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The Screening of the Second-Site Suppressor Mutations of The Int. J. Cancer: 121, 559–566 (2007) ' 2007 Wiley-Liss, Inc. The screening of the second-site suppressor mutations of the common p53 mutants Kazunori Otsuka, Shunsuke Kato, Yuichi Kakudo, Satsuki Mashiko, Hiroyuki Shibata and Chikashi Ishioka* Department of Clinical Oncology, Institute of Development, Aging and Cancer, and Tohoku University Hospital, Tohoku University, Sendai, Japan Second-site suppressor (SSS) mutations in p53 found by random mutation library covers more than 95% of tumor-derived missense mutagenesis have shown to restore the inactivated function of mutations as well as previously unreported missense mutations. some tumor-derived p53. To screen novel SSS mutations against We have shown the interrelation among the p53 structure, the common mutant p53s, intragenic second-site (SS) mutations were function and tumor-derived mutations. We have also observed that introduced into mutant p53 cDNA in a comprehensive manner by the missense mutation library was useful to isolate a number of using a p53 missense mutation library. The resulting mutant p53s 16,17 with background and SS mutations were assayed for their ability temperature sensitive p53 mutants and super apoptotic p53s. to restore the p53 transactivation function in both yeast and Previous studies have shown that there are second-site (SS) mis- human cell systems. We identified 12 novel SSS mutations includ- sense mutations that recover the inactivated function of tumor- ing H178Y against a common mutation G245S. Surprisingly, the derived mutations, so-called intragenic second-site suppressor G245S phenotype is rescued when coexpressed with p53 bearing (SSS) mutations.18–23 Analysis of the molecular background of the H178Y mutation. This result indicated that there is a possibil- ity that intragenic suppressor mutations might restore the protein the SSS mutation should be interesting because the recovery of function in an intermolecular manner. The intermolecular mecha- mutant p53 function might be useful for cancer treatment, espe- cially the design of small molecules that restore mutant p53 func- nism may lead to novel strategies for restoring inactivated p53 24 function and tumor suppression in cancer treatment. tion. ' 2007 Wiley-Liss, Inc. The purpose of this study is the isolation of SSS mutations against the 10 most common tumor-derived p53 mutations, Key words: p53; second-site suppressor mutation; intragenic V157F, R175H, C176F, G245S, R248Q, R248W, R249S, R273C, suppressor; transactivation R273H and R282W using our comprehensive p53 mutation library (see above), and a yeast-based functional assay, and the investiga- tion of the underlying molecular mechanism. We isolated 12 Tumor-suppressor p53 protein is a 393 amino-acid nuclear pro- mutations against G245S and 1 mutation against R273H, and have tein that acts as a tetramer. It is divided into 3 domains, an NH2- shown that G245S phenotype is rescued when coexpressed with terminal domain containing a transactivation domain, a central p53 bearing the H178Y mutation. The underlying molecular core DNA-binding domain and a COOH-terminal domain contain- mechanism of the SSS mutation described here clearly differed ing a tetramerization domain.1–6 Among these, the core DNA- from previous knowledge. Further understanding of the inter-p53 binding domain is well conserved and retains high homology mechanism may lead to novel strategies for restoring inactivated between a variety of lower species and humans7 as well as human p53 function and tumor suppression in cancer therapeutics. p53 homologues.8,9 The structure of the core DNA-binding do- main has been resolved by X-ray crystallographic analysis.2 The p53 forms a homotetramer through the tetramerization domain Material and methods and the p53 tetramer forms a sequence-specific DNA-binding Preparation of gap-repair vectors interface. The p53 protein is activated by a variety of cellular The pLSC53A25-based mutant p53 expression vectors with 1 of stresses including DNA damage and hypoxia, and is phosphoryl- the 10 most common tumor-derived p53 mutations, V157F, ated and acetylated after translation. The activated p53 binds to R175H, C176F, G245S, R248Q, R248W, R249S, R273C, R273H the specific DNA sequence in the regulatory region of downstream or R282W15 were digested by Bsu36I and StuI for the plasmids genes, resulting in cellular events including cell-cycle arrest and with V157F, R175H and C176F (Supplementary Fig. 1a)orby apoptosis. So far, a number of downstream genes involved in cell- NcoI and Bsu36I for the remaining 7 plasmids (Supplementary cycle, apoptosis, DNA-repair, angiogenesis and p53 stability have Fig. 1b). The linearized plasmids were separated by agarose-gel been identified. The loss of p53 function therefore fails to activate electrophoresis, purified with Gfx (Pharmacia) and used as gap- these genes after cellular stresses and is thought to be a critical repair vectors for each of the background mutations. cause of carcinogenesis and/or tumor progression. Although the frequency of TP53 mutations differs among tumor types, 50% of tumors contained the TP53 mutation.10–12 The Preparation of SS mutations published mutations have been summarized in the 2 major TP53 The p53 mutation library containing 2314 p53 missense muta- mutation databases that contain more than 20,000 mutations.13,14 tions was constructed through 96-well formatted site-directed mu- According to the databases, 74% of mutations are missense muta- tagenesis,15 and was used for PCR templates of SS mutations. For tions. So far, 1,200 distinct missense mutations have been reported background mutations, V157F, R175H and C176F, the p53 frag- and there are mutation hot spots at residues R175, G245, R248, ments covering codons from 192 to 354 were amplified from the R273 and R282. These residues reside in the L2 or L3 loop, or the yeast library using a set of primers, OKO-04 (50-CCCCACCAT- LSH motif and may be particularly critical because the stability of the structures is thought to depend on interactions among side- This article contains supplementary material available via the Internet at chains, or between side chains and the backbone structure. Amino http://www.interscience.wiley.com/jpages/0020-7136/suppmat. acid substitutions at these sites are therefore more likely to destroy Grant sponsors: Ministry of Education, Science, Sports and Culture, the DNA-binding interface than the core b-sandwich structure. Gonryo Medical Foundation. Recently, we have used a comprehensive site-directed mutagen- *Correspondence to: 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Fax: 181-22-717-8548. E-mail: [email protected] esis technique and a yeast-based functional assay to construct, ex- Received 9 August 2006; Accepted after revision 13 February 2007 pressed and evaluated 2,314 p53 mutants representing all possible DOI 10.1002/ijc.22724 amino-acid substitutions caused by a point mutation throughout Published online 6 April 2007 in Wiley InterScience (www.interscience. the protein (5.9 substitutions per residue).15 The TP53 missense wiley.com). Publication of the International Union Against Cancer 560 OTSUKA ET AL. GAGCGCTGCTCAGATAG-30) and LS6 (50-GCGAAGCTT- Mammalian p53 expression vectors 0 CAGTCTGAGTCAGGCCCTT-3 ) (Supplementary Fig. 1a). For Mutant p53-expression vectors, pCR259-p53MTs, with candi- G245S, R248Q, R248W, R249S, R273C, R273H and R282W, the date SSS mutations and background mutations in the p53 open p53 fragments covering codons from 110 to 225 were amplified reading frame were constructed. pCR259-p53WT and pCR259 0 from the yeast library using a set of primers, OKO-03 (5 - were control vectors expressing wild-type p53 or null p53, respec- 0 0 GCCCATGCAGGAACTGTTACACATG-3 ) and LS-5 (5 - tively.15 CGGGATCCATGGAGGAGCCGCAGTCA-30) (Supplementary Fig. 1b). Forty-eight p53 fragments were combined as a pool of Cell culture and transfection the PCR fragments. A TP53-deficient human osteosarcoma cell line, Saos-2, was Screening SSS mutations using the yeast assay cultured in RPMI1640 medium, supplemented with 10% heat- inactivated (56°C, 30 min) fetal calf serum (JRH Bioscience) in The linearized gap vector with the background mutation was the presence of 5% CO2 at 37°C. For luciferase assays, the cells cotransformed with the pooled PCR fragments (see above) into were grown to 60–90% confluence in 96-well tissue culture plates. the yeast haploid strain, YPH499 (MATa, his3D200, ade2-101, For immunoblotting, the cells were grown to the same confluence leu2D1, ura3-52, trp1-289, lys2-801) harboring p21WAF1 reporter 2 25 on 90 3 20 mm tissue culture plates. Transient transfections EGFP plasmid, pAS03G. The SS mutation was recombined into were performed using the Effectene transfection reagent (Qiagen). the vector on a solid medium-containing synthetic complete (SC) For luciferase assays, the cells were cotransfected with 12.5 ng of media lacking leucine and tryptophane (SC-leu-trp). The resulting one of the expression vectors (pCR259-p53WT, pCR259-p53MT yeast colonies expressed p53 protein with 2 amino acid substitu- or a p53-null pCR259 vector)15 or 6.25 ng of each of the 2 differ- tions derived from both the background and the SS mutation (Sup- ent expression vectors plus 85 ng of the p53-responsive luciferase plementary Figs. 1a and 1b). YPH500 (MATa, his3D200, ade2- plasmid (p21Ps-luc, pMDMPs-luc, pBAXPs-luc, pSIGMAPs- 101, leu2D1, ura3-52, trp1-289, lys2-801) harboring each of the luc p53R2Ps-luc or p53GADD45Ps-luc)15,25 and incubated for Ds-Red reporter plasmids (either pKS05R, pKS07R, pKS09R, 15 further 24 hr. For immunoblotting analysis, the cells were trans- pKS11R, pKS13R, pKS15R or pKS17R) was spread on the sur- fected with 2 lg of one of the expression vectors (a p53-null face of YPD plates. The p53-expressing YPH499 strains were pCR259, pCR259-p53WT, pCR259-p53MT, pCR259-Myc-H178Y then added to the YPH500-spread YPD plates with an autoclaved or pcDNA1.1-HA-G245S) or 1 lg each of the 2 different expres- velvet replicator and further incubated at 30°C for 12 hr.
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