units.) (B) Exposure of AF5 cells to 500 nM STSP exhibited a 4-fold activated caspase-3/7 activity after 8–10 h exposure.

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2.8.

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p53-dependent response in AF5 cells by reporter assay

Activation of a p53 promoter luciferase construct occurred in response to H2O2 and STSP treatments. Preliminary experiments showed that 200 μM H2O2 concentration was an effective concentration for the p53 reporter assay. Treatment of transfected AF5 cells with 200 μM H2O2 caused a 200–300% increase in p53 promoter activity as compared to control as early as 4 h after exposure (Fig. 8A). Although there was a slight drop in activity at 6 h, the activity was maintained for as least 8 h, eventually falling off at 10 to 12 h. STSP treatment at 250 nM also showed activation of the p53 reporter construct over the 12 h period (as seen in Fig. 8B); however, the effects were less pronounced and may be due to a higher baseline activity of control cultures exposed to the DMSO vehicle. However, the

Fig. 7 – H2O2 and STSP activate p53 and upregulate downstream targets in AF5 cells (A) AF5 cells were treated with 400 μM of H2O2 over a 24 h time period. Whole cell lysates (20 μg protein/lane) were resolved on 4–20% or 8–16% SDS-PAGE gels and processed for Western blot analysis according to Methods. (B) AF5 cells were treated with 500 nM of STSP over a 24 h time period. Whole cell lysates were resolved and blotted as described for H2O2 samples. (C) Cytochrome c release into cytosolic fractions of AF5 cells after H2O2 and STSP treatment for 8 h period. Cytosolic fractions (20 μg/lane) were isolated according to Methods and resolved on SDS-PAGE gels for Western blot analysis. The experiment was repeated twice.

proteins PUMA and Noxa remained constant with minimal fluctuation over the entire 24 h STSP treatment. Cytosolic fractions of STSP-treated AF5 cells showed an increase in the release of cytochrome c into the cytosol over an 8 h period (Fig. 7C). Time-matched vehicle controls showed little expression of total p53, P-Ser15-p53, mdm2, p21waf1, and cytochrome c and the control levels bax, Noxa, and PUMA did not change in absence of H2O2 and STSP (data not shown).

Fig. 8 – Luciferase reporter assay measures increased p53 activation during of exposure of AF5 cells to H2O2 and STSP. Luciferase and β-galactosidase activity were measured from the same lysate using a Dual-Light Kit (Tropix). Luciferase activity was normalized to β-galactosidase internal control activity and is expressed as percentage increase. ANOVA and Fishers Exact Test were performed for differences from control. (A) Transiently co-transfected AF5 cells were treated with 200 μM H2O2 for indicated time periods. A significant increase (*P < .0001, **P < .005) in p53 activity was observed as early as 4 to 8 h with levels decreasing by 10 h. (B) Transiently co-transfected AF5 cells were treated with 250 nM STSP for indicated time periods and activity was measured as previously indicated for H2O2. A significant increase of activity was also observed between 4–8 h.

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observed increased p53 activation over a 12 h period showed a similar outcome for H2O2 and STSP exposures in AF5 cells.

3.

Discussion

The purpose of this study was to determine if wt p53 activation and function are involved in neural AF5 cell death produced by model apoptotic agents H2O2 and STSP. Experimental evidence supported apoptosis by H2O2 exposure as demonstrated by subG1 DNA content, externalization of phosphatidylserine from flow cytometric and cellular immunofluorescence analyses, increased caspase-3/7 activity, cytochrome c release, and DNA fragmentation in a time-related and concentrationdependent manner. Time course studies showed increases in p53 luciferase reporter activity within 4–8 h and an alternating p53/mdm2 expression pattern over 2–12 h in which p53 and phospho-Ser15 p53 were upregulated followed by increased mdm2 and then subsequent downregulation of p53 and its phosphoisomer. STSP also produced DNA fragmentation, increased caspase-3/7 activity, and cytochrome c release as important attributes of apoptotic cell death but without phosphatidylserine externalization. Elevations in p53 protein levels, p53 reporter activity, and dependent gene expression from STSP exposure were also associated apoptotic events in a similar pattern to H2O2 but with some differences. Beginning at 2–4 h, STSP increased p53 and P-Ser15-p53 that was sustained for up to 24 h. p21waf1 levels were also rapidly elevated for up to 12 h by STSP but returned to control by 24 h. The shorter and less intense increase in mdm2 at 2–6 h after STSP compared to H2O2 (4–12 h) may be partially responsible for prolonged elevation of p53 levels by STSP. Despite some temporal differences, both H2O2 and STSP exposures produced cellular events consistent with apoptosis in rat neural AF5 cells that were associated with increased p53 activation. A notable difference between the two agents in the process of apoptosis is the increase in Annexin V binding with H2O2 but not STSP. A common feature of apoptosis is the loss of plasma membrane asymmetry and externalization of phosphatidylserine for recognition by phagocytic cells in eliminating apoptotic cells in vivo (Balasubramanian and Schroit, 2003). Oxidation of phosphatidylserine during apoptosis with agents such as H2O2 precedes its externalization in the plasma membrane (Tyurina et al., 2004a,b). In some cell types such as neonatal cardiomyocytes, both STSP and H2O2 can produce phosphatidylserine-positive cells and DNA fragmentation (Monceau et al., 2004). STSP may produce a certain level of intracellular oxidation since STSP-induced apoptosis in some primary cortical neuronal preparations can be blunted by pretreatment with antioxidants like superoxide dismutase/ catalase mimetics (Pong et al., 2001). However, the potential for oxidation in AF5 cells by STSP exposure (compared to H2O2) under our experimental conditions may be insufficient for phosphatidylserine externalization and substantial Annexin V binding. Increased p53 protein expression and apoptosis has also been reported after H2O2 and STSP exposure for other neural cell lines including MN9D (Choi et al., 1992; Oh et al., 1998), PC12 (Jiang et al., 2003; Wang et al., 2002), and SHSY5Y (De Sarno et al., 2003; Luetjens et al., 2001) frequently used to study

neurodegenerative diseases such as Parkinson's disease. In one report, apoptosis was compared from an oxidative stress and kinase inhibition with STSP in murine neural stem cells (C17.2 line) (Tamm et al., 2004). Investigators found that exposure up to 12 h to the oxidant stressor 2,3-dimethoxy-1,4naphthoquinone (DMNQ), released cytochrome c, activated caspase-3 and -9, and translocated bax in temporal association with DNA fragmentation, Annexin V binding, and intact plasma membranes. Similarly, STSP exposure activated caspase-3, produced DNA fragmentation, and Annexin V binding but with some secondary necrosis as evidenced by PI-positive stained cells and trypan blue leakage. Although these results are in general agreement with those of the present study, differences such as negative phosphatidylserine externalization may be due to intrinsic differences between these cell lines. The involvement of p53 in regulating BH3-only proapoptotic proteins such as Noxa and PUMA has been increasingly recognized as important in neural degeneration and neuronal cell death (Chandra et al., 2005; Nakano and Vousden, 2001; Oda et al., 2000; Yu et al., 2001). Once activated, BH3-only proteins (e.g. Bim, PUMA, Noxa, Bmf, Bad, Bid) target and neutralize prosurvival Bcl-2-like proteins (Danial and Korsmeyer, 2004) followed by the actions of proapoptotic proteins (Bax and Bak) that permeabilize organelles such as the mitochondria to release cytochrome c for caspase activation and initiation of the apoptotic program (Schuler et al., 2000). Noxa appears to selectively interact with the Bcl-2 family prosurvival factor Mcl-1, while PUMA binding is more general and efficiently antagonizes all the Bcl-2-like proteins (Chen et al., 2005a,b,c). This might explain the observation that upregulation of PUMA alone (unlike Noxa) can induce neuronal cell death (Cregan et al., 2004). Expression of Noxa and PUMA has been reported as key cell death factors in primary neuronal culture studies (Aleyasin et al., 2004; Cregan et al., 2004; Erster et al., 2004) in SH-SY5Y neuroblastoma cells (Reimertz et al., 2003; Yakovlev et al., 2004), in the rodent neuronal hybrid ND7 line (Hudson et al., 2005), and in mouse motor neurons in vivo after axotomy (Kiryu-Seo et al., 2005). Further studies will clarify the role of p53-regulated expression of Noxa and PUMA and involvement of other BH3-only proteins in mechanisms of AF5 cell death. H2O2 exposure is used to model what is a complex and diverse process of de novo formation of reactive-oxygenspecies-mediated cell death and neural degeneration (Valencia and Moran, 2004). The effects of oxidative stress in wt p53 expressing AF5 cells (400 μM, 48 h) appear comparable to other cell line models used for neurodegenerative studies. Similar concentrations in PC12 cells (e.g. 100 μM H2O2 for 12 h) (Wang et al., 2001) at 200 μM were effective in producing cell death at 24 h in SHSY5Y cells (De Sarno et al., 2003; Zhang et al., 1997). H2O2 exposure of MN9D cells revealed that a 500 μM concentration over a 24 h time period caused substantial cell death (Oh et al., 1998). Differences in metabolic behavior, growth characteristics, culture conditions (fetal bovine serum concentration, medium/growth factor composition), and gene expression patterns are likely to play important roles in ROSmediated cytotoxic responses. The influence of FBS concentration as an important determinant of H2O2-induced apoptosis has also been reported by others (Demelash et al., 2004)

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who also noted that higher amounts of H2O2 were needed to produce apoptosis at normal FBS concentrations (5–10%) than in low serum conditions (i.e. 0.5%). Alterations in signaling and apoptotic mechanisms in tumor lines containing wt p53 typically require careful consideration because of multiple pathway dysregulation (Wang et al., 2004). Staurosporine (STSP) is a microbial alkaloid with broad spectrum kinase inhibitory activity that produces apoptosis in a wide variety of cell types (Bertrand et al., 1994; Budd et al., 2000; Prehn et al., 1997), including neuronal cells (Ahlemeyer et al., 2000; Canzoniero et al., 2004; Chen et al., 2005a,b,c; Dawson, 2000; Krohn et al., 1999; Thomas and Mayle, 2000; Zhang et al., 2004). The mechanism of STSPmediated apoptosis is incompletely understood and may involve alterations in mitochondrial membrane potential and permeability transition pore (Charlot et al., 2004; Precht et al., 2005), intracellular Ca++ levels (Canzoniero et al., 2004), mitochondrial release of cytochrome c into cytoplasm (Krohn et al., 1999) or the extracellular space (Ahlemeyer et al., 2000), the cell cycle (Yamasaki et al., 2003), and changes in specific kinase activities (Chen et al., 2005a,b,c; Yamasaki et al., 2003; Zhang et al., 2004), particularly direct activation of nuclear endonucleases such as L-DNase II (Belmokhtar et al., 2000), endonuclease G (Zhang et al., 2003), and a chymotrypsin-like serine protease (O'Connell et al., 2006). In the current study, STSP-mediated apoptosis was evident from DNA fragmentation and caspase-3/7 activity that was associated with p53-mediated gene expression and reporter activity. Though cytotoxicity of STSP was pronounced over 250–1000 nM (Fig. 1), the magnitude of p53 reporter activity with STSP was less than with H2O2. The data suggest mechanistic differences in production of AF5 apoptosis by H2O2-mediated oxidative stress and kinase inhibition with a greater p53-dependent transcriptional response during oxidative injury. Furthermore, we observed that 400 and 800 μm H2O2 killed most (∼ 75%) AF5 cells at 48 h while about 72–75% cells survived at 250–1000 nM STSP. This is consistent with a report in SH-SY5Y neuroblastoma cells in which DNA damage by etoposide produced a marked, p53-dependent apoptotic response that was inhibited by cycloheximide while staurosporine-mediated apoptosis still occurred during protein synthesis inhibition suggesting minimal dependence upon p53-induced genes (Yakovlev et al., 2004). The complexity of STSP action in p53-mediated apoptosis appears in a recent report comparing DNA damaging agents and STSP in murine neural stem cells (Akhtar et al., 2006). They found that STSPinduced death was not associated with transactivation of p53 target genes like DNA damaging agents, but instead changes in effector molecules Bax and Bak, Apaf-1, and caspase-9 (downstream of p53) were common to both DNA damage and STSP pathways. These findings are not entirely inconsistent with our results in which p53, P-Ser15-p53, and bax induction occurs with both H2O2 and STSP. Differentiation capabilities of AF5 cells from a progenitor astrocytic-like state to a post-mitotic neuronal cell state are a distinguishing feature of this cell type for modeling in vivo chronic neurodegenerative disorders (Truckenmiller et al., 2002). The presence of the tyrosine hydroxylase (TH) marker in confluent, non-differentiated AF5 cells may provide insight into Parkinson's disease. Studies have shown that modulation

11

of components such as cytokines (Ling et al., 1998; Potter et al., 1999) and ascorbic acid (Yan et al., 2001) within growth media during differentiation may increase TH expression as well as the development of other mature dopaminergic markers such as Nurr and the dopamine transporter (Chung et al., 2002; Haas and Wree, 2002). The rat origin of AF5 cells maximizes its utility for other in vivo rat models of Parkinson's disease using agents such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), lipopolysaccharide, 6-hydroxydopamine, and rotenone (Gao et al., 2003; Sherer et al., 2003; Xu et al., 2003; Youdim et al., 2004). Evaluation of the balance between apoptotic proteins such as bax/PUMA/Noxa and other prosurvival factors like bcl2 (Zhong et al., 1993) will be an important determinant for cell death. Future studies will be conducted to further explore the potential use of differentiated AF5 cells as an in vitro model for Parkinson's disease.

4.

Experimental procedures

4.1.

Cell culture and reagent treatment

Establishment of the AF5 rat mesencephalic cell has been described previously (Truckenmiller et al., 1998). Cultures were maintained in 95% air/5% CO2 at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 2 mM Lglutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin. Prior to treatment, cells were seeded at 4 × 104/cm2, unless otherwise indicated. Since we found AF5 cell viability and attachment were sensitive to prolonged exposure to low serum levels (<1% FBS), we conducted all experiments at 10% FBS. After overnight culture, cells were treated with indicated concentrations of H2O2 (0, 100, 200, 400, and 800 μM) in complete medium or in separate experiments with STSP (0, 250, 500, and 1000 nM) dissolved in DMSO. Control cells for H2O2 were exposed to ddH2O and for STSP experiments were exposed to a final concentration of 0.4% DMSO. After 2 h of exposure to H2O2, the treated media was replaced with untreated, prewarmed media for removal of any H2O2 oxidized constituents (Chen et al., 2000).

4.2.

Cell viability assay

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to determine the effects of H2O2 and STSP on AF5 cell viability and proliferation according to the manufacturer's protocol from the Celltiter 96 Aqueous One Solution Cell Proliferation Assay™ (Promega, Madison WI). In metabolically active cells, MTS is reduced by dehydrogenases into an aqueous-soluble formazan product. Absorbance of formazan was measured directly at 490 nm from 96-well assay plates without additional processing. The quantity of the formazan product in time-matched control cells was used as a measure of total viable cells for comparison to treated AF5 cells. Cells were seeded at 4 × 104 cells/cm2 and treated with H2O2 for 24 and 48 h or with STSP for 24 h. Samples were analyzed in triplicate. Results are expressed as a percentage mean of viable cells with untreated control cells set at 100%.

12 4.3.

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Propidium iodide (PI) analysis by flow cytometry

Following treatment with H2O2 or STSP, cells were collected by trypsinization (Gibco, Grand Island, NY) and centrifuged at 1000 rpm (100×g) for 5 min. The cell pellet was resuspended in 200 μl of PBS, fixed in 70% ethanol, and then stored at 4 °C overnight prior to analysis. Fixed cells were centrifuged at 1000 rpm for 5 min and resuspended in 1 ml of PBS containing 5 μg/ml PI (Boehringer Mannheim, Indianapolis, IN) and 50 μl of 10 mg/ml RNAse (Roche, Indianapolis, IN). Cells were analyzed on FACSort flow cytometer (BD Bioscience, Palo Alto, CA) at 488 nM absorbance using CellQuest™ Software. PI staining was used for measuring the distribution of AF5 cells within the cell cycle. SubG1 peaks on DNA histograms from hypodiploid DNA represent dead cells which may indicate apoptosis with corroborating biochemical data (Telford et al., 1992) but may also contain some necrotic cells.

4.4.

Annexin V binding by flow cytometry

Following treatment, cells were processed according to the ApoAlert™ Annexin V protocol (BD Bioscience, Palo Alto, CA). Briefly, cells were trypsinized and then gently washed with serum-containing media. Cells were then rinsed with 1× binding buffer and resuspended in 200 μl of 1× binding buffer. Five microliters of anti-Annexin V and 10 μl of PI were added. Samples were then incubated at room temperature for 5– 15 min in the dark and analyzed by flow cytometry at 488 nM absorbance using Cell Quest™ Software. Plots distinguished dead cell populations as late apoptosis/early necrosis in quadrant III (Annexin V+/PI+), necrosis in quadrant IV (Annexin V−/PI+), and the apoptotic population (Annexin V+/PI−) in quadrant II from the normal cell population (Annexin V−/PI−) in quadrant I. The position of the quadrant marker was set to distinguish these cell populations from each other.

4.5. PI and Annexin V analyses by immunofluorescence staining Triple immunofluorescent staining with anti-Annexin V, propidium iodide (PI), and 4,6-diamidino-2-phenylindole (DAPI) was performed to characterize cell death as apoptosis or necrosis in AF5 cells. A reduction in cell density compared to cell culture was required for proper cell adherence and visual clarity of the cells so that slight adjustments were made for H2O2 (0–300 μM) and STSP (0–250 nM) concentrations. Although AF5 cells were examined hourly up to 6 h, a time of 4 h after treatment was selected for optimal staining. Briefly, cells were plated overnight on 18 mm, 1.5 nm thick German borosilicate glass coverslips (Warner Instruments Inc., Hamden, CT) at 2 × 104 cells/cm2 in 12-well tissue culture plates. After treatment with H2O2 or STSP, cells were washed with 500 μl of phosphate-buffered saline (PBS, pH 7.4) then rinsed with 500 μl of Annexin binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). A volume of 150 μl of Annexin binding buffer was applied to cells, and final dilutions of 1:20 Alexa Fluor 488 conjugated to anti-Annexin V (Invitrogen, Carlsbad, CA) and 1:20 PI (Clontech, Palo Alto, CA) were added to this buffer volume for a 15-minute incubation. Cells were washed with binding buffer and fixed

with 2% paraformaldehyde. Coverslips were then mounted onto glass slides with Prolong™ mounting media containing DAPI (Invitrogen, Carlsbad, CA). Fluorescence microscopy was performed with a Leica DMRBE microscope (Wetzlar, Germany) equipped with epifluorescence and motorized Zcontrol. The excitation/emission values for anti-Annexin V Alexa Fluor 488 conjugate, PI, and DAPI were 495/519, 590/617, and 346/442 nm, respectively. Digital images were acquired using a SpotRT™ cooled, charged-couple device (CCD) camera (Diagnostic Instruments, Sterling Heights, MI) under the control of Metamorph™ software (Universal Imaging Co., Downingtown, PA). Digital fluorescent images were captured as black and white monochrome images and were pseudocolored using Metamorph software.

4.6.

DNA fragmentation

Genomic DNA was isolated from both floating and attached cells using Suicide-Track DNA Ladder Isolation Kit™ (Oncogene, La Jolla, CA) as described by the manufacturer. DNA pellets were dissolved in 20 μl of TE buffer (10 mM Tris pH 7.5, 1 mM EDTA). DNA samples were separated by electrophoresis on a 1.5% agarose gel for 1 h at 80 V. The gel was examined and photographed with an ultraviolet gel documentation system.

4.7.

Caspase-3/7 activity assay

A quantitative enzymatic activity assay was performed according to instructions supplied with the Apo-ONE Homogeneous Caspase-3/7 Assay™ (Promega, Madison WI). Protease activity of caspase-3/7 was monitored using a profluorescent substrate, rhodamine 110 (Z-DEVD-R110), with an optimized bifunctional cell lysis/activity buffer. Samples containing 1 × 105 cells were lysed and assayed at 499 nM absorbance on the Fmax Fluorescence Microplate Reader (Molecular Devices, Sunnydale, CA). Specific inhibition of caspase-3/7 was achieved by the addition of caspase inhibitor AC-DEVD-CHO.

4.8.

Western blot analysis

For measuring protein expression levels, treated cultures were washed with ice-cold PBS and lysed in 10 volumes of an isotonic lysis buffer (150 mM NaCl, 0.5% NP-40, 0.1% SDS, 0.5% Na deoxycholate, 25 mM Tris pH 8.0) supplemented with a cocktail containing protease and phosphatase inhibitors (Calbiochem, San Diego, CA) and 1 mM PMSF (Sigma, St. Louis, MO). Nuclear DNA was carefully fragmented by progressive needle shearing on ice with 19-, 22,- and 25gauge needles in a 3-ml syringe. DNA was pelleted by centrifugation in a refrigerated microfuge at 15,000 rpm (12,000×g) for 20 min at 4 °C. The supernatant was transferred to new Eppendorf tubes and stored at −80 °C until use. After measuring protein content using the BCA protein assay (Pierce, Rockford, IL), 10–25 μg of protein from each sample was separated on 4–20% or 8–16% gels by SDS-PAGE, blotted onto nitrocellulose membranes, and processed for Western blot analysis using the following mono- or polyclonal antibodies: p53 (pAb122) (BD Bioscience, Palo Alto, CA);

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p21waf1 (C-19), mdm2 (SMP-14), and bax (P-19) (Santa Cruz Biotechnology, Santa Cruz, CA); PUMA and anti-p53 phosphoserine-15 (Cell Signaling, Beverly, MA); Noxa (Imgenex, San Diego, CA); and cytochrome c (BD Bioscience, San Jose, CA). Specific bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL) as recommended by the manufacturer. The Mitochondria/Cytosol fractionation kit (BioVision, Mountain View, CA) was employed to prepare enriched cytosolic fractions.

4.9.

Luciferase reporter assay

To confirm p53 activation, 1 × 106 AF5 cells were transiently co-transfected with 1 μg of p53 luciferase reporter plasmid (Clontech, Palo Alto, CA) and 250 ng pSV-βgal (Promega, Madison, WI) internal control plasmid using FuGene6 (Roche, Indianapolis, IN) according to the manufacturer's recommendations. Cells were treated with 200 μM H2O2 or 250 nM STSP for indicated time periods. Luciferase and β-galactosidase activities were measured from the same lysate using a Dual-Light Kit (Tropix, Foster City, CA). Luciferase activity was normalized to β-galactosidase internal control activity and is expressed as the percentage changed compared to control.

4.10.

Statistical analysis

Analysis of variance (ANOVA) was performed to evaluate differences in multiple concentrations and times, and the Fishers Exact Test was used to determine differences of treatment groups from control.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.brainres.2006.07.024. REFERENCES

Ahlemeyer, B., Fischer, D., Kissel, T., Krieglstein, J., 2000. Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate. Neurosci. Res. 37, 245–253. Akhtar, R.S., Geng, Y., Klocke, B.J., Roth, K.A., 2006. Neural precursor cells possess multiple p53-dependent apoptotic pathways. Cell Death Differ. Available online publication doi:10.1038/sj.cdd.4401879. Alexi, T., Borlongan, C.V., Faull, R.L., Williams, C.E., Clark, R.G., Gluckman, P.D., Hughes, P.E., 2000. Neuroprotective strategies for basal ganglia degeneration: Parkinson's and Huntington's diseases. Prog. Neurobiol. 60, 409–470. Aleyasin, H., Cregan, S.P., Iyirhiaro, G., O'Hare, M.J., Callaghan, S.M., Slack, R.S., Park, D.S., 2004. Nuclear factor-(kappa) B modulates the p53 response in neurons exposed to DNA damage. J. Neurosci. 24, 2963–2973. Anglade, P., Vyas, S., Hirsch, E.C., Agid, Y., 1997. Apoptosis in dopaminergic neurons of the human substantia nigra during normal aging. Histol. Histopathol. 12, 603–610. Balasubramanian, K., Schroit, A.J., 2003. Aminophospholipid asymmetry: a matter of life and death. Annu. Rev. Physiol. 65, 701–734.

13

Behl, C., 1999. Alzheimer's disease and oxidative stress: implications for novel therapeutic approaches. Prog. Neurobiol. 57, 301–323. Belmokhtar, C.A., Torriglia, A., Counis, M.F., Courtois, Y., Jacquemin-Sablon, A., Segal-Bendirdjian, E., 2000. Nuclear translocation of a leukocyte elastase Inhibitor/Elastase complex during staurosporine-induced apoptosis: role in the generation of nuclear L-DNase II activity. Exp. Cell Res. 254, 99–109. Bertrand, R., Solary, E., O'Connor, P., Kohn, K.W., Pommier, Y., 1994. Induction of a common pathway of apoptosis by staurosporine. Exp. Cell Res. 211, 314–321. Bonini, P., Cicconi, S., Cardinale, A., Vitale, C., Serafino, A.L., Ciotti, M.T., Marlier, L.N., 2004. Oxidative stress induces p53-mediated apoptosis in glia: p53 transcription-independent way to die. J. Neurosci. Res. 75, 83–95. Budd, S.L., Tenneti, L., Lishnak, T., Lipton, S.A., 2000. Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons. Proc. Natl. Acad. Sci. U. S. A. 97, 6161–6166. Canzoniero, L.M., Babcock, D.J., Gottron, F.J., Grabb, M.C., Manzerra, P., Snider, B.J., Choi, D.W., 2004. Raising intracellular calcium attenuates neuronal apoptosis triggered by staurosporine or oxygen-glucose deprivation in the presence of glutamate receptor blockade. Neurobiol. Dis. 15, 520–528. Chandra, D., Choy, G., Daniel, P.T., Tang, D.G., 2005. Bax-dependent regulation of Bak by voltage-dependent anion channel 2. J. Biol. Chem. 9, 19051–19061. Charlot, J.F., Pretet, J.L., Haughey, C., Mougin, C., 2004. Mitochondrial translocation of p53 and mitochondrial membrane potential (Delta Psi m) dissipation are early events in staurosporine-induced apoptosis of wild type and mutated p53 epithelial cells. Apoptosis 9, 333–343. Chen, G.G., Sin, F.L., Leung, B.C., Ng, H.K., Poon, W.S., 2005a. Differential role of hydrogen peroxide and staurosporine in induction of cell death in glioblastoma cells lacking DNA-dependent protein kinase. Apoptosis 10, 185–192. Chen, L., Willis, S.N., Wei, A., Smith, B.J., Fletcher, J.I., Hinds, M.G., Colman, P.M., Day, C.L., Adams, J.M., Huang, D.C., 2005b. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell. 17, 393–403. Chen, Q., He, Y., Yang, K., 2005c. Gene therapy for Parkinson's disease: progress and challenges. Curr Gene Ther. 5, 71–80. Chen, Q.M., Liu, J., Merrett, J.B., 2000. Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts. Biochem. J. 347, 543–551. Choi, H.K., Won, L., Roback, J.D., Wainer, B.H., Heller, A., 1992. Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions. Proc. Natl. Acad. Sci. U. S. A. 89, 8943–8947. Chung, S., Sonntag, K.C., Andersson, T., Bjorklund, L.M., Park, J.J., Kim, D.W., Kang, U.J., Isacson, O., Kim, K.S., 2002. Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons. Eur. J. Neurosci. 16, 1829–1838. Cohen, G.M., 1997. Caspases: the executioners of apoptosis. Biochem. J. 326, 1–16. Cregan, S.P., Arbour, N.A., Maclaurin, J.G., Callaghan, S.M., Fortin, A., Cheung, E.C., Guberman, D.S., Park, D.S., Slack, R.S., 2004. p53 activation domain 1 is essential for PUMA upregulation and p53-mediated neuronal cell death. J. Neurosci. 24, 10003–10012. Danial, N.N., Korsmeyer, S.J., 2004. Cell death: critical control points. Cell 116, 205–219. Datta, K., Babbar, P., Srivastava, T., Sinha, S., Chattopadhyay, P., 2002. p53 dependent apoptosis in glioma cell lines in

14

B RA IN RE S EA RCH 1 11 2 (2 0 0 6 ) 1 –1 5

response to hydrogen peroxide induced oxidative stress. Int. J. Biochem. Cell Biol. 34, 148–157. Dawson, G., 2000. Mechanisms of apoptosis in embryonic cortical neurons (E6 and E7) in culture involve lipid signalling, protein phosphorylation and caspase activation. Int. J. Dev. Neurosci. 18, 247–257. De Sarno, P., Shestopal, S.A., King, T.D., Zmijewska, A., Song, L., Jope, R.S., 2003. Muscarinic receptor activation protects cells from apoptotic effects of DNA damage, oxidative stress, and mitochondrial inhibition. J. Biol. Chem. 278, 11086–11093. De Simone, R., Ajmone-Cat, M.A., Tirassa, P., Minghetti, L., 2003. Apoptotic PC12 cells exposing phosphatidylserine promote the production of anti-inflammatory and neuroprotective molecules by microglial cells. J. Neuropathol. Exp. Neurol. 62, 208–216. Degen, W.G., Pruijn, G.J., Raats, J.M., van Venrooij, W.J., 2000. Caspase-dependent cleavage of nucleic acids. Cell Death Differ. 7, 616–627. Demelash, A., Karlsson, J.O., Nilsson, M., Bjorkman, U., 2004. Selenium has a protective role in caspase-3-dependent apoptosis induced by H2O2 in primary cultured pig thyrocytes. Eur. J. Endocrinol. 150, 841–849. Emerit, J., Edeas, M., Bricaire, F., 2004. Neurodegenerative diseases and oxidative stress. Biomed. Pharmacother. 58, 39–46. Erster, S., Mihara, M., Kim, R.H., Petrenko, O., Moll, U.M., 2004. 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. 24, 6728–6741. Farso, M.C., Carroll, F.Y., Beart, P.M., 2006. Establishment of primary cultures of rat olfactory bulb under serum-free conditions for studies of cellular injury. Cell Tissue Res. 323, 343–349. Freed, W.J., Zhang, P., Sanchez, J.F., Dillon-Carter, O., Coggiano, M., Errico, S.L., Lewis, B.D., Truckenmiller, M.E., 2005. Truncated N-terminal mutants of SV40 large T antigen as minimal immortalizing agents for CNS cells. Exp Neurol. 191 (Suppl. 1), S45–S59. Gao, H.M., Liu, B., Zhang, W., Hong, J.S., 2003. Synergistic dopaminergic neurotoxicity of MPTP and inflammogen lipopolysaccharide: relevance to the etiology of Parkinson's disease. FASEB J. 13, 1957–1959. Granville, D.J., Carthy, C.M., Jiang, H., Shore, G.C., McManus, B.M., Hunt, D.W., 1998. Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy. FEBS Lett. 437, 5–10. Greene, L.A., Tischler, A.S., 1976. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U. S. A. 73, 2424–2428. Haas, S.J., Wree, A., 2002. Dopaminergic differentiation of the Nurr1-expressing immortalized mesencephalic cell line CSM14.1 in vitro. J. Anat. 201, 61–69. Hirsch, E.C., Hunot, S., Faucheux, B., Agid, Y., Mizuno, Y., Mochizuki, H., Tatton, W.G., Tatton, N., Olanow, W.C., 1999. Dopaminergic neurons degenerate by apoptosis in Parkinson's disease. Mov. Disord. 14, 383–385. Hudson, C.D., Morris, P.J., Latchman, D.S., Budhram-Mahadeo, V.S., 2005. Brn-3a transcription factor blocks p53 mediated expression of pro-apoptotic target genes, Noxa and Bax, in-vitro and in-vivo to determine cell fate. J. Biol. Chem. 280, 11851–11858. Jiang, B., Liu, J.H., Bao, Y.M., An, L.J., 2003. Hydrogen peroxide-induced apoptosis in pc12 cells and the protective effect of puerarin. Cell. Biol. Int. 27, 1025–1031. Kiryu-Seo, S., Hirayama, T., Kato, R., Kiyama, H., 2005. Noxa is a critical mediator of p53-dependent motor neuron death after nerve injury in adult mouse. J. Neurosci. 25, 1442–1447. Krohn, A.J., Wahlbrink, T., Prehn, J.H., 1999. Mitochondrial

depolarization is not required for neuronal apoptosis. J. Neurosci. 19, 7394–7404. Leist, M., Nicotera, P., 1998. Apoptosis, excitotoxicity, and neuropathology. Exp. Cell Res. 239, 183–201. Ling, Z.D., Potter, E.D., Lipton, J.W., Carvey, P.M., 1998. Differentiation of mesencephalic progenitor cells into dopaminergic neurons by cytokines. Exp. Neurol. 149, 411–423. Luetjens, C.M., Lankiewicz, S., Bui, N.T., Krohn, A.J., Poppe, M., Prehn, J.H., 2001. Up-regulation of Bcl-xL in response to subtoxic beta-amyloid: role in neuronal resistance against apoptotic and oxidative injury. Neuroscience 102, 139–150. Martin, L.J., Al-Abdulla, N.A., Brambrink, A.M., Kirsch, J.R., Sieber, F.E., Portera-Cailliau, C., 1998. Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis. Brain Res. Bull. 46, 281–309. Martinou, I., Missotten, M., Fernandez, P.A., Sadoul, R., Martinou, J.C., 1998. Bax and Bak proteins require caspase activity to trigger apoptosis in sympathetic neurons. NeuroReport 9, 15–19. Miyashita, T., Reed, J.C., 1995. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293–299. Mochizuki, H., Goto, K., Mori, H., Mizuno, Y., 1996. Histochemical detection of apoptosis in Parkinson's disease. J. Neurol. Sci. 137, 120–123. Monceau, V., Belikova, Y., Kratassiouk, G., Charue, D., Camors, E., Communal, C., Trouve, P., Russo-Marie, F., Charlemagne, D., 2004. Externalization of endogenous annexin A5 participates in apoptosis of rat cardiomyocytes. Cardiovasc. Res. 64, 496–506. Morrison, R.S., Kinoshita, Y., Johnson, M.D., Guo, W., Garden, G.A., 2003. p53-dependent cell death signaling in neurons. Neurochem. Res. 28, 15–27. Nakano, K., Vousden, K., 2001. PUMA, a novel proapoptotic gene, is induced by p53. Mol. Cell 7, 683–694. O'Connell, A.R., Holohan, C., Torriglia, A., Lee, B.W., Stenson-Cox, C., 2006. Characterization of a serine protease-mediated cell death program activated in human leukemia cells. Exp. Cell Res. 312, 27–39. Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino, T., Taniguchi, T., Tanaka, N., 2000. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53 induced apoptosis. Science 288, 1053–1058. Oh, J.H., Choi, W.S., Kim, J.E., Seo, J.W., O'Malley, K.L., Oh, Y.J., 1998. Overexpression of HA-Bax but not Bcl-2 or Bcl-XL attenuates 6hydroxydopamine-induced neuronal apoptosis. Exp. Neurol. 154, 193–198. Pahlman, S., Odelstad, L., Larsson, E., Grotte, G., Nilsson, K., 1981. Phenotypic changes of human neuroblastoma cells in culture induced by 12-O-tetradecanoyl-phorbol-13-acetate. Int. J. Cancer 28, 583–589. Patel, T., Gores, G.J., Kaufmann, S.H., 1996. The role of proteases during apoptosis. FASEB J. 10, 587–597. Pong, K., Doctrow, S.R., Huffman, K., Adinolfi, C.A., Baudry, M., 2001. Attenuation of staurosporine-induced apoptosis, oxidative stress, and mitochondrial dysfunction by synthetic superoxide dismutase and catalase mimetics, in cultured cortical neurons. Exp. Neurol. 171, 84–97. Potter, E.D., Ling, Z.D., Carvey, P.M., 1999. Cytokine-induced conversion of mesencephalic-derived progenitor cells into dopamine neurons. Cell Tissue Res. 296, 235–246. Precht, T.A., Phelps, R.A., Linseman, D.A., Butts, B.D., Le, S.S., Laessig, T.A., Bouchard, R.J., Heidenreich, K.A., 2005. The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis. Cell Death Differ. 12, 255–265. Prehn, J.H., Jordan, J., Ghadge, G.D., Preis, E., Galindo, M.F., Roos, R.P., Krieglstein, J., Miller, R.J., 1997. Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis. J. Neurochem. 68, 1679–1685.

BR A IN RE S EA RCH 1 1 12 ( 20 0 6 ) 1 –1 5

Reimertz, C., Kogel, D., Rami, A., Chittenden, T., Prehn, J.H., 2003. Gene expression during ER stress-induced apoptosis in neurons: induction of the BH3-only protein Bbc3/PUMA and activation of the mitochondrial apoptosis pathway. J. Cell Biol. 162, 587–597. Schuler, M., Bossy-Wetzel, E., Goldstein, J.C., Fitzgerald, P., Green, D.R., 2000. p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release. J. Biol. Chem. 275, 7337–7342. Sherer, T.B., Kim, J.H., Betarbet, R., Greenamyre, J.T., 2003. Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation. Exp. Neurol. 179, 9–16. Sleeper, E., Tamm, C., Frisen, J., Zhivotovsky, B., Orrenius, S., Ceccatelli, S., 2002. Cell death in adult neural stem cells. Cell Death Differ. 9, 1377–1378. Tamm, C., Robertson, J.D., Sleeper, E., Enoksson, M., Emgard, M., Orrenius, S., Ceccatelli, S., 2004. Differential regulation of the mitochondrial and death receptor pathways in neural stem cells. Eur. J. Neurosci. 19, 2613–2621. Telford, W.G., King, L.E., Fraker, P.J., 1992. Comparative evaluation of several DNA binding dyes in the detection of apoptosisassociated chromatin degradation by flow cytometry. Cytometry 13, 137–143. Thomas, C.E., Mayle, D.A., 2000. NMDA-sensitive neurons profoundly influence delayed staurosporine-induced apoptosis in rat mixed cortical neuronal cultures. Brain Res. 884, 163–173. Truckenmiller, M.E., Tornatore, C., Wright, R.D., Dillon-Carter, O., Meiners, S., Geller, H.M., Freed, W.J., 1998. A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53induced growth arrest and immortalizes primary mesencephalic cells. Cell Tissue Res. 291, 175–189. Truckenmiller, M.E., Vawter, M.P., Zhang, P., Conejero-Goldberg, C., Dillon-Carter, O., Morales, N., Cheadle, C., Becker, K.G., Freed, W. J., Tornatore, C., Wright, R.D., Meiners, S., Geller, H.M., 2002. AF5, a CNS cell line immortalized with an N-terminal fragment of SV40 large T: growth, differentiation, genetic stability, and gene expression. Exp. Neurol. 175, 318–337. Tyurina, Y.Y., Serinkan, F.B., Tyurin, V.A., Kini, V., Yalowich, J.C., Schroit, A.J., Fadeel, B., Kagan, V.E., 2004a. Lipid antioxidant, etoposide, inhibits phosphatidylserine externalization and macrophage clearance of apoptotic cells by preventing phosphatidylserine oxidation. J. Biol. Chem. 279, 6056–6064. Tyurina, Y.Y., Tyurin, V.A., Zhao, Q., Djukic, M., Quinn, P.J., Pitt, B.R., Kagan, V.E., 2004b. Oxidation of phosphatidylserine: a mechanism for plasma membrane phospholipid scrambling during apoptosis? Biochem. Biophys. Res. Commun. 324, 1059–1064. Valencia, A., Moran, J., 2004. Reactive oxygen species induce different cell death mechanisms in cultured neurons. Free Radical Biol. Med. 36, 1112–1125. Wang, R., Xiao, X.Q., Tang, X.C., 2001. Huperzine A attenuates hydrogen peroxide-induced apoptosis by regulating expression of apoptosis-related genes in rat PC12 cells. NeuroReport 12, 2629–2634. Wang, R., Zhou, J., Tang, X.C., 2002. Tacrine attenuates hydrogen peroxide-induced apoptosis by regulating expression of apoptosis-related genes in rat PC12 cells. Brain Res. Mol. Brain Res. 107, 1–8.

15

Wang, S., Konorev, E., Kotamraju, S., Joseph, J., Kalivendi, S., Kalyanaraman, B., 2004. Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms. Intermediacy of H(2)O(2)- and p53-dependent pathways. J. Biol. Chem. 279, 25535–25543. Whittemore, S.R., Snyder, E.Y., 1996. Physiological relevance and functional potential of central nervous system-derived cell lines. Mol. Neurobiol. 12, 13–38. Wu, G.S., 2004. The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol. Ther. 3, 156–161. Xiang, H., Kinoshita, Y., Knudson, C.M., Korsmeyer, S.J., Schwartzkroin, P.A., Morrison, R.S., 1998. Bax involvement in p53-mediated neuronal cell death. J. Neurosci. 18, 1363–1373. Xu, Y., Sun, S., Cao, X., 2003. Effect of levodopa chronic administration on behavioral changes and fos expression in basal ganglia in rat model of PD. J. Huazhong Univ. Sci. Technol. Med. Sci. 23, 258–262. Yakovlev, A.G., Di Giovanni, S., Wang, G., Liu, W., Stoica, B., Faden, A.I., 2004. BOK and NOXA are essential mediators of p53-dependent apoptosis. J. Biol. Chem. 279, 28367–28374. Yamasaki, F., Hama, S., Yoshioka, H., Kajiwara, Y., Yahara, K., Sugiyama, K., Heike, Y., Arita, K., Kurisu, K., 2003. Staurosporine-induced apoptosis is independent of p16 and p21 and achieved via arrest at G2/M and at G1 in U251MG human glioma cell line. Cancer Chemother. Pharmacol. 51, 271–283. Yan, J., Studer, L., McKay, R.D., 2001. Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors. J. Neurochem. 76, 307–311. Youdim, M.B., Stephenson, G., Ben Shachar, D., 2004. Ironing iron out in Parkinson's disease and other neurodegenerative diseases with iron chelators: a lesson from 6hydroxydopamine and iron chelators, desferal and VK-28. Ann. N. Y. Acad. Sci. 1012, 306–325. Yu, J., Zhang, L., Hwang, P.M., Kinzler, K.W., Vogelstein, B., 2001. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol. Cell 7, 673–682. Zhang, L., Zhao, B., Yew, D.T., Kusiak, J.W., Roth, G.S., 1997. Processing of Alzheimer's amyloid precursor protein during H2O2-induced apoptosis in human neuronal cells. Biochem. Biophys. Res. Commun. 235, 845–848. Zhang, Y., Dawson, V.L., Dawson, T.M., 2000. Oxidative stress and genetics in the pathogenesis of Parkinson's disease. Neurobiol. Dis. 7, 240–250. Zhang, J., Dong, M., Li, L., Fan, Y., Pathre, P., Dong, J., Lou, D., Wells, J.M., Olivares-Villagomez, D., Van Kaer, L., Wang, X., Xu, M., 2003. Endonuclease G is required for early embryogenesis and normal apoptosis in mice. Proc. Natl. Acad. Sci. U. S. A. 100, 15782–15787. Zhang, B.F., Peng, F.F., Zhang, W., Shen, H., Wu, S.B., Wu, D.C., 2004. Involvement of cyclin dependent kinase 5 and its activator p35 in staurosporine-induced apoptosis of cortical neurons. Acta Pharmacol. Sin. 25, 1105–1111. Zhong, L.T., Sarafian, T., Kane, D.J., Charles, A.C., Mah, S.P., Edwards, R.H., Bredesen, D.E., 1993. bcl-2 inhibits death of central neural cells induced by multiple agents. Proc. Natl. Acad. Sci. U. S. A. 90, 4533–4537.