Amyloid-β precursor-like protein APLP1 is a novel p53 transcriptional target gene that augments neuroblastoma cell death upon genotoxic stress

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Abstract

The tumor suppressor p53 is a key modulator of the cellular stress response, inducing cell-cycle arrest, apoptosis, senescence and cell differentiation. To evaluate further the molecular mechanism underlying p53 function, the transcriptional profiles of proliferating and senescent WI-38 cells, both wild-type p53 expressers and counterparts with an inactivated p53, were compared by DNA microarray analysis. In particular, the amyloid-β precursor-like protein 1 (APLP1) is induced in senescent cells in a p53-dependent manner. APLP1 was confirmed to be a novel transcriptional target of p53 by in vivo and in vitro characterization of a p53 responsive element found in the first intron of the APLP1 gene locus. APLP1 knockdown experiments demonstrate that APLP1 is required for the proliferation of fibroblastic and epithelial cells. Moreover, depletion of APLP1 expression diminishes stress-induced apoptosis of neural cells, whereas ectopic APLP1 expression augments apoptosis. Based on these data, a mechanism is proposed whereby p53-dependent induction of APLP1 is involved in neural cell death, and which may exacerbate neuronal cell loss in some acute or chronic neurodegenerative disorders.

Introduction

The tumor suppressor p53 is a key modulator of the cellular stress response. In response to DNA damage, hypoxia, viral infection or oncogene activation, the protein is stabilized and activated. This leads to diverse biological effects, such as cell-cycle arrest, apoptosis, senescence, differentiation and anti-angiogenesis (Vousden and Lu, 2002). p53 is the most frequently altered gene in human cancer (Hainaut et al., 1997). Absence of functional p53 allows cellular immortalization and predisposes cells to neoplastic transformation (Ryan et al., 2001).

p53 mediates many of its key functions largely by the transactivation or transrepression of its target genes (Bargonetti and Manfredi, 2002; Vousden and Lu, 2002). Most tumor-derived mutants of p53 are defective in DNA binding and transactivation, supporting a critical role for transactivation in p53's ability to suppress neoplasia. A number of p53 responsive genes have been identified and characterized to participate downstream in different p53 cellular pathways, such as growth arrest, cellular differentiation or apoptosis. p53-dependent G1 arrest is primarily mediated by p21WAF1, a cyclin-dependent kinase inhibitor (el-Deiry et al., 1992). Many p53 target genes have been proposed to play roles in apoptosis, such as Bax, Bid and PUMA (p53-upregulated modulator of apoptosis), transmembrane proteins Perp, Fas and Killer/Dr5, and PIGs proteins. Bax, Bid and PUMA proteins induced by p53 activate an intrinsic pathway to induce cell death. Fas and Killer/Dr5 induce cell apoptosis through an extrinsic pathway. The PIGs, encode proteins that are generally connected to the redox state of the cell, which may suggest that p53 activation affects the production of reactive oxygen species and causes cell death (Polyak et al., 1997). The diverse proapoptotic genes transactivated by p53 may suggest that these genes may play a role in different contexts of p53-dependent apoptosis.

Many acute or chronic neurodegenerative diseases such as ischemic stroke, Alzheimer's disease and Parkinson's disease result from degeneration and death of specific populations of neurons. Although the genetic and nongenetic factors that initiate these disorders differ between the various diseases, a biochemical cascade of events executing the cell death process appears to be shared (Culmsee and Mattson, 2005). Interestingly, activation of p53 has been observed frequently in acute neuronal injury as well as in chronic neurodegenerative disorders. Elevated levels of p53 messenger RNA (mRNA) and protein have been detected in brain tissue derived from patients diagnosed with neurodegenerative diseases and from animal models with such disorders. This suggests a role for p53 in neuron death during neurodegeneration (Morrison and Kinoshita, 2000).

Alzheimer's is a neurodegenerative disease characterized by senile plaques containing amyloid-β peptide (Aβ1−42) deposits, and these deposits are considered to be a major cause of the progressive degeneration and death of neurons (Mattson, 2004). Neurotoxic Aβ1−42 is a proteolytic product from amyloid precursor protein (APP), which has two paralogues, namely amyloid-β precursor-like protein 1 (APLP1) and protein 2 (APLP2). APP and APLP2 are expressed in tissues throughout the body, while APLP1 expression appears to be limited to the brain (Wasco et al., 1992). Knockout mice studies have revealed that the functions of APP and its two paralogues are redundant (Heber et al., 2000). Indeed, both APLP1 and APLP2 have been found to accumulate in the senile plaques of Alzheimer's patients' brains (Bayer et al., 1997; McNamara et al., 1998). The specific contribution of APLP1 and APLP2 to the pathogenesis of Alzheimer's remains unclear. Notably, in mice overexpressing the Aβ1−42, elevated p53 levels were observed in those neurons associated with DNA strand breaks (LaFerla et al., 1996; de la Monte et al., 1997). These results raise the possibility that there are functional connections between p53 and APP or its paralogues and neural cell death.

In this work, the transcriptional profiles were compared of senescent and proliferating WI-38 human primary fibroblasts with their counterparts where p53 had been inactivated by DNA microarray analysis. Specific genes regulated by p53 during senescence were identified, in particular the APLP1. Various assays demonstrated that APLP1 is transactivated directly by p53. In light of the putative role of APLP1 in Alzheimer's disease, it was tested whether APLP1 influences neural cell death. Ectopic APLP1 expression was found to enhance neuroblastoma cell death, whereas its depletion diminished cell death upon genotoxic stress. These results identify a novel p53 target gene that potentially influences neural cell death.

Results

Induction of the APLP1 in senescent cells is p53 dependent

Normal human primary fibroblasts undergo replicative senescence after a limited number of cell divisions. Endogenous p53 can be inactivated using the GSE56 polypeptide that works by a negative-dominant mechanism (Ossovskaya et al., 1996). This approach was taken to inactivate p53 and was found to extend effectively the replicative life span of WI-38 human primary cells (Figure 1a), suggesting that p53 is activated during cellular senescence and inhibits cell growth. To delineate further the role of p53 in cellular senescence, DNA microarray analysis was used to compare the gene-expression profiles from four types of WI-38 cells: proliferating ‘wild-type’ cells (infected with control virus), senescent wild-type cells and their ‘p53 inactive’ (GSE56-infected) counterparts. Clusters of genes were observed that are either upregulated or downregulated during senescence only when p53 is wild type (Tables 1 and 2 in Supplementary data). In particular, it was notable that APLP1, a paralogue of Alzheimer's protein APP, is induced in senescent cells in a manner similar to the bona fide p53 target gene p21waf1, as judged by our cluster analysis.

Figure 1
figure1

Induced amyloid-β precursor-like protein 1 (APLP1) expression in senescent WI-38 cells is p53 dependent. (a) Life-span curves of empty vector (puro) and GSE56-infected WI-38 cells. Mean population doublings (PDLs) were calculated as detailed in the Material and methods section. (b) Quantitative PCR analysis of gene expression in proliferating (p22V), senescent (p30V), proliferating GSE56 expressing (p22G) and senescent GSE56-expressing (p30G) WI-38 cells. Values shown are means±s.d. of duplicate readings and represent expression patterns normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. (c) Immunoblot analysis of protein expression in whole cell lysates from proliferating (p22), senescent (p30) cells, proliferating GSE56 expressing (p22G) and senescent GSE56-expressing (p30G) WI-38 cells. β-Tubulin expression was used as a control for protein loading.

To confirm that the induction of APLP1 gene expression in senescent cells is dependent on p53, the levels of APLP1 mRNA in proliferating and senescent WI-38 cells, as well as in their GSE56-infected counterparts, were compared by quantitative reverse transcription (qRT)–PCR. As shown in Figure 1b, the level of APLP1 mRNA is significantly induced in wild-type senescent cells (p30V), but not in senescent cells where p53 has been inactivated (p30G). This is in contrast to the situation for APP and APLP2, two other members of the APP family, which exhibit slightly increased mRNA expression during replicative senescence, but in a p53-independent manner. Immunoblot analysis revealed that the APLP1 protein level is induced in senescent cells, and that this induction is absent in GSE56-infected cells (Figure 1c). The basal level of APLP1, different from p21Waf1, is not strongly affected by p53. It is noteworthy that APLP1 protein undergoes post-translational glycosylation (Eggert et al., 2004), giving rise to several bands, as was detected in this study. Western blot and RT–PCR analyses were used to confirm that GSE56 infection abrogates p53 activity; indeed massive p53 protein stabilization was observed, but p53 transactivation activity was eliminated completely, as indicated by reduced RNA and protein expression of p21Waf1 (Figures 1b and c).

Together, these experiments indicate that APLP1 expression is induced in a p53-dependent manner during replicative senescence.

APLP1 is induced by genotoxic stress in a p53-dependent manner

It is well accepted that activated p53 induces the expression of specific target genes (el-Deiry, 1998). p53 can be activated by various stimuli, therefore it was interesting to examine whether genotoxic stress, the archetypal p53 inducer, results in elevated APLP1 expression. To that end, APLP1 expression was examined in wild type (infected with control virus) and p53 inactive (GSE56-infected) proliferating WI-38 cells following exposure to doxorubicin (Dox). An increase in APLP1 mRNA levels is observed only in control cells following Dox exposure, and not in the GSE56-infected cells. A similar mRNA pattern is observed for p21Waf1 (Figure 2a). In contrast, APP and APLP2 mRNA levels do not change detectably in response to Dox. Immunoblot analysis revealed that the amount of APLP1 protein accumulates in a relatively slow manner following Dox treatment, and that this induction requires p53 activity, since it is absent in GSE56-infected cells (Figure 2b). As expected, p21Waf1 protein is induced only in treated control cells (Figure 2b). In addition, p53-dependent induction of APLP1 upon stress was also abolished by p53-specific RNAi in WI-38 proliferating cells under stress condition (Supplementary Figure S1). These results indicate that APLP1, but not APP and APLP2, is regulated specifically by p53 during genotoxic stress.

Figure 2
figure2

Amyloid-β precursor-like protein 1 (APLP1) expression is induced by genotoxic stress in a p53-dependent manner in several cell types. (a) Quantitative real-time (qRT)–PCR analysis of gene expression in vector or GSE56-infected proliferating WI-38 fibroblasts following exposure to doxorubicin (Dox; 0.2 μg/ml) for 24 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used for normalization. (b) Immunoblot analysis of protein expression in vector or GSE56 infected proliferating WI-38 fibroblasts following exposure to Dox (0.2 μg/ml) at the indicated times. β-Tubulin expression was used as a control for protein loading. (c) qRT–PCR analysis of gene expression in MCF7-puro and MCF7-p53RNAi cells treated with Dox (0.2 μg/ml) for 24 h. (d) Immunoblot analysis of protein expression in MCF7 cells treated with Dox (0.2 μg/ml) at the indicated times. (e) qRT–PCR analysis of APLP1 expression in SK-N-SH puro and SK-N-SH E6 cells treated with Dox (0.1 μg/ml) or etoposide (Etp; 10 μ M) for 14 h. (f) Immunoblot analysis of protein expression in SK-N-SH cells treated with Dox (0.1 μg/ml) or Etp (10 μ M) at the indicated times.

To corroborate these data, similar experiments were performed using a different cellular system. MCF7 is a breast cancer cell line characterized to possess wild-type p53. A counterpart cell line (p53RNAi) was created by transfection with specific p53 RNAi (Brummelkamp et al., 2002). APLP1 expression was assessed in these cells before and after treatment with Dox. APLP1 mRNA increases in MCF7-puro cells following Dox exposure. In contrast, there is no significant alteration in the level of APLP1 mRNA in MCF7-p53RNAi cells (Figure 2c). Similarly, at the protein level, APLP1 induction is only evident in Dox-treated MCF7-puro cells (Figure 2d). The functionality of p53 RNAi was validated by the significantly reduced levels of p53 and p21Waf1 protein in MCF7-p53RNAi cells following Dox treatment (Figures 2c and d). These results using the MCF7 system confirm that APLP1 induction after genotoxic stress is p53 dependent.

Previously, it was reported that APLP1 is a brain-specific protein (Kim et al., 1995). Therefore, it was important to check APLP1 expression in a neural system, and so similar experiments were carried out using SK-N-SH neuroblastoma cells, which established from a bone metastasis and possess a wild-type p53. Viral oncoprotein E6 expression was used to create a counterpart cell line where p53 is inactive, as this viral protein triggers p53 degradation (Scheffner et al., 1990). As shown in Figure 2e, the level of APLP1 mRNA is induced in SK-N-SH-puro cells (infected with empty virus) following treatment with either Dox or etoposide (Etp). However, no alteration in APLP1 levels is observed in E6-infected cells. Consistently, APLP1 protein level is induced after treatment only in SK-N-SH control cells (Figure 2f). As described above, the p53 status of each cell line was checked by examining p53 and p21Waf1 induction upon stress, which as expected is absent in E6-infected cells (Figures 2e and f).

Taken together, these data demonstrate that APLP1 expression is induced upon genotoxic stress in a p53-dependent manner in various cell types.

APLP1 gene is a direct target of p53

The p53-dependent induction of APLP1 mRNA in various cell types pointed to the possibility that p53 transactivates APLP1 gene expression directly. Almost all genes transactivated by p53 contain one or more p53 responsive elements (p53REs), with a sequence that conforms generally to the consensus defined by el-Deiry et al. (1992). Therefore, the APLP1 gene locus was screened at the sequence level for potential p53REs (NCBI human genomic database). Several were found in the APLP1 gene locus; the three best candidates were designated as p53RE-1, p53RE-2 and p53RE-3 (Figure 3a), and were analysed further. To verify whether p53 binds to these APLP1 loci in vivo, chromatin immunoprecipitation (ChIP) assays were performed. MCF7 cells, control and Dox treated, were subjected to cross-linking procedures. Cross-linked protein–DNA complexes were immunoprecipitated using either an anti-p53 antibody, or an anti-APLP1 antibody, which served as a negative (nonspecific binding) control. The precipitated DNA was amplified by PCR using primers specific to each of the three potential p53REs. As seen in Figure 3b, only APLP1 p53RE-3 is amplified specifically from anti-p53 immunoprecipitated complexes. The two other genomic regions (p53RE-1 and p53RE-2) are each amplified equivalently from anti-p53 and control antibody immunoprecipitated complexes. The integrity and specificity of this ChIP assay is validated by amplification of the p21Waf1 promoter only from complexes immunoprecipitated by anti-p53 (Figure 3b). These results suggest that the p53RE-3, which is located in the first intron of the APLP1 gene, is a functional p53-binding site. It is noteworthy that the left part of p53RE-3 shares homology with the p53RE consensus, while the right part is less homologous due to three nucleotides inserted within the core region.

Figure 3
figure3

Amyloid-β precursor-like protein 1 (APLP1) is a direct transcriptional target of p53. (a) Schematic representation of APLP1 promoter constructs. Genomic fragments containing the APLP1 promoter, the first exon, the partial first intron and combinations were inserted into the basic luciferase reporter plasmid PGL3. The sequences of three potential p53 responsive elements (p53Res; spacer sequences are omitted) in the APLP1 gene locus and a mutation of p53RE-3 are detailed in the insert panel. The arrows indicate the approximate position of three potential p53REs in the APLP1 gene locus. The boxes indicate the first exon of APLP1 gene. (White box represents untranslated region and black box represents coding region.) (b) Chromatin immunoprecipitation (ChIP) assay in MCF7 cells which treated with (+) or without (−) 0.2 μg/ml doxorubicin (Dox) for 24 h. (c) Various APLP1 reporters were co-transfected with a wild-type p53 expression plasmid or empty vector control plasmid into H1299 cells. The reporter activity was measured 48 h after transfection and is normalized to p53-independent β-galactosidase activity to correct for transfection efficiency. (d) The wild-type APLP1-Junc or the mutated APLP1-Junc-Mut reporter were co-transfected with increasing amounts (2.5 and 10 ng, respectively) of wild-type p53 expression plasmid into H1299 cells. (e) The APLP1-Junc reporter was co-transfected with wild-type p53 or p53-22,23, R175H p53 or R273H p53 mutant expression plasmids into H1299 cells.

Having established that p53 binds directly to the APLP1 gene in vivo via the p53RE-3, it was necessary to verify the functionality of this site. To this end, various APLP1 genomic fragments (Figure 3a) were inserted into a PGL3 basic luciferase reporter plasmid, and their ability to confer p53-dependent luciferase activity was measured. As seen in Figure 3c, each of the APLP1 reporters containing p53RE-3 (1.2Kbpro-Junc or Junc constructs) is induced by coexpression of wild-type p53. APLP1 reporters without the p53RE-3 region are not induced and interestingly are inhibited slightly by p53 coexpression. The capacity of p53RE-3 to confer p53-dependent luciferase activity is abrogated if three nucleotides are changed in its left part (GGACATGTCC to GGAtccGTCC; Figure 3a), indicating that this locus within the APLP1-Junc reporter indeed represents the p53-binding site (Figure 3d). The p53RE-3 in the APLP1-Junc reporter does not mediate transcriptional induction for p53 mutants, such as the transcriptionally inactive mutant p53-22, 23 and several p53 core mutants (Figure 3e). This corroborates further that p53RE-3 is a bona fide locus capable of mediating specifically wild-type p53 transactivation.

In all, these results indicate that APLP1 is a direct transcriptional target of p53, where p53 binding is mediated by a specific p53RE-3 in the first intron of the APLP1 gene locus.

APLP1 expression is required for fibroblastic and epithelial cell proliferation

Although APLP1 is considered a neural-specific protein, this study has demonstrated that it is induced in a p53-dependent manner in cultured fibroblastic and epithelial cell lines, during senescence or in response to genotoxic stress. To illuminate the biological role of APLP1 in these cell types, its expression was modulated ectopically and the phenotypic consequences were evaluated. Unexpectedly, ectopic expression of APLP1 in WI-38 cells has no detectable effect on cell proliferation or life span (Figure 4a). Moreover, genotoxic stress induces apoptosis in APLP1 overexpressing cells to the same degree as in control cells overexpressing green fluorescent protein (GFP, data not shown).

Figure 4
figure4

Amyloid-β precursor-like protein 1 (APLP1) is required for fibroblastic and epithelial cell proliferation. (a) Life-span curves of green fluorescent protein (GFP) and APLP1-infected WI-38 cells. Immunoblot analysis demonstrating ectopic APLP1 expression is shown in the insert panel. (b) Immunoblot analysis of protein expression in WI-38 cells infected with LacZi, APLP1 RNAi-1 or RNAi-2 producing plasmids was selected. (c) Growth curves of LacZi, APLP1–RNAi-1 and APLP1–RNAi-2 infected proliferating WI-38 cells. (d) Life-span curves of LacZi, APLP1–RNAi-1 and APLP1–RNAi-2-infected WI-38 cells. (e) Growth curves of various MCF7-infected cells. MCF7 puro and p53RNAi cells were infected with APLP1 antisense (APLP1-AS) or GFP-producing plasmids.

As no phenotypic changes were detected upon APLP1 overexpression, another approach was taken whereby APLP1 expression was reduced using RNAi method, and any consequent phenotypic changes were assessed. Two different RNAi constructs (APLP1–RNAi-1 and APLP1–RNAi-2) were created using pRetroSuper vector, each designed to knockdown APLP1 expression when infected into WI-38 cells. Western blot analysis indicates that the endogenous APLP1 protein level is reduced successfully by APLP1–RNAi-1, but is not affected by APLP1–RNAi-2. Indeed, APLP1–RNAi-2-infected cells exhibit similar levels of APLP1 protein as LacZi-infected control cells (Figure 4b). Strikingly, depletion of APLP1 expression by APLP1–RNAi-1 reduces dramatically the proliferation of proliferating WI-38 cells (Figure 4c). This inhibitory effect on growth is not attributable to induction of classical growth inhibitory genes, since there are no detectable differences in the protein levels of p21Waf1, p16 and p27 in APLP1–RNAi-1 cells (Figure 4b). Furthermore, when approaching senescence, APLP1–RNAi-1 cells perform fewer population doublings (PDLs) than control cells (Figure 4d). Similar results were obtained with MCF7 cells. Notably, depletion of APLP1 expression by antisense (APLP1-AS) causes a reduction in cell proliferation both in MCF7-puro cells and in MCF7-p53RNAi cells, where p53 itself has been depleted by RNAi (Figure 4e). This implies that the inhibitory growth effect due to APLP1 depletion is p53-independent.

In summary, the studies using APLP1 RNAi or antisense method suggest that APLP1 is required for fibroblastic and epithelial cell growth under normal culture conditions that is independent on the function of p53.

Knockdown of APLP1 expression diminishes stress-induced cell death in neuroblastoma cells

APLP1 is a neural-specific protein and its function in neural cells remains unclear (Wasco et al., 1992; Bayer et al., 1997). Based on our observations that p53-dependent regulation of APLP1 occurs in neural cells, it is interesting to delineate the neural function of this protein. To that end, RNAi methods were employed to knockdown APLP1 expression in neuroblastoma cells and consequent phenotypic changes were evaluated. APLP1–RNAi-1 expression reduces significantly APLP1 protein expression in two human neuroblastoma cell lines, SK-N-SH and CHP134. No effect was found with APLP1–RNAi-2 or with the LacZ RNAi (Figures 5b and e). Unexpectedly, depletion of APLP1 in SK-N-SH and CHP134 cells has no significant effects on cell proliferation (data not shown). This is in contrast to our findings described above, where depletion of APLP1 in both WI-38 and MCF7 cells causes a significant reduction in cell proliferation. However, when APLP1-depleted SK-N-SH cells are exposed to Dox or Etp, these cells exhibit less apoptosis (15–20%) than control cells (LacZ RNAi or APLP1–RNAi-2 cells) as assessed by WST1 survival assays, which measures the metabolic activity of viable cells (Figure 5a). An assay for apoptosis, where caspase cleavage of PARP1 protein is evaluated by immunoblot analysis corroborates that stress-induced apoptosis is reduced in APLP1-depleted SK-N-SH cells as compared to control cells (Figure 5b). Additionally, flow cytometric analysis of propidium iodide (PI)-stained cells, which reveals apoptotic cells as a result of their sub-G1 DNA content, confirms that APLP1–RNAi-1 SK-N-SH cells undergo less stress-induced apoptosis, since their sub-G1 population is significantly smaller than the sub-G1 population of control cells (Figure 5c). Depletion of APLP1 does not alter stress induced p53 accumulation or transcriptional activation of the p53 target p21waf1, suggesting that APLP1 might act downstream of p53 (Figure 5b and data not shown).

Figure 5
figure5

Knockdown of amyloid-β precursor-like protein 1 (APLP1) expression by RNAi reduces stress-induced neuroblastoma cell death. (a) WST1 cell survival assay. SK-N-SH neuroblastoma cells infected with LacZi, APLP1–RNAi-1 or APLP1–RNAi-2 producing plasmids were treated either with or without 50 ng/ml doxorubicin (Dox) or 5 μ M etoposide (Etp) for 24 h, then subjected to the WST1 assay. The readouts of treated cells are normalized to those of the nontreated cells, which were set to 100%. These data represent the average and standard deviation of independent experiments (a P-value <0.05 is indicated by *n=3–5). (b) Immunoblot analysis of protein expression in SK-N-SH cells treated with 0.1 μg/ml Dox for 24 h. β-Tubulin expression was used as a control for protein loading. (c) Quantitative analysis of the sub-G1 population by flow cytometric analyses (FACS). Various SK-N-SH cells were treated with or without Dox (0.1 μg/ml) for 24 or 36 h. Cells were fixed, propidium iodide (PI) stained and subjected to FACS. (d) CHP134 neuroblastoma cells infected with LacZi, APLP1–RNAi-1 or APLP1–RNAi-2 producing plasmids were treated either with or without Dox or Etp at the indicated concentration for 24 h, then subjected to the WST1 cell survival assay (a P-value <0.05 is indicated by #n=3). (e) Immunoblot analysis of protein expression in CHP134 cells were treated either with or without 40 ng/ml Dox or 2 μ M Etp for 24 h.

In CHP134 neuroblastoma cells, depletion of APLP1 results in a more pronounced resistance to stress-induced cell death. There is a 30% increase in Dox resistance and a 20% increase in Etp resistance in comparison to control cells according to the WST1 assay (Figure 5d). This resistance to stress-induced cell death is manifested by the partial cleavage of PARP1 protein observed in APLP1-depleted cells, in contrast to the complete cleavage observed in the two control cell lines under the same stress conditions (Figure 5e). There is a poor p21Waf1 and APLP1 induction in response to stress (Figure 5e), which may be due to the documented cytoplasmic sequestration of wild-type p53 in this cell line (Moll et al., 1996).

Overall, these data suggest that depletion of APLP1 in neuroblastoma cells reduces apoptosis following genotoxic stress, but does not influence detectably cell proliferation.

Ectopic expression of APLP1 enhances stress-induced neuroblastoma cell death

To explore further the role of APLP1 in neuroblastoma cell death upon genotoxic stress, APLP1 was overexpressed ectopically and any effects on apoptosis monitored. SK-N-SH cells were infected with a virus encoding APLP1 and a stable producer cell line selected for further analysis. After exposure to Dox, the APLP1 overexpressing cells exhibit enhanced cell death (1520%), when compared to GFP-infected control cells, according to WST1 assays (Figure 6a).

Figure 6
figure6

Ectopic expression of amyloid-β precursor-like protein 1 (APLP1) enhances stress-induced cell death of neuroblastoma cells. (a) WST1 cell survival assay. SK-N-SH cells infected with green fluorescent protein (GFP), APLP1 or zinc-acid domain deleted APLP1 (APLP1-ZA) expression plasmids were treated either with or without 50 ng/ml doxorubicin (Dox) or 5 μ M etoposide (Etp) for 24 h, and then subjected to the WST1 assay. The readouts of treated cells are normalized to those of the nontreated cells. This data represents the average and standard deviation of independent experiments (a P-value <0.01 is indicated by *n=4). (b) Immunoblot analysis of protein expression in SK-N-SH cells were treated either with or without Dox (0.1 μg/ml) for 20 h. (c) Quantitative analysis of the sub-G1 population by flow cytometric analyses (FACS). Various SK-N-SH cells were treated either with or without Dox (0.1 μg/ml) for 24 h. A P-value <0.05 is indicated by #(n=3).

Similar to the APP paralogue, APLP1 also appears to undergo proteolytic processing by various enzymes such as α-, β-, γ-secretase and caspases (Eggert et al., 2004; Li and Sudhof, 2004). The C-terminal proteolytic product of APP generated by caspase cleavage has been shown to be toxic to cells (Galvan et al., 2002). However, comparable caspase-cleaved products of APLP1 were not detected in the present study, making it unlikely that this is a feature of APLP1 activity (data not shown). It has been reported that the acidic domain of APP is essential for its ability to inhibit heme oxygenase activity and for targeting APP protein to mitochondrial compartments (Takahashi et al., 2000; Anandatheerthavarada et al., 2003). It is likely that the analogous domain is functionally significant. To test this premise, an APLP1 mutant where the zinc acidic domain had been deleted (APLP1-ZA) was introduced into cells and apoptosis monitored. It was observed that ectopic expression of APLP1-ZA does not affect stress-induced cell death (Figure 6a). This result suggests that the zinc and acidic domains are indeed required for APLP1's role in apoptosis. This finding was further corroborated using PARP1 and flow cytometric analyses (FACS) (Figures 6b and c). PARP1 is cleaved more effectively and the sub-G1 population is larger in APLP1 overexpressing cells as compared with APLP1-ZA overexpressing cells following stress treatment (Figure 6b). Specifically, the APLP1-ZA overexpressing cells behaved like the control cells.

Taken together, these results suggest that the enhancement of apoptosis induced by ectopic APLP1, is dependent on the integrity of its zinc acid domain.

Discussion

This study identifies the APLP1 gene as a novel target gene for the tumor suppressor protein p53. Modulation of APLP1 protein expression affects the neuroblastoma cell death upon stress. Importantly, APLP1 expression is associated with Alzheimer's disorders (McNamara et al., 1998; Bayer et al., 1999). Such a functional relationship between p53 and APLP1 might shed light on the pathogenesis of these disorders.

The conclusion that APLP1 is a novel direct transcriptional target of p53 is based on the following three criteria. First, exposure of primary fibroblasts, breast carcinoma or neuroblastoma cells to genotoxic stress induces an increase in APLP1 mRNA and protein that is paralleled by increases in p21WAF1 mRNA and protein. This induction of APLP1 is wild-type p53 dependent, since cells where p53 has been inactivated by various methods, such as GSE56 polypeptide, p53-specific RNAi or the oncoprotein E6, show consistently a striking reduction in the stress-induced expression of APLP1. Second, in silico analysis of the APLP1 gene locus identifies three potential p53-binding sites that conform to the consensus defined by el-Deiry et al. (1992). It is well established that p53 activates its targets by binding to one or more p53REs, commonly located in promoter or intron regions. The p53RE consensus is two copies of the 10 base-pair motif RRRCWWGYYY separated by 0–13 bp. ChIP assays indicate that the genomic region containing p53RE-3 is bound by p53 in vivo, suggesting that this indeed represents a functional p53-binding site. Finally, in vitro reporter assays demonstrate that p53RE-3 does confer specifically p53-dependent expression.

Having discovered that transcription of APLP1 is induced by wild-type p53 during senescence, it was interesting to find out if APLP1 expression is induced by p53 in other circumstances and to investigate the functional role of APLP1. APLP1 is indeed induced in a p53-dependent manner in response to genotoxic stress in fibroblastic (WI38), epithelial (MCF7) and neural (SK-N-SH and CHP134) cell types. Unexpectedly, ectopic APLP1 expression has no significant effects on cell proliferation or apoptosis in fibroblastic and epithelial cell types. However, in neural cell types, where APLP1 is expressed exclusively (Kim et al., 1995; Lorent et al., 1995), the function of APLP1 is in stress-induced apoptosis. Ectopic APLP1 expression enhances stress-induced cell death. In a complimentary approach, where RNAi was used to reduce APLP1 expression, neuroblastoma cells exhibited the reduction of stress-induced apoptosis.

Many proapoptotic genes regulated by p53 have been found to play an important role in neural apoptosis. For example, the Bcl-2 family protein Bax is a major mediator of p53-dependent neuronal apoptosis (Cregan et al., 1999). Other p53-regulated proteins, such as PUMA and Noxa that are characterized to be involved in endoplasmic reticulum stress, and Bid that is considered to influence glutamate toxicity and ischemia, may play also key roles in the specific case of neuronal apoptosis (Ward et al., 2004). Notably, in the present study, ectopic expression of APLP1 alone does not induce neuroblastoma apoptosis, but only enhances cell death upon stress stimuli, which implies that APLP1 is not a neural cell death executor, but more likely a co-effector. Interestingly, APLP1 is a transmembrane protein at the plasma membrane (Wasco et al., 1992), which relates it to several p53 targets such as KILLER/DR5, Fas and PERP (el-Deiry, 1998; Attardi et al., 2000). This raises the possibility that APLP1 may be functionally analogous to KILLER/DR5 and Fas, and serves as a membrane receptor to receive either autocrine or paracrine signals. Depletion of APLP1 causes neuroblastoma cells to acquire resistance for genotoxic stress. Thus, loss or reduction of APLP1 expression may contribute to chemotherapy resistance of neuroblastoma.

More research is required to understand the mechanism by which APLP1 enhances stress-induced cell death. It has been reported that the acidic domain of APP can interact with heme oxygenase and inhibit its enzymatic activity; thus neurotoxicity is augmented by heme or H2O2 treatments (Takahashi et al., 2000). Deletion of the zinc and acidic domain from APLP1 does diminish its potency as a cell death promoter, suggesting that APLP1 may promote cell death by certain mechanisms similarly to APP. It is interesting to note that APLP1 may also be involved in Alzheimer's disease. Accumulation of APLP1 has been found in the senile plaques and in aged brains (Bayer et al., 1997; McNamara et al., 1998). Based on our results, we suggest that induction of APLP1 expression by p53, which responds to some intrinsic stress in brain, may further enhance neuronal apoptosis and augments neurodegeneration.

In the case of fibroblastic and epithelial cells, proliferation was reduced when APLP1 expression was reduced by RNAi or antisense. The results argue that APLP1 is required for the proliferation in epithelial and fibroblastic cell types. Such a conclusion may be supported by a report that the APLP1 gene knockout mouse survives but with a slight growth deficit (Heber et al., 2000). Moreover, it has been shown that APP protein is required for the proliferation of epithelial cells and some human carcinomas (Pietrzik et al., 1998; Meng et al., 2001; Ko et al., 2004). However, these data raise the question, why is the proliferation of neural cell types unaffected when APLP1 expression is disturbed? Since neural cells rarely divide in vivo, it may be that the function of APLP1 is cell type specific.

In conclusion, APLP1 is a novel direct transcriptional target of the p53 tumor suppressor. APLP1 is a tissue-specific target; indeed it is required for cell proliferation in fibroblastic and epithelial cells, but not for neural cell proliferation. However, in neural cells, APLP1 expression influences stress-induced cell death.

Materials and methods

Cell culture

Primary human embryonic lung fibroblasts (WI-38, ATCC) were grown in minimum essential medium supplemented with 10% fetal calf serum (FCS), 1 mM Sodium Pyruvate, 2 mM L-glutamine. Cells were trypsinized and seeded at a cell density of 1 × 105 cells/plate for proliferating cells and at a cell density of 2 × 105 cells/plate for near senescent cells and were grown for 1 week. The cumulative mean PDLs were calculated using the formula: PDLs=log (cell output/cell input)/log 2. MCF7 breast carcinoma, SK-N-SH and CHP134 human neuroblastoma cells (provided by Dr Ute M Moll, Stony Brook University, New York, USA), and Phoenix cells (Clontech, Mountain View, CA, USA) were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 2 mM L-glutamine. H1299 cells (ATCC) were grown in RMPI supplemented with 10% FCS. All the cells were maintained in a humidified incubator at 37 °C and 5% CO2.

Microarray hybridization, RNA isolation and quantitative PCR

DNA microarray hybridization and processing was carried out as described previously (Milyavsky et al., 2005). Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and transcribed using the cDNA Synthesis Kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Quantitative PCR was performed using the ABI Prism 7900 sequence detection system (Perkin-Elmer, Foster City, CA, USA). The qRT–PCRs were performed in duplicate, and the results were averaged. The following primer pairs, forward and reverse respectively, were used to detect the indicated gene: APLP1 (IndexTermAAGGGTCCACAGAACAAGATG and IndexTermGCATTCACCTTTCGCTCATA), APP (IndexTermCTTCCCGTGAATGGAGAGTT and IndexTermTCAACAGGCTCAACTTCGTT), APLP2 (IndexTermCTCGCTTCCAGAAGGCTAAG and IndexTermATGGCTTGGAAGTGCTGAAT), p21WAF1 (IndexTermGGCAGACCAGCATGACAGATT and IndexTermGCGGATTAGGGCTTCCTCTT) and glyceraldehyde-3-phosphate dehydrogenase (IndexTermACCCACTCCTCCACCTTTGA and IndexTermCTGTTGCTGTAGCCAAATTCGT). Each primer is described 5′ to 3′.

Immunoblot analysis

Immunoblot analysis was performed using standard procedures. Cells were lysed in NP-40 buffer (50 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP-40) supplemented with protease Inhibitor cocktail (Roche, Mannhein, Germany) for 20 min on ice to extract total protein. Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). Polyclonal anti-p53 (produced in our laboratory), antibodies for the CDK inhibitors p21Waf1, p16 and p27 from Santa Cruz, polyclonal anti-APLP1 (Calbiochem, Beeston, Nottingham, UK), monoclonal anti-PARP (Biomol, Polymouth Meeting, PA, USA) and monoclonal anti-β-tubulin (Sigma, St Louis, MI, USA) were used for immunoblotting. Horseradish peroxidase anti-mouse and anti-rabbit (Sigma) were used as secondary antibodies. The signal was detected by the super-signal-enhanced chemiluminescence system (Pierce).

APLP1 promoter reporters and luciferase assay

To generate the APLP1 promoter reporter constructs, individual APLP1 genomic fragments obtained either by PCR from WI-38 genomic DNA or by subrestriction, were cloned into the pGL3-basic luciferase reporter vector (Promega). The APLP1-3.2pro fragment was obtained by Sac1 restriction cleavage of the PCR products generated using the following primers (forward-IndexTermCCATTCTCTTGGCCTGAAACAC and reverse-IndexTermCGCGCAGAAGCAGCAGCAATAG). The APLP1-1.2pro promoter comprises the Sph1/Sac1 restriction fragment of APLP1-3.2pro. The APLP1-Junc fragment was produced by Sac1/Xho1 restriction cleavage of PCR products generated using the following primers (forward-IndexTermGGGTCTAAAGAGGGTGAGAGTC and reverse-IndexTermGTTCCTATGCTCGAGATGGG). APLP1-Exo1 contains the Sac1/Bssh2 restriction fragment from APLP1-Junc. Wild-type p53 (pC53-SN3), p53-175R/H and p53-273R/H plasmids were provided by Dr B Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD, USA). The p53-22,23 mutant plasmid was provided by Dr A Levine (The Rockefeller University, New York, NY, USA).

Luciferase assays were performed in p53 null H1299 cells as described previously (Tang et al., 2004).

APLP1 expression and RNAi constructs and retroviral infection

The APLP1 cDNA was amplified by PCR using the following primers (forward-IndexTermGAGGGCGCAAGGGCCGGGACA and reverse-IndexTermGGGCCGGGTCAGGGTCGTTCC) and cloned into the pWZL-blasticidin retroviral vector (kindly provided by Dr W Hahn, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA). The APLP1 zinc acidic domain-deleted mutant (APLP1-ZA, 194-300 aa deletion) was created by Sph1 restriction of the pWZL-APLP1 plasmid, followed by self-ligation. The APLP1 antisense (APLP1-AS) construct was created by cloning the BamH1/EcoR1 restricted APLP1 cDNA fragment into the pWZL vector. The RNAi-producing plasmid was constructed as described previously (Tang et al., 2004). APLP1 RNAi target sequences are IndexTermGTAGGATGCGCCAGATTAA (APLP1–RNAi-1) and IndexTermCACTCATCGGAGATTCAGA (APLP1–RNAi-2), respectively.

Retroviral infection was performed as described previously (Tang et al., 2004). Infected cells were selected as follows: 1 μg/ml puromycin (for 1 week), 400 μg/ml G418 (for 2 weeks) or 5 μg/ml blasticidin (for 1 week).

ChIP

ChIP was performed as described previously (Stambolsky et al., 2006). DNA–protein complexes were immunoprecipitated using anti-p53 or anti-APLP1 antibodies, the latter serving as a control for nonspecific binding. The precipitated DNA was subjected to PCR amplification using primers specific to each of the three potential p53REs of the APLP1 gene and the p53RE in the promoter of p21Waf1. The following primer pairs, forward and reverse respectively, were used to detect APLP1 promoter sequences relative to the exon-1 starting site: +250 bp region (IndexTermCGCTGCTGCTGCCACTATTG and IndexTermGTTCCTATGCTCGAGATGGG); −1000 bp region (IndexTermCCTTCCACCTCAGCCTCCCAAGTA and IndexTermATGGCTGGGCACAGTGGCTCAT); and −3000 bp region (IndexTermCCATTCTCTTGGCCTGAAACAC and IndexTermGATGGTAGAGCCCTCAGCATA). The primers used for the detection of p21 promoter sequences, which amplify the region near the p53-binding site, were: forward-IndexTermGCACTCTT-GTCCCCCAG and reverse-IndexTermTCTATGCCAGAGCTCAACAT.

WST1 proliferation assay and FACS analysis

Cell survival in response to genotoxic stress treatments was assessed using a WST1 assay according to the manufacturer's instructions (Roche). The WST1 reagent was added and incubated with the cells for 1 h before reading the plate. Each treatment was conducted in triplicate. For FACS analysis, cells were collected and fixed in 70% ethanol in Hank's balanced salt solution (HBSS). The following day, cells were washed and resuspended in phosphate-buffered saline buffer containing 50 μg/ml PI, 50 μg/ml RNAse A and 0.1% Triton. Flow cytometry was performed using a Becton Dickinson flow cytometer. Statistical analyses were carried out by Student's t-test.

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Acknowledgements

This research was supported by a Center of Excellence grant from the Flight Attendant Medical Research Institute (FAMRI), EC FP6 grant LSHC-CT-2004-503576 and Yad Abraham Center for Cancer Diagnosis and Therapy. This publication reflects our views and not necessarily those of the European Community. The EC is not liable for any use that may be made of the information contained herein. VR is the incumbent of the Norman and Helen Asher Professorial Chair Cancer Research at the Weizmann Institute.

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Correspondence to V Rotter.

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Supplementary Information accompanies the paper on the Oncogene Web site (http://www.nature.com/onc).

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Keywords

  • p53
  • APLP1
  • transcription
  • apoptosis
  • senescence
  • neuroblastoma and Alzheimer's disease

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