Suppressive activities of OGG1 and MYH proteins against G:C to T:A mutations caused by 8-hydroxyguanine but not by benzo[a]pyrene diol epoxide in human cells in vivo

Arito Yamane1,3, Kazuya Shinmura1, Noriaki Sunaga1, Takayuki Saitoh1,3, Satoru Yamaguchi1, Yumi Shinmura1, Kimio Yoshimura2, Hirokazu Murakami4, Yoshihisa Nojima3, Takashi Kohno1 and Jun Yokota1,5

1 Biology Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo, Japan
2 Cancer Information and Epidemiology Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo, Japan
3 Third Department of Internal Medicine, Gunma University School of Medicine, 39-15 Showa-machi 3-chome, Maebashi, Japan
4 School of Health Sciences, Faculty of Medicine, Gunma University School of Medicine, 39-15 Showa-machi 3-chome, Maebashi, Japan

5 To whom correspondence should be addressed Email: jyokota{at}gan2.ncc.go.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
8-Hydroxyguanine (8OHG), an oxidatively damaged base, and benzo[a]pyrene-diol-epoxide (BPDE), a metabolite of benzo[a]pyrene found in cigarette smoke, are thought to be major causes for G:C to T:A transversions in DNA of human cells. In this study, we assessed the abilities of OGG1, MYH and APE1 proteins, which are components of a base excision repair pathway, to suppress G:C to T:A transversions caused by 8OHG or BPDE by a bacterial suppressor tRNA (supF) forward mutation assay using a shuttle plasmid, pMY189. The introduction of a single 8OHG residue at position 159 of the supF gene and treatment with BPDE led to a 65- and 34-fold increase in mutation frequencies of the pMY189 plasmid, respectively, after replication in the NCI-H1299 human lung cancer cell line. G:C to T:A transversions were predominantly induced in these plasmids. Both the mutation frequency of the 8OHG-containing plasmid in NCI-H1299 cells and the occurrence of G:C to T:A transversions at position 159 in the supF gene were significantly reduced by overexpression of OGG1 and MYH proteins, but not by that of APE1 protein. In contrast, neither mutation frequency nor the occurrence of G:C to T:A transversion of the BPDE-treated plasmid was reduced by overexpression of OGG1, MYH and APE1 proteins. These results indicate that OGG1 and MYH function as suppressors for G:C to T:A transversions by 8OHG but not by BPDE in human cells.

Abbreviations: BER, base excision repair; BPDE, benzo[a]pyrene-diol-epoxide; IPTG, isopropyl-ß-D-thiogalactopyranoside; MF, mutation frequency; 8OHG, 8-hydroxyguanine; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactose.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
G:C to T:A transversion occupies a significant fraction of mutations in oncogenes and tumor suppressor genes in human cancers, especially in lung cancers (1,2). 8-Hydroxyguanine (8OHG), an oxidatively damaged base, and benzo[a]pyrene-diol-epoxide (BPDE), a metabolite of benzo[a]pyrene, a carcinogen in cigarette smoke, are thought to be major causes for G:C to T:A transversions in DNA of human cells (3,4). Thus, genes playing principal roles in suppressing mutagenesis by these adducts need to be defined for the development of novel ways of cancer prevention as well as for further understanding of human carcinogenesis.

8OHG in double-stranded DNA can pair with adenine in DNA replication, resulting in the formation of an 8OHG:A mispair, and this mispair leads to G:C to T:A transversion after the next DNA replication (3,5). A base excision repair (BER) pathway is supposed to be involved in the repair of 8OHG (69). OGG1 and MYH proteins have both DNA glycosylase and AP lyase activities to excise 8OHG opposite cytosine and adenine opposite 8OHG, respectively (812). APE1 protein, an apurinic/apyrimidinic (AP) endonuclease, has a 3'-phosphodiesterase activity to remove the 3' blocking end, which is formed by glycosylase reactions of OGG1 and MYH proteins, for the subsequent gap filling by DNA polymerase ß (13,14). In addition, APE1 protein has been indicated to enhance the glycosylase and/or AP lyase activities of OGG1 (1517). Therefore, it is predicted that all the OGG1, MYH and APE1 proteins function as suppressors for G:C to T:A transversion caused by 8OHG in human cells. In fact, we recently showed that human OGG1 protein has an activity to suppress G:C to T:A transversion caused by 8OHG in DNA of human cells by bacterial suppressor tRNA (supF) forward mutation assays (18). However, it is still unclear whether human MYH and APE1 proteins also have activities to suppress G:C to T:A transversion caused by 8OHG in DNA.

BPDE is considered to be a major carcinogen in tobacco smoke on the basis of its mutagenicity and carcinogenicity (4). BPDE forms adducts preferentially at the N2 position of guanine (4). Similar to the case of 8OHG, the guanine–BPDE adduct formed in double-stranded DNA can also pair with adenine in DNA replication, resulting in the formation of a guanine–BPDE adduct:A mispair, and this mispair leads to G:C to T:A transversion after the next DNA replication (1921). Although guanine–BPDE adducts have been indicated to be repaired by a nucleotide excision repair (NER) pathway (6,7,2224), several studies have also suggested the involvement of other repair pathways, such as a BER pathway, in the repair of guanine–BPDE adducts (22,24). Blakey and Douglas (22) compared the DNA lesions of CHO cells treated with benzo[a]pyrene in the presence or absence of inhibitors for NER, and suggested that mechanisms independent of NER also play a role in the repair of guanine–BPDE adducts. Recently, Braithwaite et al. (24) indicated that some BPDE adducts are labile and result in AP sites, which are likely to be repaired by BER employing AP endonucleases. Thus, we can predict that proteins involved in the repair of 8OHG in BER, such as OGG1, MYH and APE1, also function as suppressors for G:C to T:A transversions induced by BPDE adducts. However, to our knowledge, their abilities to suppress G:C to T:A transversions induced by BPDE adducts have not been investigated.

In this study, we examined suppressive activities of OGG1, MYH and APE1 proteins against G:C to T:A transversions caused by 8OHG or BPDE in human cells by supF forward mutation assays. pMY189 plasmids carrying 8OHG and BPDE adducts, respectively, were replicated in H1299 human lung cancer cells with or without overexpression of OGG1, MYH and APE1 proteins, and frequencies and spectra of mutations generated in the supF gene of these plasmids were compared. The results indicated that MYH and OGG1, but not APE1, were suppressors for G:C to T:A transversions caused by 8OHG. On the other hand, neither OGG1, MYH nor APE1 were indicated to be suppressors for G:C to T:A transversions caused by BPDE adducts.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells, bacterial strains, helper phage and plasmids
A lung cancer cell line, NCI-H1299, was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. NCI-H1299 had no mutation for the coding regions of the OGG1, MYH and APE1 genes (data not shown). This cell line was homozygous for the Arg46, Ser326 and Glu308 alleles at non-synonymous polymorphic sites of the OGG1 gene, for Pro18, Val22, Gly25, Gln324 and Gly382 alleles of the MYH gene and for Ile64 and Glu148 alleles of the APE1 gene. A plasmid pMY189 and an indicator Escherichia coli strain KS40/pKY241 were used for the supF forward mutation assay as reported previously (18,25). pMY189 is a shuttle vector containing the bacterial suppressor tRNA (supF) gene. KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 with genotype lacZ (am), CA7070, lacY1, hsdR, hsdM, {Delta}(araABC-leu)7679, galU, galK, rpsL, thi. Plasmid pKY241 contains a chloramphenicol resistance marker and the gyrA (amber) gene. Escherichia coli KS40/pKY241 cells carrying the active supF gene are sensitive to nalidixic acid and form blue colonies on LB plates containing 5-bromo-4-chloro-3-indolyl-ß-D-galactose (X-gal) and isopropyl-ß-D-thiogalactopyranoside (IPTG), whereas the cells carrying the mutated supF gene form white colonies on plates containing nalidixic acid, X-gal and IPTG. Escherichia coli XL1-Blue MRF' (Stratagene, La Jolla, CA) and R408 Helper Phage (Stratagene) were used to prepare single-stranded pMY189 DNA for the construction of double-stranded pMY189 DNA with or without a 8OHG:C base pair site.

Construction of double-stranded plasmid with or without a 8OHG:C base pair site
Two kinds of plasmids, pMY189-8OHG, containing a single 8OHG-cytosine pair at nucleotide position 159 of the supF gene and pMY189-G, containing a guanine-cytosine pair at the same position, were prepared according to the method described previously (18). Briefly, 30 µg of single-stranded plasmid pMY189 and a 5-fold molar excess of 5'-phosphorylated 24mer oligonucleotide with or without a single 8OHG at nucleotide position 159 of the supF gene [5'-CGACTTCGAA-8OHG (or G)-GTTCGAATCCTTC-3'], were annealed in a 140 µl reaction mixture containing 10 mM Tris–HCl, pH 7.9, 50 mM NaCl, 10 mM MgCl2 and 1 mM DTT at 65°C for 15 min and cooled to room temperature. Forty units T4 DNA polymerase (Takara, Tokyo, Japan), 600 µM deoxynucleotide triphosphate (dNTPs), 36 Weiss units T4 DNA ligase (New England Biolabs, Beverly, MA) and 1 mM ATP were added to the reaction mixture and it was incubated at 37°C for 4 h. Closed circular pMY189-8OHG was isolated by CsCl–ethidium bromide density gradient centrifugation.

Preparation of plasmids with BPDE adducts
The pMY189 double-stranded plasmid DNA was prepared as described (18,25). Treatment with BPDE was performed in 200 µl of reaction mixture containing 10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 30 µg of double-stranded pMY189. Eight microliters of 1 or 0 mM (±)-anti-BPDE (the National Cancer Institute Chemical Carcinogen Reference Standard Repository, Rockville, MD) in a tetrahydrofuran solution containing 0.5% triethylamine was added to the reaction mixture and the mixture was incubated for 90 min at room temperature with protection from light. Unbound BPDE was removed by ethanol precipitation three times.

SupF forward mutation assay
A total of 1 x 106 cells were seeded onto 100-mm tissue culture plate and transfected with pMY189 plasmid using DMRIE-C reagent after 16 h incubation. After 48 h, the plasmid was recovered using a QIAprep Spin Miniprep kit (Qiagen). The plasmid solution was treated with 20 U of DpnI to eliminate unreplicated plasmids, which retained a bacterial methylation pattern. The DNA was purified with a Microcon and Micropure-EZ filter (Millipore, Bedford, MA). Aliquots of purified DNA solution were used to transform the E.coli KS40/pKY241 indicator strain by electroporation using a Gene Pulser apparatus (BioRad, Hercules, CA). Transformants were plated onto LB agar plates containing 50 µg/ml nalidixic acid, 150 µg/ml ampicillin, 30 µg/ml chloramphenicol, 2 mM IPTG and 80 µg/ml X-gal. White colonies on this plate were counted as supF mutants. Mutation frequencies were calculated as the number of supF mutants per the total number of transformants, which were counted on LB plates containing ampicillin, chloramphenicol, IPTG and X-gal. Each assay was undertaken in triplicate and mutation frequency was expressed as mean ± SD. The frequency of each type of mutation was calculated by multiplying the total mutation frequency by the proportion of mutants carrying the mutation among all mutants.

Analysis of mutations in the supF gene
Mutations in the supF gene were analyzed as described previously (18). Briefly, each mutant colony was transferred to 50 µl of LB medium with ampicillin. DNA fragments containing the supF gene were amplified from mutant plasmids by PCR. One microliter of the medium was used for PCR in 20 µl of total reaction mixture with 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, 0.5 U Taq DNA polymerase (Takara) and 300 nM forward primer (5'-TGTAAAACGACGGCCAGT-3', 30–47 bp upstream of the supF gene) and reverse primer (5'-ATCTCAAGAAGATCCTTTGATC-3', 31–52 bp downstream of the supF gene). PCR conditions were as follows: 60 s at 95°C, 60 s at 55°C, 60 s at 72°C for 35 cycles, followed by 10 min at 72°C. The sizes of PCR products were roughly determined by agarose gel electrophoresis. Mutants with larger- and smaller-sized PCR products were considered to carry deletions, insertions or gross rearrangements of the supF gene and were excluded from sequence analysis. Mutants with apparently normal-sized PCR products were sequenced to confirm whether the supF gene was mutated or not. PCR products were purified with a QIAquick PCR purification kit (Qiagen) and were directly sequenced with a BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems Japan, Tokyo, Japan) and ABI 310 Genetic Analyzer (Applied Biosystems Japan).

Construction of stable transfectants
pcDNA3.1 plasmids (Invitrogen, Carlsbad, CA) containing full length cDNAs for OGG1, MYH and APE1 were used for the construction of NCI-H1299 clone stably expressing OGG1, MYH and APE1 proteins, respectively. Stable transfectants for OGG1 were described previously (18). The vector to express nuclear type 2 MYH protein was constructed previously (26). The vector to express APE1 protein was constructed as follows. The coding region of APE1 cDNA was amplified by PCR against the pUAEH1 plasmid DNA (27) with a primer set (5'-CCGGAATTCCGGGTAACGGGAATGCCGAAGC-3' and 5'-CCGGAATTCCGGTCACAGTGCTAGGTATAGGG-3') and was subcloned into the EcoRI site of the pcDNA3.1 plasmid vector. Transfections were performed by DMRIE-C reagent (Invitrogen) according to the manufacturer's instructions. Stable transfectants of MYH and APE1 were isolated by selection with 600 µg/ml of G418 (Invitrogen).

Western blot analysis
Cellular proteins were extracted in lysis buffer containing 40 mM HEPES–KOH, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and Complete Mini, EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Aliquots of 50, 30, 20 and 10 µg of whole cell lysates were used for detection of OGG1, MYH, APE1 and {alpha}-tubulin proteins, respectively. The whole cell lysates were resolved by SDS–PAGE and electroblotted to Hybond-P (Amersham, Tokyo, Japan) membranes. Membranes were incubated with 5 µg/ml of a mouse anti-human OGG1 antibody (mAb-7E2) (28), 5 µg/ml of a rabbit anti-human MYH antibody #2 (Alpha Diagnostic International, San Antonio, TX), 1 µg/ml of a rabbit anti-Ref-1/APE1 antibody (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA) or 1 µg/ml of a mouse anti-{alpha}-tubulin antibody (Oncogene Research Products, San Diego, CA), followed by exposure to a 1:5000 dilution of a sheep anti-mouse Ig-HRP linked F(ab')2 fragment (Amersham) or a 1:5000 dilution of a donkey anti-rabbit Ig-HRP secondary antibody (Amersham). Proteins were visualized using the ECL Western blotting detection system (Amersham).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Induction of G:C to T:A transversions by 8OHG
The pMY189-8OHG plasmid, which contains a single 8OHG at position 159 of the supF gene, and the pMY189-G plasmid, which contains guanine at the same position, were introduced to and replicated for 48 h in the human lung cancer cell line, NCI-H1299. The mutation frequency (MF) in the supF gene of the pMY189-8OHG plasmid was 4.6 ± 1.0 x 10-2, whereas that in the pMY189-G plasmid was 7.1 ± 0.6 x 10-4 (Figure 1). Therefore, MF of pMY189-8OHG was 65 times higher than MF of pMY189-G. Ninety-four and 81 supF mutants for pMY189-8OHG and pMY189-G, respectively, were randomly selected and analyzed for the types of mutation by PCR amplification and sequencing analysis of the supF gene. Base substitutions in position 159 of the supF gene were detected in all 94 mutants from pMY189-8OHG, and 91 of them were G:C to T:A transversions (Table I). The frequency of G:C to T:A transversion of pMY189-8OHG in position 159 was estimated as 4.4 x 10-2 (=4.6 x 10-2 x 91/94). In contrast, among the 81 mutants analyzed for pMY189-G, no G:C to T:A transversion was detected in position 159. Therefore, the frequency of G:C to T:A transversion of pMY189-G in position 159 was estimated as <8.7 x 10-6 (<7.1 x 10-4 x 1/81). Thus, consistent with our previous study (18), G:C to T:A transversions were specifically induced at position 159, where a 8OHG:C base pair was present in the pMY189-8OHG plasmid.



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Fig. 1. Mutation frequencies in the supF gene of pMY189 plasmids with or without 8OHG replicated in parental and stably transfected NCI-H1299 cells. Data represent mean mutation frequencies of three independent experiments ± SD. *P value for the difference was <0.05, respectively, in unpaired t-test. NS, not significant for the difference was >0.05.

 

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Table I. Proportion of mutants with base substitutions in the supF gene of pMY189 with or without 8OHG

 
Abilities of OGG1, MYH and APE1 proteins to suppress G:C to T:A transversions caused by 8OHG
Three NCI-H1299 derived clones with OGG1 protein overexpression were isolated previously (18). MYH- and APE1-overexpressing and vector-transfected NCI-H1299 cells were obtained by selection of stable transfectants with vectors to express MYH and APE1 proteins and an empty vector, respectively. Expression of OGG1 and MYH proteins was detected in three OGG1-overexpressing clones, H1299-OG1, H1299-OG2 and H1299-OG3, and in three MYH-overexpressing clones, H1299-MY1, H1299-MY2 and H1299-MY3, respectively, in western blot analysis, while expression of these two proteins was undetectable in the parental NCI-H1299 cell line and three vector-transfected clones, H1299-pcDNA1, H1299-pcDNA2 and H1299-pcDNA3 (Figure 2). Expression of APE1 protein was detected not only in three APE1-overexpressing clones, H1299-AP1, H1299-AP2 and H1299-AP3, but also in the parental NCI-H1299 and vector-transfected clones. However, the APE1 protein expression levels in the APE1-overexpressing clones were estimated as being at least five times higher than those in the parental NCI-H1299 cells and the vector-transfected clones (data not shown).



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Fig. 2. Western blot analysis of OGG1, MYH and APE1 proteins in parental and stably transfected NCI-H1299 cells. {alpha}-tubulin was also analyzed to standardize the amounts of proteins loaded. pcDNA1, 2 and 3, vector transfectants; OG1, 2 and 3, OGG1 stable transfectants; MY1, 2 and 3, MYH stable transfectants; AP1, 2 and 3, APE1 stable transfectants.

 
Mutation frequencies in the supF gene of the pMY189-8OHG plasmid were examined in the NCI-H1299 derived clones described above (Figure 1). Mutation frequencies in OGG1- or MYH-overexpressing clones were significantly lower than those in vector-transfected clones. In contrast, MFs in APE1-overexpressing clones were not significantly lower than those in vector-transfected clones. Mutation frequencies in the supF gene of the pMY189-G plasmid after replication in H1299-pcDNA1, -OG1, -MY1 and -AP1 were not significantly different to each other.

SupF mutant clones were randomly picked up and were subjected to analysis of the types of mutations in the supF gene (Table I). More than 80% of mutants analyzed for pMY189-8OHG replicated in vector-transfected clones were G:C to T:A transversions at position 159. Consistent with our results (18), the proportion of G:C to T:A transversions at position 159 in the OGG1-overexpressing clones were lower than those in vector-transfected clones, and the differences were statistically significant when the proportion in H1299-pcDNA1 was used as a reference. Instead of the reduction of G:C to T:A transversions, genetic alterations other than base substitutions, including deletions, insertions and gross rearrangements, were dominantly detected among mutants from OGG1-overexpressing clones. The proportion of G:C to T:A transversions at position 159 in MYH-overexpressing clones was also lower than those in vector-transfected clones, and the differences were statistically significant when the proportion in H1299-pcDNA1 was used as a reference. Instead of the reduction of G:C to T:A transversions, G:C to C:G transversions and G:C to A:T transitions at position 159 were dominantly detected among mutants from MYH-overexpressing clones. In contrast, the proportion of G:C to T:A transversions at position 159 was not significantly different between APE1-overexpressing clones and vector-transfected clones.

Induction of G:C to T:A transversion by BPDE
pMY189-BPDE-40 and pMY189-BPDE-0 were the pMY189 plasmids treated with the solution containing 40 µM of BPDE and without BPDE, respectively. These plasmids were introduced to and replicated for 48 h in the parental NCI-H1299 cells. The mutation frequency in the supF gene of pMY189-BPDE-40 was 1.3 ± 0.3 x 10-2, whereas that of pMY189-BPDE-0 was 3.8 ± 0.9 x 10-4 (Figure 3). Therefore, MF of pMY189-BPDE-40 was 34 times higher than that of pMY189-BPDE-0. Four hundred and seventy-eight and 69 supF mutants were picked up for pMY189-BPDE-0 and pMY189-BPDE-40, respectively. The proportions of mutants with base substitutions for pMY189-BPDE-40 (56/69, 81.2%) were significantly higher than that for pMY189-BPDE-0 (8/478, 1.7%). G:C to T:A transversions were most frequent among the base substitutions detected in mutants both for pMY189-BPDE-40 and pMY189-BPDE-0, although other base substitutions including G:C to C:G and G:C to A:T were also detected (Table II). The frequency of G:C to T:A transversions in pMY189-BPDE-40 was estimated as 8.4 x 10-3 (=1.3 x 10-2 x 44/69), whereas that in pMY189-BPDE-0 was as 3.2 x 10-6 (=3.8 x 10-4 x 4/478). Therefore, the frequency of G:C to T:A transversions in pMY189-BPDE-40 was ~2600 times higher than that in pMY189-BPDE-0. This result indicated that several base substitutions, in particular G:C to T:A transversions as predominant ones, were induced by the BPDE treatment.



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Fig. 3. Mutation frequencies in the supF gene of pMY189 plasmids with or without BPDE-treatment replicated in parental and stably transfected NCI-H1299 cells. Data represent mean mutation frequencies of three independent experiments ± SD.

 

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Table II. Base substitutions in the supF gene of pMY189 plasmid with or without BPDE treatment

 
Abilities of OGG1, MYH and APE1 proteins to suppress G:C to T:A transversions caused by BPDE
Mutation frequencies in the supF gene of the pMY189- BPDE-40 plasmid after replication in H1299-pcDNA1, H1299-OG1, H1299-MY1 and H1299-AP1 cells were examined (Figure 3). Mutation frequencies of NCI-H1299 clones overexpressing OGG1, MYH or APE1 were similar to and not significantly different from those of a vector-transfected clone and parental NCI-H1299 cells. Ninety-six, 92, 94 and 96 mutants were randomly picked up for H1299-pcDNA1, H1299-OG1, H1299-MY1 and H1299-AP1, respectively, and were subjected to the analysis of mutation types in the supF gene (Table II). Proportions of mutants with base substitutions for NCI-H1299 clones overexpressing OGG1, MYH or APE1 were similar to and not significantly different from those for a vector-transfected clone and parental NCI-H1299 cells. Several types of base substitutions were detected with different proportions among mutants for each H1299 clone; however, G:C to T:A transversions were predominant in mutants for all H1299 clones as in the case of parental H1299 cells. Proportions of mutants with each type of base substitution, including those with G:C to T:A transversions, were not significantly different among H1299 clones and parental H1299 cells.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we investigated abilities of BER proteins, OGG1, MYH and APE1, to suppress G:C to T:A mutagenesis caused by 8OHG and BPDE.

We first examined suppressive activities of OGG1, MYH and APE1 proteins against G:C to T:A transversions by 8OHG. The mutation frequency of pMY189-8OHG was 65 times higher than that of pMY189-G due to the exclusive occurrence of G:C to T:A transversions at the position of 8OHG. This result was well consistent with previous findings that the presence of 8OHG paired with C in DNA causes G:C to T:A transversion in vivo (9,29,30) as well as in vitro (5). Overexpression of OGG1 protein led to the decrease in MFs of pMY189-8OHG by reducing G:C to T:A transversion at the position of 8OHG (Figure 1), as shown in our previous study (18). Overexpression of the MYH protein also led to the decrease in MFs of pMY189-8OHG. The proportion of G:C to T:A transversions at the position of 8OHG was also decreased. In contrast, decreases in MFs of pMY189-8OHG were not observed in APE1-overexpressing clones and the proportion of G:C to T:A transversions at the 8OHG position were not significantly different among APE1-overexpressing clones, parental NCI-H1299 cells and vector-transfected clones. Thus, it was indicated that MYH, but not APE1, has a suppressive activity against G:C to T:A transversion caused by 8OHG:C mispair in DNA of human cells, and that the activity of MYH was comparable with that of OGG1.

G:C to T:A transversions were reduced both in MYH- and OGG1-overexpressing clones; however, the mutation spectra for the resultant supF-mutants were different between MYH- and OGG1-overexpressing clones. In MYH-overexpressing clones, G:C to C:G and G:C to A:T mutations were produced, whereas, in OGG1-overexpressing clones, deletions and other gross rearrangements were produced. These results were probably due to the difference of MYH and OGG1 proteins in the mode of 8OHG-repair. When DNA containing 8OHG:C pair is replicated, guanine and thymine are incorporated opposite 8OHG, generating 8OHG:G and 8OHG:T mispairs, although adenine and cytosine are predominantly incorporated (29,30). Consistent with this fact, in parental and vector-transfected H1299 cells, G:C to C:G and G:C to A:T mutations occurred in addition to G:C to T:A mutations (Table I). MYH has a specific DNA glycosylase activity to excise the adenine base from an 8OHG:A pair; therefore, 8OHG:G and 8OHG:T mispairs remain unrepaired and could lead to producing G:C to C:G and G:C to A:T mutations in MYH-overexpressing clones. This prediction was supported by the fact that frequencies of G:C to C:G and G:C to A:T substitutions, which were estimated from mutation frequencies multiplied by proportions of each type of base substitutions, were similar between vector-transfected clones and MYH-overexpressing clones (e.g. the frequency of G:C to C:G substitution in H1299-pcDNA1 were 5.7 x 10-4 [= 1.8 x 10-2 x 3/94] and that in H1299-MY1 were 8.6 x 10-4 [= 5.1 x 10-3 x 16/95]). A possible reason for the overproduction of mutants with deletions and other gross rearrangements in OGG1-overexpressing clones would be the following, as we previously discussed (18). The presence of 3'-blocking ends at AP sites, which were left after the removal of 8OHG by OGG1 protein, could lead to double strand breaks underlying deletions and gross rearrangements.

The result of APE1 was unexpected, as APE1 protein was thought to show suppressive activity both by removing the 3' blocking ends, which were generated at AP sites by OGG1 and/or MYH, for the subsequent gap filling (13,14) and by enhancing the glycosylase and/or AP-lyase activity of OGG1 protein (1517). The reason for this result might be the abundant amounts of APE1 protein in parental NCI-H1299 cells. In contrast to the fact that the amounts of endogenous OGG1 and MYH proteins were less than the level to be detected by western blot analysis, endogenous APE1 proteins were readily detectable (Figure 2). Thus, the effects of exogenous APE1 protein were masked by the activities of endogenous APE1 protein. Alternatively, parental NCI-H1299 cells contain only a small amount of endogenous OGG1 and MYH proteins, thus, overexpression of APE1 alone did not lead to the mutation suppression due to inefficient generation of AP sites to be processed by APE1 protein.

We next addressed the suppressive activity of OGG1, MYH and APE1 proteins against G:C to T:A transversions by BPDE. The mutation frequency of pMY189 plasmid treated with BPDE was 34 times higher than that of untreated plasmid. Although BPDE treatment led to the induction of several types of genetic alterations, base substitutions were predominantly induced. In particular, G:C to T:A transversions were predominant among several types of base substitutions induced (Table II). This result is well consistent with previous studies using shuttle plasmids treated with BPDE (19,20,31,32), and indicates that G:C to T:A transversions were predominantly induced by BPDE-adducts formed on the pMY189 plasmid. Thus, BPDE-treated pMY189 plasmid was subjected to assess the suppression activities of BER proteins against G:C to T:A transversions caused by BPDE. Neither the mutation frequency nor the proportions of mutants with base substitutions for the BPDE-treated plasmid was significantly reduced by overexpression of any of the OGG1, MYH or APE1 proteins. Several types of base substitutions were detected among mutants for each of the H1299 clones with OGG1, MYH or APE1 overexpression, as in the case of parental H1299 cells. G:C to T:A transversions were predominant in mutants for all these H1299 clones, and proportions of G:C to T:A transversion among mutants were not significantly reduced by overexpression of OGG1, MYH and APE1. Thus, G:C to T:A transversions by BPDE adducts were unlikely to be suppressed by OGG1, MYH and APE1 protein expression. Proportions of mutants with each base substitution were different among H1299 clones (Table II). As BPDE leads to the formation of multiple types of adducts in DNA (4), this result might suggest that each BER protein repaired a subset of BPDE–adducts formed on the plasmid. However, this result might also be due to the bias in picking up mutant colonies, as such differences were also observed in proportions of each base substitution between parental and vector-transfected H1299 cells. Thus, we could not conclude that either OGG1, MYH or APE1 protein have suppressive activities against mutations induced by BPDE, including G:C to T:A transversions. BPDE–adducts in DNA have been suggested to lead to AP site formation (24), therefore, it was conceivable that APE1 protein suppressed the mutagenesis caused by BPDE by repairing the AP sites. As discussed above, the abundant endogenous APE1 protein could have masked the activity of exogenous APE1 protein. Alternatively, AP sites formed from BPDE adducts might not be a major cause for mutagenesis by BPDE.

In this study, MYH and OGG1, but not APE1, were indicated as being suppressors for G:C to T:A transversions by 8OHG. On the other hand, neither OGG1, MYH nor APE1 were indicated to be suppressors for G:C to T:A transversions caused by BPDE adducts. It is noted that genetic variations of the OGG1 and MYH genes have been shown to be associated with risks of several cancers. A non-synonymous single nucleotide polymorphism (SNP) in OGG1, which causes a difference in the activity of OGG1 protein, has been reported to be associated with risks of lung and several other cancers (3340). Two genetic variations in MYH were reported as being responsible for the development of adenomatous polyposis coli (41). In addition, we recently defined a MYH SNP causing reduced translation of MYH protein (26). Thus, the present results further support the idea that these OGG1 and MYH variations contribute to cancer susceptibility by causing inter-individual differences in suppression activities against G:C to T:A transversions by 8OHG. Therefore, functional and genetic studies of OGG1 and MYH should be further undertaken for the development of novel ways of cancer prevention as well as for further understanding of human carcinogenesis.


    Acknowledgments
 
The authors would like to thank Dr J.D.Minna and A.F.Gazdar of the University of Texas Southwestern Medical Center, USA, for providing the NCI-H1299 cell line, Dr T.Matsuda of Graduate School of Global Environmental Studies, Kyoto University, Japan, for providing a shuttle vector plasmid pMY189 and for the valuable advice on supF forward mutation assay, and Dr S.Seki of the Chugoku Gakuen University, Japan, for providing plasmid pUAEH1. We also thank Dr M.Karasawa of Gunma University School of Medicine for encouragement throughout this study. This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare, from the Ministry of Education, Culture, Sports, Science and Technology for Scientific Research on Priority Areas (KAKENHI 14026064) and from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 4, 2002; revised February 20, 2003; accepted March 23, 2003.