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The p53 family and programmed cell death

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The p53 family and programmed cell death REVIEW The p53 family and programmed cell death EC Pietsch1, SM Sykes2, SB McMahon3 and ME Murphy1 1Division of Medical Sciences, Fox Chase Cancer Center, Philadelphia, PA, USA; 2Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA and 3De...

The p53 family and programmed cell death
REVIEW The p53 family and programmed cell death EC Pietsch1, SM Sykes2, SB McMahon3 and ME Murphy1 1Division of Medical Sciences, Fox Chase Cancer Center, Philadelphia, PA, USA; 2Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA and 3Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA, USA The p53 tumor suppressor continues to hold distinction as the most frequently mutated gene in human cancer. The ability of p53 to induce programmed cell death, or apoptosis, of cells exposed to environmental or oncogenic stress constitutes a major pathway whereby p53 exerts its tumor suppressor function. In the past decade, we have discovered that p53 is not alone in its mission to destroy damaged or aberrantly proliferating cells: it has two homologs, p63 and p73, that in various cellular contexts and stresses contribute to this process. In this review, the mechanisms whereby p53, and in some cases p63 and p73, induce apoptosis are discussed. Other reviews have focused more extensively on the contribution of individual p53-regulated genes to apoptosis induction by this protein, whereas in this review, we focus more on those factors that mediate the decision between growth arrest and apoptosis by p53, p63 and p73, and on the post-translational modifications and protein–protein interactions that influ- ence this decision. Oncogene (2008) 27, 6507–6521; doi:10.1038/onc.2008.315 Keywords: p53; p63; p73; apoptosis; transcription; mitochondria The p53 family The tumor suppressor p53 is vital in maintaining cellular genomic integrity and controlled cell growth (Levine, 1997; Bargonetti and Manfredi, 2002; Fridman and Lowe, 2003). Loss or gain of p53 function results in the aberrant growth of cells. Hence, both the cellular expression and the activity of p53 are tightly regulated. p53 protein has a very short half-life and thus is usually present at extremely low levels within cells. In response to stress, DNA-damaging agents and chronic mitogenic stimulation, p53 is transiently stabilized and activated. Depending on cell type, cell environment and oncogenic alterations, p53 activation leads to inhibition of cell cycle progression, induction of senescence, differentia- tion or apoptosis (Vousden and Lu, 2002). Over a decade after the identification of the tumor suppressor p53, two p53-related genes, p63 and p73, were identified (Kaghad et al., 1997; Schmale and Bamberger, 1997; Osada et al., 1998; Trink et al., 1998; Yang et al., 1998; Zeng et al., 2001). Like p53, both p63 and p73 possess an N-terminal transactivation domain (TAD), a DNA- binding domain (DBD), and a C-terminal oligomeriza- tion domain (OD) (Murray-Zmijewski et al., 2006). Although p63 and p73 demonstrate relatively little homology with p53 in their TAD and OD, both share approximately 60% similarity with the p53 DBD, including conservation of essential DNA contact re- sidues (Deyoung and Ellisen, 2007) (see Figure 1). This similarity allows p63 and p73 to regulate p53 target genes and, similar to p53, to induce cell cycle arrest and apoptosis. However, studies of knockout mice have demonstrated that, even though these proteins clearly share some activities with p53, each of these proteins also has functions that are very distinct. TP53 null mice are viable and develop normally (Donehower et al., 1992). In contrast, p63 knockout mice show severe developmental defects, including failure to develop limbs, skin and other epithelial tissue; these mice do not survive beyond a few days after birth (Mills et al., 1999). p73 knockout mice exhibit neurodevelopmental (hippocampal dysgenesis and hydrocephalus) and in- flammatory (chronic infections and excessive inflamma- tion) defects (Yang et al., 2000). Isoforms of p53 family members The p53, p63 and p73 genes are located on chromosomes 17 (17p13.1), 3 (3q27–29) and 1 (1q36), respectively, and all three genes are now known to express many differentially spliced isoforms (Murray-Zmijewski et al., 2006; Mu¨ller et al., 2006). All three genes encode two primary transcripts that are controlled by separate promoters (P1 and P2) (Figure 1). The P1 promoter of each gene is embedded in a non-coding region of exon 1. The P2 promoter of p63 and p73 is located in intron 3, whereas the P2 promoter of p53 is located in intron 4. Transcripts generated from the P1 promoter produce proteins that contain the TAD, the DBD and the OD (TAp53, TAp63 and TAp73). In contrast, in transcripts generated from the P2 promoter, the N-terminal TAD is Correspondence: Dr ME Murphy, Division of Medical Sciences, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA. E-mail: Maureen.Murphy@FCCC.edu or Dr SB McMahon, Kimmel Cancer Center, Thomas Jefferson Medical College, 233 S. 10th St. Philadelphia, PA 19107, USA. E-mail: steven.mcmahon@mail.jci.tju.edu Oncogene (2008) 27, 6507–6521 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc found to be absent (D133p53, DNp63 and DNp73). These DN-terminal variants are generally regarded as dominant-negative versions of p53 family members, as these can occupy promoter-binding sites but fail to transactivate gene expression. For both p53 and p73, additional DN variants can be generated by alternative splicing of the N terminus or alternative initiation of translation (D40p53, Dex2p73 and Dex2/3p73) (Murray- Zmijewski et al., 2006; Mu¨ller et al., 2006). Additional complexity to p53 family member isoforms is added by virtue of the fact that P1 and P2 transcripts can be spliced at the C terminus into several splice variants (a, b, g, and so on). For p53, transcription from the P1 and P2 promoters and alternative splicing of intron 9 can generate at least six p53 isoforms, including three TA variants (p53, p53b and p53bg) and three DN variants (D133p53, D133p53b and D133p53g). For p63, three TA variants (TAp63a, TAp63b and TAp63g) and three DN variants (DNp63a, DNp63b and DNp63g) have been identified. Finally, for p73, eight different isoforms from the p73 P1 promoter and P2 promoter elements have been described (TA and DN p73a, b, g, d, e, z, Z and y) (Figure 1). Tumor suppression by p53 family members p53 can be found biallelically mutated or deleted in more than 50% of human tumors, and there are p53 mutation databases containing a wealth of information in this regard (Olivier et al., 2002). Germline mutations in p53 are found in the Li–Fraumeni syndrome, a tumor-prone disorder predisposing affected individuals to tumors of the brain, breast, bone and adrenal cortex (Malkin et al., 1992). Finally, the p53 knockout mouse is predisposed to early onset tumors, chiefly thymic lymphoma, sarcoma and testicular tumors (Donehower et al., 1992). p63 and p73 map to two regions within the human genome that are often altered and deleted in cancers (Kaelin, 1999). This observation, combined with the observation that p63 and p73 mimic p53 action in tissue culture when overexpressed, suggests that p63 and p73 have tumor-suppressive properties analogous to p53. However, characterization of these proteins as bona fide tumor suppressors has not been straightforward. Studies aimed at identifying mutations within the p63 and p73 genes in human cancers have demonstrated that p53 is mutated in 50% of all human cancers, whereas p63 and p73 mutations occur rarely (Irwin and Kaelin, 2001; Melino et al., 2003; Deyoung and Ellisen, 2007). Rather, altered expression of p63 and p73 isoforms is more commonly observed (Mu¨ller et al., 2006). For example, p63 is upregulated in 80% of head and neck squamous cell carcinomas, with DNp63 being the predominant isoform overexpressed (Weber et al., 2002; Sniezek et al., 2004; Rocco et al., 2006; DeYoung et al., 2006). DNp63 overexpression is observed in more than 60% of bladder Figure 1 p53 family member isoforms. Schematic presentation of p53, p63 and p73 isoforms. Approximate location of the transactivation (TA) domain, proline-rich domain (PR), DNA-binding domain (DBD), oligomerization domain (OD) and the sterile alpha motif (SAM) are indicated. The amino acid identity between the TA, DBD and OD of p53 and p63/p73 as well as between p63 and p73 is denoted. Full-length (FL) p53 or TAp63 and TAp73 protein are transcribed from a promoter located in the non-coding region of exon 1 (P1 promoter) of the p53, p63 and p73 gene. D133p53, DNp63 and DNp73 isoforms are generated by transcription from a promoter (P2 promoter) located in intron 3 of the p63 and p73 gene or intron 4 of the p53 gene. Furthermore, DN variants are generated by alternative splicing of the N terminus (D40p53, Dex2p73 and Dex2/3p73). Alternative splicing of the C-terminal region yields additional variants for p53 (FL, b, and g), p63 (a, b, and g) and p73 (a, b, g, d, e, z, Z, and y). p53 b and g lack the oligomerization domain. p53 family and apoptosis EC Pietsch et al 6508 Oncogene carcinomas (Park et al., 2000), whereas loss of TAp63 expression occurs in bladder cancer and is associated with metastasis and poor prognosis (Urist et al., 2002; Koga et al., 2003). Similar to p63, altered expression of p73 occurs in a multitude of different cancers (Mu¨ller et al., 2006). Recent studies analysing p73 isoform expression indicate that both TAp73 and DNp73 isoforms are upregulated in ovarian cancer and rhabdo- myosarcomas (Concin et al., 2004; Cam et al., 2006), whereas exclusive upregulation of DNp73 isoforms can be observed in gliomas as well as in carcinomas of the breast and the colon (Domı´nguez et al., 2006; Wager et al., 2006). Downregulation of TAp73 is found in lymphoblastic leukemias and Burkitt’s lymphoma as a result of p73 P1 promoter methylation (Corn et al., 1999; Kawano et al., 1999). The general, but by no means definitive, consensus appears to be that either methylation-mediated silencing of the entire gene or upregulation of the dominant-negative DN variant predominates in human tumors for p53 family members. How upregulation of specific DN variants occurs in human tumors is not presently understood. Research on the predisposition of p63 and p73 knockout mice to tumor development has been conflict- ing and has added to the complexity of defining p63 and p73 as tumor suppressor proteins. p73 knockout mice do not develop spontaneous tumors (Yang et al., 2000) and heterozygous p63 mice are neither tumor prone nor do they develop tumors at an accelerated rate on exposure to chemical carcinogens (Keyes et al., 2006). In fact, p63 deficiency was found to induce a cellular senescence program, increased aging and shortened life span (Keyes et al., 2008). In contrast to these observa- tions is a study examining tumor development in aging heterozygous p63 and p73 knockout mice. This study demonstrated that p63þ /� and p73þ /� mice develop spontaneous tumors, similar to p53þ /� mice, with a median survival time of 10 months for p53þ /�, 15 months for p63þ /� and 14 months for p73þ /� mice (Flores et al., 2005). These latter data present the firmest evidence that p63 and p73 have tumor-suppres- sive properties analogous to p53. Further evidence that p63 and p73 have tumor suppressor function comes from four independent observations, they are: (1) siRNAs specific for p63 and p73 enhance the transformation potential of p53�/� mouse embryo fibroblasts (Lang et al., 2004); (2) both p63 and p73 can mediate chemosensitivity independent of p53 status by the induction of apoptosis (Irwin et al., 2000, 2003; Bergamaschi et al., 2003a; Lang et al., 2004; Gressner et al., 2005); (3) within tumor cells, mutant p53 directly binds to both p63 and p73 via the DBD, rendering p63 and p73 impaired in their ability to induce growth suppression and apoptosis (Di Como et al., 1999; Marin et al., 2000; Gaiddon et al., 2001; Strano et al., 2002); and (4) silencing mutant p53 expression by siRNA-mediated knockdown sensitizes cells to antic- ancer agents (Bergamaschi et al., 2003a). These obser- vations indicate that the inactivation of p63 and p73 confers a growth advantage to tumor cells that have mutant forms of p53. Binding of mutant p53 to p73 is influenced by a polymorphism at codon 72, which encodes either arginine or proline. p53 mutants asso- ciated with the arginine polymorphic variant bind and inactivate p73 most efficiently (Marin et al., 2000; Bergamaschi et al., 2003a). Consistent with this obser- vation, these p53 mutants demonstrate a less favorable response to chemotherapy (Bergamaschi et al., 2003a). Likewise, in osteosarcoma cells from p53R172H (equivalent to human hot spot mutant R175H) knockin mice, mutant p53 co-immunoprecipitates and function- ally inactivates p63 as well as p73, resulting in enhanced cellular transformation (Lang et al., 2004). Collectively, these observations suggest that p63 and p73 may complement, and in some situations may substitute for, the actions of p53. However, which effects p63 and p73 exert on cell proliferation and death may ultimately depend on the cellular context and genetic background, as p63 and p73 may not be required for the development and apoptosis of all cell types (Senoo et al., 2004). Apoptosis by p53 Transcription-dependent apoptosis The best understood activity of p53 is as a transcription factor that binds to the promoters and introns of target genes, and that recruits the basal transcriptional machinery to activate expression of that gene. In 1992, a paper that searched for the consensus binding site for p53 using an unbiased affinity-binding approach re- vealed that p53 bound best to two tandem repeats of the consensus 50-Pu-Pu-Pu-C-(A/T)-(T/A)-G-Pyr-Pyr-Pyr-30, where Pu is purine and Pyr is pyrimidine, separated by 0–13 bases (el-Deiry et al., 1992). Since that time, literally hundreds of p53 target genes have been identified, and each contain one or more consensus sites in their 50 promoter regions, introns and, in some cases, exons. These include p53AIP1, Apaf-1, BAX, Caspase-1, Caspase-6, cathepsin D, DINP1, DR4, DR5, Fas, IGF- BP3, NOXA, p85, PERP, PIDD, PIG3, PTEN, PUMA, Scotin, EI24/PIG8 as well as others (for review see Riley et al., 2008). Once bound to the promoter regions of these genes, p53 recruits general transcription factors as well as histone acetyltransferases (HATs), such as CREB-binding protein (CBP), p300 and PCAF. These HATs acetylate lysine residues of histones, thereby increasing the accessibility of chromatin to the trans- criptional apparatus. Two p53 target genes deserve specific mention as being particularly important in the apoptotic armamen- tarium of p53: these are PUMA and NOXA, BH-3-only members of the BCL-2 family. Mice lacking either PUMA or NOXA, or mice doubly knocked out for both genes (PUMA/NOXA double knockout) develop nor- mally and are not tumor-prone (Jeffers et al., 2003; Villunger et al., 2003; Michalak et al., 2008). The importance of PUMA to p53-dependent apoptosis is underscored by the fact that thymocytes and cells from the developing nervous system from PUMA knockout mice are almost completely impaired for p53-dependent apoptosis to levels equivalent to the p53 knock-out p53 family and apoptosis EC Pietsch et al 6509 Oncogene mouse (Jeffers et al., 2003). In contrast, apoptosis in other cell types of the PUMA knockout mouse is only partially impaired, and in these cells NOXA appears to contribute. For example, in mouse embryo fibroblasts, both PUMA and NOXA proteins have critical func- tions, as mouse embryonic fibroblasts (MEFs) from the PUMA/NOXA double knockout are most protected from cell death by etoposide to levels similar to p53 knockout MEFs (Michalak et al., 2008). Finally, there are other cell types in the mouse where even the PUMA/ NOXA double-knockout mouse is not protected from apoptosis as well as the p53 knockout mouse; this includes mature T and B cells, which are partially but not completely protected by PUMA/NOXA loss (Michalak et al., 2008). At least in these cell types, these data not only firmly implicate PUMA and NOXA proteins as critical players in the p53 apoptotic program but also suggest that the overall importance of individual p53 target genes in apoptosis is likely to be cell context and/or stress-specific. Transcription-independent apoptosis by p53 Several studies, which have examined p53-dependent apoptosis in the presence of inhibitors of RNA and protein synthesis, indicate that p53 induces apoptosis not only by transcription-dependent but also by transcription-independent mechanisms (Caelles et al., 1994; Wagner et al., 1994; Yan et al., 1997; Gao and Tsuchida, 1999). Moreover, deletion or mutation of the p53 TAD, which is critical to the induction of p53 apoptotic target genes, does not eliminate the ability of p53 to induce apoptosis (Haupt et al., 1995). Likewise, a p53 deletion mutant containing only the first 214 N- terminal amino acid residues induces apoptosis and suppresses the transformation of rat embryo fibroblasts by several oncogenes, but it does not transactivate p53 target genes (Chen et al., 1996; Haupt et al., 1997). Compelling evidence has accumulated over the last 10 years, indicating that p53 can directly activate compo- nents of the apoptotic machinery and that this involves translocation of p53 to the mitochondria. More specifically, the addition of recombinant p53 to nucle- ar-free cytosolic extract of irradiated cells leads to cytochrome C release, caspase activation and apoptosis induction (Ding et al., 1998, 2000; Schuler et al., 2000). Therefore, the activation of p53 in enucleated cytoplasts is sufficient to induce hallmarks of apoptosis. Immuno- fluorescence, electron microscopy and cell fractionation studies have demonstrated that in response to genotoxic stress, including DNA damage, hypoxia and activated oncogenes, p53 rapidly translocates to the mitochondria (Marchenko et al., 2000; Sansome et al., 2001; Mihara et al., 2003; Nemajerova et al., 2005). Mitochondrial translocation of p53 precedes loss of mitochondrial membrane potential and caspase activation, suggesting that p53 may trigger mechanisms at the mitochondria that ultimately lead to the induction of apoptosis (Marchenko et al., 2000). Indeed, mitochondrial trans- location of p53 in irradiated mouse tissues triggers an early wave of caspase activation, which occurs long before the transcriptional program of p53 is activated (Erster et al., 2004). Notably, specific targeting of p53 to the mitochondria by fusion of the p53 N terminus to the mitochondrial import leader peptide from ornithine transcarbamylase is sufficient to induce apoptosis, suppress colony formation and to allow p53 to function effectively as a tumor suppressor in vivo (Marchenko et al., 2000; Dumont et al., 2003; Talos et al., 2005; Palacios and Moll, 2006). The importance of p53 mitochondrial translocation in apoptosis is underscored by several observations. First, in cells or tissues that undergo cell cycle arrest (and not apoptosis), p53 mitochondrial translocation does not occur after exposure to genotoxic stress (Marchenko et al., 2000; Erster et al., 2004). Second, studies on the apoptotic potential of the proline and arginine p53 codon 72 polymorphic variants revealed that the proline codon 72 polymorphic variant, which is markedly impaired for apoptosis induction as compared with the arginine codon 72 variant, does not translocate to the mitochondria as efficiently as the arginine variant (Dumont et al., 2003). In a mouse model for B-cell lymphoma, p53 targeted exclusively to the mitochondria is efficiently tumor suppressive in vivo (Talos et al., 2005). In contrast, an artificial p53 chimeric mutant that is capable of robustly transactivating pro-apoptotic p53 target genes is capable of inducing senescence, but incapable of inducing apoptosis in knockin mouse embryo fibroblasts (Johnson et al., 2008). Moreover, the compound pifithrin m, which selectively blocks p53’s mitochondrial function but leaves intac
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