Maria Carmen Marin

p73 is a human p53-related protein that can induce apoptosis

The protein p53 is the most frequently mutated tumour suppressor to be identified so far in human cancers,. The ability of p53 to inhibit cell growth is due, at least in part, to its ability to bind to specific DNA sequences and activate the transcription of target genes such as that encoding the cell-cycle inhibitor p21Waf1/Cip1 (ref. 3). A gene has recently been identified that is predicted to encode a protein with significant amino-acid sequence similarity to p53 (ref. 4). In particular, each of the p53 amino-acid residues implicated in direct sequence-specific DNA binding is conserved in this protein. This gene, called p73, maps to the short arm of chromosome 1, and is found in a region that is frequently deleted in neuroblastomas. Here we show that p73 can, at least when overproduced, activate the transcription of p53-responsive genes and inhibit cell growth in a p53-like manner by inducing apoptosis (programmed cell death).

Role for the p53 homologue p73 in E2F-1-induced apoptosis

The transcription factor E2F-1 induces both cell-cycle progression and, in certain settings, apoptosis. E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. The p53-dependent pathway involves the induction by E2F-1 of the human tumour-suppressor protein p14ARF, which neutralizes HDM2 (human homologue of MDM2) and thereby stabilizes the p53 protein. Here we show that E2F-1 induces the transcription of the p53 homologue p73. Disruption of p73 function inhibited E2F-1-induced apoptosis in p53-defective tumour cells and in p53-/- mouse embryo fibroblasts. We conclude that activation of p73 provides a means for E2F-1 to induce death in the absence of p53.

A common polymorphism acts as an intragenic modifier of mutant p53 behaviour.

The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.

Chemosensitivity linked to p73 function

Most chemotherapeutic agents induce DNA damage, leading to p53 accumulation and apoptosis. The factors that determine chemosensitivity in p53-defective tumor cells are poorly understood. We found that the p53 family member p73 is induced by a wide variety of chemotherapeutic drugs. Blocking p73 function with a dominant-negative mutant, siRNA, or homologous recombination led to chemoresistance of human tumor cells and engineered transformed cells, irrespective of p53 status. Mutant p53 can inactivate p73 and downregulation of mutant p53 enhanced chemosensitivity. These findings indicate that p73 is a determinant of chemotherapeutic efficacy in humans.

Viral Oncoproteins Discriminate between p53 and the p53 Homolog p73

ABSTRACT p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.

p73 deficiency results in impaired self renewal and premature neuronal differentiation of mouse neural progenitors independently of p53

The question of how neural progenitor cells maintain its self-renewal throughout life is a fundamental problem in cell biology with implications in cancer, aging and neurodegenerative diseases. In this work, we have analyzed the p73 function in embryonic neural progenitor cell biology using the neurosphere (NS)-assay and showed that p73-loss has a significant role in the maintenance of neurosphere-forming cells in the embryonic brain. A comparative study of NS from Trp73−/−, p53KO, p53KO;Trp73−/− and their wild-type counterparts demonstrated that p73 deficiency results in two independent, but related, phenotypes: a smaller NS size (related to the proliferation and survival of the neural-progenitors) and a decreased capacity to form NS (self-renewal). The former seems to be the result of p53 compensatory activity, whereas the latter is p53 independent. We also demonstrate that p73 deficiency increases the population of neuronal progenitors ready to differentiate into neurons at the expense of depleting the pool of undifferentiated neurosphere-forming cells. Analysis of the neurogenic niches demonstrated that p73-loss depletes the number of neural-progenitor cells, rendering deficient niches in the adult mice. Altogether, our study identifies TP73 as a positive regulator of self-renewal with a role in the maintenance of the neurogenic capacity. Thus, proposing p73 as an important player in the development of neurodegenerative diseases and a potential therapeutic target.

c-Myc inhibits Ras-mediated differentiation of pheochromocytoma cells by blocking c-Jun up-regulation.

Although mutant Ras proteins were originally described as transforming oncoproteins, they induce growth arrest, senescence, and/or differentiation in many cell types. c-Myc is an oncogenic transcription factor that cooperates with Ras in cellular transformation and oncogenesis. However, the Myc-Ras relationship in cellular differentiation is largely unknown. Here, we have analyzed the effects of c-Myc on PC12-derived cells (UR61 cell line), harboring an inducible N-Ras oncogene. In these cells, Ras activation induces neuronal-like differentiation by a process involving c-Jun activation. We found that c-Myc inhibited Ras-mediated differentiation by a mechanism that involves the blockade of c-Jun induction in response to Ras signal. Accordingly, ectopically expressed c-Jun could bypass c-Myc impediment of Ras-induced differentiation and activator protein 1 activation. Interestingly, it did not rescue the proliferative arrest elicited by Ras and did not enhance the differentiation-associated apoptosis. The blockade of Ras-mediated induction of c-Jun takes place at the level of c-Jun proximal promoter. Mutational analysis revealed that c-Myc regions involved in DNA binding and transactivation are required to block differentiation and c-Jun induction. c-Myc does not seem to require Miz-1 to inhibit differentiation and block c-Jun induction. Furthermore, Max is not required for c-Myc activity, as UR61 cells lack a functional Max gene. c-Myc–inhibitory effect on the Ras/c-Jun connection is not restricted to UR61 cells as it can occur in other cell types as K562 or HEK293. In conclusion, we describe a novel interplay between c-Myc and c-Jun that controls the ability of Ras to trigger the differentiation program of pheochromocytoma cells. (Mol Cancer Res 2008;6(2):325–39)

Multiple isoforms of the tumor protein p73 are expressed in the adult human telencephalon and choroid plexus and present in the cerebrospinal fluid.

p73, a homolog of the p53 tumor suppressor, codes for full‐length transactivating (TA) and N‐terminally truncated (ΔN) isoforms, with pro‐ and anti‐apoptotic activities, respectively. We examined the expression of the main p73 isoforms in adult human and mouse telencephalon and choroid plexus by immunohistochemistry on paraffin sections, and immunoblotting (IB) of tissue extracts and cerebrospinal fluid (CSF), using antibodies against different protein domains. Cortical neurons expressed TAp73 predominantly in the cytoplasm and ΔNp73 mainly in the nucleus, with partial overlap in the cytoplasm. Highest expression was found in the hippocampus. IB showed an array of TAp73 variants in adult human cortex and hippocampus. IB of human choroid plexus and CSF using TAp73‐specific antibodies revealed the presence of a ∼90‐kDa protein whose molecular weight was reduced after N‐deglycosylation, suggesting that glycosylated TAp73 is exported into the CSF. In the mouse, high expression of TAp73 was also detected in the subcommissural organ (SCO), an ependymal gland absent in adult humans. TAp73 colocalized with anti‐fibra‐Reissner‐antibody (AFRU), which is a marker of Reissners fiber, the secreted SCO product. p73‐deficient mice had generalized cortical hypoplasia and hydrocephalus; in addition, we observed a dramatic size reduction of the choroid plexus. However, the SCOs were apparently unaltered and continued to secrete Reissners fiber. Our findings point to complex and widespread p73 activities in the maintenance of adult cortical neurons and in brain homeostasis. TAp73 in the CSF may play important roles in the maintenance of the adult ventricular wall as well as in the development of the proliferating neuroepithelium.

Regulatory feedback loop between TP73 and TRIM32

The p73 transcription factor is one of the members of the p53 family of tumor suppressors with unique biological functions in processes like neurogenesis, embryonic development and differentiation. For this reason, p73 activity is tightly regulated by multiple mechanisms, including transcription and post-translational modifications. Here, we identified a novel regulatory loop between TAp73 and the E3 ubiquitin ligase tripartite motif protein 32 (TRIM32). TRIM32, a new direct p73 transcriptional target in the context of neural progenitor cells, is differentially regulated by p73. Although TAp73 binds to the TRIM32 promoter and activates its expression, TAp73-induced TRIM32 expression is efficiently repressed by DNp73. TRIM32 in turn physically interacts with TAp73 and promotes its ubiquitination and degradation, impairing p73-dependent transcriptional activity. This mutual regulation between p73 and TRIM32 constitutes a novel feedback loop, which might have important implications in central nervous system development as well as relevance in oncogenesis, and thus emerges as a possible therapeutic target.

p73 cooperates with Ras in the activation of MAP kinase signaling cascade.

The p73 gene is capable of inducing cell cycle arrest, apoptosis, senescence, differentiation and to cooperate with oncogenic Ras in cellular transformation. Ras can be considered as a branch point in signal transduction, where diverse extracellular stimuli converge. The intensity of the mitogen-activated protein kinase (MAPK) cascade activation influences the cellular response to Ras. Despite the fundamental role of p53 in Ras-induced growth arrest and senescence, it remains unclear how the Ras/MEK/ERK pathway induces growth arrest in the absence of p53. We report here that oncogenic Ras stabilizes p73 resulting in p73 accumulation and enhancement of its activity. p73, in turn, induces a sustained activation of the MAP kinase cascade synergizing with oncogenic Ras. We also found that inhibition of p73 function modifies the cellular outcome to Ras activation inhibiting Ras-dependent differentiation. Here, we show for the first time that there is a signaling loop between Ras-dependent MAPK cascade activation and p73 function.