The p53 Network (1998)

Loss of control of genomic stability is central in the development of cancer, and p53, by regulating normal responses to DNA damage and other forms of genotoxic stress, is a key element in maintaining genomic stability. Thus, it is no surprise that functional p53 is lost in about half of all human cancers. What about the other half? One possibility is that p53-independent regulatory mechanisms have been lost. Another is that inactivation of p53-dependent pathways can occur at any of several different points and that p53 itself is merely the most common target. For example, the p53 inhibitor Mdm2 is overexpressed in tumors independently of the p53 mutation. Here, we review pathways that signalin to p53, in response to different forms of stress, and pathways that signal out, triggered by activated p53. It is clear that p53 is the central component of a complex network of signaling pathways and that the other components of these pathways pose alternative targets for inactivation. For additional recent reviews, see Refs. 1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar and 2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar. The amount of p53 protein increases in response to a variety of signals, such as damaged DNA, arrest of DNA or RNA synthesis, or nucleotide depletion. The same stimuli also activate p53, which is mostly latent in the absence of stress. The increase in the amount of protein is often achieved through an increase in the half-life, from ∼30 min in untreated cells to ∼150 min in, for example, UV-treated cells (3Maltzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (818) Google Scholar). However, an increase in the rate of translational initiation of p53 mRNA can also affect the steady-state level of the protein (for example, see Ref. 4Mosner J. Mummenbrauer T. Bauer C. Sczakiel G. Grosse F. Deppert W. EMBO J. 1995; 14: 4442-4449Crossref PubMed Scopus (267) Google Scholar). The ubiquitin pathway probably plays an important role in degrading p53 (5Maki C.G. Howley P.M. Mol. Cell. Biol. 1997; 17: 355-363Crossref PubMed Scopus (300) Google Scholar), and evidence for a ubiquitin-independent mechanism of degradation has also been presented (6Kubbutat M.H.G. Vousden K.H. Mol. Cell. Biol. 1997; 17: 460-468Crossref PubMed Scopus (277) Google Scholar). Recent evidence has also shown that the Mdm2 protein, which binds to p53, accelerates its degradation, possibly through the ubiquitin pathway (7Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3750) Google Scholar, 8Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2860) Google Scholar). The fact that the mdm2 gene is a transcriptional target of p53 suggests a molecular basis for the commonly observed increased metabolic half-life of mutant p53 proteins defective in transactivation. Thus, the stability of these mutant proteins appears to be due to their inability to up-regulate the expression of Mdm2, a protein involved in their degradation, rather than an intrinsic property conferring resistance to degradationper se. An increase in transactivation due to p53, with no increase in the level of the protein, was found in cells treated with low doses of UV radiation, and microinjection of an antibody to the C-terminal domain also stimulated p53-dependent transcription, even in the absence of DNA damage (9Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar). Chernov and Stark (10Chernov M.V. Stark G.R. Oncogene. 1997; 14: 2503-2510Crossref PubMed Scopus (43) Google Scholar) found that sodium salicylate, which inhibits protein kinases inhibits the activation of p53, with no significant effect on the accumulation of the protein. Several processes might be involved in activating p53 (1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar), including phosphorylation, glycosylation, binding to regulatory proteins, alternative splicing, and acetylation (11Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar). How does p53 sense signals? Several known proteins are suspects. The DNA-dependent protein kinase (DNAPK), 1The abbreviations used are: DNAPK, DNA-dependent protein kinase; PARP, poly(ADP-ribose) polymerase; PALA,N-(phosphonacetyl)-l-aspartate; NBS, Nijmegen breakage syndrome; MAP, mitogen-activated protein; MAPK, MAP kinase; CAD, carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase. a plausible candidate, binds to and is activated by broken ends of DNA (12Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1027) Google Scholar) and can phosphorylate residues 15 and 37 of p53 in a DNA-dependent manner in vitro (13Lees-Miller S.P. Sakaguchi K. Ullrich S.J. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (465) Google Scholar). The phosphorylation of serine 15 affects the transactivation and growth arrest functions of p53 in some cells (14Fiscella M. Ullrich S.J. Zambrano N. Shields M.T. Lin D. Lees-Miller S.P. Anderson C.W. Mercer W.E. Appella E. Oncogene. 1993; 8: 1519-1528PubMed Google Scholar). However, cells lacking DNAPK show no defect in the p53-mediated inhibition of the cell cycle, revealing that if DNAPK has any role in regulating p53 at all, other components must be able to compensate for its loss (15Huang L.-C. Clarkin K.C. Wahl G.M. Cancer Res. 1996; 56: 2940-2944PubMed Google Scholar). Many protein kinases have been shown to phosphorylate p53 in vitro and are candidates for upstream regulators (1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar). However, very little in vivo evidence exists for the role of phosphorylation in regulating p53. Recent work showing that p53 can be acetylated in vitro is intriguing and suggests the possibility of an additional mechanism of regulation (11Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar). However, it is still necessary to show that acetylation occurs in response to stress. Poly(ADP-ribose) polymerase (PARP) has long been known to have a role in recognizing DNA damage and in DNA repair. PARP-null Chinese hamster cells are defective in activating p53 and resistant to apoptosis induced by DNA damage (16Whitacre C.M. Hashimoto H. Tsai M.-L. Chatterjee S. Berger S.J. Berger N.A. Cancer Res. 1995; 55: 3697-3701PubMed Google Scholar). However, embryo fibroblasts from PARP-null mice have normal DNA repair and DNA damage-induced apoptosis (17Wang Z.-Q. Auer B. Stingl L. Berghammer H. Haidacher D. Schweiger M. Wagner E.F. Genes Dev. 1995; 9: 509-520Crossref PubMed Scopus (715) Google Scholar), and although there is a significant decrease in the induction of p53 protein after DNA damage or nucleotide depletion, there is no change in p53 activity or in the cellular responses to stress (18Agarwal M.L. Agarwal A. Taylor W.R. Wang Z.-Q. Wagner E.F. Stark G.R. Oncogene. 1997; 15: 1035-1041Crossref PubMed Scopus (91) Google Scholar). Therefore, although PARP is involved in increasing the amount of p53 protein in mouse fibroblasts, other signaling pathways must be more important in activating p53 in response to DNA damage, consistent with experiments showing at least two levels of 53 regulation (9Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 10Chernov M.V. Stark G.R. Oncogene. 1997; 14: 2503-2510Crossref PubMed Scopus (43) Google Scholar). Loss of ATM, the product of the ataxia telangiectasia gene, slows the induction of p53 protein in response to the DNA strand breaks caused by γ-radiation but not in response to the pyrimidine dimers caused by UV radiation (19Kastan M.B. Zhan Q. El-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2931) Google Scholar, 20Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (775) Google Scholar). Similarly, p53 is induced normally in humanATM-null cells after treatment withN-(phosphonacetyl)-l-aspartate (PALA), which blocks de novo UMP biosynthesis, or adriamycin, which damages DNA. 2M. L. Agarwal and G. R. Stark, unpublished results. p53 and ATM may both be components of complexes that function in recombination (21Hawley R.S. Friend S.H. Genes Dev. 1996; 10: 2383-2388Crossref PubMed Scopus (65) Google Scholar). Similarly, the gene product involved in Nijmegen breakage syndrome (NBS) has also been placed upstream of p53 in the pathway that responds to ionizing radiation but not in the responses to other DNA-damaging agents (22Jongmans W. Vuillaume M. Chrzanowska K. Smeets D. Sperling K. Hall J. Mol. Cell. Biol. 1997; 17: 5016-5022Crossref PubMed Scopus (103) Google Scholar). Because the defects in p53 induction inATM-null, NBS-null, and PARP-null cells are partial or selective for certain kinds of DNA damage, these gene products are involved in some but not all of the signals. Double or triple knock-outs should have a more profound (perhaps even a complete) defect in p53 induction in response to DNA damage. Similar partial defects in p53 signaling have been observed in Fanconi anemia syndrome (FAS) and Bloom's syndrome (BLS) fibroblasts, suggesting that many pathways regulate p53 (20Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 23Rosselli F. Ridet A. Soussi T. Duchaud E. Alapetite C. Moustacchi E. Oncogene. 1995; 10: 9-17PubMed Google Scholar). Recently a role for oncogenic Ras and the mitogen-activated protein (MAP) kinase pathway in p53 modulation and function has been revealed in both human and rodent cells. High expression of Ras or activation of the Mos/MAPK pathway induces wild-type p53 levels and causes a permanent growth arrest, similar to cellular senescence (24Fukasawa K. Vande Woude G.F. Mol. Cell. Biol. 1997; 17: 506-518Crossref PubMed Scopus (107) Google Scholar, 25Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3994) Google Scholar). Cells lacking p53 can tolerate high levels of MAPK and display loss of p53-dependent cell cycle arrest and enhanced genomic instability (24Fukasawa K. Vande Woude G.F. Mol. Cell. Biol. 1997; 17: 506-518Crossref PubMed Scopus (107) Google Scholar). In a cell line defective in the MAP kinase pathway and in p53 expression, increased expression of the MAP kinase ERK2 restores the normal levels of p53, clearly placing ERK2 in a pathway that regulates the steady-state level of p53. 3M. L. Agarwal, R. Chilakamarti, W. R. Taylor, A. Agarwal, and G. R. Stark, manuscript in preparation. MAPK has been shown to phosphorylate residue 73 or 83 of murine p53 in vitro, and this phosphorylation may be important in stabilizing the protein (26Milne D.M. Campbell D.G. Caudwell F.B. Meek D.W. J. Biol. Chem. 1994; 269: 9253-9260Abstract Full Text PDF PubMed Google Scholar). Other kinases, such as DNAPK II, cyclin A-Cdc2, and cyclin B-Cdc2, are known to phosphorylate the p53 protein in vitro and may play a role in stabilizing it (14Fiscella M. Ullrich S.J. Zambrano N. Shields M.T. Lin D. Lees-Miller S.P. Anderson C.W. Mercer W.E. Appella E. Oncogene. 1993; 8: 1519-1528PubMed Google Scholar, 27Wang Y. Prives C. Nature. 1995; 376: 88-91Crossref PubMed Scopus (326) Google Scholar). The mechanisms of p53 induction in response to different types of stress are still largely unknown. p53 is involved in several different aspects of cell cycle arrest, apoptosis, control of genome integrity, and DNA repair (1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar, 2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). How does it regulate so many different processes? p53 is a tetramer that can bind to specific sequences and thus transactivate a group of genes (reviewed in Ref. 1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar; for example, p21/waf1,gadd45, mdm2, cyclin G,bax, and IGF-BP3). Several groups have found that active p53 is sensed differently at different promoters, resulting in differential DNA binding and transactivation (for example, see Ref.28Lohrum M. Scheidtmann K.H. Oncogene. 1996; 13: 2527-2539PubMed Google Scholar). p53 can also inhibit the expression of some genes (for example, see topoisomerase IIa (29Wang Q. Zambetti G.P. Suttle D.P. Mol. Cell. Biol. 1997; 17: 389-397Crossref PubMed Google Scholar)). Furthermore, some p53-dependent phenotypes do not involve transcriptional regulation at all (for example, see Ref. 30Caelles C. Helmberg A. Karin M. Nature. 1994; 370: 220-223Crossref PubMed Scopus (835) Google Scholar). Antibodies recognizing the C terminus of p53 prevent serum-stimulated fibroblasts from entering S phase (31Mercer W.E. Nelson D. DeLeo A.B. Old L.J. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6309-6312Crossref PubMed Scopus (184) Google Scholar). This result, originally interpreted as evidence that a positive function of p53 was required, posed a paradox when overexpression of wild-type p53 was found to cause growth arrest (32Michalovitz D. Halevy O. Oren M. Cell. 1990; 62: 671-680Abstract Full Text PDF PubMed Scopus (690) Google Scholar). The paradox was resolved when it was found that these antibodies activate rather than inhibit p53 (9Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar). It is now understood more clearly that p53 mediates G1 arrest in response to DNA damage caused by UV or γ-radiation, chemotherapeutic drugs, or nucleotide deprivation (33Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 34van Laar T. Steegenga W.T. Jochemsen A.G. Terleth C. van der Eb A.J. Oncogene. 1994; 9: 981-983PubMed Google Scholar, S.P. Clarkin K.C. A. A. Wahl G.M. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). The in p53-dependent G1 arrest is by with γ-radiation, which in normal fibroblasts causes p53-dependent arrest with induction of A. S.P. Clarkin K. Wahl G.M. Genes Dev. 1994; 8: PubMed Scopus Google Scholar). The of this arrest on the inability of these cells to repair even a of DNA so that the activating signal Clarkin K.C. Wahl G.M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). In of from a with wild-type p53, causes a Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), the line and the line which also have wild-type p53, to arrest in G1 H. C.G. Cancer Res. 1995; 55: Google Scholar). may that may involve the inactivation of components upstream or of p53, the cellular response to DNA damage to For example, p53 to on the of which binds to and inhibits kinases, of thus the of and the of any of these components may have an effect similar to that of p53 itself in the pathway from p53 is involved in a that blocks the of DNA when the has been the DNA of embryo fibroblasts was after treatment with or other of it was found that normal fibroblasts arrest with a of DNA, fibroblasts DNA of or S. B. 1995; PubMed Scopus Google Scholar). might through or might be by S In a murine cell line with wild-type p53, causes arrest, by G1 A.J. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). p53 is induced after is The that the p53 induced in response to damage blocks S phase was also by DNA in fibroblasts to or A. S.H. S.P. G. Wahl G.M. Cancer Res. 1997; Google Scholar). fibroblasts from mice do not their DNA when treated with consistent with the that G1 arrest in response to a of agents is in these cells C. S.J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). Therefore, p53 must also mechanisms to arrest cells in G1 and thus to inhibit in response to fibroblasts from mice more than two to with more than two and K. T. R. S. Vande Woude G.F. 1996; PubMed Scopus Google Scholar). p53 is with and thus may affect S.J. E. J. Cell. 1994; PubMed Scopus Google Scholar). of may signal p53 which in cause arrest in or It is intriguing that MAP kinase and both of p53, are also to (1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar, D.M. Campbell D.G. Caudwell F.B. Meek D.W. J. Biol. Chem. 1994; 269: 9253-9260Abstract Full Text PDF PubMed Google Scholar, Y. Jr., J. Biol. 1997; PubMed Scopus Google Scholar, G. S.J. J. van A.J. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar) p53, MAP kinase is important for Y. Jr., J. Biol. 1997; PubMed Scopus Google Scholar). In both human and mouse fibroblasts, overexpression of wild-type p53 can inhibit N. F. M. Oncogene. 1995; 10: Google Scholar, M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). Recent show that this property of p53 is important in a cell cycle that when DNA is R. Taylor, M. L. Agarwal, A. Agarwal, D. W. and G. R. Stark, for In cells in which of is very p53 plays a role in mouse embryo fibroblasts do not in In mouse embryo fibroblasts to entering with and high levels of However, occur and is the DNA. these responses are to prevent the of damaged or DNA. p53 plays a role in apoptosis in certain cell cells of such as DNA damage, of growth and expression of or can also cause p53-dependent apoptosis E. D. J. L. A. Oren M. Nature. 1991; PubMed Scopus Google Scholar, R.G. M.L. Nature. 1993; PubMed Scopus (2294) Google Scholar, S.W. Genes Dev. 1993; PubMed Scopus Google Scholar, M. E. Genes Dev. 1993; PubMed Scopus Google Scholar, A.J. N. Genes Dev. 1994; 8: PubMed Scopus Google Scholar). p53 must be able to function as a to the but p53-mediated apoptosis does not transcriptional inhibition of by or by does not affect p53-dependent apoptosis C. Helmberg A. Karin M. Nature. 1994; 370: 220-223Crossref PubMed Scopus (835) Google Scholar, A.J. N. Genes Dev. 1994; 8: PubMed Scopus Google Scholar). Furthermore, of protein p53-dependent apoptosis in the absence of transactivation Y. W.E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the proteins and are transcriptional targets of p53, suggesting that transactivation by p53 is important in apoptosis in some In the proteins and the protein can prevent p53-mediated apoptosis M. E. Genes Dev. 1993; PubMed Scopus Google Scholar, L. E. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). p53 induces apoptosis in some cell types but cell cycle arrest in in response to the same the mechanisms of such responses are not of cause cells that p53-dependent cell cycle arrest to apoptosis the role of in these cellular responses K. T. Vogelstein B. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). Several such as the of DNA damage and the levels of p53, also affect the cell cycle arrest and apoptosis X. L.J. L. Prives C. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). the p53 and pathways may be important in the responses to DNA damage. For example, the inactivation of in the loss of and induction of apoptosis after DNA damage Jacks T. Nature. 1994; PubMed Scopus Google Scholar). This might be by the of which when overexpressed on its can apoptosis D.M. Jr., Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). Furthermore, overexpression of blocks p53-mediated apoptosis Y. S. E. Vousden K.H. Oren M. Genes Dev. 1995; 9: PubMed Scopus Google Scholar). Thus, modulation of and through p53 signaling in response to DNA damage may play a central role in the cell cycle arrest and p53-dependent cell cycle control in of cells. is a used to In most or such as or which inhibit the of nucleotide for the of target genes products the carbamyl-P synthetase/aspartate in the of and for Chernov M.V. Agarwal M.L. Taylor W.R. Stark G.R. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). However, normal cells Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar) and cell such as M.E. M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar), do not to resistant in these The function of p53 is lost the of B. Nature. 1990; PubMed Scopus Google Scholar) and in the of cells D.M. A.J. Genes Dev. 1991; PubMed Scopus Google Scholar), that p53 can be the for gene embryo fibroblasts from mice are for gene A. J. E. Jacks T. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar), and human cells from as as lost their of wild-type p53 A. J. E. Jacks T. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, Y. Wahl G.M. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). of cells with or activated Ras p53-dependent cell cycle control and these cells to for gene M.E. M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). What signal is as a of the mechanism of gene that activate p53-dependent pathways and prevent the of The of as an in which broken DNA is an of cells G.R. Cancer Res. 1993; PubMed Scopus Google Scholar, A. E. F. G. M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The of DNA damage in regulating of gene was with cells with a mutant of Y. Chernov M.V. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). these cells are with at low active p53, the cells for gene of p53 by at a very in the of all cells DNA. cell can resistance to by mechanisms other than gene in which is by the most common mechanism in rodent cells. from several different human cell do not DNA at all or increase the of as Stark M.B. Agarwal A. Agarwal M.L. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). However, in both p53-dependent pathways are still The of pyrimidine caused by a signal for p53 induction any DNA damage occurs S.P. Clarkin K.C. A. A. Wahl G.M. Genes Dev. 1996; 10: PubMed Scopus Google Scholar), the cells and from Recent work has shown that or cells to the p53-dependent cell cycle arrest caused by DNA damage, these cells for gene Chernov M.V. Y. Agarwal M.L. Stark G.R. Mol. Cell. Biol. PubMed Scopus Google Scholar). This the fact that p53-dependent pathways can be at any of several different points p53 signaling pathways with and known to the cell cycle in components upstream or of p53 may be to inactivation of p53 all or a of the pathway from and to of cell cycle genomic and the development of In the protein which has a high of and functional to p53, may be important target for inactivation the development of M. H. A. L. A. A. Cell. 1997; 90: Full Text Full Text PDF PubMed Scopus Google Scholar). It to be if affects on or from p53 or if it is a central component of its signaling