Jean-Christophe Bourdon

Mdm2-Mediated NEDD8 Conjugation of p53 Inhibits Its Transcriptional Activity

The only reported role for the conjugation of the NEDD8 ubiquitin-like molecule is control of the activity of SCF ubiquitin ligase complexes. Here, we show that the Mdm2 RING finger E3 ubiquitin ligase can also promote NEDD8 modification of the p53 tumor suppressor protein. Mdm2 is itself modified with NEDD8 with very similar characteristics to the autoubiquitination activity of Mdm2. By using a cell line (TS-41) with a temperature-sensitive mutation in the NEDD8 conjugation pathway and a p53 mutant that cannot be NEDDylated (3NKR), we demonstrate that Mdm2-dependent NEDD8 modification of p53 inhibits its transcriptional activity. These findings expand the role for Mdm2 as an E3 ligase, providing evidence that Mdm2 is a common component of the ubiquitin and NEDD8 conjugation pathway and indicating the diverse mechanisms by which E3 ligases can control the function of substrate proteins.

p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress.

p63, p73 and p53 compose a family of transcription factors involved in cell response to stress and development. p53 is the most frequently mutated gene in cancer (50%) and loss of p53 activity is considered to be ubiquitous to all cancers. Recent publications may have a profound impact on our understanding of p53 tumour suppressor activity. p63, p73 and p53 genes have a dual gene structure conserved in drosophila, zebrafish and man. They encode for multiple p63, p73 or p53 proteins containing different protein domains (isoforms) due to multiple splicing, alternative promoter and alternative initiation of translation. In this review, we describe the different isoforms of p63, p73, p53 and their roles in development and cancer. The changes in the interactions between p53, p63 and p73 isoforms are likely to be fundamental to our understanding in the transition between normal cell cycling and the onset of tumour formation.

The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor

The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53‐dependent promoters and that endogenous p68 and p53 co‐immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53‐dependent apoptosis, but does not influence p53 stabilisation or expression of non‐p53‐responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53‐dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock‐down does not significantly affect NF‐κB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity.

p53 isoforms Δ133p53 and p53β are endogenous regulators of replicative cellular senescence

The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumour progression in vivo. We show that the human p53 isoforms Δ133p53 and p53β function in an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53β and diminished Δ133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA (short interfering RNA)-mediated knockdown of endogenous Δ133p53 induced cellular senescence, which was attributed to the regulation of p21WAF1 and other p53 transcriptional target genes. In overexpression experiments, whereas p53β cooperated with full-length p53 to accelerate cellular senescence, Δ133p53 repressed miR-34a expression and extended the cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (that is, elevated p53β and reduced Δ133p53) was observed in vivo in colon adenomas with senescent phenotypes. The increased Δ133p53 and decreased p53β isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma.

p53 and its isoforms in cancer

p53, p63 and p73 are members of the p53 gene family involved in development, differentiation and response to cellular stress. p53 gene is a transcription factor essential for the prevention of cancer formation. The p53 pathway is ubiquitously lost in human cancer either by p53 gene mutation (60% of cancers) or by lost of cell signalling upstream and downstream of p53 in the remaining cancers expressing WTp53 gene. As p53 pathway inactivation is a common denominator to all cancers, the understanding of p53 tumour suppressor activity is likely to bring us closer to cancer therapy. However, despite all the experimental evidences showing the importance of p53 in preventing carcinogenesis, it is difficult in clinical studies to link p53 status to cancer treatment and clinical outcome. The recent discovery that p53 gene encodes for nine different p53 proteins (isoforms) may have a profound impact on our understanding of p53 tumour suppressor activity. Studies in several tumour types have shown that the nine different p53 isoforms are abnormally expressed in tumour tissues compared to normal cells. p53 protein isoforms modulate p53 transcriptional activity and cell fate outcome in response to stress. Regulation of p53 function in normal and tumour tissues in man is likely to be more complex than has been hitherto appreciated. Therefore, the tumour p53 status needs to be determined more accurately by integrating p53 isoform expression, functional p53 mutation analysis and a panel of antibodies specific of p53 and of its target genes.

Further characterisation of the p53 responsive element – identification of new candidate genes for trans -activation by p53

The p53 protein is known to trans-activate a number of genes by specific binding to a consensus sequence containing two decamers of the type: PuPuPuCA/TT/AGPyPyPy. In order to identify new p53 trans-activated genes, we defined a set of criteria for computer search of p53-responsive elements. Based on experimental data, we proposed an extended consensus sequence composed of the two decamers of the El-Deiry consensus sequence flanked by two additional ones. A maximum of 3 bp substitutions was accepted for the two decamers of the El-Deiry consensus sequence, as well as for each additional decamer, except when the two decamers of the El-Deiry consensus sequence are contiguous. In this case, each additional decamer is allowed to bear one base insertion or deletion between the median C and G. This set of criteria was validated by identifying within the promoter region of the IGF-BP3 gene the existence of a novel p53-responsive element whose functional significance was verified. By limiting our computer search to Vertebrate genes involved in cell cycle regulation, cellular adhesion or metastatic processes and to gene families most often found in HOVERGEN database, 7785 gene sequences were first analysed. Among the oncogenes, kinases, proteases and structural proteins, 55 new genes were selected; six of them were retrieved in more than one species

Biological functions of p53 isoforms through evolution: lessons from animal and cellular models

The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of ‘p53 isoforms’, including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.

Human and mouse Fas (APO-1/CD95) death receptor genes each contain a p53-responsive element that is activated by p53 mutants unable to induce apoptosis

p53 is a tumor suppressor protein that induces apoptosis at least in part through its ability to act as a sequence-specific transactivator. This work reports that intron 1 of the mouse Fas death receptor gene contains a p53-responsive element (p53RE) that matches the p53 consensus sequence and that is located between nucleotides +1704 and +1723 from the transcription initiation site. This element is specifically bound by p53 and functions as a p53-dependent enhancer in mammalian or in yeast reporter gene assays. Contrary to bax, another known pro-apoptotic p53-target gene, both mouse and human FASp53REs are still activated by the discriminatory p53 mutants Pro-175 and Ala-143, a class of mutants unable to induce apoptosis. We propose that p53-dependent up-regulation of Fas does not induce apoptosis per se but sensitizes the cell to other pro-apoptotic signal(s). The functional conservation of p53-dependent Fas up-regulation argues strongly in favor of its biological importance and suggests that murine models may be used to study further the in vivo role of Fas in the p53 response.

p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish.

p53 is a well-known tumor suppressor and is also involved in processes of organismal aging and developmental control. A recent exciting development in the p53 field is the discovery of various p53 isoforms. One p53 isoform is human Delta133p53 and its zebrafish counterpart Delta113p53. These N-terminal-truncated p53 isoforms are initiated from an alternative p53 promoter, but their expression regulation and physiological significance at the organismal level are not well understood. We show here that zebrafish Delta113p53 is directly transactivated by full-length p53 in response to developmental and DNA-damaging signals. More importantly, we show that Delta113p53 functions to antagonize p53-induced apoptosis via activating bcl2L (closest to human Bcl-x(L)), and knockdown of Delta113p53 enhances p53-mediated apoptosis under stress conditions. Thus, we demonstrate that the p53 genetic locus contains a new p53 response gene and that Delta113p53 does not act in a dominant-negative manner toward p53 but differentially modulates p53 target gene expression to antagonize p53 apoptotic activity at the physiological level in zebrafish. Our results establish a novel feedback pathway that modulates the p53 response and suggest that modulation of the p53 pathway by p53 isoforms might have an impact on p53 tumor suppressor activity.

Uncovering the role of p53 splice variants in human malignancy: a clinical perspective

Thirty-five years of research on p53 gave rise to more than 68,000 articles and reviews, but did not allow the uncovering of all the mysteries that this major tumor suppressor holds. How p53 handles the different signals to decide the appropriate cell fate in response to a stress and its implication in tumorigenesis and cancer progression remains unclear. Nevertheless, the uncovering of p53 isoforms has opened new perspectives in the cancer research field. Indeed, the human TP53 gene encodes not only one but at least twelve p53 protein isoforms, which are produced in normal tissues through alternative initiation of translation, usage of alternative promoters, and alternative splicing. In recent years, it became obvious that the different p53 isoforms play an important role in regulating cell fate in response to different stresses in normal cells by differentially regulating gene expression. In cancer cells, abnormal expression of p53 isoforms contributes actively to cancer formation and progression, regardless of TP53 mutation status. They can also be associated with response to treatment, depending on the cell context. The determination of p53 isoform expression and p53 mutation status helps to define different subtypes within a particular cancer type, which would have different responses to treatment. Thus, the understanding of the regulation of p53 isoform expression and their biological activities in relation to the cellular context would constitute an important step toward the improvement of the diagnostic, prognostic, and predictive values of p53 in cancer treatment. This review aims to summarize the involvement of p53 isoforms in cancer and to highlight novel potential therapeutic targets.