The cause or consequence of overexpression of p73 (refs 1, 2), the structural and functional homologue of the tumour-suppressor gene product p53 (refs 3, 4), in human cancers is poorly understood. Here, we report a role for p73 in supporting cellular growth through the upregulation of AP-1 transcriptional activity. p73 suppresses growth when overexpressed alone, but synergises with the proto-oncogene c-Jun to promote cellular survival. Conversely, silencing of p73 expression compromises cellular proliferation. Molecular analysis revealed that expression of the AP-1 target-gene product cyclinD1 (ref. 5) is reduced concomitant with p73, but not p53, silencing. Moreover, cyclinD1 was induced by p73 expression in a c-Jun-dependent manner, and was required for p73-mediated cell survival. Furthermore, c-Jun-dependent AP-1 transcriptional activity was augmented by p73 and, consistently, induction of endogenous AP-1 target genes was compromised in the absence of p73. Chromatin immunoprecipitation and electrophoretic mobility shift analysis indicated that p73 enhanced the binding of phosphorylated c-Jun and Fra-1, another AP-1 family member, to AP-1 consensus DNA sequences, by regulating c-Jun phosphorylation and Fra-1 expression. Collectively, our data demonstrates a novel and unexpected role of p73 in augmenting AP-1 transcriptional activity through which it supports cellular growth.
Although p73 is a structural and functional homologue of the tumor-suppressor gene p53, it is not mutated in many human cancers as p53. Besides, p73 was shown to be activated by only a subset of signals that activate p53, such as gamma-irradiation and cisplatin, but not by other common genotoxic stress-inducing agents such as ultraviolet (UV) irradiation, although many of these signals are also capable of inducing p53-independent cell death. Using a p73-specific antibody, we confirmed that c-Abl is required for cisplatin-induced p73 upregulation, and further demonstrate that the p73 protein is upregulated by UV irradiation and other stress stimuli including sorbitol, hydrogen peroxide, nocodazol, and taxol. These stress signals upregulate both p73 mRNA and increases the stability of p73, indicating that p73 is regulated transcriptionally and posttranslationally. Cells stably expressing the dominant-negative p73 inhibitor protein (p73DD) and p73(-/-) fibroblasts are more resistant than control cells to apoptosis induced by these stress signals, suggesting that p73 contributes to apoptosis induction. Together, the data demonstrate that several stress signals can signal to p73 in vivo, which raises the possibility of eradicating cancers with an unmutated p73 gene by activating them with stress-inducing agents or their mimetics.
p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or loss of function models, in human cancers where it is hyper-expressed is unclear. We demonstrate here that both TAp73 and DNp73 are able to specifically transactivate the expression of the anti-apoptotic member of the caspase family, caspase-2S. Neither p53 nor TAp63 has this property, and only the p73β form, but not the p73α form, has this competency. Caspase-2 promoter analysis revealed that a non-canonical, 18 bp GC-rich Sp-1-binding site-containing region is essential for p73β-mediated activation. However, mutating the Sp-1-binding site or silencing Sp-1 expression did not affect p73β's transactivation ability. In vitro DNA binding and in vivo chromatin immunoprecipitation assays indicated that p73β is capable of directly binding to this region, and consistently, DNA binding p73 mutant was unable to transactivate caspase-2S. Finally, DNp73β over-expression in neuroblastoma cells led to resistance to cell death, and concomitantly to elevated levels of caspase-2S. Silencing p73 expression in these cells led to reduction of caspase-2S expression and increased cell death. Together, the data identifies caspase-2S as a novel transcriptional target common to both TAp73 and DNp73, and raises the possibility that TAp73 may be over-expressed in cancers to promote survival.
The molecular mechanisms regulating cell death during mitosis are poorly understood. We show here a critical role for p73, but not p53, in regulating mitotic cell death induced by various means. Prolonged mitotic arrest and the activation of spindle checkpoint are required for mitotic death, which occurs before mitotic exit and which can be ameliorated by accelerated mitotic exit. Absence or silencing of p73 expression abrogated mitotic death without accelerating mitotic exit, and was independent of BubR1 and Mad2, the loss of which promotes mitotic exit. However, the absence of p73 reduced mitotic death by compromising the expression of the proapoptotic BH3-only protein Bim and thereby affecting cytochrome c release and caspase activation. p73 was found to induce bim expression through direct binding to regulatory elements in intron 1. Congruently, mitotic cell death was rescued to similar extents by silencing either bim or p73 expression. Taken together, the data show an important role for the p73-Bim axis in regulating cell death during mitosis that is independent of p53.
Reactivation of telomerase is a feature in many cancer cells. Telomerase activation inhibits telomere shortening, thereby preventing cell cycle arrest and apoptosis activated by shortened telomeres or chromosomal rearrangements. The tumor-suppressor gene product, p53, was previously shown to transcriptionally suppress the activation of the catalytic subunit of telomerase (hTERT). Here we have evaluated the role of p73 in hTERT regulation. We found that ectoptic expression of p73, in contrast to p73␣ or p53, in p53 null H1299 cells does not lead to suppression of hTERT transcription. However co-expression of p73␣ or p73 together with p53 abolished p53-mediated hTERT suppression. This phenomenon was found to be dependent on the DNA binding ability of p73. We also show that p53-mediated suppression of hTERT transcription requires a minimum threshold level of p53, and p73 abrogates p53-mediated suppression by reducing p53 levels through the activation of HDM2. Moreover, p53-mediated hTERT suppression was not relieved by p73 in cells depleted of HDM2 through small interfering RNAmediated gene silencing. In addition, knockdown of HDM2 in MCF7 cells, which express moderately high levels of p73 and p53, resulted in the reduction of endogenous hTERT levels. Finally, knockdown of p73 in MCF7 cells resulted in increased p53 protein levels and a concomitant decrease in hTERT levels. Together, our data indicate a plausible way by which p73, through HDM2, can oppose p53 tumor suppressor function, thereby possibly contributing to tumorigenesis.
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