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Carcinogenesis vol.18 no.10 pp.2019–2021, 1997 SHORT COMMUNICATION Simple identification of dominant p53 mutants by a yeast functional assay Alberto Inga1, Sara Cresta1, Paola Monti1, Anna Aprile, Gina Scott2, Angelo Abbondandolo1,3, Richard Iggo4 and Gilberto Fronza1,5 1CSTA-Mutagenesis Laboratory, National Institute for Research on Cancer (IST), Genova, Italy, 2School of Medicine, University of Leeds, Leeds, UK, 3Chair of Genetics, University of Genova, Italy and 4Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland 5To whom correspondence should be addressed Analysis of families with germline p53 mutations shows that the mutant p53 allele behaves as a dominant oncogene at the genetic level, although it behaves as a recessive oncogene at the cellular level, since tumours invariably show mutation or loss of both wild-type alleles. At the biochemical level it is possible that some clinically important mutant p53 proteins may be carcinogenic through a dominant mechanism. We show that p53 mutants can be readily classified according to their dominant potential using a simple yeast functional assay. Wild-type p53 is constitutively expressed from a TRP1 vector, p53 mutants are expressed from an otherwise identical LEU2 vector and net transcriptional activity is scored using an ADE2based reporter. Twenty seven p53 mutants were tested: 19 were recessive, i.e. gave white colonies, and eight showed dominant activity, i.e. gave pink/red colonies. This simple assay should facilitate studies on p53 dominance. p53 alleles inactivated by missense mutations are seen in ~50% of all human tumours (1,2). Tumour-derived mutants are unable to function as sequence-specific transcription factors, generally because they contain mutations in the DNA binding domain which reduce the affinity for DNA (3,4). The high level of p53 protein commonly seen in tumours probably reflects the presence of a persistent p53 activating signal and loss of the mdm2-induced p53 degradation feedback loop (5,6). In a normal cell p53 activation leads to cell cycle arrest or apoptosis, but in a tumour containing mutant p53 the cells can continue to divide because mutant p53 is unable to transactivate its target genes. While this abortive activation/ impaired degradation model provides a satisfactory explanation for the high level p53 expression seen in tumours (5,6), it does not rule out the possibility that a selective growth advantage may be conferred by mutated p53 proteins, either because dominant inhibition of wild-type protein could facilitate progression from the heterozygous to the homozygous mutant state or because the mutant protein has a novel growth promoting activity (7). Although superficially similar, there is an important biological distinction between these two models, since the former implies only loss of p53 function as a transcription factor, whereas the latter suggests that mutants have gained new functions. Tumour development is marked by a long period of coexistence of wild-type and mutant protein in the same cell, either following an initial somatic mutagenic event or from © Oxford University Press birth in the case of germline mutations (2). Since p53 exists as a stable tetramer in the cell (8) and since in most cases both alleles are equally expressed and equally stable, heterozygous mutant cells should contain a mixture of wild-type, mixed and mutant tetramers in the ratio 1:14:1, provided the mutant protein is able to form oligomers with the wild-type. The extent to which p53 mutants really do abrogate wild-type p53 function in the heterozygous state is controversial and depends on the particular assay employed (4,9–12), but there is certainly the potential for strong dominant inhibition of wild-type p53 through tetramerization with mutant protein in heterozygous cells. It is possible to classify p53 mutations into those that introduce adverse contacts and actively prevent DNA binding by changing residues that physically interact with DNA (contact mutants) and those that interfere with the orientation of the contact residues by changing the overall structure of the DNA binding domain (structural mutants) (8). Since physiological p53 binding sites in genomic DNA frequently contain imperfect matches to the p53 consensus, some subunits of a tetramer frequently do not make a full set of bonds with DNA. Indeed, a difference in affinity between the various natural p53 targets has been described (13). Hence, a reduction in the number of available protein contact sites is not a priori grounds for mixed tetramers being inactive in the cell. It has been shown that wild-type p53 can form stable tetramers with DNA contact mutant proteins and that mixed tetramers can bind specific DNA sequences. For example, mixed tetramers containing 248W mutant p53 bind DNA well (10), which illustrates how a reduced affinity, due to loss of the four hydrogen bonds between Arg248 and the minor groove, need not detract from high affinity binding by remaining wild-type subunits in the tetramer, provided the mutant residue (tryptophan) can be accommodated in the structure. Despite the fact that only one out of 16 tetramers in a heterozygous cell are fully wild-type, some studies have shown near normal p53 responses in heterozygous cells. This may partly reflect the ability of p53 to accumulate to high levels after activation, an effect which could negate even a substantial reduction in the starting level of wild-type tetramers. Many of the problems with mammalian studies derive from the difficulty of achieving stable low level p53 expression in a reproducibly defined genetic background and results are frequently clouded by concerns over the amount of p53 produced, which often far exceeds any relevant physiological level. For instance, differences with transcription assays and transformation assays probably reflect the fact that very small amounts of p53 give saturating levels of transcriptional activation, whilst CMV promoter-derived p53 overexpression is necessary for growth inhibition. Dominance is an important issue in transformation assays, where incoming p53 mutants must inactivate both resident wild-type alleles. A further problem with mammalian transfection studies is that it is difficult to achieve exactly equal expression of both alleles. This can be overcome by use 2019 A.Inga et al. Table I. Dominance analysis of p53 mutants in strain yIG397 of Saccharomyces cerevisiae Mutantb Mutation Codon Amino acid Dominancec Domaind CCNU- and UV-induced mutations never found in human tumoursa 1 CCC→ATC 72 Pro→Ilee No Out 2 CCT→CTT 98 Pro→Leu No Preceeds S1 3 GGG→GAG 117 Gly→Asp No L1 4 AAG→GAG 139 Lys→Glu No S29–S3 5 ACG→AGG 170 Thr→Arg No L2 6 GAG→AAG 224 Glu→Lys No S7–S8 7 GAC→GTC 228 Asp→Val No S7-S8 8 ACC→TCC 230 Thr→Ser No S8 9 GGT→GAT 262 Gly→Asp No S9–S10 10 CTG→CCG 323 Leu→Pro No Out CCNU-induced and UV-induced mutations already found in human tumours 11 ACC→ATC 155 Thr→Ile No S3–S4 12 CGC→CCC 156 Arg→Pro No S4 13 CCC→CAC 177 Pro→His Yes H1 14 CGA→TGA 213 Arg→Gln No S6–S7 15 CCC→CTC 219 Pro→Leu No S7 16 TCC→TTC 241 Ser→Phe Yes L3 17 TGC→TAC 242 Cys→Tyr Yes L3 18 GGC→AGC 245 Gly→Ser Yes L3 19 GGC→GAC 245 Gly→Asp Yes L3 20 GAA→GAT 258 Glu→Lys No S9–S10 21 GAC→AAC 259 Asp→Asn No S9–S10 22 GGA→AGA 266 Gly→Arg No S10 23 CGT→TGT 273 Arg→Cys Yes S10 24 CCT→TCT 278 Pro→Ser No H2 25 GGG→GAG 279 Gly→Glu Yes H2 26 GAG→AAG 281 Asp→Asn Yes H2 27 GAA→AAA 286 Glu→Lys No H2 aAccording to EMBL p53 database, May 13 1996, 5174 mutants entered (18). bNot Fig. 1. Schematic representation of dominance analysis of p53 mutants in strain yIG397. Double transformant clones (Leu1, Trp1), where only wildtype p53 homotetramers are present (A), express the ADE2 gene, giving rise to white (Ade1) colonies on minimal plates lacking leucine and tryptophan but containing sufficient adenine for adenine auxotrophs to grow and turn red. Double transformants clones expressing wild-type p53 and mutant p53 proteins form white (Ade1) or pink/red (Ade–) colonies and were interpreted as expressing recessive (B) or dominant (C) p53 mutant proteins respectively. For simplicity, the situation in which mixed heterotetramers containing three wild-type and one mutant protein (3:1) is represented. of bicistronic vectors and studies using this approach have shown that wild-type p53 is generally dominant over mutant p53 (12). To get over some of the problems with mammalian studies we have modified the ADE2-based yeast p53 functional assay (14) to allow straightforward characterization of the dominance potential of p53 mutants. In this assay, human p53 cDNA is cloned by homologous recombination in vivo (gap repair) into a yeast expression vector. To determine whether the p53 clone can activate transcription, the yeast strain contains a transcription reporter (Figure 1A) where the ADE2 gene is regulated by a p53-responsive promoter. With this system, wild-type p53 gives white colonies and mutant p53 gives red colonies. To test dominance, two p53 cDNA must be expressed together in the reporter strain. The existing expression vector has a LEU2 marker; a new p53 expression vector, named pTS76, was therefore constructed which is identical with the first but has a TRP1 marker. To do this, the 4.9 kb PvuI fragment (bp 2968–7964) containing the pADH1 wild-type p53 cDNA from pLS76 (15) was ligated to the 2.9 kb PvuI 2020 underlined, CCNU-induced mutants (17); underlined, UV-induced mutants (unpublished data). cTransformants (Leu1Trp1) forming white (Ade1) or red/pink (Ade–) colonies were classified as recessive or dominant mutants respectively. dLocalization of the mutation according to the topological diagram of the core domain of p53 (19). eAmino acid change never found, but this is a site of a known polymorphism. fragment (bp 5973–1736) containing the CEN/ARS TRP1 region from pLS89 (16). pTS76 was used to express wildtype p53, while different p53 mutants were expressed from pLS76-derived LEU2 vectors. The haploid strain yIG397 (MATa ade2-1 leu2-3,112 trp1-1 his3-11,15 can1-100, ura3-1 URA3 3xRGC::pCYC1::ADE2), containing the ADE2 reporter gene under p53 control, was transfected with equimolar amounts of both vectors by electroporation. Double transformants were selected on minimal plates lacking leucine and tryptophan but containing sufficient adenine (5 mg/l) for adenine auxotrophs to grow and turn red. Double transformant clones (Leu1, Trp1) giving rise to white (Ade1) or pink/ red (Ade–) colonies were interpreted as expressing recessive (Figure 1B) or dominant (Figure 1C) mutations respectively. Generally, .100 independent transformants were analysed and their phenotype was invariably homogeneous. A panel of p53 mutations obtained through application of the yeast p53 functional assay to the field of mutagenesis (17) was used to validate the dominance assay. This included 18 CCNU-induced and nine UV-induced mutants (Table I). About 20% of the experimentally induced p53 mutants selected so far in yeast (19 out of 82) (17; unpublished data) contain base pair substitutions never found in human tumours or cell lines Dominant p53 mutatants in yeast (18; EMBL database, May 13 1996, 5174 mutations entered). Such mutations, although completely inactive for transactivation (red colonies), are apparently not selected for in vivo during the carcinogenic process. This could reflect mutagen specificity, repair specificity or some biological activity of the mutants. In particular, if dominance plays an important part in vivo, one would not expect to see recessive mutations in tumours. Dominance analysis revealed that all the CCNU- and UVinduced mutations not found in human tumours were indeed recessive (Table I). Double transformants of eight out of the 17 mutants already found in tumours showed instead pink/red colonies and were considered dominant. However, colony size and staining intensity suggested the presence of a minimal wild-type p53 activity. All dominant mutants affected key amino acids that are essential for stabilization of the DNA binding surface of the p53 core domain or for direct interaction between p53 and DNA (19). Recently another selection scheme (URA3-based) for spontaneous dominant p53 mutations in yeast was described (20). The analysis of that spectrum showed that p53 dominant mutations correlate well with mutational hotspots in human cancer. Only two out of the 41 dominant mutations described (20) have not been observed in tumours, a number significantly lower than that found by selecting mutants irrespective of their dominant phenotype (19 out of 82, P 5 0.01, Fisher’s exact test) (17; unpublished data). The results with the ADE2 selection method are generally consistent with those obtained by the URA3-based selection. Dominant mutations were found only in the group of mutants already found in tumours (Table I; P 5 0.012, Fisher’s exact test). Selection for dominance thus may play a role in determining the mutation spectrum seen in human tumours. It should be noted, however, that this is not evidence for ‘gain of function’ in the sense of acquisition of novel biochemical activities favouring tumour cell growth, since it can be adequately explained by mutant-induced loss of function of the wild-type allele. The ability to classify mutants simply according to their dominant potential at the biochemical/cellular level should facilitate studies on Li–Fraumeni syndrome and tumour progression from the heterozygous to the homozygous state and it may in future provide important information to the clinician about the likelihood of response to chemotherapy and p53 gene therapy. 5. Haupt,Y., Maya,R., Kazaz,A. and Oren,M. (1997) Mdm2 promotes the rapid degradation of p53. Nature, 387, 296–299. 6. Kubbutat,M.H.G., Jones,S.N. and Vousden,K.N. (1997) Regulation of p53 stability by mdm2. Nature 387, 299–303. 7. Hann,B.C. and Lane,D.P. (1995) The dominating effect of mutant p53. Nature Genet., 9, 221–222. 8. Arrowsmith,C.H. and Morin,P. (1996) New insights into p53 function from structural studies. Oncogene, 12, 1379–1385. 9. Waterman,J.L.F., Waterman,M.J.F. and Halazonetis,T.D. (1996) An engineered four-stranded coil substitutes for the tetramerization domain of wild-type p53 and alleviates transdominant inhibition by tumor-derived p53 mutants. Cancer Res., 56, 158–163. 10. Bargonetti,J., Reynisdottir,I., Friedman,P.N. and Prives,C. (1992) Sitespecific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev., 6, 1886–1898. 11. Kern,S.E., Pietenpol,J.A., Thiagalingam,S., Seymour,A., Kinzler,K.W. and Vogelstien,B. (1992) Oncogenic forms of p53 inhibits p53-regulated gene expression. Science, 265, 827–830. 12. Frebourg,T., Sadelain,M., Ng,Y.-S., Kassel,J. and Friend,S.H. (1994) Equal transcription of wild-type and mutants p53 using bicistronic vectors results in the wild-type phenotype. Cancer Res., 54, 878–881. 13. Wang,Y. and Prives,C. (1995) Increased and altered DNA binding of human p53 by S and G2/M but not G1 cyclin-dpendent kinases. Nature, 376, 88–91. 14. Flaman,J.M. et al. (1995) A simple p53 functional assay for screening cell lines, blood, and tumors. Proc. Natl Acad. Sci. USA, 92, 3963–3967. 15. Ishioka,C., Frebourg,T., Yan,Y-X., Vidal,M., Friend,S.H., Schmidt,S. and Iggo,R. (1993) Screening patients for heterozygous p53 mutations using a functional assay in yeast. Nature Genet., 5, 124–129. 16. Scharer,E. and Iggo,R. (1992) Mammalian p53 can function as a transcription factor in yeast. Nucleic Acids Res., 7, 1539–1545. 17. Inga,A., Iannone,R., Monti,P., Molina,F., Bolognesi,M., Abbondandolo,A., Iggo,R. and Fronza,G. (1997) Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology. Oncogene, 14, 1307–1313. 18. Hollstein,M., Shomer,B., Greenblatt,M., Soussi,T., Hovig,E., Montesano,R. and Harris,C.C. (1996) Somatic point mutation in the p53 gene of human tumours and cell lines updated compilation. Nucleic Acids Res., 24, 141–146. 19. Cho,Y., Gorina,S., Jeffrey,P.D. and Pavletich,N.P. (1994) Crystal structure of a p53 tumor suppressor–DNA complex: understanding tumorigenic mutations. Science, 265, 346–355. 20. Brachmann,R.K., Vidal,M. and Boeke,J.D. (1996) Dominant-negative p53 mutations selected in yeast hit cancer hot spots. Proc. Natl Acad. Sci. USA, 93, 4091–4095. Received on April 21, 1997; revised on June 18, 1997; accepted on June 18, 1997 Acknowledgements We thank Drs P.Menichini, P.Campomenosi and L.Ottaggio for helpful discussions. This work was partially supported by contract CHRX-CT94-0581 from the Commission of the European Communities and by the Italian Association for Cancer Research (AIRC). References 1. Greenblatt,M.S., Bennett,W.P., Hollstein,M. and Harris,C.C. (1994) Mutations in the p53 tumor suppression gene: clues to cancer etiology and molecular pathogenesis. Cancer Res., 54, 4855–4878. 2. Ko,L.J. and Prives,C (1996) p53: puzzles and paradigm. Genes Dev., 10, 1054–1072. 3. Ory,K., Legros,Y., Auguin,C. and Soussi,T. (1994) Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation. EMBO J., 13, 3496–3504. 4. Pietenpol,J.A., Tokino,T., Thiagalingam,S., El-Deiry,W.S., Kinzler,K.W. and Vogelstein,B. (1994) Sequence-specific transcriptional activation is essential for growth suppression by p53. Proc. Natl Acad. Sci. USA, 91, 1998–2002. 2021