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Originally published In Press as doi:10.1074/jbc.M400376200 on April 22, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28706-28714, July 2, 2004
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The Proapoptotic Gene SIVA Is a Direct Transcriptional Target for the Tumor Suppressors p53 and E2F1*

Andre Fortin{ddagger}, Jason G. MacLaurin, Nicole Arbour§, Sean P. Cregan, Neena Kushwaha||, Steven M. Callaghan, David S. Park**, Paul R. Albert{ddagger}{ddagger}, and Ruth S. Slack§§

From the Ottawa Health Research Institute, Neuroscience Centre and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

Received for publication, January 13, 2004 , and in revised form, April 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease and in neurodegeneration. The p53 signaling cascade is complex, and the mechanism by which p53 induces apoptosis is cell type-dependent. Using DNA microarray analysis, we have found a striking induction of the proapoptotic gene, SIVA. SIVA is a proapoptotic protein containing a death domain and interacts with members of the tumor necrosis factor receptor family as well as anti-apoptotic Bcl-2 family proteins. SIVA is induced following direct p53 gene delivery, treatment with a DNA-damaging agent camptothecin, and stroke injury in vivo. SIVA up-regulation is sufficient to initiate the apoptotic cascade in neurons. Through isolation and analysis of the SIVA promoter, we have identified response elements for both p53 and E2F1. Like p53, E2F1 is another tumor suppressor gene involved in the regulation of apoptosis, including neuronal injury models. We have identified E2F consensus sites in the promoter region, whereas p53 recognition sequences were found in intron1. Sequence analysis has shown that these consensus sites are also conserved between mouse and human SIVA genes. Electrophoretic mobility shift assays reveal that both transcription factors are capable of binding to putative consensus sites, and luciferase reporter assays reveal that E2F1 and p53 can activate transcription from the SIVA promoter. Here, we report that the proapoptotic gene, SIVA, which functions in a broad spectrum of cell types, is a direct transcriptional target for both tumor suppressors, p53 and E2F1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease (1–6) and in neurodegeneration (7). Studies have demonstrated that p53 protein levels are up-regulated following excitotoxicity, hypoxia, and ischemia (2, 3, 8) and that forced up-regulation of p53 alone is sufficient to cause neuronal cell death (5, 9–11). Consistent with this, mice carrying a p53 null mutation exhibit reduced brain damage following excitotoxicity and stroke (5, 6, 12). Finally, recent studies have shown that p53-blocking peptides are neuroprotective following acute brain injury and may serve as potential therapeutic agents (13). Taken together, these studies demonstrate the importance of p53 as a key apoptotic factor following neuronal injury and underscores the necessity to uncover the mechanisms by which p53 induces the death of postmitotic neurons.

The p53 signaling cascade is complex, and it has become increasingly clear that the mechanisms by which p53 induces apoptosis vary depending on the tissue type (reviewed by Prives and Hall (14)). For example, recent studies have demonstrated that, in certain cell types, p53 can induce apoptosis exclusively at the mitochondrial level through direct interaction with Bcl-2 family proteins (15). In contrast, other cell types such as postmitotic neurons exposed to DNA-damaging agents require the transcriptional activation domain of p53 for death to occur.1 In postmitotic neurons, we and others (16–18) have demonstrated that p53-mediated cell death involves a Bax-dependent, caspase3 activation involving induction of APAF1. In proliferating cells, Bax was shown to be a direct target for p53 (19), however no significant p53-mediated Bax up-regulation was found in neurons (16, 17, 20). Although we have found that p53 transcriptional activity is essential for the induction of neuronal cell death, the downstream targets responsible for Bax activation remain unknown. To identify the regulatory targets by which p53 induces neuronal cell death, we conducted DNA microarray analysis using postmitotic neurons undergoing p53-mediated apoptosis. Using this approach, we and others (21–23) have previously identified APAF1 as a key intermediate in the apoptotic signaling cascade that is directly activated by p53. We have now used DNA microarray analysis to identify p53 target genes involved in neuronal injury and have consistently found a striking induction of the proapoptotic gene, SIVA.

SIVA is a proapoptotic protein that was originally identified through its association with the cytoplasmic tail of CD27, a member of the tumor necrosis factor receptor (TNFR)2 superfamily (24–27). CD27 lacks a death domain, thus yeast-2-hybrid assays were performed to identify the mechanism by which CD27 may induce apoptosis (28–31). SIVA, the CD27-interacting protein was found to contain a death domain homology region, a box-B-like ring finger, and a zinc finger-like domain (27). More recently SIVA was also found to interact with an additional TNFR family protein, GITR (32). Consistent with its structural domains, forced expression of SIVA has been shown to induce apoptosis when expressed in a number of different cell lines (27).

In addition to its interaction with TNFR superfamily members, SIVA has also been shown to interact with anti-apoptotic Bcl-2 family members (33). There are two SIVA splice variants, SIVA-1 and SIVA-2, of which SIVA-1 retains exon2, which is believed to be critical for apoptotic activity (34), although a recent study suggests that both splice forms can induce apoptosis in a similar fashion (35). Exon2 is comprised of an amphipathic helical structure, known as the SAH domain, which is structurally similar to the BH3 domain of Bcl-2 family proteins (33). Consistent with an endogenous interaction with Bcl-2 family proteins, SIVA has been localized to the cytoplasm and mitochondria and was recently shown to directly bind to Bcl-XL through this amphipathic domain. Mutation of the SAH domain prevents interaction with anti-apoptotic Bcl-2 family proteins and abrogates its ability to induce apoptosis (33). This, as well as studies demonstrating interactions with TNFR family proteins, suggests that SIVA may function through multiple mechanisms that may be dependent on the cell type and apoptotic stimulus. Consistent with a key role in regulating apoptosis in tumor cells, SIVA is up-regulated in response to UV radiation and oxidative stress in a number of cell types (33, 36). Recently, SIVA was identified through DNA microarray analysis as a p53-induced and DNA damage-induced gene following treatment of colon carcinoma cells with a topoisomerase I inhibitor (37). In addition, examination of certain types of cancer has revealed a down-regulation of the SIVA gene along with p53 suggesting that SIVA itself may have a potential role as a tumor suppressor due to its proapoptotic function (38).

The role of the proapoptotic gene, SIVA, in regulating the death of neuronal cells has not yet been explored. Using DNA microarray analysis to identify p53 target genes involved in neuronal cell death, we have identified SIVA as a p53 target. In addition to its proapoptotic role in the immune system and in tumor cells, we now demonstrate that SIVA also functions in injury-induced apoptosis of postmitotic neurons. By isolation and analysis of the SIVA promoter, we have identified response elements for both E2F1 and p53. Like p53, E2F1 is a tumor suppressor gene (39, 40) involved in the regulation of apoptosis, and its involvement has been demonstrated in a number of neuronal injury models (41–47). We have identified E2F consensus sites in the promoter region, whereas recognition sequences for p53 were found in the first intron. Furthermore, these consensus sites are also conserved in the human SIVA gene where E2F sites were also located within the promoter and p53 sites were again found in the first intron. Electrophoretic mobility shift assays revealed that both transcription factors are capable of binding their putative consensus sites, and luciferase reporter assays revealed that E2F1 and p53 can activate transcription of the SIVA promoter at these sites. Here, we report that the proapoptotic gene SIVA is a direct transcriptional target for both tumor suppressors, p53 and E2F1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Primary Neuronal Cultures and Cell Lines—Cortical and cerebellar granule neurons were cultured as described previously (17, 23). Murine SN48 cells were maintained in Dulbecco's modified Eagle's medium (Wisent Inc., St. Bruno, Quebec, Canada) supplemented with 10% fetal calf serum (Wisent Inc.) at 37 °C in 5% CO2 (23). To culture progenitor cells, the epidermal ectoderm was removed from E12.5 mouse embryos, and the telencephalic neuroepithelia was dissected and transferred to a 1.5-ml Eppendorf tube containing 400 µl of serum-free stem cell media with 10 ng/ml basic fibroblast growth factor and 2 µg/ml heparin as previously described (48, 49). Neuroepithelia were mechanically dissociated, and single cells were plated at a density of 50,000 cells/ml in uncoated 60-mm Nunclon plates (Invitrogen). Primary neurospheres were expanded for 3–4 days. 7 days post-plating, the neurospheres were pelleted and all but 1–2 ml of media was removed. Neurospheres were triturated to generate a single cell suspension that was centrifuged and resuspended in Neurobasal medium containing B-27 supplement, N-2 supplement, 0.5 mM L-glutamine, 20 ng/ml platelet-derived growth factor, 1% nondialyzed fetal bovine serum, and 50 units/ml penicillin/streptomycin (Invitrogen). Cells were plated in Nunc 4-well (2 x 105 cells/well) dishes (Invitrogen) coated with poly-L-ornithine (Sigma-Aldrich).

Recombinant Adenovirus Infection and Camptothecin Treatment—cDNA for SIVA was a kind gift from Dr. Kanteti V. S. Prasad (27). cDNA for wild type p53, p53-{Delta}V (deletion of conserved box V: residues 270–286), and p53-173L (point mutation at residue 173 to Leu) were kind gifts from Dr. Karen Vousden (50, 51). The p53 double transactivation mutant p53-DM (mutations L22E, W23S, W53F, and E54S) was a kind gift from Dr. Xinbin Chen (52). Recombinant adenoviral vectors carrying the SIVA-GFP, GFP, and wild type or mutant p53 expression cassettes were constructed, purified, and titered as described previously (53). Prior to use, all recombinant adenovirus vectors were tested and confirmed to be wild type-free. All experiments were performed at a multiplicity of infection (m.o.i.) of 50 plaque-forming units/cell. Recombinant adenoviral vectors were added to cell suspensions immediately before plating for primary neuronal cultures and 24 h following plating for progenitor cell cultures. Cortical neurons were treated with 10 µM camptothecin (Sigma-Aldrich) 2 days after plating. For viability tests the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide survival assay (Cell Titer Kit, Promega, Madison, WI) that measures the mitochondrial conversion of the tetrazolium salt to a blue formazan salt was used as described previously (9).

DNA Microarray Analysis—For p53 microarray analysis, cortical neurons were infected at an m.o.i. of 20 with recombinant adenovirus vectors carrying an expression cassette for either a full-length human p53 (Ad-p53), a transcriptionally defective p53 (Ad-p53-DM), or a DNA-binding mutant p53 (Ad-p53-{Delta}V). For E2F1 microarray analysis, neural precursor cells were infected at 50 m.o.i. with adenoviral vectors carrying E2F1 (Ad-E2F1) or the control vector Ad-GFP. Total RNA was extracted at 48 (p53) or 72 (E2F1) h post-infection using Tripure isolation reagent according to the manufacturer's instruction (Roche Diagnostics). RNA was sent to the Ottawa Genome Centre Affymetrix GeneChip Facility for analysis.

Surgical Procedures—All animal procedures conformed to guidelines endorsed by the Canadian Institutes of Health Research and were approved by the Animal Care Committee of the University of Ottawa. Male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) weighing 20–22 g were subjected to 2 h of middle cerebral artery occlusion as previously described (23). Following reperfusion for 48 or 72 h, brains were removed and the striatum and cortex from the ipsilateral and contralateral sides were dissected separately and tissue was flash frozen using liquid nitrogen prior to protein extraction.

Semiquantitative RT-PCR Analysis—Total RNA was isolated from cells using Tripure isolation reagent according to the manufacturer's instructions (Roche Diagnostics). Pilot experiments were done to determine the linear range of amplification with respect to quantity of starting template and PCR cycles using mouse-specific primers: SIVA forward (5'-CGCCCATCGCTTGTTCATCGTG-3') and SIVA reverse (5'-CCGCAGCCCCAGCAGGTGTAT-3'). 6–12 ng of total RNA was used for cDNA synthesis and targeted gene amplification using the Super-Script One-Step RT-PCR kit (Invitrogen). cDNA synthesis was carried out at 50 °C for 30 min followed by a 2-min initial denaturation step at 94 °C. This was followed by 35 cycles (SIVA) or 25 cycles (glyceraldehyde-3-phosphate dehydrogenase) at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min. Primers were designed to amplify nucleotides 329–437 of the SIVA transcript and 139–740 of the glyceraldehyde-3-phosphate dehydrogenase transcript. The resulting product was sequenced and confirmed to be SIVA.

Western Blot Analysis—Western blot analysis was performed as described previously (17) with antibodies against p53 (1C12 [PDB] , Cell Signaling Technology, Beverly, MA) and {beta}-actin (SC-1616, Santa Cruz Biotechnologies, Santa Cruz, CA) as a loading control.

Electrophoretic Mobility Shift Assay—EMSAs were performed on total protein extracts as described previously (23), with the following modifications. In brief, cells were harvested, centrifuged, and extracted in lysis buffer and assayed by the method of Bradford (Bio-Rad Laboratories protein assay reagent). 10–20 µg of total cell lysate was incubated with an excess of indicated 32P-labeled double-stranded DNA probes (60,000 cpm/0.2 ng of DNA). Oligonucleotides used included 5'-AGTCTAGACATGGCCTGGCGTCGTGGCTTGTTT-3' (p53-BS1) and 5'-GTCTATGCAAGCCTGGACATGAGT-3' (p53-BS2) corresponding to the p53 binding consensus sequences located between +752 to +780 and +897 to +917, respectively, and 5'-CAGAGCCTTCAGGCTTTTCGCGCGCT-3' (E2F-BS1) and 5'-CGCCCTTGGCCTTTTCCCGCGCC-3' (E2F-BS2) corresponding to the E2F binding consensus sequences located between -392 to -372 and -296 to -280, respectively, from the transcription start site (+1) referenced from the longest published SIVA sequence (GenBankTM accession number AF033114 [GenBank] ). The binding reaction (25 µl) was carried out at room temperature for 20 min in binding buffer with 0.1 µg of sonicated herring sperm DNA, and, for p53 binding, 1 µl of p53 Ab-1 monoclonal antibody was added to the binding buffer (OP03L; Oncogene Research Products). To control for binding specificity, a 100-fold excess of unlabeled oligonucleotide for BS1 and BS2 (p53 and E2F) was added to the binding reaction, and the mixture was incubated for 20 min before the addition of labeled probe. Furthermore, supershifts were performed with a p53-specific antibody FL393 (Santa Cruz Biotechnology, Inc.) and an E2F1-specific antibody C20-X (Santa Cruz Biotechnology, Inc.). Complexes were resolved on a 5% polyacrylamide, 1x Tris-glycine gel, dried, and visualized by autoradiography.

SIVA Promoter Luciferase Reporter Assays—The SIVA luciferase reporter construct (pGL3b-SIVA) was generated by subcloning a murine SIVA gene fragment (-440 to +1770) containing the putative promoter, exon1 and intron1 into the SmaI site of pGL-3 basic (Promega). P53 binding site deletion constructs were generated by excising p53-BS1 (+752 to +780) with BsmB1 and PvuII, p53-BS2 (+897 to +917) with PvuII and EcoR1, and p53-BS1 and p53-BS2 (+752 to +917) with BsmB1 and EcoR1. SN48 cells were transfected by calcium phosphate precipitation as previously described (23) with some modifications. Briefly 15 µg/plate of luciferase construct, 3 µg/plate of either the pCMV-p53, the pCMV-E2F1, the DNA binding mutant pCMV-p53-173L, or the empty pCMV vectors, and 2 µg/plate of pPGK-LacZ vector as an internal standard. After 4 h, cells were passaged into three wells of a 6-well dish/10-cm plate (Sarstedt, Inc., Newton, NC) and incubated for 20 h with fresh medium supplemented with 40 mM Hepes buffer (Sigma-Aldrich) before assaying for luciferase activity. After 24 h cells were washed once with phosphate-buffered saline and lysed in the wells with 200 µl/well of 1x reporter lysis buffer (Promega). Cells were collected by scraping and were subjected to one freeze-thaw cycle followed by centrifugation. Supernatants were collected and assayed for luciferase activity using a BioOrbit 11250 luminometer. A portion of the harvested cell extract was assayed for {beta}-galactosidase activity based on the conversion of 4-methylumbelliferyl-D-galactoside (Sigma-Aldrich) to the highly fluorescent molecule methylumbelliferone. Cell extract (10 µl) was incubated in the dark with 30 µl of 0.3 mM 4-methylumbelliferyl-D-galactoside, 15 mM Tris-HCl, pH 8.8, for 30 min after which time a stop solution was added. After addition of 2 ml of Z-buffer, fluorescence was quantified using a PerkinElmer Life Sciences LS50 luminescence spectrofluorometer at 350-nm excitation and 450-nm emission settings. The ratio of luciferase to {beta}-galactosidase activity was determined in triplicate samples and normalized to vector-transfected extracts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SIVA Is Induced during p53-mediated Neuronal Cell Death—Although previous studies in proliferating cells have identified Bax as a direct transcriptional target for p53 (19), we and others have found no induction of Bax in response to p53 in postmitotic neurons (16, 17, 20). Presently, little is known regarding the molecules that regulate neuronal cell death in the p53 apoptotic cascade. To identify the mechanism by which p53 induces the death of postmitotic neurons, DNA microarray analysis was performed using Affymetrix gene chip arrays. This analysis was conducted in primary cortical neurons by direct p53 overexpression using adenoviral vector-mediated gene delivery of a GFP control vector (Ad-GFP), wild type p53 (Ad-p53), a transactivation defective mutant (Ad-p53-DM), and a p53 DNA-binding mutant (Ad-p53-{Delta}V) that fails to induce apoptosis. To verify the reliability of the GeneChip data, three known p53-responsive genes were used as a basis for comparison, including APAF1 (21, 23), PERP (54), and Bax (19). Evaluation of previously characterized p53-responsive genes revealed a reproducible 3.5-fold induction of APAF1, a 6.8-fold increase in PERP, but no significant change in Bax mRNA expression in neurons expressing wild type p53 (Table I). Furthermore, no induction of SIVA mRNA was found in response to mutant p53 lacking either the DNA binding domain or both transactivation domains. These data suggest that SIVA may serve as an important p53 target gene involved in several injury-induced modes of neuronal cell death.


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  		TABLE I
SIVA is a p53-Inducible gene in neurons Microarray analysis of RNA extracted from primary cortical neurons 48 h after infection with either Ad-p53, the transcriptionally defective mutant Ad-p53-DM, or the DNA binding mutant Ad-p53-{Delta}V. "Fold change" represents the ratio of gene expression in cells transduced with Ad-GFP versus either Ad-p53 or Ad-p53 mutants. Accession numbers indicate the sequence used as probes in the microarray analysis. Data represent the average of two independent determinations.

 
To confirm results obtained with DNA microarray analysis, we examined SIVA mRNA levels by reverse transcription (RT)-PCR under similar conditions. Consistent with DNA microarray data, primary cerebellar granule neurons infected at 50 m.o.i. with Ad-p53 exhibited a significant increase in SIVA mRNA levels relative to cells infected with Ad-LacZ (Fig. 1A). This increase in SIVA mRNA levels was evident as soon as p53 became stably expressed, usually at 36–48 h following Ad-p53 infection (results not shown). To verify that SIVA could also be up-regulated in response to endogenous p53 activity, we treated cortical neurons with 10 µM camptothecin a DNA-damaging agent that has been shown previously to induce p53-dependent death (16). Cortical neurons treated with camptothecin responded with a rapid increase in p53 protein that was first detectable at 2 h and continued to increase for 4 h after which the levels became stabilized (Fig. 1B). In wild type neurons, camptothecin caused SIVA induction as early as 6 h following treatment demonstrating that SIVA up-regulation is an early event in the apoptotic cascade co-incident with the increase in p53 protein (Fig. 1C). This induction, however, was not observed in p53-deficient cells treated under identical conditions (Fig. 1C). Finally, to ask whether SIVA induction occurs in vivo following neuronal injury in which the involvement of endogenous p53 has previously been demonstrated (3, 55), we examined the brains of mice subjected to a model of focal ischemia. Brains were removed from mice subjected to 2 h middle cerebral artery occlusion followed by 48 and 72 h of reperfusion. This procedure generates an infarct in the striatum and cortex on the side of the brain ipsilateral to the occluded middle cerebral artery. Extracts from brain tissue revealed that SIVA mRNA is induced in the affected ipsilateral forebrain relative to the control tissue obtained from the contralateral hemisphere (Fig. 1D). Together, these results indicate that increased levels of SIVA transcript correlate directly with p53 activation and suggest the possibility that this proapoptotic gene may be a direct transcriptional target of p53 in the regulation of neuronal cell death.



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  		FIG. 1.
p53-mediated induction of SIVA mRNA in neurons. A, SIVA expression was analyzed using semiquantitative RT-PCR from RNA extracted from cerebellar granule neurons 48 h after infection with Ad-p53 or Ad-LacZ. B, protein was extracted from cortical neurons at the indicated times following treatment with 10 µM camptothecin and assayed for p53 levels by Western blot analysis. C, RNA was extracted from cortical neurons at the indicated times after treatment with 10 µM camptothecin and SIVA expression was analyzed using semiquantitative RT-PCR. D, SIVA expression was analyzed using RT-PCR from RNA extracted from ipsilateral and contralateral forebrain from mice subjected to 2 h of middle cerebral artery occlusion and following the indicated hours of reperfusion. The figures are a representative of three (n = 3) independent experiments.

 
Up-regulation of SIVA Is Sufficient to Induce Cell Death in Neural Precursor Cells and Postmitotic Neurons—Before exploring the possibility that p53 may directly activate the SIVA gene, we first determined whether SIVA up-regulation alone would be sufficient to induce the death of primary neural precursor cells and postmitotic neurons. A recombinant adenovirus vector was constructed carrying an expression cassette with full-length SIVA-1 tagged with GFP. Primary cerebellar granule neurons or neural precursor cells were infected at 50 m.o.i. with Ad-SIVA-GFP or a control adenoviral vector carrying GFP only. SIVA expression was verified by Western blot analysis (data not shown). Postmitotic neurons infected with Ad-SIVA-GFP underwent apoptosis that was first evident by 72 h post-infection. The rate of cell death progressed such that by 120 h more that 72% of the SIVA expressing cerebellar granule neurons had died relative to only 33% of control cultures (Fig. 2A). Similarly, increased expression of SIVA was also sufficient to induce the death of neural precursor cells treated under identical conditions (Fig. 2B). Cell death was evident by 72 h post-infection and progressed rapidly leaving only 23% of the cells surviving at 120 h. These results demonstrate that SIVA is up-regulated in response to p53 following neuronal injury and that SIVA induction alone is sufficient to activate the apoptotic cascade in neural tissue.



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  		FIG. 2.
SIVA expression is sufficient to induce neuronal cell death. Neuronal cells were infected with 50 m.o.i. of Ad-SIVA-GFP or Ad-GFP/Ad-LacZ control. Survival was measured by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at the indicated times and shown as a percentage of uninfected controls. A, cerebellar granule neurons. Data represent the mean ± S.E. from four (n = 4) independent experiments. B, neural precursor cells. Data represent the mean ± S.E. from three (n = 3) independent experiments.

 
The Regulatory Sequences of the SIVA Gene Contain p53 and E2F1 Response Elements—To understand the mechanism by which the SIVA gene is regulated, we used PromoterInspector (Genomatix) software to identify the putative promoter region for the SIVA gene. Analysis of the SIVA promoter sequence with MatInspector (Genomatix) software revealed the existence of two putative E2F consensus binding sites (56) at -392 to -372 bp and -296 to -280 bp and two putative p53 consensus sequences (57) within intron 1 at 752–780 bp and 897–917 bp (Fig. 3A). These are referenced from the transcription start site (+1), which refers to the longest published sequence of SIVA (GenBankTM accession number AF033114 [GenBank] ). Because the SIVA gene exhibited a striking induction during p53-mediated neuronal apoptosis, we assayed whether p53 was able to bind the consensus sites and activate transcription on the SIVA promoter. Oligonucleotides corresponding to the p53 consensus sites were synthesized, and electrophoretic mobility shift assays (EMSAs) were performed. Protein was extracted from neurons 48 h following adenovirus-mediated p53 delivery. The EMSA demonstrates that neuronal extracts exhibit p53-DNA binding activity at both putative binding sites (Fig. 3B). The specificity of p53 binding at these consensus sites was supported by two control experiments: (a) DNA binding activity could be competed out by incubation with excess unlabeled probes, and (b) the complexes were supershifted by the addition of a p53-specific antibody. The results of these experiments demonstrate that p53 can bind the two p53 consensus sites in the first intron of the SIVA gene.



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  		FIG. 3.
Specific binding of p53 to SIVA promoter elements in neuronal extracts. A, map of the SIVA fragment isolated from mouse genomic DNA containing two putative E2F consensus binding sequences within the promoter region and two putative p53 consensus binding sequences within intron1. B, EMSA: Protein was extracted from cerebellar granule neurons 48 h after infection with Ad-p53. p53 binding activity to the SIVA p53 response elements was assayed by electrophoretic mobility shift assay. Binding reactions were carried out with neuronal extracts (10 µg of protein) and the indicated oligonucleotides in the presence of p53 antibody (Ab1). Supershift with an antibody directed against p53 (FL393) was carried out to further confirm the composition of the complex on the SIVA p53 response elements.

 
p53 Can Activate Transcription from the SIVA Promoter—Although the above experiments demonstrate that p53 can directly bind the putative consensus sequences, we next asked whether p53 binding could activate transcription from the SIVA promoter. To test this, primers were designed to isolate 550 bp of the SIVA promoter as well as exon1 and intron1, which contains the p53 response elements. This insert was isolated by PCR and cloned into the pGL3b luciferase reporter plasmid. To examine whether p53 could directly activate transcription, four different SIVA-luciferase reporter constructs were tested (Fig. 4A), including the full-length SIVA insert (pGL3b-SIVA), a truncated insert missing the first p53 consensus sequence (pGL3b-SIVA-{Delta}BS1), a truncated insert missing the second p53 recognition sequence (pGL3b-SIVA-{Delta}BS2), and a truncation deleting both proposed p53 recognition sequences (pGL3b-SIVA-{Delta}{Delta}). Cultured SN48 neuronal cell lines were transfected with one of the SIVA promoter/luciferase constructs, a plasmid containing PGK-LacZ to control for transfection efficiency, and one of the following plasmids containing wild type p53, mutant p53, or empty vector. Cells cotransfected with the intact SIVA luciferase construct (pGL3b-SIVA) and wild type p53 exhibited a dramatic 12-fold increase in luciferase activity (Fig. 4B). As controls, we used p53 mutants that failed to induce SIVA mRNA, based on DNA microarray analysis (Table I). In contrast to wild type p53, there was no induction of luciferase activity following cotransfection with the DNA binding mutant p53-173L or the double mutant (p53-DM) lacking both transactivation domains of p53 (Fig. 4B). To determine the responsiveness of the individual p53 DNA binding sites, cells were cotransfected with each of the SIVA promoter constructs carrying deletions in the p53 consensus sites together with an expression vector for wild type p53. Deletion of p53-BS1 exhibited a 35% reduction in luciferase activity, however, still retaining activity. In contrast, deletion of p53-BS2 alone or deletion of both binding sites revealed complete loss of p53 responsiveness (Fig. 4C). From these results, we conclude that the SIVA gene is a direct transcriptional target for p53, and, of the two p53 consensus sites, BS2 is the most important to confer p53 responsiveness.



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  		FIG. 4.
Activation of the SIVA promoter by p53 in a neuronal cell line. A, p53 responsiveness of the SIVA gene was tested using luciferase reporter constructs (pGL3b; Promega) consisting of the luciferase gene fused to a DNA fragment containing both p53 binding sites (BS1 and BS2), or truncated fragments deleted for one or both p53 recognition sequences. SN48 cells were cotransfected with the indicated luciferase reporter construct, a PGK-LacZ reporter construct and an expression plasmid for either wild type p53, DNA binding-defective p53-173L, transactivation mutant p53-DM, or empty vector as control (B) or an expression plasmid for either wild type p53 or empty vector (C). Luciferase activity was measured in cell lysates obtained 24 h after transfection and normalized to {beta}-galactosidase activity. The -fold increase indicates the ratio of normalized luciferase activity of each SIVA construct in the presence of p53 expression vector versus empty vector control. Data represent the mean ± S.E. of triplicate samples from five (n = 5) independent experiments (except "a," which represents two (n = 2) independent experiments).

 
SIVA Is Directly Induced by E2F1—Because putative E2F response elements were found on the SIVA promoter and E2F1 has previously been shown to be a key proapoptotic protein involved in neuronal injury (41–47), we asked whether E2F1 could also directly modulate transcription of the SIVA gene in neurons. We first addressed whether E2F1 had any effect on SIVA-1 mRNA levels by conducting DNA microarray analysis and RT-PCR. Consistent with previous results, E2F1 induced mRNA expression for c-Myb (10.5-fold), DHFR (7.9-fold), caspase3 (2.9-fold), and APAF1 (2.5-fold). In addition, our results revealed a 2.8-fold induction of SIVA-1 mRNA in response to E2F1 (Table II). Results from DNA microarray analysis were confirmed by RT-PCR, which also demonstrated a dramatic induction of SIVA-1 mRNA in response to E2F1 (Fig. 5A). To ask whether E2F1 was capable of binding the SIVA-1 promoter at the two consensus sequences, EMSA was performed as for p53. Our results show that E2F1 is capable of binding both consensus E2F sites on the SIVA promoter (Fig. 5B). Again, specificity was confirmed by competition with unlabeled probe and supershift with an E2F1-specific antibody. Although our results demonstrate that E2F1 could bind the E2F consensus sites on the SIVA promoter, we next examined whether E2F1 could activate transcription. Luciferase reporter assays were performed with the -440 to 1771 bp fragment containing the SIVA promoter, the first exon and intron as described above as well as the luciferase construct pGL3b-SIVA-{Delta}{Delta} missing both p53 DNA consensus binding sequences. SN48 cells were cotransfected with pGL3b-SIVA or pGL3b-SIVA-{Delta}{Delta} along with expression plasmids for E2F1 or empty control vector and LacZ. Our assays revealed that E2F1 was capable of inducing a 2.6-fold activation of the SIVA promoter relative to the control vector alone (Fig. 5C). Deletion of both p53 consensus binding sites from the luciferase construct did not alter the ability of E2F1 to induce luciferase activity from the SIVA promoter demonstrating that SIVA can be regulated independently of p53. Based on our findings, that E2F1 can induce SIVA mRNA in neurons, the SIVA promoter contains E2F consensus sequences, E2F1 is capable of binding these sites, and the SIVA promoter is activated 2.6-fold by E2F1, we conclude that SIVA is also a direct transcriptional target for E2F1 in postmitotic neurons.


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  		TABLE II
SIVA is an E2F1-inducible gene in neurons Microarray analysis of RNA extracted from neural precursor cells 48 h after infection with either Ad-GFP or Ad-E2F1. "Fold change" represents the ratio of gene expression in cells transduced with Ad-GFP versus Ad-E2F1. Accession numbers indicate the sequence used as probes in the microarray analysis.

 



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  		FIG. 5.
SIVA is a direct target for E2F1. A, RNA was extracted from cerebellar granule neurons 72 h after infection with Ad-E2F1 or Ad-LacZ and analyzed for SIVA or glyceraldehyde-3-phosphate dehydrogenase expression using semiquantitative RT-PCR. B, protein was extracted from cerebellar granule neurons 72 h after infection with Ad-E2F1. E2F1 binding activity to the SIVA promoter was assayed by electrophoretic mobility shift assay. Binding reactions were carried out with neuronal extracts (10 µg of protein) and the indicated oligonucleotides. Supershift with an antibody directed against E2F1 (C20X) was carried out to further confirm the presence of E2F1 binding to the SIVA promoter. C, SN48 cells were cotransfected with the indicated luciferase reporter construct (pGL3b-SIVA contains the intact SIVA promoter; pGL3b-SIVA-{Delta}{Delta} contains the SIVA promoter lacking both p53 consensus binding sites), a PGK-LacZ reporter construct, and an expression plasmid for wild type E2F1 or empty vector as control. Luciferase activity and -fold increase were determined as above. Data represent the mean ± S.E. of triplicate samples from three (n = 3) independent experiments.

 
E2F1 and p53 Regulatory Elements Are Conserved in the Human SIVA Gene—Similar to the mouse, analysis of the promoter region of the human SIVA gene with the Genomatix software revealed four E2F consensus binding sequences but no p53 binding sites in the promoter region (Fig. 6A). Further analysis of the human SIVA gene also uncovered putative p53 consensus binding sequences in introns 1, 2, and 3 (Fig. 6B), which are also similar to those found in mouse genomic DNA.



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  		FIG. 6.
Putative E2F1 and p53 consensus binding sequences are located in human SIVA gene. DNA consensus binding sites within the human SIVA gene were identified using Genomatix software. A, four putative E2F binding sites fitting the general consensus site TTTSSCGC (56) were identified in a potential promoter region. B, putative p53 binding sites closely resembling the p53 consensus binding half site, RRRCWWGYYY (57), were identified in introns 1, 2, and 3.

 
In summary, the results of our studies demonstrate that the proapoptotic gene SIVA is induced following p53-mediated neuronal injury and that SIVA induction alone is sufficient to induce the death of neurons. Moreover, we have cloned the SIVA promoter and identified response elements for E2F1 and p53. By EMSA and luciferase reporter assays, we show that both transcription factors are capable of binding their respective consensus sites and activating transcription on the SIVA promoter. Taken together, our results show that the proapoptotic gene SIVA is a direct transcriptional target for the tumor suppressors p53 and E2F1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A dramatic induction of the SIVA gene resulting from p53-mediated apoptosis prompted us to pursue the regulation of the SIVA gene in neuronal injury. Previous studies have revealed that SIVA is induced by oxidative stress (36) and p53-mediated death induced by genotoxic agents (37). Studies with colon carcinoma have revealed that SIVA is up-regulated in response to treatment with a DNA-damaging agent, topotecan, and that cells lacking p53 fail to induce SIVA (37). Our studies with neuronal cell types also revealed a considerable induction of SIVA in response to forced expression of p53, in response to up-regulation of endogenous p53 induced by camptothecin, and following stroke injury in vivo. Using p53 deletion mutants, our results show that the transactivation domains at residues 22 and 23, and 53 and 54, as well as the DNA binding domains of p53 are essential for SIVA to be induced. These observations strongly suggested to us that SIVA might be a direct transcriptional target for p53. Isolation and analysis of the regulatory sequences of the SIVA gene, including the promoter and the first exon and intron revealed two p53 response elements within the first intron. Luciferase reporter assays revealed a striking 12-fold induction of the SIVA gene in response to wild type p53. To determine which of the consensus sites were most important to confer p53 responsiveness deletion analysis was performed. Deletion mutations of each of the p53 consensus sites revealed that the second p53 binding site, p53-BS2, was most important and was most likely responsible for p53-mediated SIVA activation. The fact that deletion of this second recognition sequence resulted in complete loss of activity is consistent with this interpretation. As a p53-responsive gene, SIVA was shown to be induced by many injury models involving p53, not only in neuronal cells but also in tumor cells, and cells of the immune system. Taken together, SIVA is a direct transcriptional target for p53 that is induced in a broad scope of tissue types.

SIVA induction was also found in response to E2F1, another proapoptotic transcription factor (45). E2F1 is a cell cycle regulatory gene that, when overexpressed in cells, can induce S-phase entry or apoptosis (58–62). Mice deficient in E2F1 develop tumors in a number of tissues, including uterine, lung, and lymphatic, thus demonstrating a tumor suppressor function for E2F1 (39, 40). We and others have shown E2F1 plays a prominent role in the regulation of neuronal cell death (45–47, 63, 64). Specifically, neurons deficient for E2F1 are more resistant to injury by {beta}-amyloid (45), oxygen glucose deprivation (65), K+ deprivation (42), and mice lacking E2F1 show significant protection from stroke injury (64). Because stroke injury has been shown to involve both E2F1 and p53, it is not surprising that we find a significant induction of SIVA gene expression in brains following focal ischemia. Interestingly, the presence of both E2F and p53 response elements on the promoter of a proapoptotic gene has been previously shown. Moroni et al. (21) have demonstrated that the promoter of APAF1 is regulated by both p53 and E2F1. Recently, the BH3-only proteins PUMA and Noxa known to be direct p53 targets have also been shown to be regulated by E2F1 (66). It appears that genes like APAF1, PUMA, Noxa, and SIVA are part of a growing list of genes that may be important in regulating the death response initiated by these tumor suppressors.

E2F1-mediated apoptosis is thought to proceed through both p53-dependent and p53-independent pathways. Thus, the potential for the convergence of both the p53 and E2F1 pathways to induce neuronal cell death exists following brain injury. Recent research, in proliferating cells, has demonstrated that interaction of E2F1 with the p53 pathway could involve transcriptional up-regulation of E2F1 target genes such as p14/p19ARF, which affect p53 accumulation (67, 68), E2F1-induced phosphorylation of p53 (69) or direct E2F1-p53 complex formation (70). All are believed to enhance the apoptotic activity of p53. In neurons, SIVA regulation could be complemented by this synergy between E2F1 and the p53 pathways. Not all models of neuronal cell death, however, require both pathways. {beta}-Amyloid-evoked neuronal cell death, which is mediated by E2F1, does so in a p53-independent manner (45). We have also shown here that, following DNA damage, SIVA is not up-regulated in p53-deficient neurons. Western blot analysis demonstrated that E2F1 was not induced at early time points following treatment with camptothecin and was only up-regulated after 16 h, long after apoptotic events have been initiated (data not shown). These results suggest that E2F1 is not involved in the regulation of SIVA in this model of neuronal injury. Whether SIVA regulation is an independent event or a consequence of cross-talk between these two apoptotic pathways is likely a function of the initiating mechanism.

Although SIVA has been shown to play a role in regulating apoptosis through the TNFR signaling cascade, the involvement of SIVA in neuronal injury has not yet been described. SIVA contains a death domain and therefore functions in TNFR signaling in cells of the immune system (27). More recently SIVA has also been shown to interact with GITR, another member of the TNFR superfamily expressed in T cells. Although SIVA was first characterized in cells of the immune system and was thought to be involved in T cell homeostasis, expression analysis revealed that SIVA is expressed in most tissues (34). In addition to normal tissues, SIVA is also expressed in tumor cells exposed to genotoxic agents that induce a p53 death response. Because CD27 is expressed primarily in T and B cells, the mechanism by which SIVA signals apoptosis in other cell types such as neurons or tumor cells remains unknown. Presently, it is unknown whether SIVA interacts with any TNFR family members expressed in the nervous system. There are a number of TNFR subtypes expressed in CNS neurons, including TNFR1, p55-TNFR, TNFRII, and the p75 low affinity neurotrophin receptor (71). It has also been reported that TNF signaling is increased in stroke and is believed to have a major impact on the extent of brain injury (71, 72). Based on studies in the immune system it is likely that SIVA may also be involved in TNF signaling following injury, however the interacting TNF targets in the CNS remain unknown.

In addition to interactions with members of the TNFR super family, SIVA-1 has also been shown to directly interact with antiapoptotic members of the Bcl-2 family proteins. SIVA has no BH3-like domain, however the site of interaction with Bcl-XL was found to be through a 20-amino acid long amphipathic helix known as the SAH domain (33). Disruption of this interaction by mutation of this site mitigated the apoptotic response. Thus, it is believed that SIVA may induce cell death by sequestering anti-apoptotic Bcl-2 family proteins and thereby allowing the proapoptotic family members to function. The molecules with which SIVA interacts to induce apoptosis may vary depending on the cell type, and future studies should elucidate its mechanism of action in the CNS.

In summary, SIVA is a proapoptotic gene that is expressed in a broad scope of tissues, including cells of the immune system, tumor cells, and in the CNS. SIVA is induced in response to p53 and E2F1, two tumor suppressor genes that function in cell cycle regulation and apoptosis. Up-regulation of SIVA in itself is sufficient to induce the apoptotic cascade implicating its importance in injury-induced apoptosis. Indeed, a number of tumor tissues have revealed significantly low levels of SIVA suggesting that SIVA may also play an important role in protection against cancer. By isolation of the mouse SIVA promoter, we have identified consensus binding sites for both p53 and E2F1 and have shown that these factors are able to bind their respective sites. Moreover, we have shown that p53 and E2F1 binding can activate the SIVA promoter demonstrating that SIVA is a direct transcriptional target for these tumor suppressor genes. Presently, a SIVA-deficient mouse has not been developed, however, future studies involving deletion of SIVA will reveal the extent of its involvement in different modes of cell death. The fact that SIVA is highly induced by p53 and E2F1 in a broad spectrum of cell types and that SIVA up-regulation is sufficient to induce the death cascade speak to its importance not only in protection against tumorigenesis but also in brain damage following acute injury.


   FOOTNOTES
 
* This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) and Heart and Stroke Foundation of Ontario (to R. S. S.). Adenoviral vectors were generated by the Viral Vector Core Facility supported by grants from the Canadian Stroke Network (to R. S. S. and D. S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by the Canadian Stroke Network. Back

§ Supported by an Ontario Neurotrauma studentship. Back

Supported by a CIHR postdoctoral fellowship. Current address: Robarts Research Institute, Cell Biology Group, P. O. Box 5015, 100 Perth Dr., London, Ontario N6A 5K8, Canada. Back

|| Supported by a CIHR studentship. Back

** A CIHR/Novartis Chair in Neuroscience. Back

{ddagger}{ddagger} A GlaxoSmithKline professor. Back

§§ To whom correspondence should be addressed: Ottawa Health Research Institute, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada. Tel.: 613-562-5800 (ext. 8459); Fax: 613-562-5403; E-mail: rslack{at}uottawa.ca.

1 S. P. Cregan, N. A. Arbour, J. G. MacLaurin, S. M. Callaghan, A. Fortin, E. C. C. Cheung, D. S. Park, and R. S. Slack, unpublished observation. Back

2 The abbreviations used are: TNFR, tumor necrosis factor receptor; GFP, green fluorescent protein; m.o.i., multiplicity of infection; Ad, adenovirus; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; DM, double transactivation mutant. Back


   ACKNOWLEDGMENTS
 
We are grateful to Jacqueline Vanderluit and Kelly McClellan for critical reading of the manuscript, to Alexandre Anawati for help with cell culture and luciferase assays, and to Elizabeth Keramaris for technical support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Li, Y., Chop, M., Zhang, Z. G., Zaloga, C., Niewenhuis, L., and Gautam, S. (1994) Stroke 25, 849-855; discussion 855-846[Abstract]
  2. Sakhi, S., Bruce, A., Sun, N., Tocco, G., Baudry, M., and Schreiber, S. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7525-7529[Abstract/Free Full Text]
  3. McGahan, L., Hakim, A. M., and Robertson, G. S. (1998) Brain Res. Mol. Brain Res. 56, 133-145[Medline] [Order article via Infotrieve]
  4. Banasiak, K. J., and Haddad, G. G. (1998) Brain Res. 797, 295-304[CrossRef][Medline] [Order article via Infotrieve]
  5. Xiang, H., Hochman, D. W., Saya, H., Fujiwara, T., Schwartzkroin, P. A., and Morrison, R. S. (1996) J. Neurosci. 16, 6753-6765[Abstract/Free Full Text]
  6. Morrison, R. S., Wenzel, H. J., Kinoshita, Y., Robbins, C. A., Donehower, L. A., and Schwartzkroin, P. A. (1996) J. Neurosci. 16, 1337-1345[Abstract/Free Full Text]
  7. Zhang, Y., McLaughlin, R., Goodyer, C., and LeBlanc, A. (2002) J. Cell Biol. 156, 519-529[Abstract/Free Full Text]
  8. Halterman, M. W., Miller, C. C., and Federoff, H. J. (1999) J. Neurosci. 19, 6818-6824[Abstract/Free Full Text]
  9. Slack, R. S., Belliveau, D. J., Rosenberg, M., Atwal, J., Lochmuller, H., Aloyz, R., Haghighi, A., Lach, B., Seth, P., Cooper, E., and Miller, F. D. (1996) J. Cell Biol. 135, 1085-1096[Abstract/Free Full Text]
  10. Miller, F. D., Pozniak, C. D., and Walsh, G. S. (2000) Cell Death Differ. 7, 880-888[CrossRef][Medline] [Order article via Infotrieve]
  11. Johnson, M. D., Kinoshita, Y., Xiang, H., Ghatan, S., and Morrison, R. S. (1999) J. Neurosci. 19, 2996-3006[Abstract/Free Full Text]
  12. Crumrine, R. C., Thomas, A. L., and Morgan, P. F. (1994) J. Cereb. Blood Flow Metab. 14, 887-891[Medline] [Order article via Infotrieve]
  13. Culmsee, C., Zhu, X., Yu, Q. S., Chan, S. L., Camandola, S., Guo, Z., Greig, N. H., and Mattson, M. P. (2001) J. Neurochem. 77, 220-228[Medline] [Order article via Infotrieve]
  14. Prives, C., and Hall, P. A. (1999) J. Pathol. 187, 112-126[CrossRef][Medline] [Order article via Infotrieve]
  15. Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P., and Moll, U. M. (2003) Mol. Cell 11, 577-590[CrossRef][Medline] [Order article via Infotrieve]
  16. Xiang, H., Kinoshita, Y., Knudson, C. M., Korsmeyer, S. J., Schwartzkroin, P. A., and Morrison, R. S. (1998) J. Neurosci. 18, 1363-1373[Abstract/Free Full Text]
  17. Cregan, S. P., MacLaurin, J. G., Craig, C. G., Robertson, G. S., Nicholson, D. W., Park, D. S., and Slack, R. S. (1999) J. Neurosci. 19, 7860-7869[Abstract/Free Full Text]
  18. Keramaris, E., Stefanis, L., MacLaurin, J., Harada, N., Takaku, K., Ishikawa, T., Taketo, M. M., Robertson, G. S., Nicholson, D. W., Slack, R. S., and Park, D. S. (2000) Mol. Cell. Neurosci. 15, 368-379[CrossRef][Medline] [Order article via Infotrieve]
  19. Miyashita, T., and Reed, J. C. (1995) Cell 80, 293-299[CrossRef][Medline] [Order article via Infotrieve]
  20. Johnson, M. D., Xiang, H., London, S., Kinoshita, Y., Knudson, M., Mayberg, M., Korsmeyer, S. J., and Morrison, R. S. (1998) J. Neurosci. Res. 54, 721-733[CrossRef][Medline] [Order article via Infotrieve]
  21. Moroni, M. C., Hickman, E. S., Denchi, E. L., Caprara, G., Colli, E., Cecconi, F., Muller, H., and Helin, K. (2001) Nat. Cell Biol. 3, 552-558[CrossRef][Medline] [Order article via Infotrieve]
  22. Cregan, S. P., Fortin, A., MacLaurin, J. G., Callaghan, S. M., Cecconi, F., Yu, S. W., Dawson, T. M., Dawson, V. L., Park, D. S., Kroemer, G., and Slack, R. S. (2002) J. Cell Biol. 158, 507-517[Abstract/Free Full Text]
  23. Fortin, A., Cregan, S. P., MacLaurin, J. G., Kushwaha, N., Hickman, E. S., Thompson, C. S., Hakim, A., Albert, P. R., Cecconi, F., Helin, K., Park, D. S., and Slack, R. S. (2001) J. Cell Biol. 155, 207-216[Abstract/Free Full Text]
  24. Gravestein, L. A., Blom, B., Nolten, L. A., de Vries, E., van der Horst, G., Ossendorp, F., Borst, J., and Loenen, W. A. (1993) Eur. J. Immunol. 23, 943-950[Medline] [Order article via Infotrieve]
  25. Camerini, D., Walz, G., Loenen, W. A., Borst, J., and Seed, B. (1991) J. Immunol. 147, 3165-3169[Abstract]
  26. Smith, C. A., Farrah, T., and Goodwin, R. G. (1994) Cell 76, 959-962[CrossRef][Medline] [Order article via Infotrieve]
  27. Prasad, K. V., Ao, Z., Yoon, Y., Wu, M. X., Rizk, M., Jacquot, S., and Schlossman, S. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6346-6351[Abstract/Free Full Text]
  28. Kobata, T., Agematsu, K., Kameoka, J., Schlossman, S. F., and Morimoto, C. (1994) J. Immunol. 153, 5422-5432[Abstract]
  29. Agematsu, K., Kobata, T., Sugita, K., Freeman, G. J., Beckmann, M. P., Schlossman, S. F., and Morimoto, C. (1994) J. Immunol. 153, 1421-1429[Abstract]
  30. Hintzen, R. Q., Lens, S. M., Lammers, K., Kuiper, H., Beckmann, M. P., and van Lier, R. A. (1995) J. Immunol. 154, 2612-2623[Abstract]
  31. Jacquot, S., Kobata, T., Iwata, S., Morimoto, C., and Schlossman, S. F. (1997) J. Immunol. 159, 2652-2657[Abstract]
  32. Spinicelli, S., Nocentini, G., Ronchetti, S., Krausz, L. T., Bianchini, R., and Riccardi, C. (2002) Cell Death Differ. 9, 1382-1384[CrossRef][Medline] [Order article via Infotrieve]
  33. Xue, L., Chu, F., Cheng, Y., Sun, X., Borthakur, A., Ramarao, M., Pandey, P., Wu, M., Schlossman, S. F., and Prasad, K. V. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6925-6930[Abstract/Free Full Text]
  34. Yoon, Y., Ao, Z., Cheng, Y., Schlossman, S. F., and Prasad, K. V. (1999) Oncogene 18, 7174-7179[CrossRef][Medline] [Order article via Infotrieve]
  35. Py, B., Slomianny, C., Auberger, P., Petit, P. X., and Benichou, S. (2004) J. Immunol. 172, 4008-4017[Abstract/Free Full Text]
  36. Cao, C., Ren, X., Kharbanda, S., Koleske, A., Prasad, K. V., and Kufe, D. (2001) J. Biol. Chem. 276, 11465-11468[Abstract/Free Full Text]
  37. Daoud, S. S., Munson, P. J., Reinhold, W., Young, L., Prabhu, V. V., Yu, Q., LaRose, J., Kohn, K. W., Weinstein, J. N., and Pommier, Y. (2003) Cancer Res. 63, 2782-2793[Abstract/Free Full Text]
  38. Okuno, K., Yasutomi, M., Nishimura, N., Arakawa, T., Shiomi, M., Hida, J., Ueda, K., and Minami, K. (2001) Dis. Colon Rectum 44, 295-299[CrossRef][Medline] [Order article via Infotrieve]
  39. Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996) Cell 85, 537-548[CrossRef][Medline] [Order article via Infotrieve]
  40. Field, S. J., Tsai, F. Y., Kuo, F., Zubiaga, A. M., Kaelin, W. G., Jr., Livingston, D. M., Orkin, S. H., and Greenberg, M. E. (1996) Cell 85, 549-561[CrossRef][Medline] [Order article via Infotrieve]
  41. Hou, S. T., Callaghan, D., Fournier, M. C., Hill, I., Kang, L., Massie, B., Morley, P., Murray, C., Rasquinha, I., Slack, R., and MacManus, J. P. (2000) J. Neurochem. 75, 91-100[CrossRef][Medline] [Order article via Infotrieve]
  42. O'Hare, M. J., Hou, S. T., Morris, E. J., Cregan, S. P., Xu, Q., Slack, R. S., and Park, D. S. (2000) J. Biol. Chem. 275, 25358-25364[Abstract/Free Full Text]
  43. Wang, F., Corbett, D., Osuga, H., Osuga, S., Ikeda, J. E., Slack, R. S., Hogan, M. J., Hakim, A. M., and Park, D. S. (2002) J. Cereb. Blood Flow Metab. 22, 171-182[CrossRef][Medline] [Order article via Infotrieve]
  44. Osuga, H., Osuga, S., Wang, F., Fetni, R., Hogan, M. J., Slack, R. S., Hakim, A. M., Ikeda, J. E., and Park, D. S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10254-10259[Abstract/Free Full Text]
  45. Giovanni, A., Keramaris, E., Morris, E. J., Hou, S. T., O'Hare, M., Dyson, N., Robertson, G. S., Slack, R. S., and Park, D. S. (2000) J. Biol. Chem. 275, 11553-11560[Abstract/Free Full Text]
  46. Park, D. S., Morris, E. J., Bremner, R., Keramaris, E., Padmanabhan, J., Rosenbaum, M., Shelanski, M. L., Geller, H. M., and Greene, L. A. (2000) J. Neurosci. 20, 3104-3114[Abstract/Free Full Text]
  47. Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., and Greene, L. A. (1998) J. Cell Biol. 143, 457-467[Abstract/Free Full Text]
  48. Reynolds, B. A., Tetzlaff, W., and Weiss, S. (1992) J. Neurosci. 12, 4565-4574[Abstract]
  49. Tropepe, V., Sibilia, M., Ciruna, B. G., Rossant, J., Wagner, E. F., and van der Kooy, D. (1999) Dev. Biol. 208, 166-188[CrossRef][Medline] [Order article via Infotrieve]
  50. Rowan, S., Ludwig, R. L., Haupt, Y., Bates, S., Lu, X., Oren, M., and Vousden, K. H. (1996) EMBO J. 15, 827-838[Medline] [Order article via Infotrieve]
  51. Crook, T., Marston, N. J., Sara, E. A., and Vousden, K. H. (1994) Cell 79, 817-827[CrossRef][Medline] [Order article via Infotrieve]
  52. Zhu, J., Zhang, S., Jiang, J., and Chen, X. (2000) J. Biol. Chem. 275, 39927-39934[Abstract/Free Full Text]
  53. Cregan, S. P., MacLaurin, J., Gendron, T. F., Callaghan, S. M., Park, D. S., Parks, R. J., Graham, F. L., Morley, P., and Slack, R. S. (2000) Gene Ther. 7, 1200-1209[CrossRef][Medline] [Order article via Infotrieve]
  54. Ihrie, R. A., Reczek, E., Horner, J. S., Khachatrian, L., Sage, J., Jacks, T., and Attardi, L. D. (2003) Curr. Biol. 13, 1985-1990[CrossRef][Medline] [Order article via Infotrieve]
  55. Watanabe, H., Ohta, S., Kumon, Y., Sakaki, S., and Sakanaka, M. (1999) Brain Res. 837, 38-45[CrossRef][Medline] [Order article via Infotrieve]
  56. Slansky, J. E., and Farnham, P. J. (1996) Curr. Top. Microbiol. Immunol. 208, 1-30[Medline] [Order article via Infotrieve]
  57. Levine, A. J. (1997) Cell 88, 323-331[CrossRef][Medline] [Order article via Infotrieve]
  58. Nevins, J. R. (1992) Science 258, 424-429[Abstract/Free Full Text]
  59. Wu, C. L., Classon, M., Dyson, N., and Harlow, E. (1996) Mol. Cell. Biol. 16, 3698-3706[Abstract]
  60. Asano, M., Nevins, J. R., and Wharton, R. P. (1996) Genes Dev. 10, 1422-1432[Abstract/Free Full Text]
  61. Fueyo, J., Gomez-Manzano, C., Yung, W. K., Liu, T. J., Alemany, R., McDonnell, T. J., Shi, X., Rao, J. S., Levin, V. A., and Kyritsis, A. P. (1998) Nat. Med. 4, 685-690[CrossRef][Medline] [Order article via Infotrieve]
  62. Kowalik, T. F., DeGregori, J., Schwarz, J. K., and Nevins, J. R. (1995) J. Virol. 69, 2491-2500[Abstract]
  63. Giovanni, A., Wirtz-Brugger, F., Keramaris, E., Slack, R., and Park, D. S. (1999) J. Biol. Chem. 274, 19011-19016[Abstract/Free Full Text]
  64. MacManus, J. P., Koch, C. J., Jian, M., Walker, T., and Zurakowski, B. (1999) Neuroreport 10, 2711-2714[Medline] [Order article via Infotrieve]
  65. Gendron, T. F., Mealing, G. A., Paris, J., Lou, A., Edwards, A., Hou, S. T., MacManus, J. P., Hakim, A. M., and Morley, P. (2001) J. Neurochem. 78, 316-324[CrossRef][Medline] [Order article via Infotrieve]
  66. Hershko, T., and Ginsberg, D. (2004) J. Biol. Chem. 279, 8627-8634[Abstract/Free Full Text]
  67. Robertson, K. D., and Jones, P. A. (1998) Mol. Cell. Biol. 18, 6457-6473[Abstract/Free Full Text]
  68. Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden, K. H. (1998) Nature 395, 124-125[CrossRef][Medline] [Order article via Infotrieve]
  69. Rogoff, H. A., Pickering, M. T., Debatis, M. E., Jones, S., and Kowalik, T. F. (2002) Mol. Cell. Biol. 22, 5308-5318[Abstract/Free Full Text]
  70. Hsieh, J. K., Yap, D., O'Connor, D. J., Fogal, V., Fallis, L., Chan, F., Zhong, S., and Lu, X. (2002) Mol. Cell. Biol. 22, 78-93[Abstract/Free Full Text]
  71. Martin-Villalba, A., Herr, I., Jeremias, I., Hahne, M., Brandt, R., Vogel, J., Schenkel, J., Herdegen, T., and Debatin, K. M. (1999) J. Neurosci. 19, 3809-3817[Abstract/Free Full Text]
  72. Barone, F. C., and Feuerstein, G. Z. (1999) J. Cereb. Blood Flow Metab. 19, 819-834[Medline] [Order article via Infotrieve]

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