Simulated sunlight and benzo[a]pyrene diol epoxide induced mutagenesis in the human p53 gene evaluated by the yeast functional assay: lack of correspondence to tumor mutation spectra
Jung-Hoon Yoon1,
Chong-Soon Lee2 and
Gerd P. Pfeifer1,3
1 Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA, USA and
2 Department of Biochemistry, College of Natural Sciences, Yeungnam University, Kyongsan, 712-749, Korea
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Abstract
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Many mutations in the p53 gene destroy the transcriptional transactivation function of the p53 protein. This function of p53 can be determined in a yeast assay using a p53 responsive reporter gene. The yeast assay could hold promise for the identification of mutagens implicated in human cancer if the p53 mutational spectra obtained with this assay would match human tumor mutation data. Ultraviolet (UV) light from the sun and polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are strongly implicated in the spectrum of p53 mutations found in human non-melanoma skin cancers and smoking-associated lung cancers, respectively. We have used these two model mutagens to assess the feasibility of using the p53 yeast assay in cancer epidemiology. After treatment of CpG methylated p53 DNA with a solar UV simulator or with benzo[a]pyrene diol epoxide (BPDE), the modified p53 sequences were assayed in yeast for mutational outcome. As expected, BPDE produced predominantly G to T transversions and simulated sunlight produced mostly C to T transitions at dipyrimidine sites in the p53 coding sequence. However, the preferentially mutated p53 sequences (hotspots) in the yeast assay were completely different from those in the mutational spectra found in human lung and skin cancers. The data indicate that this assay is not a reliable measurement of p53 mutagenesis in human tissues and that, perhaps, transcriptional activation is not the primary function of p53 in tumor suppression.
Abbreviations: BPDE, (+/) anti-benzo[a]pyrene-7,8 dihydrodiol-9,10-epoxide; CPD, cyclobutane pyrimidine dimer; mCpG, 5-methylcytosine guanine dinucleotide; PAH, polycyclic aromatic hydrocarbon
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Introduction
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The p53 tumor suppressor protein plays an important role in DNA damage responses such as in cell cycle arrest, in apoptosis and in activating certain DNA repair systems. Disabling p53 function by mutations in the p53 coding sequence leads to the development of tumors. More than 50% of all human tumors contain a mutation in the p53 gene (1,2). p53 is one of the most common mutational targets in human cancers reported to date (3,4). The accumulation and analysis of the mutation data gives information on the tissue specificity of human p53 tumor-associated mutations (4,5). The analysis of human p53 mutational spectra provides a good opportunity to understand the etiology, epidemiology and pathogenesis of human cancers (1,2).
Cigarette smoking is the major risk factor for development of lung cancer (6,7). Among the many carcinogens present in cigarette smoke, benzo[a]pyrene and other carcinogenic polycyclic aromatic hydrocarbons (PAH) have been implicated in lung cancer (8,9). The metabolically activated form of benzo[a]pyrene, (+/) anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) forms stable N2-guanine adducts and consequently, these adducts can lead to G to T transversions (1013). This G to T transversion is thought to be a molecular signature for cigarette smoke mutagens (8). The p53 mutation spectrum in lung cancer is different from mutational spectra in other cancers (8,9). According to the human p53 tumor mutation database, >30% of the mutations in lung cancer are G to T transversions and six dominant mutation hotspots were identified (8). There is an increased frequency of G to T transversions in the p53 gene of lung cancers from smokers compared with lung cancers from non-smokers and most other cancers. In addition, five of the six mutational hotspots (p53 codons 157, 158, 245, 248 and 273, but not codon 249) are guanines at methylated CpG (mCpG) sites (14). As demonstrated by mapping of DNA adducts along the human p53 gene, these five hotspots are major binding sites for BPDE and other PAH compounds (1517). The data strongly suggest that PAHs in cigarette smoke are a major risk factor for lung cancer.
Solar ultraviolet (UV) light is another known causative agent for human cancer. Solar UV light is certainly involved in the induction of non-melanoma skin tumors such as squamous cell carcinomas and most probably also plays an important role in melanoma (18,19). In human non-melanoma skin cancers, a large fraction of the p53 mutations are C to T transitions at dipyrimidine sites (2022). Moreover, >30% of all p53 mutations in skin tumors are C to T transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG (14). These sequences include several mutational hotspots along the p53 gene (codons 196, 213, 245, 248 and 282) (23). As all CpGs are methylated along the p53 coding sequence in human tissues (24), these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-PymCG. There are two major types of DNA photoproducts, i.e. CPDs (cyclobutane pyrimidine dimers) and (64) photoproducts formed by UVB irradiation (25). It has been reported that CPDs form preferentially at dipyrimidines containing 5-PymCG when UVB or natural sunlight (23,26) were used for irradiation and several of these sites are repaired slowly (27). In our previous studies, CPDs were at least 2040 times more frequent than any other DNA photoproduct when DNA or cells were irradiated with simulated sunlight (28). In addition, using photoproduct-specific photolyases transfected into mouse cells containing mutational reporter genes, we found that CPDs are responsible for the vast majority of UVB-induced mutations in mouse cells (29). The data suggest that CPDs are the most important DNA damage products implicated in the development of human skin cancer.
Mutations in the p53 gene cannot be identified in cell cultures treated with mutagens with the exception of mutations at selected restrictions sites, which can serve in an assay in which cleavage is inhibited by a mutation (30,31). Thus, a complete mutational spectrum of the p53 gene cannot be determined directly. A system has been developed that allows scoring of mutations in a p53 cDNA vector that is assayed for transcriptional transactivation of a reporter gene in yeast (32,33). This assay has been used initially to analyze p53 mutations in tumor samples from patients (32). Several reports have adopted this assay for measuring mutagen-induced mutations. In this case, the p53 cDNA vector is treated in vitro with a DNA damaging agent, and is then transfected into yeast to score for inactivating mutations. It is hoped that this assay would, at least to some extent, reproduce the mutational spectrum in tumors. Experiments were conducted with chloroethyl-cyclohexyl-nitroso-urea (34), UV light (35,36) and other mutagenic agents. However, conditions somewhat inappropriate for comparisons with p53 mutational spectra in vivo (254 nm UVC irradiation and an unmethylated p53 plasmid) were used in these publications.
We set out to verify whether the yeast assay can generate results that are relevant for molecular epidemiology by using two model mutagens. In this study, we used the yeast p53 functional assay to analyze the mutation spectra of BPDE and solar UV light in a CpG methylated human p53 gene. Even though assayed in yeast cells, this system may provide a direct analysis of BPDE and solar UV light mutagenesis within the p53 gene. We found that BPDE induced predominantly G to T transversions and solar UV light induced predominantly C to T transitions at dipyrimidines containing 5'-TC or 5'-CC. We then compared the p53 mutational spectra (types of base substitutions and positions of hotspots) obtained with the p53 vector in yeast with p53 mutational spectra in human tumors. We found that, compared with the p53 mutational spectra in human cancer, the mutation spectra of BPDE and solar UV light obtained from the yeast assay were substantially different.
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Materials and methods
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Yeast strains and vectors
The yIG397 yeast (Saccharomyces cerevisiae) strain and the pRDI-22 gap repair vector were kindly provided by Dr Iggo (Epalinges, Switzerland). The recipient yeast strain (yIG397) is defective in adenine biosynthesis because of a mutation in the endogenous ADE2 gene, but it contains a second copy of the ADE2 gene controlled by a p53 responsive promoter (32,33,37). Basic methods for yeast manipulations were carried out as described (32). Briefly, yeast cells were grown in liquid media containing 0.1% yeast extract (Difco BRL), 0.2% peptone (Difco BRL), 0.2% dextrose, 200 µg/ml of adenine (high concentration) and 0.25% bacto agar. The pRDI-22 gap repair vector has an ADH promoter followed by homologous recombination sites for the human p53 gene (34). This vector was linearized by restriction enzymes HindIII and StuI, and then treated with calf intestinal alkaline phosphatase to block self-ligation. The cut plasmid vector was agarose gel purified.
Preparation of human p53 cDNA fragments and methylation at CpG sites
Full-length wild-type human spleen p53 cDNA (Clontech, Palo Alto, CA) was used as a template for PCR. PCR was done with Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and two primers, i.e. forward primer 5'-CGGAATTCCGATTTGATGCTGTCCC-3' (the EcoRI cutting site is underlined) and reverse primer 5'-CATCGAGCTCGACCCTTTTTGGACTT-3' (the SacI cutting site is underlined). PCR products were subcloned into a plasmid restricted with EcoRI and SacI. The plasmid was isolated, cut with the restriction enzymes to release the p53 fragment, which was then purified by agarose gel electrophoresis. Sequencing revealed that PCR had not introduced any mutation into the p53 gene. Since all of the CpG sites in the human p53 coding sequence spanning exons 5 through 9 are methylated in tissues (24) and the mutagens we used have been closely implicated in mCpG mutagenesis (14), the purified p53 cDNA fragment was methylated in vitro using the CpG-specific SssI DNA methylase according to the manufacturers instructions (New England Biolabs, Beverly, MA). Control DNA was mock-methylated in the absence of S-adenosylmethionine.
BPDE and solar UV light treatment
BPDE was purchased from the NCI repository (Midwest Research Institute; Kansas City, MO) and was dissolved in DMSO. Methylated and unmethylated p53 cDNA (1 µg) was treated with a final concentration of 0 (DMSO only), and 2 and 5 µM of BPDE. To remove free BPDE, DNA samples were column-purified by Wizard DNA clean-up kits (Promega, Madison, WI). For solar UV treatment, A 1000 W Oriel solar UV simulator was used. The emission spectrum of this instrument is shown in Yoon et al. (28). One microgram of methylated and unmethylated p53 cDNA fragments in TE buffer (pH 8.0) were irradiated on ice from the solar simulator for 30 min, 1 and 2 h.
Yeast p53 functional mutation assay
The yIG397 yeast strain (grown to an OD600 of 0.60.9) was co-transformed with a mixture of 100 ng of the pRDI-22 gapped-vector, 100 ng of the mutagen treated p53 cDNA fragment, and 100 µg of carrier DNA (herring testis) using the lithium acetate procedure according to the YEASTMAKERTM yeast transformation system kit (Clontech, Palo Alto, CA). The cells were collected, resuspended in 100 µl of TE buffer (pH 8.0) and plated on synthetic minimal medium minus leucine plus minimal adenine (5 µg/ml) and incubated for 3 days at 35°C. The yIG397 strain has an integrated vector containing an adenine reporter gene (ADE2 gene) under the control of the pCYC1 promoter linked with p53 binding sites (ribosomal gene cluster, RGC binding sites) (33). Once wild-type p53 is expressed from the gap-repaired vector, it binds to the promoter region and activates ADE2 gene expression, whereas mutants containing mutations in the p53 gene cannot activate this promoter. Wild-type colonies are big and white but mutants are small and red. The color is more intense after 2 days at 4°C. The mutant frequency was calculated as the number of red colonies divided by the total number of colonies.
Recovery of p53 plasmids from yeast and DNA sequencing
pRDI-22 vectors containing the p53 gene were isolated from transformed yeast by the glass bead lysis method. Briefly, red yeast colonies were cultured in 3 ml YPD medium for 16 h at 30°C. An aliquot of the culture medium (1.5 ml) was transferred to a tube, the yeast was collected by centrifugation at 10 000 g for 10 s and resuspended in 0.25 ml of 10 mM TrisCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1% SDS and 2% Triton X-100. After addition of 0.25 ml of phenolchloroform (1:1, v/v) and 0.2 g of acid-washed glass beads (Sigma, St Louis, MO) the cells were disrupted by vigorous vortexing for 2 min. The upper aqueous phase was transferred to a new tube following centrifugation at 12 000 g for 10 min. The DNA was precipitated by adding 25 µl of 3 M sodium acetate and 0.5 ml of ethanol. After washing with 70% ethanol and drying, the DNA was dissolved in 20 ml of TrisEDTA, pH 8.0. A 920 bp fragment of p53 was amplified by PCR with two primers: P3 (5'-GATTTGATGCTGTCCCCGGACGAT-3') and P4 (5'-CTTTTTGGACTTCA-GGTGGCAGGAGTG-3'). The PCR products were sequenced with an automated DNA sequencer. Any mutation in the DNA binding domain of p53 (codons 120300) was confirmed by sequencing the opposite strand with primers p90 (5'-CCCCTGTCATCTTCTGTCCCTTCC-3') and p330 (5'-CCC-ACGGATCTGAAGGGTGAAATA-3').
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Results
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Overview of the yeast p53 functional assay
The most attractive advantage of this assay is that the target gene is the human p53 gene. Thus, a direct determination of a mutational spectrum of mutagens in this gene can be obtained from this assay. Figure 1
shows an overview of the procedure. In this assay, transcriptional transactivation by p53 is assayed to select mutants. If a yeast cell has a mutation in the endogenous ADE2 gene, it is not able to survive in adenine deficient media. The yeast strain used for this assay contains an exogenous copy of the ADE2 gene. This ADE2 gene is controlled by human p53 because the promoter of this gene is linked with p53 binding sites (33). To select mutants, yeast cells containing mutant p53 are grown in lower concentrations of adenine. If yeast cells contain a mutation in the p53 gene, mutant p53 is not able to bind to the promoter region of the ADE2 gene for transactivation, and consequently, mutant colonies are smaller and of red color due to accumulation of an intermediate in adenine metabolism. In contrast, wild-type yeast cells grow as larger white colonies.
To more closely mimic the situation in human cells, we have introduced CpG methylation into the p53 fragment before mutagen treatment. This is of importance as methylated CpG sequences are preferential targets for BPDE and sunlight induced mutations (13,14,38). We initially noticed that a p53 expression vector, when methylated at all CpG sites, did not grow in yeast (data not shown). We then used the gap repair assay in which the unmethylated linearized expression vector backbone (pRDI-22) is co-transformed into yeast with a CpG methylated and mutagen-treated p53 cDNA fragment spanning codons 67346. In human tumors, the majority of mutations (>90%) occur along these sequences. In yeast, the gap repair vector and the p53 fragment combine by homologous recombination.
BPDE mutagenesis in the p53 gene
Methylated or unmethylated BPDE treated p53 cDNA was co-transformed with the gap repair vector into yIG397 yeast cells. Table I
shows the mutant frequencies after BPDE treatment with the methylated and unmethylated p53 gene. After BPDE treatment, the mutant frequency increased up to
4-fold. Methylated p53 had a slightly higher mutant frequency than unmethylated p53 (Table I
). This is consistent with the finding that guanines at mCpG sites are preferential targets for BPDE adduct formation (16). We selected the methylated and 5 µM BPDE treated samples and the untreated methylated samples for DNA sequencing analysis. 114 samples of 5 µM BPDE and 65 samples of no BPDE treatment were sequenced. Sixty-six percent (75/114) of the BPDE treated samples and 63% (41/65) of the no treatment samples contained mutations in the p53 sequence spanning codons 117330. The remainder of the samples may have mutations outside of the sequenced region. Figure 2
shows the mutation spectrum of p53 with no BPDE treatment. There was no defined mutation hotspot in untreated samples and all the mutations were scattered along the p53 gene. Figure 3
shows the mutation spectrum of p53 after 5 µM BPDE treatment. G to T transversions (33/75 = 44%) were the predominant types of mutations induced by BPDE treatment. C to T transitions were 27% and G to C transversions were 12% (Figure 4
). These numbers are quite similar to those obtained in other studies on BPDE mutagenesis (12,13,39). Four mutation hotspots (defined as three or more mutations at the same base position) along the p53 DNA binding domain were formed at codons 158, 170, 186 and 258 (Figure 3
). Table II
shows the amino acid changes corresponding to BPDE-induced mutations at mutation hotspots. We noticed that there is no non-transcribed strand bias of G to T mutation induction in this system.

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Fig. 2. Spontaneous mutation spectrum along the DNA sequences of the p53 gene encoding the DNA binding domain. The p53 vector was not treated with a mutagen. The numbers indicate nucleotide positions of the p53 cDNA. The mutant base is written below the sequence. -X-, Single base deletion. +A, Addition of one nucleotide.
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Fig. 3. Mutation spectrum along the p53 cDNA sequence induced by treatment of the vector with 5 µM BPDE. The numbers 351 and 951 indicate nucleotide positions of the p53 cDNA. Selected p53 codons are indicated by numbers above the sequence. The mutant DNA bases are written below the sequence. -X-, Single base deletion. +A, Addition of one nucleotide. Mutational hotspots are outlined.
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Fig. 4. Types of mutations found in the p53 gene assayed in yeast. The total number of mutations were 41 for no treatment, 75 for the 5 µM BPDE treatment, and 71 for the solar simulator treatment.
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Solar UV mutagensis in the p53 gene in yeast
Methylated and unmethylated p53 cDNA fragments were irradiated with a solar UV simulator for 0, 30 min and 2 h. After conducting the yeast functional assay, we found that the mutant frequency induced by the solar UV light treated p53 fragment was increased from 0.27% in untreated DNA to 0.98% in the 2 h irradiated DNA sample (Table III
). As with BPDE treatment, the mutant frequency of methylated p53 was slightly higher than that of unmethylated p53. We chose and sequenced the methylated and 2 h solar UV light treated and the methylated and no solar UV light treated samples. Among 71 of the 2 h solar UV light treated p53 mutant samples, 50 samples (70.4%) were C to T transitions and 12 samples (17%) had G to T transversions (Figure 4
). Six mutation hotspots (codons 194, 199, 213, 246, 271 and 306) containing the sequence context CC or TC were induced and two of them (codon 213 and 306) were overlapping mCpG sites (Figure 5
). Table II
shows the amino acid changes at these mutational hotspots. Moshinsky and Wogan (36) reported that several mutation hotspots, i.e. codons 199, 213, 246, 271 and 286 were induced by UVC light. Four of their mutation hotspots (codons 199, 213, 246 and 271) were also found in our data. However, there were some differences in mutation hotspots and mutation patterns between the data sets. Most of the C to T transition hotspots could be ascribed to a dipyrimidine sequence on the transcribed strand.

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Fig. 5. Mutation spectrum along the p53 cDNA sequence induced by treatment of the vector with the solar UV simulator for 2 h. The numbers 351 and 951 indicate nucleotide positions of the p53 cDNA. Selected p53 codons are indicated by numbers above the sequence. The mutant DNA bases are written below the sequence. -X-, Single base deletion. +G, +T, addition of one nucleotide. Tandem mutations are underlined. Mutational hotspots are outlined.
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Discussion
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So far, there is no mutation assay system to study p53 mutagenesis directly except for sequences that fall within codons, which are part of restriction enzyme cutting sites (30,31). From this viewpoint, the assay system used in this study appears to have several advantages over other mutation detection systems. Although mutagen treatment is done in vitro, this yeast p53 functional assay can be used for direct comparisons between the mutation spectra of this assay and that of the human p53 tumor database. The general mutational pattern of BPDE and solar UV light seen in the yeast system was quite similar to other assay systems, that is, BPDE induced predominantly G to T transversions and solar UV light induced C to T transitions at CC and TC sequences. We also found that BPDE and solar UV light have some preference for methylated CpGs in this assay. For example, mutations at mCpGs in untreated cells were 7/41 (=17%) of total mutations but were 26/75 (=35%) in BPDE treated cells most of them being transversions (P = 0.05;
2 test).
However, the mutation spectra of both mutagens are strikingly different from the p53 mutation database of human cancers (Figure 6
). Only one of four mutation hotspots (codon 158) for BPDE and one (codon 213) of six hotspots for solar UV light were corresponding to p53 mutation hotspots in smoking-associated lung cancer and non-melanoma skin cancer, respectively. However, the BPDE-induced hospot at codon 158 in the yeast assay involves a guanine on the transcribed strand whereas the lung cancer mutations at this codon are almost exclusively G to T transversions on the non-transcribed strand. The same applies to the codon 213 mutations induced by simulated sunlight. In the yeast assay, the dipyrimidine is on the transcribed strand while in the human skin cancer mutations, C to T transitions are observed on the non-transcribed strand. Another study, using aflatoxin B1 as a mutagen and the p53 yeast assay (40), also failed to reproduce the codon 249 mutation so commonly found in liver cancers from geographic areas where food contamination with aflatoxin is a problem.

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Fig. 6. Mutational spectra of the p53 gene in smoking-associated lung cancers and non-melanoma skin cancers. The data are from the IARC p53 mutation database (5) and from the primary literature. For lung cancers, non-smokers and occupationally exposed individuals were excluded. When displayed as G to T transversions only, the same lung cancer hotspots are identified (see ref. 8). All skin cancer hotspots are C to T transitions at dipyrimidine sequences. The open diamonds indicate the mutation hotspots found with the yeast assay. Codon 158 in lung cancer and codon 213 in skin cancer appear as hotspots in both the p53 tumor database and in the yeast assay. However, at the nucleotide level, these mutations occur at different sequence positions within the same codon.
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These results clearly show the limitations with this assay in that it does not reflect human cancer mutations. There may be several reasons for this discrepancy. The possibility cannot formally be excluded that the human tumor mutations are not the result of exposure of the founder cells to the carcinogen. It has been suggested by Cha et al. (41) and Rodin and Rodin (42) that the mutations that appear in tumor relevant genes pre-exist in non-tumor tissue and appear in tumors only as a result of selection during tumorigenesis. We do not agree with this view. We refer the reader to ref. (8) for a discussion of tobacco smoke carcinogenesis in light of the DNA damage versus the selection model. For skin cancer mutations in p53, it is hard to imagine that UV signature mutations in skin tissue (e.g. CC to TT) would not be induced by the UV component of sunlight but would somehow pre-exist, perhaps as a result of endogenous DNA damage. There are other factors that might more reasonably explain the differences between the human data and the yeast assay.
First, mutagen exposure is done under different conditions, that is, mutagen treatment is done in vitro and the target gene is not within its chromosomal context. However, it was shown that BPDE has the same sequence specificity in naked DNA and in BPDE-treated cells along the p53 gene (16) and the same is true for cyclobutane pyrimidine dimers induced by sunlight exposure (23).
Secondly, we think that these differences are related to the host cell, in which mutation fixation and selection are occurring, i.e. in yeast versus human cells. These two systems most probably involve different cellular processes related to mutagenesis such as different repair systems and lesion bypass DNA polymerases. Although S.cerevisiae does have transcription-coupled repair (43), it would seem that a strand bias of mutations (e.g. for G to T transversions on the non-transcribed strand or for C to T transitions at dipyrimidine sites) is not produced (Figures 2 and 5
). However, such a strand bias is clearly obvious for G to T transversions in smokers lung cancers and, although somewhat less pronounced, for skin cancer C to T transitions (5,8). A strand bias is also reflected in a preferential repair of BPDE adducts on the transcribed strand of the p53 gene in human cells (44) and is seen for CPD repair as well (45). Human cells have a larger collection of lesion bypass DNA polymerases (4648) than yeast and this may result in different sequence- and polymerase-dependent base incorporation events occurring at specific DNA lesions in each organism. In addition, repair and bypass of a lesion may be different when the lesion is within an extrachromosomal vector versus when it is within a chromosomal gene.
Thirdly, and probably most importantly, the yeast assay selects the mutants by only one functional defect of p53, transcriptional transactivation activity. Several of the p53 mutants commonly found at mutation hotspots in lung and in skin cancers have been shown to be defective in transcriptional activation (49). Thus, theoretically they should be scored in the yeast assay, but in fact they were rarely obtained. This leads us to propose that defects in transcription function, at least as measured through the RGC p53 binding site, are unreliable indicators of the tumorigenicity of a particular p53 mutant. One should keep in mind, however, that the yeast p53 assay does not score the potency of a p53 mutant to act in a dominant negative fashion (50). This means that in human cells with two wild-type p53 alleles, a point mutation in one allele might produce a protein that can inhibit the function of the wild-type p53 protein, presumably because the mutant is more stable and can out-compete wild-type proteins in tetrameric p53 complexes (51). The contribution of the dominant negative effect to human cancer mutations in the p53 gene is unclear. Given the apparent inability of mutant p53 to accumulate in the presence of wild-type p53, a dominant-negative effect may not naturally occur and, not surprisingly, heterozygous p53 mt/wt cells are rare (52). Nonetheless, some missense p53 mutants may be stronger dominant negatives than others and this could be a promoter-specific phenomenon (53,54). Indeed, it was shown that the mutational spectrum of spontaneous dominant-negative p53 mutants selected in a yeast assay included several cancer hotspots (55). However, also using a yeast assay, others have shown that transdominance per se fails to predict which mutations occur frequently in cancer (56). Future studies are necessary to investigate if human p53 tumor mutation spectra can be better reproduced in a yeast assay that scores dominant negative mutations arising in a mutagen-treated shuttle vector replicated in yeast and uses, perhaps, a different p53 binding site.
The other transcription-independent proposed functions of p53, just to name a few, are induction of apoptosis independently of transcription (57), exonuclease activity (58), replication inhibition (59) and inhibition of recombination (60). In addition, mutant p53 lacking transactivation function nevertheless may retain the ability to repress transcription of certain genes. As p53 has such multiple functions implicated in tumorigenesis, the yeast assay may not be able to reflect mutation spectra of the p53 gene in human cancers.
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Notes
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3 To whom correspondence should be addressed Email: gpfeifer{at}coh.org 
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Acknowledgments
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We thank Erica Mito for assistance. This work was supported by grants from the National Institutes of Health (CA84469 and ES06070) to G.P.P.
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Received August 12, 2002;
revised September 24, 2002;
accepted October 1, 2002.