Pro-apoptotic Activity Of P53 Research Articles

Acetylation of p53 in the Cerebral Cortex after Photothrombotic Stroke.

p53 expression and acetylation are crucial for the survival and death of neurons in penumbra. At the same time, the outcome of ischemia for penumbra cells depends largely on the histone acetylation status, but the effect of histone acetyltransferases and deacetylases on non-histone proteins like p53 is largely understudied. With combined in silico and in vitro approach, we have identified enzymes capable of acetylation/deacetylation, distribution, stability, and pro-apoptotic activity of p53 in ischemic penumbra in the course of post-stroke recovery, and also detected involved loci of acetylation in p53. The dynamic regulation of the acetylation of p53 at lysine 320 is controlled by acetyltransferase PCAF and histone deacetylases HDAC1 and HDAC6. The in silico simulation have made it possible to suggest the acetylation of p53 at lysine 320 acetylation may facilitate the shuttling of p53 between the nucleus and cytoplasm in penumbra neurons. Acetylation of p53 at lysine 320 is more preferable than acetylation at lysine 373 and probably promotes survival and repair of penumbra neurons after stroke. Strategies to increase p53 acetylation at lysine 320 via increasing PCAF activity, inhibiting HDAC1 or HDAC6, inhibiting p53, or a combination of these interventions may have therapeutic benefits for stroke recovery and would be promising for neuroprotective therapy of stroke.

Silver nanoparticles functionalized Paclitaxel nanocrystals enhance overall anti-cancer effect on human cancer cells

For chemotherapeutic drugs, precise tumor-targeting and high anti-cancer efficiency is equally important in order to enhance chemotherapy and reverse drug resistance. The combination of multifunctional agents to achieve synergy should be a promising strategy. In our study, we have successfully developed novel multifunctionalized drug nanocrystals to realize co-delivery of the organic drug Paclitaxel (PTX), inorganic silver nanoparticles (AgNPs) and a tumor targeting agent. To be specific, PTX nanocrystals were first prepared as a template, then coated with polydopamine (PDA). The PDA layer was utilized as the connection bridge to produce and deposit AgNPs in situ, and provide sites for tumor-targeting peptide NR1 (RGDARF) grafting. As a result, these NR1/AgNP-decorated drug nanocrystals exhibited dramatically improved cellular uptake efficiency, in vitro anti-cancer activity and an anti-migratory effect against a variety of cancer cells, which was attributable to the synergistic, or at least additive, effect of the AgNPs and PTX, enhanced cellular uptake efficiency through NR1-receptor interaction, pH-responsive drug release and the nanoscaled nature. In particular, high anti-cancer activity and low side effects from these NR1/AgNP-decorated PTX nanocrystals were well balanced in terms of good selectivity and biocompatibility. Moreover, these novel drug nanocrystals displayed strong apoptotic-inducing potency, resulting in cell membrane lysis, nuclear damage, mitochondria dysfunction, excessive ROS release and double-stranded DNA breakage. The potential acting mechanism and molecular basis of these novel drug nanocrystals is relevant to the regulation of mitochondria-mediated apoptosis with a greater Bax-to-Bcl-2 ratio and the activation of pro-apoptotic P53 and caspase 3.

Abstract 3426: A synthetic lethality approach to eradicate AML via synergistic activation of pro-apoptotic p53 by MDM2 and BET inhibitors

Abstract Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation and minimize toxicity via MDM2i, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. By integrating the results of these two screens, we found inhibition of BRD4 activates p53 and its target genes. BRD4 (Bromodomain-containing protein 4) is a member of the bromodomain and extraterminal (BET) family proteins, which has typically been reported to activate genes, such as c-MYC, BCL2 and CDK4/6. However, we unexpectedly reveal that BRD4 acts as a transcriptional repressor of p53 target genes. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type p53 in vitro, against two mouse models of AML in vivo, and against several primary human patient blasts in vitro. Synergistic cell killing was associated with synergistic activation of p53 target genes, and cell killing and p53 target gene expression. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, targetable wild-type p53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML. Citation Format: Sha Li, Anne Latif, Ashley Newcombe, Kathryn Gilroy, Neil Robertson, Xue Lei, Darren Finlay, Helen Stewart, Karina Barbosa, Brian Higgins, Tim Chevassut, Xu Huang, Mhairi Copland, Karen Keeshan, Ani Deshpande, Peter Adams. A synthetic lethality approach to eradicate AML via synergistic activation of pro-apoptotic p53 by MDM2 and BET inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3426.

BReast CAncer susceptibility gene 2 deficiency exacerbates oxidized LDL-induced DNA damage and endothelial apoptosis.

Mutations in the tumor suppressor gene BRCA2 (BReast CAncer susceptibility gene 2) predispose carriers to breast, ovarian, and other cancers. In response to DNA damage, BRCA2 participates in homology‐directed DNA damage repair to maintain genome stability. Genome‐wide association studies have identified an association between BRCA2 single nucleotide polymorphisms and plasma‐lipid levels and lipid deregulation in humans. To date, DNA damage, apoptosis, and lipid deregulation are recognized as central pathways for endothelial dysfunction and atherosclerosis; however, the role of BRCA2 in endothelial dysfunction remains to be elucidated. To determine the role of BRCA2 in endothelial dysfunction, BRCA2 was silenced in human umbilical vein endothelial cells (ECs) and assessed for markers of DNA damage, apoptosis, and endothelial function following oxidized low‐density lipoprotein (oxLDL) treatment. OxLDL was found to induce significant reactive oxygen species (ROS) production in BRCA2‐silenced ECs. This increase in ROS production was associated with exacerbated DNA damage evidenced by increased expression and activation of DNA double‐stranded break (DSB) marker γH2AX and reduced RAD51‐foci formation—an essential regulator of DSB repair. Increased DSBs were associated with enhanced expression and activation of pro‐apoptotic p53 and significant apoptosis in oxLDL‐treated BRCA2‐silenced ECs. Loss of BRCA2 in ECs was further associated with oxLDL‐induced impaired tube‐forming potential and eNOS expression. Collectively, the data reveals, for the first time, a novel role of BRCA2 as a regulator of EC survival and function in the setting of oxLDL treatment in vitro. Additionally, the data provide important clues regarding the potential susceptibility of BRCA2 mutation carriers to endothelial dysfunction, atherosclerosis, and other cardiovascular diseases.

Silencing of RUNX2 enhances gemcitabine sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the stimulation of TAp63-mediated cell death.

It has been well-known that human pancreatic cancer represents the fourth and fifth leading causes of cancer-related deaths in the United States and Japan, respectively.1, 2 Notably, pancreatic cancer is characterized by high metastatic potential, resistance to chemotherapy and thus its prognosis is extremely poor with 5-year survival <5%. At diagnosis, more than 80% cases are already advanced and non-resectable.3 Therefore, chemotherapy and/or radiotherapy is the only option. Despite improvements in the treatments, the survival rate has not been significantly ameliorated over the last few decades. For chemotherapy, a deoxycytidine analog termed gemcitabine (GEM) is the first line of standard treatment given to most of the patients bearing advanced pancreatic cancer.4 Unfortunately, GEM treatment provides limited clinical benefits, especially in advanced and metastatic disease.5 Hence, the extensive efforts to clarify the precise molecular mechanisms behind GEM-resistant phenotype of malignant pancreatic cancer and also to develop the promising strategies to enhance the efficacy of GEM should be required.

DNA damage strength modulates a bimodal switch of p53 dynamics for cell-fate control

BackgroundThe p53 pathway is differentially activated in response to distinct DNA damage, leading to alternative phenotypic outcomes in mammalian cells. Recent evidence suggests that p53 expression dynamics play an important role in the differential regulation of cell fate, but questions remain as to how p53 dynamics and the subsequent cellular response are modulated by variable DNA damage.ResultsWe identified a novel, bimodal switch of p53 dynamics modulated by DNA-damage strength that is crucial for cell-fate control. After low DNA damage, p53 underwent periodic pulsing and cells entered cell-cycle arrest. After high DNA damage, p53 underwent a strong monotonic increase and cells activated apoptosis. We found that the damage dose-dependent bimodal switch was due to differential Mdm2 upregulation, which controlled the alternative cell fates mainly by modulating the induction level and pro-apoptotic activities of p53.ConclusionsOur findings not only uncover a new mode of regulation for p53 dynamics and cell fate, but also suggest that p53 oscillation may function as a suppressor, maintaining a low level of p53 induction and pro-apoptotic activities so as to render cell-cycle arrest that allows damage repair.

Abstract 1721: Testicular germ cell tumors are hypersensitive to p53 activation based on their Oct-4/Noxa-mediated cellular context rather than on differential p53 activity.

Abstract TGCTs are highly apoptosis-prone cancers which can be cured by Cisplatin-based chemotherapy. We have previously shown that p53 plays a major role in this phenotype. Furthermore, we demonstrated that sensitivity of these tumors is dictated by p53 accumulation in general and is not restricted to Cisplatin. Hypersensitivity seems to be an intrinsic feature of pluripotent TGCTs, since both differentiation and loss of the pluripotency factor Oct-4 induce resistance. We sought to investigate the link between the Oct-4-mediated pluripotent context and the proapoptotic p53 response in these tumors. For this, we tested if loss of Oct-4 also reduces p53-mediated apoptosis upon non-genotoxic p53 activation. Indeed, RNAi-mediated silencing of Oct-4 significantly reduced sensitivity to the p53 activator Nutlin-3. Hence, we investigated a possible differential activation of p53 in the context of Oct-4-positive TGCT cells. For this, a qPCR system that allows simultaneous acquisition of 46 bona fide p53 target genes was used to detect p53’s transactivation capacity in NTERA-2D1 and 2102EP cells upon Cisplatin treatment in the presence and absence of Oct-4. In spite of p53’s central role in Cisplatin-induced apoptosis, no significant alterations in transactivation of p53 target genes were observed upon Cisplatin treatment in both cell lines. We also analyzed the cellular distribution of p53 upon Cisplatin treatment and found that p53 was not only accumulated in the nucleus but was increased to a similar extent in the cytoplasm. Importantly, RNAi-mediated silencing of Oct-4 did neither influence accumulation of p53 in the nucleus nor in the cytoplasm. These data indicate that Oct-4 does not directly modulate p53 activity but provides a cellular context that augments the proapoptotic activity of p53. We have previously proposed that Oct-4-dependent high constitutive Noxa protein levels account for the low apoptotic threshold in TGCTs. In contrast to Cisplatin or Nutlin-3, treatment with MG132 resulted in apoptosis in Oct-4-depleted NTERA-2D1 and 2102EP cells. This was due to simultaneous accumulation of p53 and Noxa, as RNAi-mediated silencing of either protein rescued the cells from death. It is of importance that p53 knockdown did not compromise Noxa accumulation upon MG132 treatment, indicating that both p53 activation and the Noxa-mediated apoptosis-prone context are required for high sensitivity. In conclusion, our data demonstrate that in spite of its impact on hypersensitivity, p53 activity is not altered in Oct-4-positive TGCT cells when compared to relatively resistant Oct-4-depleted cells. Although a robust p53 response is essential for hypersensitivity of TGCTs, its predominantly proapoptotic characteristic requires the apoptosis-prone cellular context of pluripotent Oct-4-positive TGCT cells, which includes high Noxa protein levels. Citation Format: Matthias Gutekunst, Thomas Mueller, Andrea Weilbacher, Michael A. Dengler, Moshe Oren, Walter E. Aulitzky, Heiko van der Kuip. Testicular germ cell tumors are hypersensitive to p53 activation based on their Oct-4/Noxa-mediated cellular context rather than on differential p53 activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1721. doi:10.1158/1538-7445.AM2013-1721

Runt-related transcription factor 2 (RUNX2) inhibits p53-dependent apoptosis through the collaboration with HDAC6 in response to DNA damage

Runt-related transcription factor 2 (RUNX2) is the best known as an essential protein for osteoblast differentiation. In this study, we have found for the first time that RUNX2 acts as a negative regulator for p53 in response to DNA damage. On DNA damage mediated by adriamycin (ADR) exposure, p53 as well as RUNX2 was induced at protein and mRNA level in human osteosarcoma-derived U2OS cells in association with a significant upregulation of various p53-target genes. Indirect immunostaining and co-immunoprecipitation experiments demonstrated that RUNX2 colocalizes with p53 in cell nucleus and forms a complex with p53 following ADR treatment. Chromatin immunoprecipitation assays revealed that RUNX2/p53 complex is efficiently recruited onto p53-target promoters in response to ADR, suggesting that RUNX2 might be involved in the regulation of transcriptional activation mediated by p53. Indeed, forced expression of RUNX2 resulted in a remarkable downregulation of p53-target genes. Consistent with these observations, knockdown of RUNX2 enhanced ADR-mediated apoptosis and also elevated p53-target gene expression in response to ADR. On the other hand, depletion of RUNX2 in p53-deficient human lung carcinoma-derived H1299 cells had an undetectable effect on p53-target gene expression regardless of ADR treatment, indicating that RUNX2-mediated downregulation of p53-target genes is dependent on p53. Furthermore, RUNX2/p53 complex included histone deacetylase 6 (HDAC6) and HDAC6 was also recruited onto p53-target promoters following ADR exposure. Of note, HDAC6-specific chemical inhibitor tubacin treatment enhanced ADR-mediated upregulation of p53-target gene expression, indicating that deacetylase activity of HDAC6 is required for RUNX2-mediated downregulation of p53-target gene. Taken together, our present findings strongly suggest that RUNX2 inhibits DNA damage-induced transcriptional as well as pro-apoptotic activity of p53 through the functional collaboration with HDAC6 and therefore might be an attractive therapeutic target for cancer treatment.

ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 ActivationIn Vivo

The transcription factor p53 mediates neuronal death in a variety of stress-related and neurodegenerative conditions. The proapoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1 and ASPP2. However, whether ASPP1/2 play a role in the regulation of p53-dependent neuronal death in the CNS is currently unknown. To address this, we asked whether ASPP1/2 contribute to the death of retinal ganglion cells (RGCs) using in vivo models of acute optic nerve damage in mice and rats. Here, we show that p53 is activated in RGCs soon after injury and that axotomy-induced RGC death is attenuated in p53 heterozygote and null mice. We demonstrate that ASPP1/2 proteins are abundantly expressed by injured RGCs, and that short interfering (si)RNA-based ASPP1 or ASPP2 knockdown promotes robust RGC survival. Comparative gene expression analysis revealed that siASPP-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA), Fas/CD95, and Noxa depends on p53 transcriptional activity. Furthermore, siRNA against PUMA or Fas/CD95 confers neuroprotection, demonstrating a functional role for these p53 targets in RGC death. Our study demonstrates a novel role for ASPP1 and ASPP2 in the death of RGCs and provides evidence that blockade of the ASPP-p53 pathway is beneficial for central neuron survival after axonal injury.

RUNX3 Modulates DNA Damage-mediated Phosphorylation of Tumor Suppressor p53 at Ser-15 and Acts as a Co-activator for p53

Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.

P53 codon 72 polymorphism in patients affected with ulcerative colitis

The p53 tumor suppressor protein plays a fundamental role in maintaining genomic integrity through its ability to arrest the cell cycle in G1 and induce apoptosis. The proapoptotic activity of p53 seems to be strictly related to proline-rich regions, homologous to the SH3 binding domain. In the literature, reported data suggest a role for polymorphism at codon 72 of p53 in the predisposition to neoplastic transformation, although the results are still controversial. In this study, we investigated Arg72Pro polymorphism of p53 and related this polymorphism to clinical parameters in patients affected with ulcerative colitis (UC). We studied 243 consecutive outpatients affected with well-established UC. The control group comprised 142 healthy blood donors, with age and sex comparable to those of the patients. p53 Pro/Pro was significantly related to the clinical course and duration of disease (odds ratio, 55.8 and 8.8, respectively). Nineteen of 24 patients with Pro homozygosity had a duration of disease >7 years. In contrast, 87 of 123 patients with Arg/Arg had short-standing UC (< or =7 yrs) and 66 of 96 with Arg/Pro had short-standing UC (chi-squared, 22.86; P < 0.0001). Thirty-four of 243 patients affected with UC had a positive family history for colorectal carcinoma (CRC). In those patients p53, Pro/Pro was significantly related to a family history of CRC (odds ratio, 38.1). These preliminary data suggest that polymorphism at codon 72 of the p53 gene influences the clinical course of UC, with continuous disease associated with p53 Pro homozygosity.

Fibroblast Growth Factor 1 inhibits p53-dependent apoptosis in PC12 cells

The survival activity of FGF1 and the pro-apoptotic activity of p53 were characterized in vitro and/or in vivo for different types of neurons after different stresses and in different neurodegenerative pathologies. To investigate whether or not FGF1 and p53 pathways interact in neuronal cells, we studied the effect of FGF1 on p53-dependent apoptosis in PC12 cells. We first characterized p53-dependent PC12 cell death induced by etoposide (a DNA damaging agent). We showed that etoposide increased p53 stabilization, phosphorylation (Ser-15), nuclear translocation and transcriptional activity. In particular, p53 promoted mdm2, p21, puma and noxa expression in PC12 cells. The activation of p53 initiated a classical mitochondrial apoptosis process associated with caspases activation and nuclear degradation. We demonstrated that FGF1 protected PC12 cells from p53-dependent apoptosis upstream from mitochondrial and nuclear events. FGF1 inhibited etoposide-induced p53 phosphorylation, stabilization, nuclear translocation and transcriptional activity. This study presents the first evidence that FGF1 and p53 pathways interact in neuronal cells, and that FGF1 protects neuronal cells from p53-dependent apoptosis, suggesting that alterations of FGF1/p53 crosstalk could be involved in a large range of neurons and in neurological disorders.

The tumor suppressor protein p53 is required for neurite outgrowth and axon regeneration

Axon regeneration is substantially regulated by gene expression and cytoskeleton remodeling. Here we show that the tumor suppressor protein p53 is required for neurite outgrowth in cultured cells including primary neurons as well as for axonal regeneration in mice. These effects are mediated by two newly identified p53 transcriptional targets, the actin-binding protein Coronin 1b and the GTPase Rab13, both of which associate with the cytoskeleton and regulate neurite outgrowth. We also demonstrate that acetylation of lysine 320 (K320) of p53 is specifically involved in the promotion of neurite outgrowth and in the regulation of the expression of Coronin 1b and Rab13. Thus, in addition to its recognized role in neuronal apoptosis, surprisingly, p53 is required for neurite outgrowth and axonal regeneration, likely through a different post-translational pathway. These observations may suggest a novel therapeutic target for promoting regenerative responses following peripheral or central nervous system injuries.

P53's Posttranslational Code

Phosphorylation and acetylation are posttranslational modifications that influence protein function. Not only are the structure and accessibility of chromatin influenced by these modifications but so are the transcription factors that bind DNA and regulate gene expression. The activity of p53, which is a transcriptional regulator, is controlled through posttranslational modifications that influence the stability of the protein, the localization of the protein, and its interactions with corepressors and coactivators. Knights et al. show that lysine 373 (K373) and lysine 320 (K320) appear to promote different p53 activities--K373 stimulates the proapoptotic activities of p53, and K320 promotes cell survival activities. The authors created a series of p53 mutants using glutamine (Q) to mimic acetylation at specific sites: p53Q373, p53Q320, and a mutant in which four lysines were replaced at Q320, 370, 372, and 373 (p53DM). Expression of p53Q320 or p53DM in cells that lacked endogenous p53 promoted G1 arrest and protected cells from apoptosis in response to DNA-damaging drugs (adozelesin or bizelesin) or inhibition of topoisomerase (etopside). In contrast, p53Q373 promoted transient arrest in G2/M and ultimately increased cell death in response to adozelesin, bizelesin, or etopside treatment. Coimmunoprecipitation experiments with the mutant p53 proteins or with acetylated p53 showed that acetylation of K373 or K320 promoted the interaction of p53 with different chromatin-remodeling enzymes. The data suggested that when acetylated at position K373, p53 interacted more strongly with corepressors and histone deacetylases. Microarray experiments comparing the gene-expression profiles of cells expressing wild-type p53, p53Q373, or p53Q320 showed that p53Q373 was a stronger repressor. Furthermore, the genes that were activated in the p53Q373 cells were proapoptotic, and genes that were repressed were associated with cell survival, which is consistent with the proapoptotic effect of p53Q373. The p53Q320 profile tended to be opposite that of the p53Q373 profile, with p53Q320 showing repression of genes associated with apoptosis and stimulation of genes associated with cell survival. Differences in the affinity of p53 acetylated at different sites (or the acetylation mimics) was observed at specific p53 target gene promoters in chromatin immunoprecipitation and electrophoretic mobility shift assays. p53Q320 exhibited a cytosolic localization, whereas p53Q373 was nuclear. Futhermore, the phosphorylation of sites in the N terminus of p53 was decreased in p53Q320 compared with wild-type p53, whereas p53Q373 exhibited more intense phosphorylation than did wild-type p53. Thus, the acetylation status of p53 appears to differentially control phosphorylation of p53, nuclear localization of p53, and the transcriptional activity of p53. Stimuli that promote deacetylation of K373, such as was observed in cells treated with the DNA monoalkylating agent adozelesin, can trigger a cell survival response, whereas those that promote K373 acetylation, such as bizelesin or etopside, trigger a cell death response. C. D. Knights, J. Catania, S. Di Giovanni, S. Muratoglu, R. Perez, A. Swartzbeck, A. A. Quong, X. Zhang, T. Beerman, R. G. Pestell, M. L. Avantaggiati, Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate. J. Cell Biol. 173 , 533-544 (2006). [Abstract] [Full Text]

Smaller Liver Tumors Without c-Jun

The transcription factor c-Jun controls both cell proliferation and cell survival, and not surprisingly, its activity has been linked to various human tumors. However, although it appears to be proapoptotic in some tumor types, it displays antiapoptotic activity in others, as seen in liver cancer. Eferl et al. found that in a mouse model of liver carcinoma, deletion of c-Jun did not affect proliferation of liver tumor cells, but caused an increase in the number of apoptotic cells and a consequent decrease in liver tumor mass. By inducing deletion of c-Jun in mice at different times after liver tumor formation, the authors determined its requirement for tumor cell survival only in early stages of cancer development. Hepatocytes lacking c-Jun had increased expression of the tumor suppressor protein p53 and were also more sensitive to cell death induced by exposure to tumor necrosis factor (TNF). Hepatocytes lacking both c-Jun and p53 regained resistance to TNF-induced apoptosis, which suggests that c-Jun antagonizes the proapoptotic and antiproliferative activities of p53. Human liver tumors express high levels of c-Jun, and use of inhibitors in the early stages of this particular cancer may be therapeutically effective. R. Eferl, R. Ricci, L. Kenner, R. Zenz, J.-P. David, M. Rath, E. F. Wagner, Liver tumor development: c-Jun antagonizes the proapoptotic activity of p53. Cell 112 , 181-192 (2003). [Online Journal]

A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1.

p53 exerts its tumor suppressor effects by activating genes involved in cell growth arrest and programmed cell death. The p53 target genes inducing growth arrest are well defined whereas those inducing apoptosis are not fully characterized. Proapoptotic activity of p53 was shown to involve several genes like Bax, Noxa and Puma, which may function in the release of cytochrome c from the mitochondria. Cytochrome c associates with Apaf-1 and caspase 9 to form the apoptosome. Genetic and cellular data indicate that Apaf-1 deficiency abrogates the apoptotic effect of p53 and substitutes for p53 loss in promoting tumor formation. Here we show that Apaf-1, the mammalian homologue of C. elegans CED4, is a direct target of p53 as demonstrated by gel shift analysis of the target site sequence in the presence of p53 and by Apaf-1 promoter-luciferase assays. We also show that the p53 activation of the Apaf-1 luciferase construct can be enhanced by the putative tumor suppressor gene product, Zac-1, a transcription factor that has previously been shown to inhibit cell proliferation. Furthermore, we demonstrate that Zac-1 is a possible direct target of p53 since the sequence upstream to the first coding exon of Zac-1 contains a p53 recognition site and the luciferase construct containing this region is activated by p53. These results suggests the existence of a tightly controlled self amplifying mechanism of transcriptional activation leading to apoptosis by p53.

Mechanisms of p53-dependent apoptosis.

Cellular stresses, such as growth factor deprivation, DNA damage or oncogene expression, lead to stabilization and activation of the p53 tumour suppressor protein. Depending on the cellular context, this results in one of two different outcomes: cell cycle arrest or apoptotic cell death. Cell death induced through the p53 pathway is executed by the caspase proteinases, which, by cleaving their substrates, lead to the characteristic apoptotic phenotype. Caspase activation by p53 occurs through the release of apoptogenic factors from the mitochondria, including cytochrome c and Smac/DIABLO. Released cytochrome c allows the formation of a high-molecular weight complex, the apoptosome, which consists of the adapter protein Apaf-1 and caspase 9, which is activated following recruitment into the apoptosome. Active caspase 9 then cleaves and activates the effector caspases, such as caspases-3 and -7, which execute the death program. Released Smac/DIABLO facilitates caspase activation through repression of the IAP caspase inhibitor proteins. The release of mitochondrial apoptogenic factors is regulated by the pro- and anti-apoptotic Bcl-2 family proteins, which either induce or prevent the permeabilization of the outer mitochondrial membrane. The mechanism by which p53 signals to the Bcl-2 family proteins is unclear. It was shown that some of the pro-apoptotic family members, such as Bax, Noxa or PUMA, are transcriptional targets of p53. In addition, transcription-independent, pro-apoptotic activities of p53 have been described. The elucidation of the p53-dependent pathway, resulting in mitochondrial outer membrane permeabilization through the pro-apoptotic Bcl-2 family proteins, is a key to unveiling the mechanism of stress-induced apoptosis.

A minimal Bcl-x promoter is activated by Brn-3a and repressed by p53

The Brn-3a transcription factor stimulates the expression of the anti-apoptotic Bcl-2 and Bcl-x proteins and protects neuronal cells from apoptosis. Here we show that a minimal Bcl-x promoter is activated by Brn-3a and that this stimulation is prevented by the pro-apoptotic p53 protein. Both these effects are mediated via Bcl-x promoter sequences, which are indistinguishable from those required for minimal basal promoter activity. A newly described upstream Bcl-x promoter is also activated by Brn-3a with this activation being prevented by p53. Hence, Brn-3a-mediated activation of two distinct Bcl-x promoters and of the Bcl-2 promoter is blocked by p53 whereas this is not observed for Brn-3a activated promoters derived from genes not involved in inhibiting apoptosis. p53 therefore appears to specifically target the activation by Brn-3a of promoters derived from genes with an anti-apoptotic effect and this may be involved in the pro-apoptotic activity of p53.

Mechanisms of p53-dependent apoptosis

Cellular stresses, such as growth factor deprivation, DNA damage or oncogene expression, lead to stabilization and activation of the p53 tumour suppressor protein. Depending on the cellular context, this results in one of two different outcomes: cell cycle arrest or apoptotic cell death. Cell death induced through the p53 pathway is executed by the caspase proteinases, which, by cleaving their substrates, lead to the characteristic apoptotic phenotype. Caspase activation by p53 occurs through the release of apoptogenic factors from the mitochondria, including cytochrome c and Smac/DIABLO. Released cytochrome c allows the formation of a high-molecular weight complex, the apoptosome, which consists of the adapter protein Apaf-1 and caspase 9, which is activated following recruitment into the apoptosome. Active caspase 9 then cleaves and activates the effector caspases, such as caspases-3 and -7, which execute the death program. Released Smac/DIABLO facilitates caspase activation through repression of the IAP caspase inhibitor proteins. The release of mitochondrial apoptogenic factors is regulated by the pro- and anti-apoptotic Bcl-2 family proteins, which either induce or prevent the permeabilization of the outer mitochondrial membrane. The mechanism by which p53 signals to the Bcl-2 family proteins is unclear. It was shown that some of the pro-apoptotic family members, such as Bax, Noxa or PUMA, are transcriptional targets of p53. In addition, transcription-independent, pro-apoptotic activities of p53 have been described. The elucidation of the p53-dependent pathway, resulting in mitochondrial outer membrane permeabilization through the pro-apoptotic Bcl-2 family proteins, is a key to unveiling the mechanism of stress-induced apoptosis.

P53 facilitates pRb cleavage in IL-3-deprived cells: novel pro-apoptotic activity of p53.

In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis. Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53. Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb. Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis.