Susan Erster

In Vivo Mitochondrial p53 Translocation Triggers a Rapid First Wave of Cell Death in Response to DNA Damage That Can Precede p53 Target Gene Activation

ABSTRACT p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. We recently showed that wild-type p53 rapidly translocates to mitochondria in response to multiple death stimuli in cultured cells. Mitochondrial p53 physically interacts with antiapoptotic Bcl proteins, induces Bak oligomerization, permeabilizes mitochondrial membranes, and rapidly induces cytochrome c release. Here we characterize the mitochondrial p53 response in vivo. Mice were subjected to γ irradiation or intravenous etoposide administration, followed by cell fractionation and immunofluorescence studies of various organs. Mitochondrial p53 accumulation occurred in radiosensitive organs like thymus, spleen, testis, and brain but not in liver and kidney. Of note, mitochondrial p53 translocation was rapid (detectable at 30 min in thymus and spleen) and triggered an early wave of marked caspase 3 activation and apoptosis. This caspase 3-mediated apoptosis was entirely p53 dependent, as shown by p53 null mice, and preceded p53 target gene activation. The transcriptional p53 program had a longer lag phase than the rapid mitochondrial p53 program. In thymus, the earliest apoptotic target gene products PUMA, Noxa, and Bax appeared at 2, 4, and 8 h, respectively, while Bid, Killer/DR5, and p53DinP1 remained uninduced even after 20 h. Target gene induction then led to further increase in active caspase 3. Similar biphasic kinetics was seen in cultured human cells. Our results suggest that in sensitive organs mitochondrial p53 accumulation in vivo occurs soon after a death stimulus, triggering a rapid first wave of apoptosis that is transcription independent and may precede a second slower wave that is transcription dependent.

ΔNp73, A Dominant-Negative Inhibitor of Wild-type p53 and TAp73, Is Up-regulated in Human Tumors

p73 has significant homology to p53. However, tumor-associated up-regulation of p73 and genetic data from human tumors and p73-deficient mice exclude a classical Knudson-type tumor suppressor role. We report that the human TP73 gene generates an NH2 terminally truncated isoform. ΔNp73 derives from an alternative promoter in intron 3 and lacks the transactivation domain of full-length TAp73. ΔNp73 is frequently overexpressed in a variety of human cancers, but not in normal tissues. ΔNp73 acts as a potent transdominant inhibitor of wild-type p53 and transactivation-competent TAp73. ΔNp73 efficiently counteracts transactivation function, apoptosis, and growth suppression mediated by wild-type p53 and TAp73, and confers drug resistance to wild-type p53 harboring tumor cells. Conversely, down-regulation of endogenous ΔNp73 levels by antisense methods alleviates its suppressive action and enhances p53- and TAp73-mediated apoptosis. ΔNp73 is complexed with wild-type p53, as demonstrated by coimmunoprecipitation from cultured cells and primary tumors. Thus, ΔNp73 mediates a novel inactivation mechanism of p53 and TAp73 via a dominant-negative family network. Deregulated expression of ΔNp73 can bestow oncogenic activity upon the TP73 gene by functionally inactivating the suppressor action of p53 and TAp73. This trait might be selected for in human cancers.

Monoubiquitylation promotes mitochondrial p53 translocation

A major function of the p53 tumor suppressor is the induction of a pleiotropic apoptotic program in response to stress through transcription‐dependent and ‐independent mechanisms. In particular, this includes a direct apoptotic role of p53 at the mitochondria. Stress‐induced p53 translocation to the mitochondria with subsequent outer membrane permeabilization is a common early component in p53‐mediated apoptosis in normal and transformed cells. However, the mechanism of p53 delivery to the mitochondria remains unknown. Here, we show that the cytoplasm contains a separate and distinct p53 pool that is the major source for p53 translocation to the mitochondria upon its stress‐induced stabilization. Using various manipulations that enhance or diminish p53 ubiquitylation, our data provide evidence that Mdm2‐mediated monoubiquitylation of p53 greatly promotes its mitochondrial translocation and thus its direct mitochondrial apoptosis. On the other hand, p53 does not require Mdm2 as a shuttler. Upon arrival at the mitochondria, our data suggest that p53 undergoes rapid deubiquitylation by mitochondrial HAUSP via a stress‐induced mitochondrial p53–HAUSP complex. This generates the apoptotically active non‐ubiquitylated p53. Taken together, we propose a novel model for mitochondrial p53 targeting, whereby a distinct cytoplasmic pool of stabilized monoubiquitylated p53, generated in resting cells by basal levels of Mdm2‐type ligases, is subject to a binary switch from a fate of inactivation via subsequent polyubiquitylation and degradation in unstressed cells, to a fate of activation via mitochondrial trafficking.

WT p53, but Not Tumor-derived Mutants, Bind to Bcl2 via the DNA Binding Domain and Induce Mitochondrial Permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 ± 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.

Transdominant ΔTAp73 Isoforms Are Frequently Up-regulated in Ovarian Cancer. Evidence for Their Role as Epigenetic p53 Inhibitors in Vivo

Despite strong homology, the roles of TP53 and TP73 in tumorigenesis seem to be fundamentally different. In contrast to TP53, tumor-associated overexpression of TP73 in many different cancers, combined with virtual absence of inactivating mutations and lack of a cancer phenotype in the TP73 null mouse are inconsistent with a suppressor function but instead support an oncogenic function. The discovery of NH2-terminally truncated p73 isoforms, collectively called ΔTAp73, is now the focus of intense interest because they act as potent transdominant inihibitors of wild-type p53 and transactivation-competent TAp73. Therefore, establishing deregulated ΔTAp73 expression in tumors could be the crucial link to decipher which of the two opposing roles of this bipolar gene is the biologically relevant one. This study is the largest to date and encompasses 100 ovarian carcinomas with complete expression profile of all NH2-terminal isoforms, discriminating between TAp73 and ΔTAp73 (ΔNp73, ΔN′p73, Ex2p73, and Ex2/3p73) by isoform-specific real-time reverse transcription-PCR. We find that the set of NH2-terminal p73 isoforms distinguishes ovarian cancer patients from healthy controls and thus is a molecular marker for this diagnosis. Ovarian cancers strongly and almost universally overexpress ΔN′p73 compared with normal tissues (95% of cancers). About one-third of tumors also exhibit concomitant up-regulation of the antagonistic TAp73, whereas only a small subgroup of tumors overexpress ΔNp73. Thus, deregulation of the E2F1-responsive P1 promoter, rather than the alternate P2 promoter, is mainly responsible for the production of transdominant p53/TAp73 antagonists in ovarian cancer. Tumor stage, grade, presence of metastases, p53 status, and residual disease after resection are significant prognostic markers for overall and recurrence-free survival. A trend is found for better overall survival in patients with low expression of ΔN′p73/ΔNp73, compared with patients with high expression. A strong correlation between deregulated ΔTAp73 and p53 status exists. p53 wild-type cancers exhibit significantly higher deregulation of ΔN′p73, ΔNp73, and Ex2/3p73 than p53 mutant cancers. This data strongly supports the hypothesis that overexpression of transdominant p73 isoforms can function as epigenetic inhibitors of p53 in vivo, thereby alleviating selection pressure for p53 mutations in tumors.

p53's mitochondrial translocation and MOMP action is independent of Puma and Bax and severely disrupts mitochondrial membrane integrity.

p53s apoptotic program consists of transcription-dependent and transcription-independent pathways. In the latter, physical interactions between mitochondrial p53 and anti- and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators are central. Using isogenic cell systems with defined deficiencies, we characterize in detail how mitochondrial p53 contributes to mitochondrial permeabilization, to what extent its action depends on other key Bcl2 family members and define its release activity. We show that mitochondrial p53 is highly efficient in inducing the release of soluble and insoluble apoptogenic factors by severely disrupting outer and inner mitochondrial membrane integrity. This action is associated with wild-type p53-induced oligomerization of Bax, Bak and VDAC and the formation of a stress-induced endogenous complex between p53 and cyclophilin D, normally located at the inner membrane. Tumor-derived p53 mutants are deficient in activating the Bax/Bak lipid pore. These actions are independent of Puma and Bax. Importantly, the latter distinguishes the mitochondrial from the cytosolic p53 death pathway.

The post-translational phosphorylation and acetylation modification profile is not the determining factor in targeting endogenous stress-induced p53 to mitochondria

The post-translational phosphorylation and acetylation modification profile is not the determining factor in targeting endogenous stress-induced p53 to mitochondria

Stress-Induced p53 Runs a Direct Mitochondrial Death Program: Its Role in Physiologic and Pathophysiologic Stress Responses In Vivo

It is now well established that a fraction of stress-induced wtp53 protein rapidly translocates to mitochondria in immortalized and transformed cells in culture. Mitochondrial p53 interacts with anti-apoptotic proteins of the Bcl 2 family at the outer mitochondrial membrane, resulting in membrane permeabilization, release of death effectors such as cytochrome C and subsequent rapid apoptosis. The significance and relevance of this direct mitochondrial p53 program to the overall p53-mediated stress response in vivo is underlined by a number of recent studies in animals and primary cells. They all support a role for this direct pathway in the physiologic and pathophysiologic response to genotoxic and hypoxic insults and occur precisely in those tissues where p53 plays a critical role in mediating apotpotis rather than cell cycle arrest.

DeltaNp73 stabilises TAp73 proteins but compromises their function due to inhibitory hetero-oligomer formation

Since inhibitory interactions of two proteins often lead to their stabilization, we asked whether DNp73 can affect TAp73 protein levels. We show here that DNp73 isoforms indeed stabilise TAp73 proteins but, in doing so, inhibit their transactivation function. Individual p73 isoforms with variant N- and C-termini differ only moderately in their halflives. However, when co-expressed in various cell types, TAp73 a and b proteins become markedly stabilized by DNp73a in a dose-dependent manner. Similar results were seen with DNp73beta, albeit the effect is weaker. In contrast, p53 protein fails to accumulate via DNp73. Using tetramerization-deficient mutants, we find that the ability of DNp73 to mediate TAp73 accumulation largely depends on its tetramerization domain and correlates with its activity to function as a dominant-negative inhibitor of TAp73. In the ongoing debate whether TAp73 is a relevant tumor suppressor, we suggest that increased TAp73 protein levels should be interpreted with caution when levels are the only criteria that can be used to deduce TAp73 activity. This is particularly the case in primary tumors where functional studies are not possible.

Deregulated expression of ΔNp73α causes early embryonic lethality

The tumor suppressor protein p53 shares considerable structural and functional homology with its family members p63 and p73. For example, full-length human p73 (TAp73) shares 63% amino-acid identity with the DNA-binding region of p53, conserving all DNA contact residues, and 38% and 29% identity with the p53 tetramerization and transactivation domains, respectively. Human TAp73, or its homolog TAp63, is required for p53-mediated transcriptional activation of apoptotic genes in fibroblasts. However, neither TAp63 nor TAp73 are inactivated by mutations in human cancers, excluding them from being categorized as classic Knudsentype tumor suppressors. In fact, p73 has been shown to be overexpressed in many different human cancers. This apparent contradiction in p73 activity may lie in the fact that the p73 gene produces diverse N-terminal isoforms with distinct or even opposing properties. Aside from the proapoptotic transactivation-competent TAp73 isoform, the N-terminally truncated transactivation-deficient DNp73 is biologically important and might play a role as an oncogene in human cancers. DNp73 protein is either generated from the P1 promoter by alternate Exon 30 splicing or via an alternate P2 promoter in Intron 3. Moreover, alternate splicing at the C-terminus results in at least six different C terminal variants a f (of which a is the full-length version) that are thought to differ in their transcriptional potency but whose biological differences remain to be fully elucidated. Importantly, the DNp73 isoform is frequently upregulated in various human cancers, including ovarian cancer, breast and gynecological cancers, hepatocellular carcinoma, lung cancer, gastric cancer, thyroid cancer and neuroblastoma. Overexpression of DNp73 is an independent prognostic marker for reduced survival in lung cancer patients and in neuroblastoma patients. In cell-based assays, DNp73a displays oncogenic properties. DNp73a immortalizes primary mouse embryo fibroblasts (MEFs) in vitro and cooperates with oncogenic Ras in inducing MEF-derived fibrosarcomas in vivo. Together, these results suggest that in cancer tissues, DNp73 overexpression plays an oncogenic role and functionally abrogates any concomitant increase in TAp73. We were interested in further characterizing the role of DNp73. In addition to its inhibition of TAp73, DNp73 functions as a dominant-negative inhibitor of wild-type p53 and most likely of TAp63, and thus is a potent pan inhibitor of the p53 gene family. There are no studies reported to date examining the in vivo effects of homozygous compound knockouts of individual family members. Although the loss of any one of these genes alone does not interfere with viability, it creates a significantly disrupted phenotype that is genespecific. Deficiency of p53 leads to a strongly increased tumor susceptibility at young age, but the majority of p53-null mice survive to adulthood, indicating that p53 function is not essential for embryonic development. Only a small percentage of young, mainly female p53 null mice exhibit neural tube closure defects due to neuronal overgrowth with exencephaly of the midbrain. Interestingly, this exencephalic phenotype resembles that of animals with targeted mutations in other members of the mitochondrial death pathway such as APAF-1, caspase-9 and caspase-3. p63-deficient mice, on the other hand, show grave developmental defects in limb and craniofacial formation and epithelial morphogenesis but do survive until a few days after birth. p73 deficient mice exhibit a milder developmental phenotype, with defects specifically within the central nervous system (hippocampus) and the innate immune system. They survive to birth, but most p73-null mice die within 2 months after birth due to infection, with about 25% reaching adulthood. Aged p63þ / and p73þ / mice show a somewhat higher spontaneous tumor predisposition that is about half of the predisposition conferred by a p53þ / status. Defining the phenotype of compound disruptions in the gene family will be important. Mice with heterozygous compound null alleles of any two of the three family members reveal the cooperativity of family members in tumor suppression. However, homozygous compound double or even triple knockout mice have not been reported, possibly because they are difficult or even impossible to generate. Importantly, DNp73 acts as a dominant-negative pan inhibitor of p53, TAp73 and TAp63. Thus, we chose overexpression of DNp73 as an alternate way to create functional compound knockout animals of the p53 gene family. We, therefore, set out to generate mice overexpressing the a variant of human DNp73. Initially, we used constitutive overexpression of Flagtagged DNp73a driven by a CMV promoter. As negative control, mice expressing an inactive (tetramerization-incompetent but DNA-binding competent) mutant form of DNp73a (L322P) were also created (Figure 1a). These constructs were first verified for protein expression in the p53-null human cancer cell line H1299. As shown in Figure 1b, a clear Flag signal was obtained from cells transfected with wild-type and mutant DNp73 constructs, but not from cells transfected with green fluorescent protein (GFP). Cell Death and Differentiation (2006) 13, 170–173 & 2006 Nature Publishing Group All rights reserved 1350-9047/06