The tumore suppressor p53

181

Biochimica et Biophysica Acta, 1155 (1993) 181-205 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-419X/93/$06.00

BBACAN 87270

The tumor suppressor p53 Lawrence A. Donehower

a

and Allan Bradley b

a Division of Molecular Virology and b Institute for Molecular Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX (USA)

(Received 8 January 1993)

Contents I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The p53 gene and protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The p53 gene and its transcript . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The p53 protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. The involvement of p53 in human and animal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p53 mutations and human spontaneous cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. p53 mutations and an inherited human cancer syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. p53 mutations and tumorigenesis in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Biological activities of p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mutant p53 and transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Biological activities of wild-type p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mutant p53 and immortalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Biochemical activities of p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p53 interactions with viral oncoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. p53 intbi:actionswith cellular proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. p53 oligomerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Transcriptional regulation by p253 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. p53 and DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. p53 conformational alterations and regulation of cell growth . . . . . . . . . . . . . . . . . . . . . . . . . VI. The role of p53 in the normal cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

181 182 182 183 184 184 186 187 189 189 190 191 191 191 192 193 194 195 196 198 200

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

200

I. Introduction T h e seemingly u b i q u i t o u s p r e s e n c e of p53 m u t a t i o n s in h u m a n t u m o r s has t h r u s t this previously u n h e r a l d e d t u m o r s u p p r e s s o r g e n e into the limelight. W h i l e n o t all h u m a n t u m o r s have p53 m u t a t i o n s , this g e n e is by far the most f r e q u e n t l y m u t a t e d of all the k n o w n t u m o r s u p p r e s s o r genes a n d o n c o g e n e s [1-3]. Discovered in 1979 c o m p l e x e d to the SV40 large T a n t i g e n in SV40t r a n s f o r m e d r o d e n t cells [4,5], the p53 p r o t e i n was initially classified as a t u m o r antigen. T r a n s f e c t i o n of the molecularly c l o n e d p53 g e n e into r o d e n t embryo

Correspondence to: L.A. Donehower, Division of Molecular Virology, Baylor College of Medicine, Houston, TX, USA.

fibroblasts suggested that p53 was a n o n c o g e n e as it was c a p a b l e of i m m o r t a l i z i n g these cells by itself or t r a n s f o r m i n g t h e m in c o n j u n c t i o n with the ras oncog e n e [6-9]. O n l y in the last few years has it b e c o m e clear that wild-type p53 b e h a v e s as a negative growth r e g u l a t o r or t u m o r s u p p r e s s o r gene. T h e earlier transfection studies d e m o n s t r a t i n g o n c o g e n i c p r o p e r t i e s of p53 were m i s l e a d i n g b e c a u s e m u t a n t forms of p53 were used [10]. M a n y m u t a n t forms of p53 are i n d e e d capable of b e h a v i n g in a n o n c o g e n i c m a n n e r [1]. T h e e v i d e n c e for the t u m o r suppressor activity of wild-type 53 is n o w conclusive. T r a n s f e c t i o n of wildtype p53 into t u m o r cell lines r e d u c e s or t e r m i n a t e s cell growth a n d division [11-15]. Loss of wild-type p53 alleles is exceedingly c o m m o n in h u m a n tumors; the first p53 allele may i n c u r a p o i n t m u t a t i o n , while the

182 remaining wild type allele is often lost in the progression of the tumor [2,3,16]. While co-transfection of mutated p53 and ras causes transformation of rodent embryo fibroblasts in culture [8,9], addition of wild-type p53 DNA to mutant p53 and ras results in a marked decrease in transformed colonies [17,18]. Human families with Li-Fraumeni syndrome, an inherited predisposition to cancer, have a mutated germ line p53 gene [19,20]. Some mice with Friend virus-induced erythroleukemia have rearranged or deleted p53 alleles in their tumor cells [21,22]. Finally, we have found that p53-deficient mice generated by gene targeting methods, with two p53 null alleles, develop normally, but are susceptible to tumors at a young age [23]. In this review, we will discuss the structure of the p53 gene and protein, the biological and biochemical activities of the p53 protein, and the role of p53 in transformation in vitro and in tumorigenesis in vivo. Special emphasis will be placed on some of the exciting new data which elucidates the potential role of wildtype p53 in the normal cell. While this review is extensive, it is not meant to be exhaustive, and those readers desiring further information should consult other recent reviews (1-3,24-29) or the primary papers. II. The p53 gene and protein II-A. The p53 gene and its transcript

The human p53 gene spans about 20 kb of genomic DNA and contains 11 exons (Fig. 1) and has been localized to the short arm of chromosome 17 (17p13) [30-35]. The mouse gene is on chromosome 11 [36,37]. PF1 Binding Site

NF-1 like Binding Site

I TGGCGACTATCCAGI

The mouse, human, and Xenopus laeuis p53 genes have similar 11 exon structures and identical exon boundaries, although the size of the 10 introns may vary considerably among these three species [31,38]. p53 cDNAs (generally ranging in size from 2-3 kb) have also been cloned from rat [39,40], chicken [41,42], monkey [43], hamster [31] and trout [31]. However, attempts to detect p53 related genes in invertebrate animals such as Drosophila, sea urchin, and yeast have so far been unsuccessful [31]. It is possible that p53 genes or genes with p53-1ike function exist in invertebrates since human p53 has been demonstrated to be functionally active as a transcription factor and negative growth regulator when introduced into yeast [4447]. Several important cis elements have been identified within the p53 gene which clearly regulate its expression. Some of these elements are shown in Fig. 1. Unlike typical genes transcribed by RNA polymerase II, p53 contains no TATAA or CAAT boxes [48,49]. Perhaps because of this and a potential stem loop structure in the 5' part of the gene [50], attempts to map the transcriptional start site have yielded quite different results. In the human gene, the major transcription start is at position -114 from the 3' end of exon 1 [51]. Data for the mouse gene indicate a major start site at -216 (+ 105 in Fig. 1) with at least two other alternative start sites [48,50]. Assays of 5' p53 sequences revealed enhancer activities 5' to the transcription start site, and unexpectedly, also within intron 1 [52,53]. High level expression from the 5' p53 promoter requires a helix-loop-helix recognition motif (CACGTG) at position + 70 within exon 1 p53 Responsive Element

HLHBindingSite

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E2 E3 E4

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Fig. 1. Structure of the murine p53 gene. The 11 exon murine p53 gene spanning approximately 16 kilobases of DNA is shown at the bottom. Each exon is marked E1 through E l l . The non-coding regions of exons 1 and 11 are shaded. P1 represents the major p53 promoter and P2 is a downstream promoter in intron 1 described in the human p53 gene [52]. The element shown as I4+ is an intron 4 element described by Lozano and colleagues [61,62] which positively regulates p53 expression. The key transcriptional regulatory region upstream of and within exon 1 is enlarged at the top of the figure with key regulatory sequences identified. The arrows represent the currently known sites for transcriptional initiation [50].

183 [54]. Reisman et al. [55] have shown that c-Myc/Max heterodimers bind to this recognition site and activate transcription from the mouse p53 promoter. In addition to this site, Ginsberg et al. [56] have identified a potential nuclear factor-1 (NF-1)-binding site at position +5 in exon 1 by DNAse I footprinting assays. They also identified at position - 6 4 another factor binding site which they termed the PF1 (p53 factor 1)-binding site. This site, which is conserved between mouse and human p53 genes, contains an element which is similar to the APl-binding site but does not bind AP1 efficiently as measured by competition binding assays. The PFl-binding site appears to be responsive to serum and to c-jun, a component of AP1, indicating that PF1 binding may be involved in mediating the response of the p53 gene to serum stimulation. Lozano and colleagues [57] have identified a site which may be involved in autoregulation of p53 expression. p53 promoter constructs co-transfected with wild-type or mutant p53 expression plasmids showed a 10-20-fold activation of transcription by wild-type p53 and no effect by mutant p53. Deletion mapping was used to minimize the p53 response element to sequences between + 55 and + 66 [57]. In addition to its 5' transcriptional control elements, p53 appears to contain at least two intron elements which are involved in its expression. For the human gene, Reisman et al. [52] have shown that a strong promoter, called p53p2, exists about 1000 bp downstream of the first exon within intron 1. The activity of the p53p2 promoter is significantly greater than the normal p53 promoter upstream of exon 1. Transcripts initiated from this promoter have been demonstrated in some cell lines by primer extension and appear to be increased in HL-60 cells induced to undergo terminal differentiation [58]. Whether this transcript represents an alternative p53 transcript or a separate gene transcript remains unclear. In the mouse p53 gene an

intron 1-initiated transcript of about 1.3 kb has been identified but apparently it is in the antisense orientation with respect to the p53 gene [58]. This antisense transcript is induced in mouse erythroleukemia ceils following differentiation and may act to downregulate p53 RNA expression through a post-transcriptional mechanism [58,59]. The importance of intron 4 in expression of p53 was demonstrated by Hinds et al. [60], who showed p53 cDNA expression constructs were approximately 10fold decreased in protein expression compared to genomic constructs or cDNA constructs containing only intron 4. Lozano and Levine [61] extended this finding in transgenic mice showing that p53 cDNA transgenes expressed very poorly, but transgenes containing cDNAs with intron 4 generated high levels of p53 transcripts in spleen cells. Recently, Beenken et al. [62] have shown protein binding to specific intron 4 sequences which is lost when point mutations are made in the putative binding site. The role of this binding site is unclear since it does not behave as a transcriptional enhancer in standard assays and does not have similarity with any elements known to be involved in splicing [61]. The mRNA transcribed from the p53 gene varies between 1.8 and 3.0 kb, depending on the species [31]. The 3' untranslated region is long and varies in size from 800 nucleotides for mice to 1800 bp for Xenopus laevis [38]. Alternative splicing has been reported for p53 but the biological significance of these observations remains unclear [63-65]. The level of p53 mRNA in normal cells appears to be highest in undifferentiated stem cells, spleen cells, cells undergoing rapid embryonic development (particularly during midgestation in the mouse) and other rapidly proliferating cell types [66-69]. In vitro, the level of p53 mRNA appears to be downregulated in tumor cell lines induced to differentiate [58,59] and upregulated in quiescent fi-

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Fig. 2. Structural features of the murine p53 protein. The 387 amino-acid murine p53 protein contains five highly conserved domains labelled I - V [31].The protein is also subdivided into three major regions based on charge. Serine phosphorylation sites and their corresponding protein kinases are shown above the protein [91-96]. The domains of the protein involved in oligomerization and binding to other viral and cellular proteins are also indicated. The phosphoserine-linked 5.8S rRNA covalently attached to p53 is shown [90].

184 broblasts induced to divide by serum stimulation [70]. These and other data suggest that the proliferative state of the cell is correlated with p53 mRNA levels. II-B. The p53protein

The cloning and sequencing of p53 cDNAs from a variety of species has allowed researchers to analyze structural and evolutionary features of the p53 protein. Soussi et al. [31] showed that some regions of the p53 protein are highly conserved at the amino-acid level throughout vertebrate evolution (Fig. 2). These conserved regions fall into five widely spaced amino-acid clusters termed domains I to V (Fig. 2). In some of these domains the sequence conservation among all the species is perfect over stretches of 14 or more amino acids, indicating a high degree of functional importance. Sequences between the conserved domains are much less conserved. Interestingly, as will be discussed in greater detail later, the mutations in p53 associated with human tumors tend to cluster in the conserved domains II through V [1-3]. Computer searches for characteristic motifs, such as zinc fingers and ATP-binding sites, have been negative [31]. However, amino-acid sequence analysis shows that the amino terminus of p53 is highly acidic. This is consistent with findings that the amino terminus shows transactivating function in yeast and mammalian systems [71-74]. In contrast, the carboxy terminus has a highly basic profile. Again, the basicity correlates with the fact that both DNA binding domains [75] and nuclear localization signals [76] are located in this C-terminal region (Fig. 2). Finally the middle region of the protein has a hydrophobic profile, particularly in the highly conserved domains II through V, suggesting an important conformational role. Indeed, point mutations in the highly conserved hydrophobic domains confer an altered conformation on the protein, as measured by the change in binding to conformationspecific monoclonal antibodies. As will be discussed later, it is these point mutations in the conserved domains that are the mutations most often associated with in vitro transformation activity and with human tumors [1-3]. The ability of p53 to form stable complexes with viral oncoproteins such as SV40 large T antigen was the basis for the discovery of this cellular protein [4,5]. The T antigen-binding domain of p53 has been localized to the region of p53 comprising the highly conserved domains III, IV, and V [77]. In addition to its ability to bind heterologous proteins, p53 is able to oligomerize with itself [78-83]. The capability to oligomerize is tightly correlated with transformation potential [83]. Recently, Sturzbecher et al. [78], Milner and colleagues [80,84] and Shaulian et al. [83] have demonstrated the minimal oligomerization domain to

be in the carboxy terminal region of the protein. In fact, Shaulian et al. [83] have demonstrated that even p53 miniproteins containing only amino acids 302-360 are capable of forming oligomers. The p53 protein is phosphorylated on multiple serine residues. Casein kinase II, which is stimulated in mitogen-induced cells, phosphorylates a C-terminal serine in mouse p53 in vitro [85-87] and can form a stable complex with p53 [85,87]. Interestingly, this serine has also been implicated as an attachment site for a covalently linked 157 nucleotide 5.8S rRNA [88-90]. Phosphorylation at the C-terminal serine appears to be a prerequisite for attachment of this RNA as it has been found covalently linked to phosphoserine [90]. The role of this attached RNA in mediating p53 function is unclear. The p34 cdc2 kinase, a key cell cycle control kinase, phosphorylates serine 315 in human p53 (equivalent to serine 312 in mouse) [91-93]. This cdc2 phosphorylation site is adjacent to the primary nuclear localization signal in p53 [76] and suggests a mechanism by which cdc2 could regulate entry of p53 into the nucleus during the cell cycle [91]. Finally, Wang and Eckhart [94] have mapped several in vivo phosphorylation sites in the amino terminal segment of mouse p53 to serine residues 7, 9, 18, and 37. Two of these sites, residues 7 and 18, have been shown to be phosphorylated by DNA activated protein kinase (DNA-PK) [95]. However, Milne et al. [96] also reported phosphorylation of serines 7, 9, and 12 by a purified casein kinase I-like enzyme. The role of phosphorylation in regulating p53 is beginning to be elucidated (see Section V.D) III. The involvement of p53 in h u m a n and animal cancer

I l i A . p53 mutations and human spontaneous cancers

The number of papers describing p53 mutations in various human tumors has grown explosively in the last three years. Several fine reviews deal in detail with the spectrum of p53 lesions in these tumors [1-3], so we will not go into great detail here on this issue. However, the extensive study of these mutations has revealed some interesting patterns that will be discussed. The first hint that p53 genetic lesions might be involved in human cancer came from early studies showing high levels of p53 in human tumor cell lines and human tumor tissues [97]. Since the mutant protein is usually much more stable than the wild-type protein, these results suggested an alteration in regulation or structure of the p53 gene in these cell lines. A second type of finding was that of the presence of anti-p53 antibodies in a fraction of cancer patients (with the absence of such antibodies in control individuals) [97]. Again, with the advantage of hindsight, such

185 antibodies may have arisen due to the altered conformation or novel epitopes of mutated p53 generated in these tumors. The first genetic evidence for p53 alterations in human tumors came from a study by Masuda et al. [98] indicating rearragements in the p53 gone in 3 of 6 osteosarcomas analyzed. Also suggestive of p53 alterations were studies by Vogelstein and colleagues and several other groups showing allelic losses on chromosome 17p, the site of the p53 gone, in colon carcinomas and other types of human tumors [99-105]. However, the seminal discovery by the Vogelstein group [106] of point mutations (resulting in amino-acid substitutions) in p53 in colon carcinomas accompanied by the loss of the second (presumably wild type) allele resulted in a subsequent avalanche of papers describing similar genetic phenomena in other human tumor types [1-3]. Follow-up studies [107] revealed that greater than 75% of colon carcinomas had 17p chromosome losses and 86% of these tumors had a mutation in the remaining p53 allele. The observed p53 mutations were relatively rare in early stages of tumor development (e.g., adenomatous stages) and were usually associated with the transition from benign to malignant growth (i.e., the carcinoma stage) [108]. The data were consistent with the idea that the wild-type p53 gone is a tumor suppressor which inhibits colorectal tumor growth and that its loss may be a key genetic event which ultimately leads to malignancy. The work on colon carcinomas by the Vogelstein group was soon followed by analyses of p53 mutations in other tumor types. Nigro et al. [109] found that a diverse array of human tumors have mutated or lost p53 alleles. Interestingly, while the usual pattern seems to be that of a point mutation accompanied by loss of the remaining wild type allele, a number of exceptions to the pattern can occur. For example, a small number of tumors were observed which retained both p53 alleles, one of which was mutated. It was hypothesized that such a tumor represents an intermediate stage in malignancy and that further growth selection events would result in clones which had lost the remaining wild-type allele [108]. As the number of reports of tumor-associated p53 mutations increased, it became clear that the substitutions were clustering within certain 'hot-spots' within the p53 gene. The vast majority of the mutations were in exons 5 to 8 between codons 130 and 290 [1-3]. As previously mentioned, the regions of highest mutability correlated extremely well with the evolutionarily conserved domains II-V identified previously by Soussi et al. [31]. Even the mutations which fell between the highly conserved domains tended to be in highly conserved codons. Three codons, 175, 248, and 273 stood out as extreme 'hot-spot' codons in colon and breast tumors (Fig. 3).

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Fig. 3. (A) The distribution of p53 mutations in human breast tumors, colon tumors, liver tumors, and lung tumors. The horizontal bar shows a schematic representation of the 393 amino-acid codons of the human gone. Vertical lines emanating from the horizontal line represent the frequency of mutations observed at a particular codon for each tumor type. The mutation hotspots are indicated by the tallest lines. Only nucleotide substitutions resulting in missense or nonsense mutations are included in these plots. The data for the 107 breast tumors was compiled from Refs. 109, 125, 274-287. Data for the 99 colon tumors was taken from Refs. 3, 107, 109, 288-292. Data for the 63 liver tumors was obtained from Refs. 3, 110, 111, 293-297. Finally, data for the 155 lung tumors was compiled from Refs. 3, 109, 298-311. (B) The distribution of p53 mutations in all four types of human tumors shown in Fig. 3A. The data from Fig. 3A are added together to generate a composite distribution for 424 human tumors.

Of all the mutational events observed in the p53 gone in human tumors, the most commonly observed are transitions (C: G to T: A and G : C to A: T) at CpG dinucleotides [2,3]. CpG dinucleotides are associated with spontaneous mutations. Indeed, the three hot-spot mutation sites mentioned above at codons 175, 248, and 273 are predominantly G : C to A : T or C : G to T : A transitions within CpG dinucleotides. While it is these types of apparently spontaneous CpG mutations which occur most frequently in the bulk of human

186 tumor types, there is another class of G : C to T : A transversion mutation which occurs quite frequently in lung cancer and hepatocellular carcinoma, and to a lesser but significant extent in breast cancer and esophageal cancer. The most dramatic example of this type of transversion mutation was the finding of genetic lesions in the same codon (codon 249) at the third position in all eight cases of liver tumors with p53 mutations from a population of individuals from the Qidong region of China [110]. A similar finding from African liver tumors showed three of ten individuals with codon 249 mutations [111]. The likely exposure of these afflicted individuals to aflatoxin B1, a common food contaminant in these regions and a potent liver carcinogen, has been used to explain the occurence of this particular type of mutation [110,111]. Many of the p53 mutations in these liver tumors were G to T transversions, which is the type of mutation generated by aflatoxins in mutagenesis experiments. The fact that the tumor types with the highest rate of transversions (e.g., liver and lung tumors) were also the tumors most likely to be affected by environmental mutagenic agents indicates that the examination of mutational spectra in the p53 gene may provide another powerful tool for the examination of the molecular mechanisms of mutagenesis and carcinogenesis [2,3]. The high frequency of p53 mutations in human cancers also has important clinical implications. A number of groups have found that the presence or absence of high levels of p53 in a tumor tissue as measured by immunohistochemistry can be a very good prognostic indicator of patient survival. In breast tumors [112,113], colorectal tumors [114,115], lung tumors [116,117], gastric tumors [114,118], and brain tumors [119], the presence of high levels of p53 is a significant predictor of shortened patient survival time. The benefit of such a prognostic tool is that those patients with tumors found to be p53-positive could be targeted for agressive therapeutic intervention [120]. While it may appear from the above studies that p53 mutations are virtually ubiquitous in human tumors, it should be noted that even in tumor types with the highest rate of p53 mutations there are a fraction in which p53 mutations or allele losses have not been found [107,109]. And there are some classes of tumors in which few or no p53 mutations have ever been observed [121-124]. There are three possible explanations for these failures to find p53 lesions. First, adequate testing of the tumor may not have been performed. In many of the studies, reverse transcriptase based polymerase chain reaction assays are performed on the tumor mRNA with primers which only amplify exon 5-8 sequences. While the bulk of the p53 lesions do fall within these exons, it is obviously possible to miss upstream or downstream coding changes and those changes which may occur in non-coding regulatory

sequences. Thus, determinations of the percentage of tumors with p53 mutations are likely to be somewhat underestimated. The second reason for a failure to observe overt p53 mutations in a tumor might be due to a mechanism recently described by Moll et al. [125] for human breast cancer. They found that 37% of the breast tumors that they analyzed had a pattern of intense cytoplasmic staining when exposed to a p53-specific monoclonal antibody, suggesting nuclear exclusion of p53. Sequencing of the p53 genes in seven of these particular tumors revealed that six were wild type. This was in contrast to the tumors with an intense nuclear staining pattern which consistently contained p53 genes with missense and nonsense mutations. The authors suggested that some breast tumors could functionally inactivate p53 without structurally altering it by sequestering the protein in the cytoplasm, away from its normal site of action in the nucleus [125]. While the mechanisms by which this sequestration of p53 takes place remain unclear, one possible scenario is that increased production of the mdm-2 oncogene product (via gene amplification) could functionally inactivate p53 by binding to it (see section V.B. for further discussion of mdm-2). Finally, the third explanation for a failure to detect p53 mutations in a tumor may simply be that p53 in these tumors remain structurally and functionally intact. In the study cited above by Moll et al. [125], three tumors were observed which lacked any p53 staining and which contained only wild-type p53 genes. Presumably, in these cases, p53-independent pathways of tumor progression have occurred. While it remains to be seen what percentage of tumors with wild-type p53 have cytoplasmic sequestration of the protein, it is likely that a sizable percentage of such tumors have normal p53 activity. III-B. p53 mutations and an inherited human cancer syndrome The high frequency of p53 mutations in spontaneous tumors led two groups to investigate the possibility that germ line mutations in the p53 gene could be responsible for any of the known inherited cancer syndromes. Malkin et al. [19] and Srivastava et al. [20] first demonstrated that families with Li-Fraumeni syndrome transmit a mutated form of p53 in their germ line. LiFraumeni syndrome is a relatively rare inherited autosomal dominant condition characterized by a greatly increased susceptibility to a variety of tumor types, the most frequent being breast carcinomas, soft tissue sarcomas, brain tumors, osteosarcomas, and leukemias [126-128]. Affected family members have a 50% probability of developing cancer by the age of 30, in contrast to a 1% probability in the general population. The criteria for identification of a family with Li-

187 Fraumeni syndrome consists of a family member with a childhood sarcoma and two first degree relatives with cancer before the age of 45 [127]. This definition may be a fairly stringent one and is likely to miss a significant number of families who have germ line p53 mutations. In addition, not all the families which meet the Li-Fraumeni criteria contain a germ line p53 mutation. At least one Li-Fraumeni family has been identified in which linkage analysis has demonstrated that the p53 gene segregates independently of the gene conferring increased cancer risk [129]. Malkin et al. [130], using different criteria for classification of increased cancer susceptibility, have found that 4 of 59 (6.8%) children and young adults with multiple independently arising neoplasms had germ line p53 mutations. None of these patients had a family history indicative of Li-Fraumeni syndrome. Toguchida et al. [131] showed that 8 of 196 (4%) patients with sarcoma had germ line p53 lesions, while none of 200 controls had germ line p53 alterations, suggesting that the frequency of mutant p53 alleles in the general population may be relatively low. In the first studies of p53 mutations in the LiFraumeni syndrome families, one remarkable aspect was the position of the mutations within the p53 gene. All were point mutations localized to exon 7 and between codons 242 and 258 [19,20]. Recent analyses have demonstrated germ line mutations in exons 4 through 9 [130-132]. In addition, four families have been identified with p53 mutations resulting in a prematurely terminated protein, indicating that nonsense mutations as well as missense mutations can confer the susceptibility to tumors [130]. The clinical characteristics and spectrum of cancers in the patients with germ line p53 nonsense mutations were not appreciably different than those in the patients with missense mutations [130]. The presence of a number of nonsense germ line mutations and a relatively limited spectrum of missense mutations (when compared to the range of somatic p53 mutations in spontaneous tumors) led to speculation that only those p53 mutations which lead to loss of function might be compatible with development, while the more oncogenic forms of p53 which complex wild-type p53 and act in a dominant negative fashion may be incompatible with development. However, subsequent characterization of some of the biochemical properties of mutant p53 proteins from LiFraumeni patients revealed that while six out of seven different Li-Fraumeni proteins (each with a single amino-acid substitution) lost growth inhibitory activity, some of the proteins displayed characteristics typical of dominant oncogenic p53 proteins, such as reactivity with the mutant conformation-specific monoclonal antibody PAb240 and enhanced binding to hsc70 [133]. However, that these particular inherited p53 mutations express a strong dominant oncogenic protein remains to be conclusively demonstrated.

A recent report by Lane and collaborators [134] suggests that another form of inherited cancer can occur in which family members inherit a predisposition to accumulate high levels of wild-type p53 in their cells. The high levels of accumulated p53 occur in the nuclei of both normal and tumor tissues. The individuals in the studied family had a history of multiple cancers with an early age of onset. The data suggest that these individuals may be inheriting a mutation in another gene which somehow acts to stabilize the wild type p53. Presumably, the stabilization may be functionally equivalent to loss of wild-type p53 activity (which turns over quite rapidly under normal conditions).

III-C. p53 mutations and tumorigenesis in animal models The first animal studies to link p53 with tumorigenesis in vivo were a result of transfection studies initiated by Wolf et al. [135], who demonstrated that transfection of mutant p53 into Abelson murine leukemia virus-transformed cells conferred dramatically increased tumorigenicity on those cells when implanted in syngeneic mice. While these results were intriguing, the animal studies with the greatest impact involved Friend virus-induced erythroleukemia. Approximately 30% of leukemic clones isolated from mice infected with Friend virus complex (which contains the replication-competent helper virus Friend murine leukemia virus and the replication-defective spleen focus forming virus) contained rearrangements, deletions, or proviral insertions in the p53 gene [21]. Many of the clones had lost both copies of the wild-type allele or had incurred point mutations in the non-rearranged allele [21,22,136-139]. One of the interesting features of the proviral insertions was that the provirus had clearly inactivated the p53 gene and prevented its normal expression [22,140]. This contrasts with the other murine retrovirus-induced leukemias in which a dominant-acting oncogene would be activated by integration adjacent to the proto-oncogene [141]. Together with the deletions and rearrangements of p53 in these tumors, these proviral inactivations provided some of the first clues that p53 loss was an important event in tumor formation and that wild-type p53 might be a tumor suppressor gene. Subsequent experiments by Lavigueur and Bemstein [142] suggested that p53 loss was connected with later, malignancy associated stages of erythroleukemia development. The development in 1989 of a transgenic p53 mouse by Lavigueur et al. [143] played an important role in the formation of ideas about the role of p53 in tumor development. Two transgenic mouse lines containing oncogenic mutant p53 transgenes (point mutations in codons 135 and 193) in multiple copies were developed. Incidentally, neither a wild-type p53 gene nor a construct containing a carboxy-truncated form of p53

188 (p44) could not be established in the mouse germline, suggesting that ectopically expressed wild type and some forms of mutant p53 are inimical to development [143,144]. The transgenic mice expressed very high levels of mutant p53 R N A and protein (in contrast to the low levels of endogenous wild-type p53 protein). Almost 30 percent of the mice developed tumors of varied type, with a high incidence of lung adenocarcinomas, osteosarcomas, and lymphomas [143,144]. There appeared to be little correlation between the level of mutant p53 expression in a particular tissue and its susceptibility to tumor development. Since p53 is a tumor suppressor gene, it might be expected that the endogenous wild-type p53 in the transgenic mice would be sufficient to prevent tumor development. However, the high rate of tumor development in these mice provided some of the first in vivo evidence that mutant p53 overexpression might act in a dominant negative manner to functionally inactivate the endogenous wild-type p53. That the relative levels of mutant and wild-type expression may be important for this dominant negative effect is illustrated by another set of p53 transgenic mouse lines developed by Lozano and Levine [145]. Perhaps because their transgenic mice contained a mutant p53 driven by a different promoter (SV40), their relative levels of p53 expression were much closer to wild-type p53 levels in many tissues [145]. These mice did not develop tumors, suggesting that mutant p53 may not be an efficient dominant oncogenic protein or that even low levels of residual wild type p53 are sufficient to retard tumor formation. Another type of animal model which has provided further insights into the activities of p53 in tumorigenesis is the p53-deficient mouse developed by us [23]. This mouse was developed by gene targeting techniques in embryonic stem cells so that one of the endogenous wild-type p53 alleles was converted to a null allele by gene disruption. Following standard protocols, mice were eventually generated that contained a germ line p53 null allele and a p53 wild type allele. When these heterozygous mice were crossed, approximately 25% of the resulting offspring were homozygous for the null allele. These homozygous mice appeared to be morphologically normal. Careful molecular analyses of the homozygote tissues revealed that they expressed no intact p53 RNA or protein. This surprising result indicated that p53 is not essential for development. As will be discussed later, if the hypothesized role of p53 (as a negative regulator of cell division in response to D N A damage) is correct, then its absence, while not immediately critical to cell cycle progression, may have other profound long term effects. Such long term effects soon became obvious. The homozygous mice had an extremely high susceptibility to the development of early tumors. By the age of 6 months, 74% of the homozygotes had developed tumors and by ten months

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Weeks Fig. 4. Incidence of tumors in p53-deficientmice. Mixed C57B1/6× 129/Sv mice with two (p53- / - ), one (p53+ / - ), or no (p53+ / + ) null p53 germ line alleles were monitored for tumors over 15 months. 60 p 5 3 - / - , 97 p53 + / - , and 95 p53 + / + animals were maintained in this study. The percentage of animals of each p53 genotype which remained tumor free were measured over time.

all of these mice had died or developed tumors (Fig. 4). The spectrum of tumors observed were fairly diverse, although lymphomas predominated. Interestingly, the heterozygous mice generated in these experiments also displayed an elevated level of tumor susceptibility, although the tumors in this group developed at a later age in a lower fraction of the mice. By 15 months of age, 27% of the heterozygotes had developed tumors of various types (Fig. 4; M. Harvey, A.B. and L.D., unpublished data). However, instead of lymphomas, osteosarcomas and soft tissue sarcomas were the predominant tumor type in the heterozygotes. Clearly, these p53-deficient mice provide definitive proof that p53 behaves as a tumor suppressor gene. In addition, the heterozygous mice may serve as a useful model for the Li-Fraumeni syndrome, since a number of the affected families have a p53 null mutation in their germ line [131] and develop a high number of soft tissue sarcomas [19]. Since the association of p53 mutations in so many spontaneous human tumors has been reported, it is not surprising that p53 mutations have been found in some carcinogen-induced animal tumors. The ability to manipulate the conditions and stages of tumorigenesis in animal models has obvious advantages. In addition, the use of F1 interspecific hybrid mice and mixed strain mice by the Balmain group and others can facilitate detection of relevant mutations during the stages of progression. Using F1 hybrids between Mus musculus and Mus spretis, Burns et al. [146] were able to identify putative tumor suppressor loci through loss of heterozygosity of restriction fragment length polymorphisms in the tumor DNA. Their model was the classic

189 mouse skin carcinogenesis system in which benign tumors (papillomas) are readily induced by a topical dose of a chemical carcinogen (DMBA). Secondary application of a tumor promoter (TPA) results in a fraction of the papillomas converting to malignant but well differentiated squamous cell carcinomas. They found loss of heterozygosity of chromosome 11 markers (the site of the mouse p53 gene) in 4/13 mouse skin carcinomas, but not in any premalignant skin papillomas. Sequencing of the remaining p53 allele in the carcinomas with allele loss revealed nonsense mutations or amino-acid substitutions in highly conserved p53 codons, some of which were identical to mutations found in human tumors. The correlation of p53 mutations with the progression from the premalignant papilloma state to the malignant carcinoma state is consistent with the observation of increased p53 mutations in the progression from adenomas to colon carcinomas in humans [108]. Other types of carcinogen-induced tumorigenesis have demonstrated the involvement of p53 mutations in animal tumors [147-152] and future studies will undoubtedly reveal further examples of such events. In a different animal model, p53 alterations may occur prior to progression from premalignant to malignant states. The preneoplastic lesions of the mammary gland, hyperplastic alveolar nodules, were found to contain overexpression of p53 in four of five transplantable hyperplastic lines [153]. Three of the four overexpressing hyperplastic lines had mutations in p53. Immunocytochemistry revealed that only about 10-50% of the cells in the outgrowths overexpressed p53. Progression to malignant tumors was associated with an increase in the proportion of cells overexpressing p53 to 100%. Thus, it appears that p53 overexpression or mutation may be either an early or late tumorigenic event, depending on the model system. The final example of a useful animal model revealing new aspects of p53 involvement in tumorigenesis is based on the mouse prostate reconstitution model developed by Thompson and colleagues [154]. Reconstituted mouse prostate cells infected with a ras-containing retroviral vector became hyperplastic but did not progress to carcinoma. These hyperplastic cells contained p53 mutations. In contrast, prostate cells infected with ras-myc containing vectors formed carcinomas which were associated with increased levels of stabilized p53 protein. Surprisingly, no mutations in the p53 genes in these carcinomas were observed. The authors speculated that expression of the activated myc gene somehow bypasses the need for p53 mutation by neutralizing the tumor suppressor activity of normal p53. This result is reminiscent of the cytoplasmic normal p53 accumulation seen in the human breast cancers by Levine and coworkers [125], and this model may provide a useful tool for analysis of the mechanisms of wild-type p53 sequestration in tumors.

IV. Biological activities of p53 IV-A. M u t a n t p53 and transformation

The first clear evidence that p53 was associated with transformation was obtained from in vitro cell transformation experiments. Co-transfection of mutant p53 and the activated ras oncogene onto primary rat embryo fibroblasts resulted in transformed colonies [8,9]. Transfection of mutant p53 alone onto rat embryo fibroblasts or established (immortalized) fibroblasts was insufficient for generation of transformed foci. The transformed cells induced by mutant p53 plus ras were tumorigenic when inoculated into rodents. Concurrent with these experiments, it was demonstrated that transfection of mutant p53 alone onto rat primary chondrocytes was sufficient to immortalize these cells, which normally undergo senescence after about 30 doublings [6]. This result was confirmed in experiments with rat embryo fibroblasts [7]. Since immortalization is commonly held to be an important step in tumorigenesis [155,156], the ability of mutant p53 to mediate this process provides at least one biological mechanism for its role in transformation. Apparently, overexpression of the mutant form of the protein is sufficient to override the endogenous wild-type p53 in a dominant negative manner by forming complexes with wild-type protein and functionally inactivating it. These early transformation and immortalization studies were performed under the mistaken belief that a wild-type p53 gene had been utilized for the experimental protocols. Consequently, it was hypothesized that p53 was an oncogene by virtue of its ability to provide a positive growth effect when overexpressed. However, early experiments by Jenkins et al. [157] suggested that mutant forms of p53 had oncogenic potential. The demonstration by Hinds et al. [60] that in fact the p53 clones in many of the previous experiments were mutant clones led to an abrupt reevaluation of the role of p53 in tumorigenesis. Finlay et al. [17] and Eliyahu et al. [18] showed that mutant but not wild-type forms of p53 could cooperate with ras to transform rat embryo fibroblasts. When wild-type clones of p53 were added to mutant p53 plus ras in the transformation assay, there was an active suppression of transformation by wild-type p53 as measured by transformed foci numbers. The few foci which did appear in the presence of wild-type p53 were shown to have lost the expression of the wild-type protein or to have the wild-type protein change to a mutant form. Nor was this suppressive effect of wild-type p53 limited to mutant p53-associated transformation. Cells transfected with adenovirus E1A plus ras or myc plus ras formed far fewer transformed colonies in the presence of wild-type p53. These in vitro experiments, in conjunction with the observations of loss of wild type p53

190 in a host of human and animal tumors, led the investigators to postulate that the p53 gene was a tumor suppressor gene and not an oncogene. Further insights into the tumor suppressor activities of wild-type p53 were facilitated by the discovery of a temperature sensitive mutant of p53 [158]. The p53val135 mutant, in conjunction with ras, can elicit transformation at 37.5°C, but at 32.5°C suppresses transformation [158,159]. Moreover, at 32.5°C, cells with p53val135 were greatly inhibited in their ability to proliferate, but this inhibition of proliferation and transformation was reversible upon temperature upshift, indicating that suppression by wild-type p53 was not merely a general lethal effect but a real growth arrest [158]. Cells arrested at 32.5°C were blocked in the G1 phase of the cell cycle [159]. The mutant conformation (recognized by PAb240) was the predominant form at 37° and 39.5°C, while the wild type conformation (recognized by PAb246) was predominant at 32.5°C [159]. Interestingly, at 37.5°C, the mutant form of p53 was found primarily in the cytoplasm, while at 32.5°C, the wild-type form was predominantly nuclear and had a shorter half life [159,160,161]. The data indicate that the primary targets for growth arrest are in the cell nucleus. Recent studies suggest that all mutant p53s are not equal in their transforming activities. Mutants can be divided into two major classes: (i) loss of function mutants which have merely lost their ability to suppress transformation and are weakly transforming or nontransforming [162]; and (ii) dominant oncogenic mutants, which, in addition to losing their tumor suppressor function, can actively participate in the transformation of cells. This second group of mutant p53s can cooperate with ras to transform rodent embryo fibroblasts, presumably in part as a result of their dominant negative activity in complexing and inactivating wildtype protein [1,2]. Mutant p53 genes isolated from human colorectal carcinomas were all capable of cooperating with ras in transforming primary rat cells, but the efficiencies of transformation varied [163]. In addition, other properties, such as the protein half life and ability to bind hsc70 were variable and not correlated with transforming ability [162,163]. IV-B. Biological activities of wild-type p53 A number of studies have shown that overexpression of wild-type p53 in tumor cells can have one of three results: (i) suppression of cell proliferation [11-15]; (ii) induction of apoptosis (programmed cell death) [164,165]; or [3] induction of differentiation [166,167]. Which pathway the tumor cell enters is probably dependent on the tumor cell type and levels of wild-type p53 expressed. As described previously, the tumor cells which are suppressed in their proliferation by p53

appear to be blocked in late G1 (11-15,168,169). Induction of apoptosis by overexpressed wild-type p53 in tumor lines may be a frequent event as it has been described by several groups in murine erythroleukemia cells [170,171], a myeloid leukemic cell line [164,172] and a human colon tumor cell line [165]. In all cases, when exogenously added wild-type p53 was induced, the cell lines (which contained no endogenous p53) began dying in a manner characteristic of apoptosis. It is believed that apoptosis is a distinct genetically programmed pathway for induction of cell death and is different in many characteristics from generalized necrotic cell death induced by injury or toxicity [173,174]. The colon tumor cells containing the wildtype p53 gene (inducible via a metallothionein promoter) were injected into nude mice and formed tumors in the absence of inducer, but when the inducer was then added (zinc chloride fed to the animals) the tumors regressed until the tumors were totally eliminated [165]. The cells induced to die in this case again had the characteristics of apoptotic cells. Interestingly, wild-type p53-induced apoptosis in the myeloid leukemic cells could be greatly reduced by administration of the cytokine IL-6 [164], which normally induces differentiation in this particular cell line. The authors hypothesized that wild-type p53 might mediate apoptosis in proliferating myeloid progenitor cells unless the appropriate differentiation signal is present. A balance between proliferation and apoptosis would maintain low quantities of these cells until needed in greater numbers [164]. Two recent studies have analyzed in greater depth the ability of wild-type p53 to mediate apoptosis. Ryan et al. [170] transfected the temperature sensitive p53Val135 into murine erythroleukemia ceils and found that induction of wild-type p53 in synchronized cells resulted in cell death after G1 arrest. Synchronized cells allowed to pass out of G1 prior to wild-type p53 induction continued to cycle until subsequent arrest in G1; loss of viability occurred following G1 arrest. Transfection of myeloid leukemic cells with p53val135 by Yonish-Rouach et al. [172] did not result in measurable growth arrest following wild-type p53 induction. However, apoptosis did occur in these cells following entry into G1, suggesting that p53-mediated cell death is not dependent on growth arrest. The interplay of p53 and differentiation suggested above is further confirmed by studies indicating that wild-type p53 itself may be sufficient to induce differentiation in some cases. Two recent papers reported that a pre-B cell line (L12) [166] and an acute erythroid CML cell line (K562) [167] retained their ability to proliferate, albeit at a reduced rate, when wild-type p53 was added to them. These cell lines with wild-type p53 had a higher proportion of cells in G1, longer doubling times, and lower levels of tumorigenicity than

191 their non-transfected parental counterparts. Strikingly, each cell line with wild-type p53 showed markers of increased differentiation. The pre-B cells were induced to produce cytoplasmic immunoglobulin heavy chain and increased levels of a B-cell surface marker, B220, which is characteristic of mature B lineage cells [166]. The erythroid acute phase CML cells with p53 expressed up to 50-fold more hemoglobin than their p53-minus parental cells [167]. IV-C. Mutant p53 and immortalization When differentiated animal cells are placed in tissue culture, they usually divide for a limited length of time and then undergo senescence, which is characterized by an absence of cellular division. In some cases, the senescent cells may remain in this state for an indefinite period of time, or they may die. In other cases, variant clones may emerge which have the ability to grow indefinitely, and these cells are called immortal. Rodent primary embryo fibroblasts, unlike human embryo fibroblasts, have a propensity to immortalize in culture with or without a preceeding senescence phase. Early studies showed that transfection of mutant forms of p53 into rodent fibroblasts (and chondrocytes) rapidly induced an immortal phenotype in the transfected cells [6,7]. Recently, studies of spontaneously immortalized mouse embryo fibroblasts revealed that these lines contained mutated or missing alleles of p53 [175,176]. Harvey and Levine [175] found that 11 clonally derived immortal BALB/c murine fibroblast lines generated by a classical 3T3 protocol (169; 3 x 105 cells plated per 60-mm dish every 3 days) all contained at least one mutant p53 allele. The types of events altering p53 structure and expression included point mutations in highly conserved regions, large deletions, and complete or partial loss of p53 mRNA expression [175]. In contrast to this result, Rittling and Denhardt [176] failed to find p53 mutations in their immortalized Swiss mouse embryo fibroblasts cultured by a 3T3 protocol. However, these investigators did observe missense mutations in immortalized fibroblasts cultured according to a 3T12 protocol (12 x 105 cells plated per 60-mm dish every 3 days). Further evidence for the importance of p53 in immortalization came from studies of SV40 large T antigen mutants, which showed that the ability of large T antigen to immortalize mouse embryo fibroblasts correlated perfectly with its ability to bind p53 [178,179]. In our own laboratory we have shown that mouse embryo fibroblasts derived from mice without functional p53 behave very differently in culture than mice with one or two normal p53 alleles (M. Harvey and L.D., unpublished data). All embryo fibroblast cultures from embryos without p53 do not enter a senescence phase and divide at a moderate to high rate indefinitely. This growth behavior is in strik-

ing contrast to cultures from mice with wild-type p53 which enter a prolonged senescence phase with essentially no cell division. Unlike murine fibroblasts, cells from many other species, such as chicken and humans, very rarely undergo immortalization. Ulrich et al. [180] showed that mortal chicken fibroblasts and erythroblasts could be induced to undergo occasional immortalization when transformed with temperat.ure sensitive v-src and L'erbB, respectively. The immortal clones which grew out all lost functional p53 at a relatively early stage following emergence from senescence [180]. Human diploid fibroblasts are extremely refractory to spontaneous immortalization. However, Bischoff et al. [181] were able to demonstrate that diploid fibroblasts from LiFraumeni syndrome patients (containing germ line p53 mutations) could undergo spontaneous immortalization in seven of eight cases. The fibroblasts that were able to escape senescence were highly abnormal karyotypically, and had a transformed morphology but were nontumorigenic in mice. Subsequent studies by the same group showed that the immortalized Li-Fraumeni fibroblasts could be transformed and made tumorigenic in nude mice by transfection of activated H-ras [182]. The immortalization of the Li-Fraumeni fibroblasts was correlated with loss of the remaining wild-type allele [183]. Experiments performed by Shay et al. [184] indicated that immortalized human diploid fibroblast lines containing inducible SV40 large T antigen could be reverted to a senescent state in the absence of inducer. When wild-type T antigen constructs or constructs defective for p53 binding were added to the reverted cells, only wild type T antigen could rescue the immortalized phenotype, arguing that loss of functional p53 (through binding to large T) is sufficient to allow immortalization. Finally, Hara et al. [185] showed that while addition of p53 antisense oligomers to human diploid fibroblast cultures did not delay their senescence, addition of p53 and Rb antisense oligomers together potentiated the senescence-delaying effects exhibited by antisense Rb alone. V. Biochemical activities of p53 V-A. p53 interactions with viral oncoproteins The discovery that the major oncoprotein of SV40, large T antigen, complexed with a cellular protein of 53 kd in transformed cells suggested immediately that this T antigen-bound protein played a significant role in the transformed phenotype [4,5186]. Subsequent studies showed that the p53 protein complexes to T antigen in large oligomers which stabilizes the p53 and increases its half life to such an extent that it is increased 100-fold or more in amount over the corresponding normal cell line [79,97,187,188]. That the ability to bind

192 p53 is tightly linked to large T's ability to transform is demonstrated by the fact that mutations in large T which abrogate p53 binding drastically reduce transforming efficiency [189]. The capacity to bind large T is restricted to wild-type p53 and some mutant p53s [133]. The binding of murine p53 to large T prevents the efficient replication of SV40 DNA by large T [190-193]. If p53 can bind to a cellular analog of T antigen involved in mediating S phase DNA replication or to sequences adjacent to cellular origins of replication, then this may explain at least one of the mechanisms by which p53 could prevent entry into S phase and prevent normal cell cycle progression (see later discussion). As p53 is a negative growth regulator which can arrest cells in late G1 [11-15], removal of a significant fraction of p53 by complexing with T antigen could tip the cell into S phase. Consequently, loss of normal p53 activity by binding to T antigen provides a cellular environment (S phase) more amenable to the replication of SV40 DNA (which requires an actively dividing cell for maximal replication efficiency). Oncoproteins of other DNA tumor viruses also form tight complexes with p53. In 1982, Sarnow et al. [194] showed that adenovirus E1B 55 kDa protein binds p53. E1B 55K protein is important for adenovirus transformation activity, as mutants in the 55K E1B gene are severely reduced in their ability to transform rodent cells [195]. Interestingly, the region of p53 which binds to E1B 55K protein resides in the amino terminal acidic domain, which is associated with the transcriptional trans-activating function of p53 [71,72,196]. In fact, it has recently been demonstrated by Yew and Berk [197] that the apparent binding of wild-type adenovirus type 2 E1B 55K and the equivalent adenovirus type 12 E1B 54K proteins to wild-type p53 severely inhibits the ability of p53 to mediate transcriptional transactivation, at the same time promoting transformation of rodent fibroblasts in conjunction with E1A. Mutants of E1B 55K and 54K which do not bind p53 allow p53 to transactivate and are severely reduced in transformation potential in cooperation with E1A. The third class of oncoproteins which have been found to complex with p53 are the E6 proteins of the oncogenic human papillomaviruses HPV-16 and HPV18. These viruses are associated with human anogenital cancers and encode two major transforming proteins, E6 and E7 [198]. Werness et al. [199] demonstrated that the E6 proteins of HPV-16 and HPV-18 form complexes with human and murine p53 protein in in vitro binding assays. Interestingly, the E6 of 'low risk' human papilloma viruses, such as HPV 6 and 11 which are only associated with benign warts, had no binding affinity for p53. Subsequent studies have indicated that even E6 proteins from 'low risk' viruses may bind p53, but at a lower avidity [200]. It is clear that 'high risk' HPV-16 and 18 E6 proteins actively stimulate degrada-

tion of bound p53 through ubiquitin-dependent proteolysis, while 'low risk' E6 proteins were unable to show any degradation effect [200,201]. These results suggest that, unlike the papovaviruses and adenovirus oncoproteins which appear to functionally inactivate p53 by binding and stabilization, the E6 proteins of oncogenic papilloma viruses, abrogate p53 function by facilitating its degradation. Recently, it has been shown that E6p53 binding is mediated by a second cellular protein, E6-AP, whose role may be to stimulate E6 and ubiquitin-dependent degradation of p53 [202]. p53 protein levels are greatly reduced also in HPVpositive cervical cancer derived cell lines and cell lines experimentaily immortalized by HPV E6 and E7 and 18 or by E6 alone, providing further in vitro evidence for the rapid degradation of p53 in E6 containing cells [203,204]. The HPV-positive cervical cancers usually contained wild-type p53 genes [203,205]. In contrast, HPV-negative cervical carcinomas were often found to contain high levels of mutant p53 protein [205-207], indicating that two routes of p53 inactivation are possible in cervical cancer: (i) in HPV-positive cases, the p53 remains wild type but is rapidly degraded; or (ii) in HPV-negative cases, the p53 gene is mutated and the protein is functionally altered. The three classes of DNA tumor viruses described above each have oncoproteins which complex with p53. In addition, it has become clear that the same viruses have oncoproteins which form complexes with the retinoblastoma (Rb) protein, another prototypical tumor suppressor [208-210]. In the case of SV40, T antigen forms a complex with RB and p53 while adenoviruses and oncogenic papillomaviruses complex p53 via separate oncoproteins than those which complex RB (Adenovirus E1A and HPV E7). Presumably, the function of both proteins must be abrogated simultaneously because in no case do the RB- and p53-binding domains in any of the oncoproteins overlap. All three viral oncoproteins may block p53 function by preventing transcriptional regulation by p53. The prevention of the normal tumor suppressor activity of p53 by viral oncoprotein binding should result in a more actively dividing cell. In addition, evidence (discussed in the previous section) suggests that mutant p53 may block apoptosis in some cells. Therefore, the loss of functional p53 could promote further mutations as a result of genetic instability fostered by continuous cell cycling. Loss of the ability of endogenous p53 to monitor for and allow correction of DNA damage (due in part to a block in apoptosis) would ensue (see Section VI). Some of the resulting mutations would be oncogenic and eventually a tumor cell could arise. V-B. p53 interactions with cellular proteins

The ability of p53 to complex with viral proteins has provided many important insights into the potential

193 role of p53 in normal cells. However, to have a fuller understanding about the pathways in the cell through which p53 acts, it will be important to identify its normal cellular protein targets. One important step in this direction was taken when Hinds et al. [158] showed that a cellular protein of 90 kD could be co-purified along with p53 following immunoprecipitation by p53specific monoclonal antibodies. This cellular p90 could complex with either wild-type or mutant p53. Barak and Oren [211] confirmed this p53 association. Momand et al. [212] were able to purify the complexed p90 to near homogeneity. Amino acid sequencing revealed that this protein was essentally identical to the product of the murine double minute 2 gene (mdm-2). Mdm-2 was first described as an amplified gene present in a spontaneously transformed murine cell line [213]. Addition of this gene to mouse cell lines enhances their tumorigenic properties [214]. The sequence of mdm-2 suggests that it may be a transcriptional regulator [212]. Of greatest interest is the fact that co-transfection of mdm-2 and p53 expression constructs results in greatly reduced trans-activation by p53 on a p53 response element (muscle creatine kinase 5' sequences) [212]. The data argue that mdm-2 acts to inhibit p53 trans-activation and consequently removes the negative regulatory effects of p53 in a manner analogous to that observed for viral oncoproteins. Support for this activity of mdm-2 was provided by Finlay [215], who showed that overexpressed mdrn-2 could immortalize primary rat cells and co-transfection of ras and mdm-2 into these cells generated transformed foci. Presumably, these oncogenic activities of mdm-2 were at least in part a result of inactivation of functional p53. The importance of mdm-2 in human cancer was recently demonstrated by Oliner et al. [204], who showed that the mdm-2 gene was amplified in over a third of 47 sarcomas studied. In five of these tumors with mdm-2 amplification no p53 mutations were observed, suggesting that the overexpression of mdm-2 effectively abrogated p53 activity and consequently p53 mutations were unnecessary due to the presumed mdm-2 complexing of available wild-type p53. Clearly, one of the exciting frontiers in the next few years will be the further study of the role of mdm-2 in regulating cell growth pathways through binding of p53. In addition to mdm-2, mutant but not wild-type forms of p53 have been shown to bind to a heat shock or chaperone protein called hsc70 [163,217-221]. Mutant proteins which show an altered conformation by virtue of their reactivity with the mutant p53-specific monoclonal antibody PAb240 often complex with hsc70 [186]. The role of this particular binding is not clear, but it has been speculated that it may chaperone or transport p53 protein into the nucleus [186]. Apparently, the altered conformation of mutant p53 prevents

a rapid dissociation from hsc70. Since the hsc70 protein has ATPase activity; it may be that higher concentrations of ATP might be needed to dissociate mutant p53 protein than wild-type p53. In fact, Hainaut and Milner [222] have characterized in vitro complexes of temperature sensitive mutant p53val135 and have shown that binding of hsc70 to p53 is dependent upon the mutant conformation and requires the C-terminal 28 amino acids of p53. In addition, change in conformation from mutant to wild type was an ATP-dependent process [222], confirming a probable role for the ATPase of hsc70. Finally, a specific peptide derived from the p53 amino terminal domain I is able to specifically bind hsc70 and inhibit the association between p53 and intact hsc70 (223; Fig. 2).

V-C. p53 oligomerization In addition to its ability to form interactions with heterologous proteins, p53 aggregates with itself to form oligomers. Early studies of wild-type p53 from mouse teratocarcinoma cells or in vitro translation of p53 mRNA in a rabbit reticulocyte lysate suggested formation of p53 dimers or tetramers [224,225]. Recent studies using non-denaturing gel electrophoresis and chemical crosslinking methods have confirmed that p53 forms tetramers and multiples of tetramers [226]. Oligomer formation occurs very rapidly [227] and is independent of conformation. Mutant p53 proteins with altered conformation (as determined by reactivity with mutant conformation-specific monoclonal antibody PAb240) can self-aggregate and complex with wild-type p53 protein [80]. A key feature of these complexes between mutant and wild-type proteins is that the wild-type protein in the complexes assumes a mutant conformation [84]. This apparent dominant negative action of mutant p53 on wild-type p53 in mixed complexes has obvious implications for a mechanism by which mutant p53 proteins could essentially abrogate normal p53 function in a cell with mutant and wild-type p53 alleles. The aggregation of mutant and wild-type proteins would be non-functional (or abnormal in function) due to the mutant conformation assumed by all the proteins in the mixed complex. Four of five mutant p53 proteins derived from non-small cell carcinomas of the lung were capable of forcing wild-type p53 into the mutant conformation in mixed complexes [84]. Interestingly, the one mutant protein without dominant negative activity was a codon 248 trp mutant, the same mutation identified in two Li-Fraumeni syndrome families [19]. The region of p53 associated with oligomerization has been localized to the carboxy terminal end. Mutants truncated at the C-terminal end by loss of 47 amino acids fail to form complexes [80]. A minimal oligomerization domain extending from amino acids

194 302 to 360 has been identified which corresponds precisely to the minimal transforming domain of p53 [83]. These C-terminal miniproteins (carrying no point mutation) transformed as efficiently as full-length mutant p53 in the ras co-transformation assays and were able to abrogate the ability of wild-type p53 to bind specifically to a DNA sequence containing a known p53 response element [83]. Sturzbecher et al. [78] have identified an amphipathic alpha helix region (codons 334-56 in the human protein) followed by a stretch of basic amino acids (codons 363-386) which appears to be essential for formation of tetramers. The major component for the self-aggregation process appears to span the alpha helix region, since substitutions in these hydophobic codons completely abolishes p53-p53 interactions. In contrast, mutations in the basic domain reduce the size of the p53 aggregates from tetramers to dimers. These oligomer-deficient mutants are unable to bind to SV40 large T antigen yet can still inhibit SV40 origin-directed DNA replication in vivo.

V-D. Transcriptional regulation by p53 In the past two years it has become clear that p53 is capable of transcriptional regulation of heterologous genes. The acidic domain of p53 is similar to that observed in many of the previously characterized transcription factors. The first functional evidence for transcriptional regulatory activity came from Fields and Jang [71] and Raycroft et al. [72]. These investigators demonstrated that fusion of the amino terminal region of p53 to a GAL4 DNA-binding domain could generate a chimeric protein which activated transcription from the GAlA promoter. Subsequent studies have localized the p53 transactivating domain to codons 20-42 [196,228]. The first mammalian gene which showed regulation by p53 was the muscle-specific creatine kinase (MCK) gene [229]. Co-transfection of mouse wild-type p53 with an MCK-CAT reporter construct (containing the 5' regulatory sequences of MCK) resulted in a 10-80fold CAT activation by wild-type 53 and no activation by mutant forms of p53 [229]. Zambetti et al. [230] showed that purified p53 directly bound to a 50 bp upstream MCK regulatory region by DNA binding assays and DNAse I protection assays. This 50 bp sequence was sufficient to confer wild-type p53 responsiveness to several minimal promoter-reporter constructs. The sequence of the p53 response element contained two 8-bp direct repeats (at nucleotides -3156 and -3149 of the MCK gene) which contained the TGCCT sequence previously identified by Kern et al. [231] as wild-type p53-binding sites. These investigators screened genomic DNA libraries for molecular clones which bound to wild-type p53. Two clones were iso-

lated by this method which bound wild-type but not mutant forms of p53. Sequencing of the bound fragments revealed the TGCCT repeats present in both clones as part of critical binding p53-binding domain. Since this initial report of specific DNA binding by p53, a second group has utilized a PCR-based reiterative selection procedure to identify a binding site for p53 slightly different from that identified earlier [232]. From a large pool of random oligonucleotides, a small subset was found to bind to p53. The consensus oligonucleotide most frequently isolated was a 20-mer, 5'GGACATGCCCGGGCATGTCC-3', which forms a perfect complementary palindrome between the two 10-base-pair subsequences which compose the 20-mer and which are imperfect repeats of each other. This sequence bound wild-type p53 in the presence of nuclear extracts but did not bind isolated p53, suggesting in this case that p53 interacts with other nuclear proteins to activate transcription. A more systematic PCR-based survey of genomic p53-binding sites by the Vogelstein group revealed a significantly less specific consensus sequence for DNA binding [233]. They found that wild-type p53 binds to two copies of the 10-basepair motif 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' separated by 0-13 base pairs [233]. The complementarity within and between the monomers shows extraordinary symetry (generating four half-sites) and suggests that p53 may interact with its target DNA sequence in at least a dimeric form, consistent with its oligomerization behavior. p53 acts in varied ways on many different cellular genes. For example, the MCK gene is upregulated directly by binding to the p53 response element in the promoter region of the MCK gene [230]. Further, when this p53 response element is grafted onto other reporter genes such as CAT, the reporter gene is transactivated in the presence of p53 [230,234,235]. The degree of transactivation in these studies is directly correlated with the ability of p53 to bind to the response element. Tumor-derived mutant p53 does not bind to the response element nor does it transactivate in these assays. Moreover, when mutant and wild-type p53 were coexpressed in the transfected cell, there was an active dominant negative effect on transcriptional activation by mutant p53 which appeared to be related to the inability of mixed mutant p53-wild-type p53 heterooligomers to bind DNA [234]. Reports of transcriptional activation of specific cellular genes by p53 have been relatively infrequent. However, the recent discovery of upregulation of the DNA damage response gene GADD45 by p53 has opened up a new area of investigation (236; see section VI). The bulk of the recent papers on transcriptional regulation by p53 focus on its ability to repress transcription in a wide variety of cellular genes. The first suggestion of transcriptional repression by p53 was

195 from Mercer et al. [11,15], who showed that glioblastoma clones with an inducible wild-type p53 gene were prevented from entering into S phase upon induction of the wild-type protein. Associated with the late G1 block by p53 was a significant reduction in S phase linked histone H3 mRNA and proliferating-cell nuclear antigen (PCNA) mRNA (PCNA is a component of the DNA replication machinery). Subsequent studies also showed that the late G1 genes B-MYB and DNA polymerase alpha genes were also repressed [169]. However, whether p53 was exerting a direct repressive effect on mRNA synthesis or an indirect effect due to its cell cycle block was unclear. Recent studies have shownthat cells co-transfected with wild-type p53 and a PCNA promoter driven CAT construct showed a 6-7-fold repression of transcription over controls [237]. Interestingly, when these co-transfection experiments were performed in Saos-2 cells (that do not express p53) with mutant p53 expression plasmids, up to an l 1-fold activation of PCNA-CAT expression over vector controls was observed [238]. Such results suggest that in addition to negatively affecting wild-type p53, mutant p53 may behave in a dominant oncogenic manner by stimulation of growth-related cellular target genes. In addition to PCNA, investigators have shown that wild-type p53 may negatively regulate a wide variety of cellular and viral promoters, including those from c-fos, beta actin, c-jun, IL-6, hsc70, Rb (retinoblastoma susceptibility gene), MDR (multidrug resistance gene), Rous sarcoma virus, HIV, HSV-1 (herpes simplex virus), SV40, and CMV (cytomegalovirus) [237-243]. Whether p53 represses genes in a manner similar to the way it activates them, by binding to a specific DNA sequence, is unclear. However, Shiio et al. [242] have identified a repeated cis-acting sequence, GGAACTGG, which confers the susceptibility to repression by wild-type p53. It was claimed that this 'repression' sequence is similar to a sequence near the SV40 origin of replication which binds p53 [193]. However, the similarity of the repression sequence to the consensus p53-response element identified by E1-Deiry et al. [233] is only partial. It is possible that p53 mediates its specific repression effect through another DNA-binding protein or that it may bind to two sets of distinct DNA elements, one which results in activation and the other resulting in a suppression effect. Evidence for repression through transcription factor binding is provided by two recent papers [244,245]. Agoff et al. [244] showed that p53 could repress transcription from the human hsp70 gene. In addition, p53 interacted with the CCAAT-binding factor (CBF), suggesting that p53 may downregulate transcription of the hsp70 gene through protein-protein interactions with CBF. In a second study, Seto et al. [245] demonstrated that wild-type (but not mutant) p53 inhibited transcription from minimal promoters

containing only the TATA sequence. Wild-type p53 bound TATA-binding protein (TBP), again suggesting that this particular protein-protein interaction plays a key role in repressing transcription from a large array of cellular genes. An exciting recent report by Hupp et al. [246] showed that bacterially produced p53 could not specifically bind its consensus DNA-binding sequence unless the C-terminal sequences were either (i) phosphorylated by casein kinase II; (ii) truncated by removal of the Cterminal 30 amino acids; (iii) incubated with C-terminal-specific monoclonal antibody PAB421; (iv) partially cleaved with trypsin; or (v) incubated with E. coli DNAK protein. The authors hypothesize that removal or alteration of the C-terminal domain of p53 by phosphorylation changes the conformation from an inactive form to an active form for DNA binding. Additionally, they hypothesized that the alteration of the C-terminal domain converts a latent or inactive higher order complex of p53 to an activated smaller oligomer form of p53. This activated DNA-binding form of p53 is presumably its active tumor suppressor form. Support for the importance of C-terminal phosphorylation in regulation of p53 activity has been recently provided by Milne et al. [247]. They found that replacement of the C-terminal serine 386 by alanine led to the elimination of p53 growth suppressor activity. Thus, the post-translational modification of this particular residue of p53 has profound effects on its functional activities and further illustrates the importance of phosphorylation in the regulation of a tumor suppressor protein.

V-E. p53 and DNA replication There has been evidence over the last several years that, in addition to its transcriptional regulatory activity, p53 may also have the ability to affect DNA replication in some contexts. Early in vivo studies showed that p53 could inhibit large T antigen-mediated SV40 DNA replication [190] and p53 could apparently compete with DNA polymerase alpha for binding to large T antigen [192]. This inhibition of SV40 DNA replication by wild-type p53 was further demonstrated in in vitro replication assays [191,248-250]. Wild-type murine, but not mutant human p53 blocks SV40 DNA replication [249]. Wang et al. [249] demonstrated that initiation of T antigen replication was inhibited by p53, specifically the origin-binding and helicase activities of the viral oncoprotein. In addition to a direct effect of p53 on SV40 DNA unwinding by T antigen, it appears that p53 may block T antigen mediated initiation of replication by binding to sequences adjacent to the late border of the SV40 replication origin [193]. In comparison, mutant forms of p53 did not bind efficiently to the origin sequences. In addition, it was shown that T antigen inhibits p53 binding to SV40 DNA [193], sug-

196 gesting a mechanism by which T antigen (in addition to its direct binding of p53) could promote cell division by preventing the inhibitory interaction of p53 with putative cellular DNA replication sequences. That p53 could directly interact with components of the cellular DNA replication apparatus was suggested by a recent study by Wilcock and Lane [250], who showed that, in herpes simplex virus type 1 (HSV-1) infected cells, p53 colocalized with several proteins (DNA polymerase alpha, PCNA, DNA ligase 1, single-stranded DNAbinding protein, and HSV-1 DNA-binding protein ICP8) associated with DNA replication. Whether p53 affects the ability of cells to divide by involvement in DNA replication clearly needs further investigation.

V-F. p53 conformational alterations and regulation of cell growth The observation that p53 was capable of altering its conformation was made possible by the development of a series of p53 monoclonal antibodies which recognize different conformational states of the protein [251254]. For example, the monoclonal antibody PAb246 recognizes wild-type murine p53 protein, while many mutated versions of the protein do not react with it [252,255]. In contrast, PAb240 is reactive with mutant forms of murine and human p53, but not wild-type p53 [253,254]. This recognition of different conformations of mutant and wild-type versions of p53 is not a fixed property of the mutant and wild-type proteins. The discovery of a temperature-sensitive mutant (p53Val 135) of p53 by Michalovitz et al. [158] was followed by the observation that this same protein could switch conformation between wild type (PAb246-reactive, PAb240-non-reactive) at low temperature and mutant (PAb240-reactive, PAb246-non-reactive) at high temperature [256]. The change in the temperature sensitive p53 from a wild-type conformation to a mutant conformation is accompanied by change in activity from that of a growth suppressor protein to that of a growth promoter [158,159]. In addition, Hainaut and Milner [222] have shown that the change in conformation of p53Val135 from wild type to mutant is ATP-independent in vitro (and associated with the ability of the new mutant protein to bind hsc70). In contrast, reversion of mutant p53Val135 from mutant to wild-type conformation is ATP-dependent. Hainaut and Milner [257] have also demonstrated that the conformational change (wild type to mutant) in a wild-type protein can be induced by chelating agents, suggesting that a divalent cation (probably zinc) stabilizes the tertiary structure of p53 in a wild-type configuration. One mechanism of action of the mutant conformation protein may be to convert co-existing wild-type p53 to a mutant conformation [27,108] and prevent its activity as a growth suppressor. Evidence for this mode

of action comes from experiments of Milner and coworkers [80,84], who demonstrated that when mutant and wild-type p53 were cotranslated in vitro, the wildtype p53 polypeptides were forced into a mutant conformation in the oligomers that were formed. However, this type of allosteric effect of mutant p53 observed in vitro may not necessarily occur in such a direct manner in vivo. Ullrich et al. [258] showed that in a human glioblastoma line containing an endogenous mutant p53 allele and an inducible wild-type allele, wild-type p53 is capable of existing in two pools. One pool is complexed wild-type and mutant p53 in a mutant conformation and the other pool consists solely of free wild-type p53 in a wild-type conformation. As G1 arrest is entered in these cells, the amount of wild-type p53 complexed with mutant p53 decreases and the free wild type complexes increase, suggesting that factors other than p53 interactions may regulate the conformation of the wild-type p53. Interestingly, in this same paper the authors demonstrated that the wild-type p53 in the free wild-type complexes was phosphorylated to a significantly higher degree than the mutant protein. Therefore, the ability of wild-type p53 to exert an antiproliferative effect is correlated with the wild-type conformational state and an increased level of phosphorylation [258]. The above experiments suggest that wild-type p53 protein can change its conformation depending on the growth or cell cycle status of the cell. Milner and coworkers have shown that this conformational change may take place in the wild-type protein even in the absence of mutant protein. Milner and Watson [259] showed that p53 conformation was independent of the cell cycle. However, growth stimulation of fibroblasts by addition of fresh medium resulted in a change of wild type p53 conformation from PAb246-reactive to PAb246-non-reactive. They had previously observed p53 changes in conformation in stimulated lymphocytes [260]. Milner [261] has put forth a model which argues that the PAb246-reactive conformation acts as a growth suppressor, while the PAb246-non-reactive form is a growth promoter. The induction of a mutant-associated conformation in the wild-type protein following growth stimulation might be interpreted merely as a loss of suppressor function rather than the gain of promoter function. However, Mercer et al. [262] have shown that microinjection of anti-p53 monoclonal antibodies into the nuclei of murine 3T3 cells at various times after re-stimulation of serum-starved cells resulted in inhibition of the growth response when the antibody was injected early after stimulation. Antisense p53 constructs have also shown to be inhibitory to cell growth [263,264], suggesting that wild-type p53 does stimulate cell growth in certain contexts (following serum stimulation) as well as suppress it. The Milner model has obvious implications for cells with mutated forms of

197 Milner [261], Ullrich et al. [29], and Hupp et al. [246]. The composite model postulates that there are actually three states of p53 activity: (i) a latent or inactive state (composed of a higher order multimer of p53); (ii) a suppressor state which mediates G1 arrest; and (iii) a promoter state which stimulates growth. The idea of a latent state for p53 is derived from the recent study of Hupp et al. [246] who showed that full DNA-binding activity (presumably consistent with full suppressor activity) was conferred on p53 only after cleavage or phosphorylation of the C-terminal domain. According to the composite model, cells cycling in a normal fashion would have most of their p53 in the inactive state. In response to a particular stress (e.g., DNA damage), casein kinase II or other kinases would phosphorylate p53 to induce G1 cell cycle arrest via transcriptional regulation of appropriate cellular target genes. After the stress is removed, the G1 arrested ceils would need an extra push to get back into a normal cell cycle. To achieve the reentry into the cell cycle, phosphatases would remove the C-terminal phosphate and, together with other cell components,

p53 found in tumor cells. Presumably, these mutated p53 proteins are abnormally stabilized in a growth promoting conformation which stimulates the tumor cell to divide. In cells with one mutant allele and one wild type allele, mutant p53 may complex with wild-type p53 and drive it into a mutant conformation. However, as Ullrich et al. [258] have shown, this apparent dominance of the mutant form in mixed complexes may be insufficient to prevent wild-type p53 from forming free complexes in the wild-type conformation in some situations. Such incomplete dominance by mutant p53 may explain why there appears to be a selection for loss of the remaining wild-type allele in many tumors with a p53 mutation. Additionally, the ability of mutant p53 to act in a growth promoter fashion suggests that it may be capable of acting on a different set of cellular targets than wild-type p53 to effect cell growth. Alternatively, mutant p53 could interact with wild-type targets but in a different manner than wild-type protein. To illustrate and integrate some of the current ideas about p53 conformational regulation we have presented Fig. 5, which draws on models presented by Promoter/Mutant Conformation

Suppressor/Wild Type Conformation

PAb421+/Pab240+

PAb421-/Pab240-

CONFORMATION CHANGE .

~

Phosphatases?

~-~

OLIGOMERIZATIONCHANGE

I t31

"~

Latent Conformation Higher Order Oligomer

Casein Kinase II

p --L_.~._]-- P

C

C

C

C

C

C

C

C

Casein Kinase I1? Other Kinases? ATP, Zn+2 xs A

Growth Mutantp53 Regu atory Activty Positive

Cellular Targets?

/

Activation of Transcriptional RegulatoryActivity

(1) Stimulation of Cellular Target Genes (via bindingto I)53 responseelement) (2) Repressionof Other Cellular Genes

through bindingto transcnptionfactors (e.g. TBF, CBF)

GROWTH STIMULATION

G1 GROWTH ARREST

INACTIVE

Fig. 5. Model for p53 conformation and oligomerization changes. Oiigomers of p53 associated at their C-termini are shown in both their wild-type suppressor (rectangles) or mutant promoter (rounded off rectangles) conformation. Higher order oligomers shown at right are considered to be in the latent or inactive state. Phosphorylation of the C-terminal serine in p53 by casein kinase II has been shown to activate site-specific DNA binding by p53 1246].Other kinases may also be important for this change. Presumably, the phosphorylation event(s) drives the p53 complex into a wild-type conformation (as measured by absence of reactivity with mutant conformation-specific monoclonal antibodies such as PAh240) or out of the higher order oligomer (if in the latent state).Based on recent studies by Hainaut and Milner [222,257], p53 may also require ATP and zinc to enter into a stable wild-type conformation. In its wild-type conformation, p53 can bind to its response element and transcriptionally activate a battery of cellular genes which act to drive the cell into G1 growth arrest, p53 may also repress other growth stimulatory genes through binding to transcriptional regulatory factors such as TBF (TATA binding factor) [245] and CBF (CCAAT binding factor) [244]. The p53 proteins in the mutant conformation modulate a growth stimulatory response, but the mechanisms of growth stimulation remain unclear. This model was adapted from previous models published by Milner [261], Ullrich et al. [29], and Hupp et al. [246]. While Hupp et al. [246] have shown that cleavage of C-terminus can also generate an active p53 from an inactive precursor, we hypothesize that casein kinase II phosphorylation represents the physiologically relevant activation step.

198 the activated suppressor p53 would be driven into a growth promoter conformation characteristic of the mutant oncogenic p53 proteins. This promoter p53 would then help drive the cell into S phase before being degraded. Once into the normal cell cycle, the need for either promoter or suppressor p53 would be lessened and the inactive form would predominate again. This model is speculative, but is consistent with at least some of the observations regarding p53 conformational changes. VI. The role of p53 in the normal cell

The ability of the p53-deficient mice to develop normally clearly demonstrates that p53 is not essential for normal cell division, cell differentiation, or embryonic development. On the other hand, the susceptibility of the mice to tumors of widely varied type argues that p53 does play some sort of global protective role that protects the cell from tumorigenic progression. With all our knowledge about the biological and biochemical activities of p53, there have been few clues about the basic function of p53 in the life of the normal cell. However, some recent studies have opened fascinating new insights which may lead to the long awaited answer to this question. The first clue to a potential role for normal p53 was obtained from experiments in 1984 which showed that treatment of mouse fibroblast lines with ultraviolet light or UV-mimetic drugs resulted in a rapid increase in p53 protein levels due in large part to increased stability of the protein [265]. This early result was recently corroborated in vivo by induction of p53 following UV-irradiation of intact human skin [266]. Kastan et al. [267], using normal bone marrow myeloid progenitor cells and ML-1 myeloblastic leukemia cells (with wild-type p53), showed similar increases in p53 protein levels in response to DNA damaging reagents such as gamma irradiation and actinomycin D. Exposure to the DNA damaging agents caused a temporary arrest in G1 of the cell cycle in cells with wild type p53 but not with mutant p53 [267]. Transfection of wild-type p53 into malignant ceils lacking functional p53 resulted in a restoration of G1 arrest following ionizing radiation. This result was further corroborated with fibroblasts derived from p53-deficient mice homozygous for a p53 null allele. Fibroblasts lacking p53 failed to arrest in G1 after ionizing radiation [236]. The above data suggests a role for p53 in cell cycle control following DNA damage. In models put forth by Kastan [236] and Lane [268], the role of p53 is to act as a sort of molecular guardian for genomic integrity. If DNA is damaged, p53 is induced, stabilized or activated and arrests the cell until the damage is repaired. If the damage cannot be repaired, the p53 might initiate programmed cell death (apoptosis) as is observed when

wild-type p53 is added to certain types of tumor cells [164,165]. Obviously, in pre-neoplastic or neoplastic cells missing functional p53, this monitoring mechanism would be inactivated and the cells would be genetically unstable due to failure to fully repair DNA damage prior to S phase entry. Presumably, mutations and chromosomal rearrangements could accumulate in the absence of the p53 monitoring function, leading to further oncogene and tumor suppressor gene alterations and malignant progression. A more direct linkage of p53 to a DNA damage response pathway was recently described in a seminal paper by Kastan et al. [236]. Cells derived from patients with the radiation-sensitive, cancer-prone disease ataxia-telangiectasia (AT), which, like p53-deficient cells, fail to arrest in G1 following ionizing radiation, did not display induction of p53 following ionizing radiation. This result suggests that at least some of the AT complementation group genes are upstream regulators of p53 in this particular DNA damage-inducible signal transduction pathway. In addition, a human growth arrest and damage-inducible gene, GADD45, which is induced in response to DNA damaging agents (including ionizing radiation) in normal cells, is not significantly upregulated in various p53-deficient cells. This latter finding argues that p53 itself is an upstream regulator of GADD45 in the same radiation-induced signal transduction pathway that controls cell cycle arrest following DNA damage. That p53 directly regulates GADD45 was supported by the ability of a p53containing nuclear factor in extracts from radiationtreated cells to bind a p53 response element found within the GADD45 gene. Kastan et al. [236] postulate that radiation-induced DNA damage results in an increase in p53 protein levels via AT gene products and this increase induces the expression of GADD45 and other effector genes which then arrest the cell in late G1 until the DNA damage is repaired. Since p53 is clearly a central component of at least one DNA damage response pathway in the cell, its loss would certainly have important implications for cellular genetic stability. In fact, some of the most compelling evidence that loss of p53 may promote genome instability was provided by studies of Livingstone et al. [269] and Yin et al. [270]. These investigators showed that gene amplification occurs in response to the uridine biosynthesis inhibitor PALA with significant frequency only in cells devoid of p53. While cells with normal p53 arrest in G1 in response to PALA, a fraction of p53-deficient cells could apparently proceed into S phase in its presence. Li-Fraumeni fibroblasts with one wild-type p53 gene were incapable of gene amplification while immortalized Li-Fraumeni fibroblasts lacking any wild-type p53 were very efficient in gene amplification. In addition, early passage embryonic fibroblasts derived from p53-deficient embryos

199 p 5 3 ( - / - ) were capable of gene amplification, in contrast to embryo fibroblasts from normal mice [269,270]. These same p53-deficient embryo fibroblasts became highly aneuploid after continued passage in culture, in contrast to cells with a full complement of p53 ([269]; M. Harvey, M. Tainsky and L.D., unpublished data), again suggesting that the loss of p53 contributes to chromosome breakage and genomic instability. Addition or induction of wild-type p53 in p53-deficient cells restored the inability of the cell to undergo gene amplification and restored a G1 cell cycle control point [270]. These findings are consistent with a process in which loss of p53 results in a failure of the cell to arrest in suboptimal growth conditions in order to maintain chromosomal integrity. (Suboptimal growth conditions are thought to be introduced by addition of PALA to the cells in these experiments, since PALA is known to reduce intracellular pools of UTP, CTP, dCTP, dGTP, and dTTP.) Livingstone et al. have postulated that p53 may behave like some of the cell cycle checkpoint genes in yeast which are dispensable during optimal growth conditions, but can arrest the growing cell in G2 or G1 under DNA damaging conditions [271,272]. Elimination of these checkpoints can result in cell death, abnormal chromosome segregation, or increased susceptibility to DNA damaging agents. Three effectors of p53-mediated G1 arrest have now been described: (i) direct overexpression of wild-type p53 [11-15]; (ii) radiation [265-267]; and (iii) PALA [269,270]. G1 arrest initiated by PALA and ionizing radiation may affect p53 through separate or overlapping pathways. Wahl and colleagues [270] postulate that inappropriate passage of cells into S phase in the presence of PALA may result in DNA strand breaks which promote subsequent gene amplification. Reduction of intracellular nucleotide pools by PALA may

ExpandedScale:

result in DNA strand breaks which trigger induction of p53 and G1 arrest. Thus, the common element by which p53 could respond to both ionizing radiation and inadequate environmental metabolites would be the DNA strand breaks induced in both situations. That p53 mediates G1 growth arrest in response to inadequate nutrients or growth factors is also suggested by results from our own laboratories in which we determined the low density growth of embryo fibroblasts derived from wild-type, p53-deficient heterozygous and homozygous mice. Embryo fibroblasts form colonies very infrequently when plated at low density, suggesting that lack of paracrine growth factors from neighboring cells prevent progression of these cells through the cell cycle. However, p53-deficient homozygous cells (completely lacking p53) are 24-times more efficient at forming colonies than wild-type cells (A. Sands, A.B. and L.D., unpublished data; Fig. 6). Therefore, under environmental conditions normally inadequate for cell division, p53-deficient fibroblasts are less readily readily blocked in progression through the cell cycle. If p53 does in fact act as a cell cycle checkpoint determinant in G1, responding to DNA damage or inadequate metabolites, what are the mechanisms which regulate this response? While Kasten et al. [236] have provided some initial clues, much work remains to to be done in elucidating the cellular signal transduction pathways which affect p53 induction. While understanding very little about the mechanism of action upstream effectors of p53 induction, we have more information on mechanisms by which p53 might mediate downstream targets in the cell cycle checkpoint pathway. The biochemical activities described earlier for p53 provide insights into how it may promote growth arrest. The best evidence so far indicates that p53 may mediate growth arrest through both positive and negative transcriptional regulation of growth re-

I/l

(1.9 x Wild-Type)' 35 .~ 3O o

15 ~ 10 ~ 5 Z n--~

n=8

(+/+)

'0

(+/-)

n=9

n=8

(+/+)

(+/-)

Genotype

n=9

(-/-)

Genotype m

Slan. I~v.

Fig. 6. Plating efficiency of mouse embryo fibroblasts derived from normal and p53-deficient mice. Early passage embryo fibroblasts from individual p53 + / + , p53 + / - , and p 5 3 - / 12 day embryos were prepared by standard procedures. For each genotype, 10000 cells were plated on each of nine 100-mm plates and incubated for 10 days prior to fixing, staining and counting. The mean number of colonies (50 cells or greater) per 100-mm plate for the three genotypes are shown on the right. On the left is an expanded scale showing the difference in plating efficiency between p53 + / + cells and p53 + / cells. The difference in plating efficiency between these two genotypes was judged to be significant (P < 0.01).

200 ÷ ionizing radiation , ~ , . ~ ( ~ )

negativegrowth ) regulatorygenes [e.g. GADD45]

m

/ inadequate ~ metabolites, growth factors

(~

DNA strand.___>

breaks

AT gene product(s)

> pS3 J protein~

= =

>

apparatus ~ positive growth 7

G1 ARREST ~,~

GTP synthesisvia / ~" IMP dehydrogenase

Fig. 7. Hypothesized cellular pathways involving p53. According to the model, adapted from Kastan et al. [236] and Lane [268] and modified to incorporate our observations, p53 responds to DNA damage or inadequate growth conditions to mediate G1 arrest. The inducing agents may cause DNA strand breaks which activate a series of response genes including the ataxia telangiectasia (AT) gene products. The AT gene products result in increased levels of p53 through increased stabilization of the protein or perhaps also through activation of the site-specific DNA-binding (suppressor) activity as shown in the model in Fig. 5. The increased levels of activated suppressor p53 then mediate G1 arrest through one or more hypothetical mechanisms shown at the right.

lated genes or by direct interaction with the DNA replication apparatus. Recently, however, another potential mechanism of action has been put forward by Sherley [273], who showed that induction of wild-type p53 in an inducible cell line resulted in a block in guanine nucleotide biosynthesis. Whether this regulation of nucleotide synthesis is a direct or indirect effect of p53-mediated growth arrest is unclear. A model is presented in Fig. 7, based in part on the models presented by Kasten et al. [236] and Lane [268], which attempts to integrate some of our knowledge into an incomplete picture of the p53-associated cell cycle checkpoint pathway. The model may be subject to future revisions as we accrue more information, but it can be seen that p53 is centrally important to this particular signal transduction pathway. The next few years promise to be exciting ones as we discover more about this multifaceted tumor suppressor protein.

Acknowledgments The authors would like to thank G.G. Lozano, Arthur Sands, Michelle Ozbun, and Mark McArthur for a critical review of the manuscripts. We are also grateful to Moshe Oren, David Reisman, G.G. Lozano, and Jo Milner with providing manuscripts to us prior to their publication. This work was supported by a grant from the National Cancer Institute, CA54897, to L.D. A.B. is an Associate Investigator of the Howard Hughes Medical Institute.

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