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Doing the right thing: feedback control and p53
Doing the right thing: feedback control and p53 Carol Prives C o l u m b i a University, New York, USA Recent evidence suggests that exposure of cells to DNA-damaging agents causes a rise in the levels of the p53 tumor suppressor protein and arrest of progression through the cell cycle, p53 may therefore resemble a member of the RAD gene class identified in yeast, RAD9, which allows cells to repair DNA before continuation of the cell cycle. The evidence that p53 is a sequence-specific, DNA-binding protein that can regulate transcription suggests several ways in which p53 might effect this growth cessation. Current Opinion in Cell Biology 1993, 5:214-218
Introduction When it was discovered that mutation within the p53-coding region is an extraordinarily frequent event in many types of human tumors (reviewed in [1]) it became clear that determining the normal function of p53 is of paramount relevance in eventually understanding the role of the protein in tumorigenesis, p53 has been defined as a tumor suppressor. This assumption is based upon several observations that include the following: a common event in many major forms of human cancer is loss of heterozygosity at the p53 locus, coupled with mis-sense mutation located within the central portion of the protein-coding region of the single remaining allele; mutant, but not wild-type, forms of p53 are oncogenic in cell transformation assays; wild-type p53 suppresses the ability of other oncogenes to mediate malignant cell transformation and can in some cases reverse the tumorigenic phenotype. Studies to determine properties of p53 in vivo led to the observations that when mammalian [2-5,6",7] and yeast [8,9] cells are induced to express high levels of wild type, but not oncogenic mutant, forms of the tumor suppressor, they arrest within the G 1 phase of the cell cycle close to the initiation of S phase. It has also been shown that cells exposed to high levels of wild-type p53 can undergo apoptosis [10,11]. The role of p53 in tumor suppression or prevention has underlined the importance of defining the p53 protein biochemically and of determining its pathway in normal cell physiology. Studies from several laboratories have converged to provide the first broad outline of the function(s) of this important protein. In this review, I discuss some of the recent observations about p53 and how they may converge to fit into a general framework. However, as is the case with many interesting new areas, several questions have yet to be answered.
Identification of a pathway that regulates p53 levels One problem that hampered the interpretation of experiments involving the expression of large quantities of p53 was their potentially non-physiological nature. It is well established that normal, non-transformed cells accumulate vanishing small levels of rapidly turning over wild-type p53 protein that in some cases are barely detectable. By contrast, for reasons not yet entirely understood, transformed cells or cells within human tumors often contain relatively high levels of mutant p53 that has a much longer half-life ([12] and references therein). Enthusiasm over experiments showing that wild-type p53 arrests growth at a specific phase of the cell cycle was thus mitigated by the possibility that cells expressing high levels of p53 might behave in an abnormal way for trivial reasons. Interestingly, however, circumstances have been identified in which transformed cells constitutively express high levels of wild-type p53 [13-15]. This suggests that a regulatory pathway exists for the control of p53 levels. Such a pathway has recently been identified through the work of Kastan and colleagues [16%17o,18..]. Their studies have not only provided exciting new insight into a possible normal role for p53 but they have also validated experiments in which ceils are manipulated to contain large amounts of wild-type p53. Maltzman and Czyzyk [19] reported that exposure of cells to ultraviolet light caused an increase in the rate of p53 synthesis. The significance of this finding was largely ignored until studies from Kastan's laboratory [16"] showed that transformed cells bearing mutant p53 were unable to arrest in G t after treatment with DNA-damaging agents such as ionizing radiation (IR). The Kastan laboratory [17"] then extended their initial observation by showing that when cells lacking the ability to express p53 were transfected with a wild-type p53 construct, they acquired the
Abbreviations AT--ataxia-telangiectasia;GADD--growth arrest DNA damage; IR--ionizing radiation.
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Feedback control and p53 Prives ability to arrest in G 1 after IR treatment. These observations became even more compelling when the authors recently showed that while several cell lines from normal patients were able to respond to IR by elevating their p53 levels, cells from patients with the inherited autosomal disease ataxia-telangiectasia (AT), which display increased sensitivity to irradiation and predisposition to cancer, fail to accumulate p53 after similar treatment [18oo]. This analysis has thus provided an intriguing link between the devastating AT disease and regulation of p53 levels.
Ways in which p53 may cause cells to arrest growth Now that a physiological state is known to exist in which normal cells express high levels of p53 and arrest growth, it has become even more relevant to imagine ways in which p53 might bring about a block to cell cycle progression. The prediction of possible routes that p53 might take to cause its inhibitory effects is greatly facilitated by revelations in recent years as to the type of protein that p53 represents, p53 had been shown to contain a strong transcriptional activation domain at its amino terminus [20-23] and to be a sequence-specific, DNA-binding protein [24,25]. More recently, the observations about its DNA-binding properties were extended such that the identification of a consensus sequence for p53 was made possible [26-,27.]. These two aspects of the p53 protein led several groups to test whether it can function as a transcriptional activator. As predicted, p53 was shown to stimulate transcription in a DNA binding dependent manner in vivo [27" 31"] and in vitro [32"]. The fact that p53 is a site-specific, DNA-binding protein that has the characteristics of a regulator of transcription suggests at least three possible ways in which it might repress cell cycle progression: activation of genes that negatively regulate cell growth; repression of genes whose products are necessary for cell cycle progression; and direct inhibition of DNA replication. Experimental evidence for all three routes has now been demonstrated.
A role for p53 activation function The Kastan group, in collaboration with the Vogelstein laboratory [18"], has found a candidate target of p53, the growth arrest DNA damage (GADD) 45 gene. Using subtractive cDNA hybridization methods, Fornace and colleagues [33] had characterized a set of GADD genes whose synthesis is increased when cells are subjected to DNA-damaging agents. Interestingly, cells of AT patients not only fail to increase p53 after IR treatment, but also fail to accumulate GADD45 [18..]. Kastan et al. [18"] have identified a p53-binding site and response element within the intron of the GADD45 gene. This provides the first link between the transcriptional activation function of p53 and a gene involved in growth arrest. It is hoped that additional genes with potential roles as growth suppressors that are activated by p53
will soon be identified. However, the discovery of the relationship between GADD45 activation and p53 levels is a most exciting start.
p53 as a transcriptional repressor
Several reporter gene co-transfection experiments have indicated that expression of a rather large number of promoter regions of cell cycle regulated genes are inhibited when cells accumulate high levels of p53 [6,34"-38"]. This is consistent with the role that p53 plays in repressing cell cycle progression. The ability of p53 to repress transcription from promoters that lack p53-binding sites in vitro has also been observed (G Farmer, C Prives, unpublished data; [39"]).
p53 inhibits DNA replication While p53 appears to arrest cells at a point in G 1 somewhat before the onset of S phase, the tumor suppressor may play an additional role in the direct regulation of DNA replication. This suggestion is supported by several disparate but provocative observations. First, in at least one cell type, p53 is found in the cytoplasm during G 1 but then moves into the nucleus at the onset of S phase and is detected there for up to 3 h [40]. Second, Lane and colleagues [41] have shown that p53 is associated with viral DNA replicative complexes in the nucleus. Third, experiments showing that p53 binds to SV40 T antigen and blocks the ability of the viral initiator to mediate the replication of plasmids bearing the SV40 replication origin in vitro [42-44], provide a mechanistic model for inhibition of DNA synthesis by p53. Fourth, it is intriguing that the first binding sites for p53 to be recognized were isolated from sequences within the vicinity of one well characterized viral [25] and two putative cellular [24] replication origins. This suggests that positioning of p53-binding sites proximal to a replication origin might reveal a direct regulatory effect by p53 on the functioning of that origin. This idea has been tested and it was found that plasmids bearing both the polyoma replication origin and p53-binding sites are defective as templates for the synthesis of DNA progeny in reactions supported by polyoma large T antigen when in the presence of wild-type but not mutant p53 protein (S Miller, C Prives, unpublished data).
How similar are the p53 and RAD9 gene products? If p53 functions in a pathway where cells respond to DNA damage by arresting growth, then it may function in a manner similar to a well characterized yeast checkpoint gene product, RAD9 [45-47]. The possibility that RAD9 and p53 have features in common has recently been discussed [48..]. Cells expressing mutant forms of RAD9 or p53 sustain a large number of mutations. However, while both proteins are functionally altered in response
215
216 Cell multiplication to DNA damage, differences as well as similarities can be noted. The HAD9 sequence bears little homology either to p53 or to other proteins [46,47]. Whereas p53 appears to function primarily in late G1, RAD9 causes arrest during G 2 [45], implying that the cascade or pathway in which HAD9 functions may be fundamentally different from that of p53. Moreover, while both products can function in the absence of new protein synthesis, levels of p53 but not HAD9 [46] increase in response to DNA damage, indicating that RAD9 function is controlled by a post-translational modification that is potentially different from that of p53. Additionally, the possible function of p53 as an inducer of apoptosis must be considered before comparing p53 to RAD9 too closely.
Does mutation of p53 initiate tumorigenesis? In keeping with p53's suggested function as a checkpoint factor and its identification as a tumor suppressor, p53 should play a role in the very early stages of tumor progression. Thus, cells bearing inactivating p53 mutations would fail to arrest and repair damaged DNA and would eventually sustain additional mutations leading to the acquisition of increasingly malignant characteristics. In keeping with the possibility that p53 normally prevents such genetic instability, compelling studies show that cells from p53 knock-out null mice [49 o.] or cells bearing mutant p53 genes [50 o.] display dramatically increased tendencies to amplify DNA when compared with normal wild-type p53-containing cells or even heterozygous cells containing wild-type and mutant forms of p53. However, despite these experiments, the prediction that p53 is an initiator of oncogenesis is not born out by examination of cancers or experimental systems. Although space here does not permit a detailed discussion, the published literature contains multiple examples showing that p53 mutation is a late event in tumor progression (e.g. [51]). Moreover, a role for mutant p53 in not only initiating but also maintaining the transformed state has been demonstrated [52"]. Importantly, Livingstone et al. [49"'] have identified wild-type p53 bearing genetically unstable tumor ceils.
Conclusion Clearly, it will be important to continue to study p53 on many fronts, as several issues remain to be resolved. What controls the levels of normal and mutant forms of p53 and why do they differ? Are there additional specific targets of p53? Does p53 function in more than one phase of the cell cycle? How do mutant p53 proteins contribute to tumorigenesis? An interesting protein binds to p53, the product of the m d m 2 gene [53..,54o.]. Does this gene have a role in the checkpoint pathway? Questions such as these are bound to keep researchers busy for years to come.
Acknowledgements This work was supported by US PHS grant CA33620.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
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The original observations that p53 can bind specifically to sites in vi ral and cellular DNA are extended in this paper. Using an ingenious screening technique, the authors succeeded in identifying a large number of human genomic binding sites for p53 and were able to identify a loose but informative consensus-binding sequence for p53. 27. •
FUNK WD, PAK DT, KARAS RH, WRIGHT WE, SHAY JW: A Transcriptionally Active DNA-binding Site for H u m a n p53 Protein Complexes. Mol Cell Biol 1992, 12:2866-2871. Using a random sequence binding site selection method, the authors identified a binding site for p53 that is consistent with that reported in [26*]. 28.
KERNSE, PIETENOLJA, THIAGALINGAM S, SEYMOUR A, KINZLER Ik'X~, VOGELSTEIN B: Oncogenic Forms of p53 Inhibit p53regulated Gene Expression. Science 1992, 256:827-830. Based on the observations that p53 contains an amino-terminal activation domain and binds specifically to DNA it was predicted that p53 functions as a transcriptional activator. This prediction was born out in co-transfection experiments, which showed that wild-type but not oncogenic mutant forms of p53 bind to and activate CAT reporter genes containing p53 binding sites. Furthermore, mutant p53s displayed a dominant-negative effect when co-transfected with wildtype p53 constructs. Additional related experiments are described in [29"-32"]. •
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C: Wild-type p53 Activates Transcription in vitro. Nature
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FORNACE AJ JR, NEBERT DW, HOLLANDER MC, LEUTHY JD, PAPATHANASIOU M, FARGNOLI J, HOLBROOK NJ: Mammalian Genes Coordinately Regulated by Growth Arrest Signals and DNA-damaging agents. Mol Cell Biol 1989, 9:41964203.
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SANTHANAMU, ANURADHAR, RAVINKUMAR S: Repression of the Interleukin 6 Gene Promoter by p53 and t h e Retinoblastoma Susceptibility Gene Product. Proc Natl Acad Sci USA 1991, 88:7605~609. See [34"]. 36. •
CHlNK V, UEDA K, PASTAN I, GOTTESMANMM: Modulation of the Promoter of the H u m a n M D R I Gene by ras and p53. Science 1992, 255:459 462. Wild-type p53 was shown to repress the MDR1 promoter. Interestingly, a mutant p53 stimulated expression from the same promoter providing the provocative suggestion that a mutant p53 can display a gain of function. (See [34"].) 37. •
KLEY N, CHUNG RY, FEY F, LOEFFLERJP, SE~INGER BR: Repression of the Basal c-fos Promoter by Wild-type p53. Nucleic Acids Res 1992 20:40834087. That the los basal promoter was inhibited by p53 in co-transfection experiments suggests a rather general mechanism for p53 repression of cellular genes. (See [34"].)
38. •
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48. °•
HARTWELLL: Defects in a Cell Cycle C h e c k p o i n t may be Responsible for the Genomic Instability of Cancer Cells. Cell 1992, 71:543-546. A provocative short article that reviews the current evidence that p53 may contribute to genetic instability in a manner analogous to the RAD9 checkpoint gene. 49. ••
LIVINGSTONELR, WHITE A, SPROUSE J, LIVANOS E, JACKS T, TESTYTD: Altered Cell Cycle Arrest and Gene Amplification Potential A c c o m p a n y Loss of Wild-type p53. Cell 1992, 70:923935.
Drug selection (PAIA) assays measuring CAD gene amplification were used to perform elegant experiments showing that primary cells from p53 knock out mice that lack both functional p53 alleles are dramatically more capable of gene amplification than are comparable cells bearing wild type p53. This implicates p53 directly as a determinant of genetic stablility. However, the fact that cell lines were identified bear ing wild type p53 that also display a high frequency gene amplification indicates that p53 is not the sole determinant of genetic instability. 50. ,.
YIN Y, TAINSKYMR, BISCHOFF FZ, STRONG LC, WAHLGM: Wildtype p53 Restores Cell Cycle Control and Inhibits G e n e Amplification in Cells with Mutant p53 Alleles Cell 1992, 70:937948. See [49"°]. 51.
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52. •
ZAMBETTIGP, OLSON D, LABOW M, LEVINE AJ: A Mutant p53 Protein is Required for Maintenance of the Transformed Phenotype in Cells Transformed with p53 Plus ras cDNAs. Proc Natl Acad Sci USA 1992, 89:3952-3956.
53.
MOMANDJ, ZAMBETTIGP, OLSON De, GEORGE DL, LE\qNE AJ:
•.
T h e m d m - 2 0 n c o g e n e Product Forms a Complex with the p53 Protein and Inhibits p53-mediated Transactivation. Cell
1992, 69:1237 1245. This paper describes biochemical and genetic characterization of the p90 product of the mdm-2 gene that binds to p53. Co transfection as says indicate that m d m 2 inhibits the ability of p53 to activate a reporter gene containing the muscle creatine kinase p53 responsive element. 54. ••
OLINERJD, KINZLERKXY¢',MELTZER PS, GEORGE DL, VOGELSTE1N B: Amplification of a Gene Encoding a p53-associated Protein in H u m a n Sarcomas. Nature 1992, 358:80-83. Human m d m 2 binds to p53 and is amplified in human tumors, suggesting that the interaction between the two proteins is necessary for normal cell regulation.
C Prives, Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Introduction When it was discovered that mutation within the p53-coding region is an extraordinarily frequent event in many types of human tumors (reviewed in [1]) it became clear that determining the normal function of p53 is of paramount relevance in eventually understanding the role of the protein in tumorigenesis, p53 has been defined as a tumor suppressor. This assumption is based upon several observations that include the following: a common event in many major forms of human cancer is loss of heterozygosity at the p53 locus, coupled with mis-sense mutation located within the central portion of the protein-coding region of the single remaining allele; mutant, but not wild-type, forms of p53 are oncogenic in cell transformation assays; wild-type p53 suppresses the ability of other oncogenes to mediate malignant cell transformation and can in some cases reverse the tumorigenic phenotype. Studies to determine properties of p53 in vivo led to the observations that when mammalian [2-5,6",7] and yeast [8,9] cells are induced to express high levels of wild type, but not oncogenic mutant, forms of the tumor suppressor, they arrest within the G 1 phase of the cell cycle close to the initiation of S phase. It has also been shown that cells exposed to high levels of wild-type p53 can undergo apoptosis [10,11]. The role of p53 in tumor suppression or prevention has underlined the importance of defining the p53 protein biochemically and of determining its pathway in normal cell physiology. Studies from several laboratories have converged to provide the first broad outline of the function(s) of this important protein. In this review, I discuss some of the recent observations about p53 and how they may converge to fit into a general framework. However, as is the case with many interesting new areas, several questions have yet to be answered.
Identification of a pathway that regulates p53 levels One problem that hampered the interpretation of experiments involving the expression of large quantities of p53 was their potentially non-physiological nature. It is well established that normal, non-transformed cells accumulate vanishing small levels of rapidly turning over wild-type p53 protein that in some cases are barely detectable. By contrast, for reasons not yet entirely understood, transformed cells or cells within human tumors often contain relatively high levels of mutant p53 that has a much longer half-life ([12] and references therein). Enthusiasm over experiments showing that wild-type p53 arrests growth at a specific phase of the cell cycle was thus mitigated by the possibility that cells expressing high levels of p53 might behave in an abnormal way for trivial reasons. Interestingly, however, circumstances have been identified in which transformed cells constitutively express high levels of wild-type p53 [13-15]. This suggests that a regulatory pathway exists for the control of p53 levels. Such a pathway has recently been identified through the work of Kastan and colleagues [16%17o,18..]. Their studies have not only provided exciting new insight into a possible normal role for p53 but they have also validated experiments in which ceils are manipulated to contain large amounts of wild-type p53. Maltzman and Czyzyk [19] reported that exposure of cells to ultraviolet light caused an increase in the rate of p53 synthesis. The significance of this finding was largely ignored until studies from Kastan's laboratory [16"] showed that transformed cells bearing mutant p53 were unable to arrest in G t after treatment with DNA-damaging agents such as ionizing radiation (IR). The Kastan laboratory [17"] then extended their initial observation by showing that when cells lacking the ability to express p53 were transfected with a wild-type p53 construct, they acquired the
Abbreviations AT--ataxia-telangiectasia;GADD--growth arrest DNA damage; IR--ionizing radiation.
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Feedback control and p53 Prives ability to arrest in G 1 after IR treatment. These observations became even more compelling when the authors recently showed that while several cell lines from normal patients were able to respond to IR by elevating their p53 levels, cells from patients with the inherited autosomal disease ataxia-telangiectasia (AT), which display increased sensitivity to irradiation and predisposition to cancer, fail to accumulate p53 after similar treatment [18oo]. This analysis has thus provided an intriguing link between the devastating AT disease and regulation of p53 levels.
Ways in which p53 may cause cells to arrest growth Now that a physiological state is known to exist in which normal cells express high levels of p53 and arrest growth, it has become even more relevant to imagine ways in which p53 might bring about a block to cell cycle progression. The prediction of possible routes that p53 might take to cause its inhibitory effects is greatly facilitated by revelations in recent years as to the type of protein that p53 represents, p53 had been shown to contain a strong transcriptional activation domain at its amino terminus [20-23] and to be a sequence-specific, DNA-binding protein [24,25]. More recently, the observations about its DNA-binding properties were extended such that the identification of a consensus sequence for p53 was made possible [26-,27.]. These two aspects of the p53 protein led several groups to test whether it can function as a transcriptional activator. As predicted, p53 was shown to stimulate transcription in a DNA binding dependent manner in vivo [27" 31"] and in vitro [32"]. The fact that p53 is a site-specific, DNA-binding protein that has the characteristics of a regulator of transcription suggests at least three possible ways in which it might repress cell cycle progression: activation of genes that negatively regulate cell growth; repression of genes whose products are necessary for cell cycle progression; and direct inhibition of DNA replication. Experimental evidence for all three routes has now been demonstrated.
A role for p53 activation function The Kastan group, in collaboration with the Vogelstein laboratory [18"], has found a candidate target of p53, the growth arrest DNA damage (GADD) 45 gene. Using subtractive cDNA hybridization methods, Fornace and colleagues [33] had characterized a set of GADD genes whose synthesis is increased when cells are subjected to DNA-damaging agents. Interestingly, cells of AT patients not only fail to increase p53 after IR treatment, but also fail to accumulate GADD45 [18..]. Kastan et al. [18"] have identified a p53-binding site and response element within the intron of the GADD45 gene. This provides the first link between the transcriptional activation function of p53 and a gene involved in growth arrest. It is hoped that additional genes with potential roles as growth suppressors that are activated by p53
will soon be identified. However, the discovery of the relationship between GADD45 activation and p53 levels is a most exciting start.
p53 as a transcriptional repressor
Several reporter gene co-transfection experiments have indicated that expression of a rather large number of promoter regions of cell cycle regulated genes are inhibited when cells accumulate high levels of p53 [6,34"-38"]. This is consistent with the role that p53 plays in repressing cell cycle progression. The ability of p53 to repress transcription from promoters that lack p53-binding sites in vitro has also been observed (G Farmer, C Prives, unpublished data; [39"]).
p53 inhibits DNA replication While p53 appears to arrest cells at a point in G 1 somewhat before the onset of S phase, the tumor suppressor may play an additional role in the direct regulation of DNA replication. This suggestion is supported by several disparate but provocative observations. First, in at least one cell type, p53 is found in the cytoplasm during G 1 but then moves into the nucleus at the onset of S phase and is detected there for up to 3 h [40]. Second, Lane and colleagues [41] have shown that p53 is associated with viral DNA replicative complexes in the nucleus. Third, experiments showing that p53 binds to SV40 T antigen and blocks the ability of the viral initiator to mediate the replication of plasmids bearing the SV40 replication origin in vitro [42-44], provide a mechanistic model for inhibition of DNA synthesis by p53. Fourth, it is intriguing that the first binding sites for p53 to be recognized were isolated from sequences within the vicinity of one well characterized viral [25] and two putative cellular [24] replication origins. This suggests that positioning of p53-binding sites proximal to a replication origin might reveal a direct regulatory effect by p53 on the functioning of that origin. This idea has been tested and it was found that plasmids bearing both the polyoma replication origin and p53-binding sites are defective as templates for the synthesis of DNA progeny in reactions supported by polyoma large T antigen when in the presence of wild-type but not mutant p53 protein (S Miller, C Prives, unpublished data).
How similar are the p53 and RAD9 gene products? If p53 functions in a pathway where cells respond to DNA damage by arresting growth, then it may function in a manner similar to a well characterized yeast checkpoint gene product, RAD9 [45-47]. The possibility that RAD9 and p53 have features in common has recently been discussed [48..]. Cells expressing mutant forms of RAD9 or p53 sustain a large number of mutations. However, while both proteins are functionally altered in response
215
216 Cell multiplication to DNA damage, differences as well as similarities can be noted. The HAD9 sequence bears little homology either to p53 or to other proteins [46,47]. Whereas p53 appears to function primarily in late G1, RAD9 causes arrest during G 2 [45], implying that the cascade or pathway in which HAD9 functions may be fundamentally different from that of p53. Moreover, while both products can function in the absence of new protein synthesis, levels of p53 but not HAD9 [46] increase in response to DNA damage, indicating that RAD9 function is controlled by a post-translational modification that is potentially different from that of p53. Additionally, the possible function of p53 as an inducer of apoptosis must be considered before comparing p53 to RAD9 too closely.
Does mutation of p53 initiate tumorigenesis? In keeping with p53's suggested function as a checkpoint factor and its identification as a tumor suppressor, p53 should play a role in the very early stages of tumor progression. Thus, cells bearing inactivating p53 mutations would fail to arrest and repair damaged DNA and would eventually sustain additional mutations leading to the acquisition of increasingly malignant characteristics. In keeping with the possibility that p53 normally prevents such genetic instability, compelling studies show that cells from p53 knock-out null mice [49 o.] or cells bearing mutant p53 genes [50 o.] display dramatically increased tendencies to amplify DNA when compared with normal wild-type p53-containing cells or even heterozygous cells containing wild-type and mutant forms of p53. However, despite these experiments, the prediction that p53 is an initiator of oncogenesis is not born out by examination of cancers or experimental systems. Although space here does not permit a detailed discussion, the published literature contains multiple examples showing that p53 mutation is a late event in tumor progression (e.g. [51]). Moreover, a role for mutant p53 in not only initiating but also maintaining the transformed state has been demonstrated [52"]. Importantly, Livingstone et al. [49"'] have identified wild-type p53 bearing genetically unstable tumor ceils.
Conclusion Clearly, it will be important to continue to study p53 on many fronts, as several issues remain to be resolved. What controls the levels of normal and mutant forms of p53 and why do they differ? Are there additional specific targets of p53? Does p53 function in more than one phase of the cell cycle? How do mutant p53 proteins contribute to tumorigenesis? An interesting protein binds to p53, the product of the m d m 2 gene [53..,54o.]. Does this gene have a role in the checkpoint pathway? Questions such as these are bound to keep researchers busy for years to come.
Acknowledgements This work was supported by US PHS grant CA33620.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
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The original observations that p53 can bind specifically to sites in vi ral and cellular DNA are extended in this paper. Using an ingenious screening technique, the authors succeeded in identifying a large number of human genomic binding sites for p53 and were able to identify a loose but informative consensus-binding sequence for p53. 27. •
FUNK WD, PAK DT, KARAS RH, WRIGHT WE, SHAY JW: A Transcriptionally Active DNA-binding Site for H u m a n p53 Protein Complexes. Mol Cell Biol 1992, 12:2866-2871. Using a random sequence binding site selection method, the authors identified a binding site for p53 that is consistent with that reported in [26*]. 28.
KERNSE, PIETENOLJA, THIAGALINGAM S, SEYMOUR A, KINZLER Ik'X~, VOGELSTEIN B: Oncogenic Forms of p53 Inhibit p53regulated Gene Expression. Science 1992, 256:827-830. Based on the observations that p53 contains an amino-terminal activation domain and binds specifically to DNA it was predicted that p53 functions as a transcriptional activator. This prediction was born out in co-transfection experiments, which showed that wild-type but not oncogenic mutant forms of p53 bind to and activate CAT reporter genes containing p53 binding sites. Furthermore, mutant p53s displayed a dominant-negative effect when co-transfected with wildtype p53 constructs. Additional related experiments are described in [29"-32"]. •
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48. °•
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Drug selection (PAIA) assays measuring CAD gene amplification were used to perform elegant experiments showing that primary cells from p53 knock out mice that lack both functional p53 alleles are dramatically more capable of gene amplification than are comparable cells bearing wild type p53. This implicates p53 directly as a determinant of genetic stablility. However, the fact that cell lines were identified bear ing wild type p53 that also display a high frequency gene amplification indicates that p53 is not the sole determinant of genetic instability. 50. ,.
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ZAMBETTIGP, OLSON D, LABOW M, LEVINE AJ: A Mutant p53 Protein is Required for Maintenance of the Transformed Phenotype in Cells Transformed with p53 Plus ras cDNAs. Proc Natl Acad Sci USA 1992, 89:3952-3956.
53.
MOMANDJ, ZAMBETTIGP, OLSON De, GEORGE DL, LE\qNE AJ:
•.
T h e m d m - 2 0 n c o g e n e Product Forms a Complex with the p53 Protein and Inhibits p53-mediated Transactivation. Cell
1992, 69:1237 1245. This paper describes biochemical and genetic characterization of the p90 product of the mdm-2 gene that binds to p53. Co transfection as says indicate that m d m 2 inhibits the ability of p53 to activate a reporter gene containing the muscle creatine kinase p53 responsive element. 54. ••
OLINERJD, KINZLERKXY¢',MELTZER PS, GEORGE DL, VOGELSTE1N B: Amplification of a Gene Encoding a p53-associated Protein in H u m a n Sarcomas. Nature 1992, 358:80-83. Human m d m 2 binds to p53 and is amplified in human tumors, suggesting that the interaction between the two proteins is necessary for normal cell regulation.
C Prives, Department of Biological Sciences, Columbia University, New York, New York 10027, USA.