Up-regulation of NFkappaB-responsive gene expression by DeltaNp73alpha in p53 null cells

Transactivation domain (TAD)-truncated p73, DeltaNp73, associates with p53, resulting in suppression of p53's functions. Using p53 null cell lines, we examined whether or not DeltaNp73 can regulate gene expression in a p53-independent manner. When DeltaNp73alpha was co-transfected with a luciferase reporter plasmid with various enhancer elements, NFkappaB-responsive luciferase gene expression was selectively up-regulated by DeltaNp73alpha, but not by other p73-isoforms with TAD and DeltaNp73beta. Deletion of the TAD endowed p73alpha with the ability to enhance the responsive gene's expression, but deletion of the N-terminal proline-rich domain (PRD) rendered the TAD-deleted p73alpha inactive. Considering the inability of DeltaNp73beta, which is the C-terminus-truncated form of DeltaNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DeltaNp73alpha to can activate NFkappaB-responsive luciferase expression. Over-expression of p53 suppressed the TAD-truncated p73alpha-mediated luciferase expression, suggesting that p53 interferes with the TAD-truncated p73alpha-mediated activation of NFkappaB. Inhibitors for NFkappaB activation reduced the TAD-truncated p73alpha-dependent NFkappaB-responsive gene expression, indicating that TAD-truncated p73alpha activates NFkappaB as does TNFalpha. In addition to the results obtained in the reporter gene assay, TAD-truncated p73alpha stimulated the translocation of NFkappaB to the nucleus and the expression of an endogenous NFkappaB-responsive gene, Bcl-XL. Taken together, these results demonstrate that TAD-truncated p73alpha can activate NFkappaB.     

Full-length p73alpha represses drug-induced apoptosis in small cell lung carcinoma cells

The p73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the NH2 terminus, two different promoters generate the full-length and the DeltaN isoforms, with or without the transactivating domain. At the COOH terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that DeltaNp73 isoforms exert a dominant-negative effect on p73 by blocking their transactivation activity and hence the ability to induce apoptosis. Considerable efforts are made to identify the functional diversity of the COOH-terminal p73 variants. In this study, we found that p73alpha inhibited drug-induced apoptosis in small cell lung carcinoma cells, whereas p73beta promoted it. p73alpha prevented Bax activation, mitochondrial dysfunction, and caspase activation. In addition, p73alpha was also able to reduce apoptosis induced by the BH3-only protein PUMA (p53 up-regulated modulator of apoptosis). Furthermore, we discovered that p73alpha is able to inhibit the pro-apoptotic effect of p73beta, demonstrating the existence of equilibrium between these two p73 isoforms. In conclusion, the reported overexpression of p73alpha in certain tumor types, and our findings that p73alpha exerts anti-apoptotic functions, indicate a potential oncogenic activity for p73.     

deltaNp73 facilitates cell immortalization and cooperates with oncogenic Ras in cellular transformation in vivo

TP73, despite significant homology to TP53, is not a classic tumor suppressor gene, since it exhibits upregulation of nonmutated products in human tumors and lacks a tumor phenotype in p73-deficient mice. We recently reported that an N-terminally truncated isoform, DeltaNp73, is upregulated in breast and gynecological cancers. We further showed that DeltaNp73 is a potent transdominant inhibitor of wild-type p53 and TAp73 in cultured human tumor cells by efficiently counteracting their target gene transactivations, apoptosis, and growth suppression functions (A. I. Zaika et al., J. Exp. Med. 6:765-780, 2002). Although these data strongly suggest oncogenic properties of DeltaNp73, this can only be directly shown in primary cells. We report here that DeltaNp73 confers resistance to spontaneous replicative senescence of primary mouse embryo fibroblasts (MEFs) and immortalizes MEFs at a 1,000-fold-higher frequency than occurs spontaneously. DeltaNp73 cooperates with cMyc and E1A in promoting primary cell proliferation and colony formation and compromises p53-dependent MEF apoptosis. Importantly, DeltaNp73 rescues Ras-induced senescence. Moreover, DeltaNp73 cooperates with oncogenic Ras in transforming primary fibroblasts in vitro and in inducing MEF-derived fibrosarcomas in vivo in nude mice. Wild-type p53 is likely a major target of DeltaNp73 inhibition in primary fibroblasts since deletion of p53 or its requisite upstream activator ARF abrogates the growth-promoting effect of DeltaNp73. Taken together, DeltaNp73 behaves as an oncogene that targets p53 that might explain why DeltaNp73 upregulation may be selected for during tumorigenesis of human cancers.     

A dominant negative form of p63 inhibits apoptosis in a p53-independent manner

Stem cells are a source of differentiated cells in multiple tissues. If genetic alterations occur in stem cells, the problem persists and malignant cancers may arise. DeltaNp63alpha-a homologue of the tumor suppressor p53-is exclusively expressed in proliferating undifferentiated epithelial cells and cancer cells of epidermal origin. Here, we show that DeltaNp63alpha antagonizes DNA damage-induced apoptosis in a p53-independent manner. We found that upon cellular injury, DeltaNp63alpha must be downregulated before apoptotic program can be activated. The 5637 cell line has abundant levels of DeltaNp63alpha and mutant p53, and it is resistant to DNA damage-induced apoptosis. The knockdown of DeltaNp63alpha by RNA interference sensitized these cells to apoptosis upon genotoxic insult. This suggests that DeltaNp63alpha plays an anti-apoptotic role regardless of the p53 status. Considering the frequent mutations of p53 in tumor cells, our results provide important implications for the treatment of cancers in which p63 is amplified.     

TAp73/Delta Np73 influences apoptotic response, chemosensitivity and prognosis in hepatocellular carcinoma

We investigated the mechanisms by which TAp73 beta and dominant-negative p73 (Delta Np73) regulate apoptosis. TAp73 beta transactivated the CD95 gene via the p53-binding site in the first intron. In addition, TAp73 beta induced expression of proapoptotic Bcl-2 family members and led to apoptosis via the mitochondrial pathway. Endogenous TAp73 was upregulated in response to DNA damage by chemotherapeutic drugs. On the contrary, DeltaNp73 conferred resistance to chemotherapy. Inhibition of CD95 gene transactivation was one mechanism by which DeltaNp73 functionally inactivated the tumor suppressor action of p53 and TAp73 beta. Concomitantly, DeltaNp73 inhibited apoptosis emanating from mitochondria. Thus, DeltaNp73 expression in tumors selects against both the death receptor and the mitochondrial apoptosis activity of TAp73 beta. The importance of these data is evidenced by our finding that upregulation of DeltaNp73 in hepatocellular carcinoma patients correlates with reduced survival. Our data indicate that Delta Np73 is an important gene in hepatocarcinogenesis and a relevant prognostic factor.     

ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DeltaNp63alpha associated with its increased phosphorylated status. Since DeltaNp63alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DeltaNp63alpha in HNSCC cells upon cisplatin exposure. We found that DeltaNp63alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397, and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DeltaNp63alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DeltaNp63alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DeltaNp63alpha degradation. Finally, we demonstrated that the DeltaNp63alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DeltaNp63alpha following DNA damage.     

Definition of the p53 functional domains necessary for inducing apoptosis

The p53 protein contains several functional domains necessary for inducing cell cycle arrest and apoptosis. The C-terminal basic domain within residues 364-393 and the proline-rich domain within residues 64-91 are required for apoptotic activity. In addition, activation domain 2 within residues 43-63 is necessary for apoptotic activity when the N-terminal activation domain 1 within residues 1-42 is deleted (DeltaAD1) or mutated (AD1(-)). Here we have discovered that an activation domain 2 mutation at residues 53-54 (AD2(-)) abrogates the apoptotic activity but has no significant effect on cell cycle arrest. We have also found that p53-(DeltaAD2), which lacks activation domain 2, is inert in inducing apoptosis. p53-(AD2(-)DeltaBD), which is defective in activation domain 2 and lacks the C-terminal basic domain, p53-(DeltaAD2DeltaBD), which lacks both activation domain 2 and the C-terminal basic domain, and p53-(DeltaPRDDeltaBD), which lacks both the proline-rich domain and the C-terminal basic domain, are also inert in inducing apoptosis. All four mutants are still capable of inducing cell cycle arrest, albeit to a lesser extent than wild-type p53. Interestingly, we have found that deletion of the N-terminal activation domain 1 alleviates the requirement of the C-terminal basic domain for apoptotic activity. Thus, we have generated a small but potent p53-(DeltaAD1DeltaBD) molecule. Furthermore, we have determined that at least two of the three domains (activation domain 1, activation domain 2, and the proline-rich domain), are required for inducing cell cycle arrest. Taken together, our results suggest that activation domain 2 and the proline-rich domain form an activation domain for inducing pro-apoptotic genes or inhibiting anti-apoptotic genes. The C-terminal basic domain is required for maintaining this activation domain competent for transactivation or transrepression.