OGG1 protein suppresses G:C->T:A mutation in a shuttle vector containing 8-hydroxyguanine in human cells

Noriaki Sunaga1,2, Takashi Kohno1, Kazuya Shinmura1, Takayuki Saitoh1, Tomonari Matsuda3, Ryusei Saito2 and Jun Yokota1,4

1 Biology Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045,
2 First Department of Internal Medicine, Gunma University School of Medicine, 39-15 Showa-machi 3-chome, Gunma 371-8511 and
3 Research Center for Environmental Quality Control, Kyoto University, 1-2 Yumihara, Otsu, Shiga 520, Japan


    Abstract
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 Materials and methods
 Results
 Discussion
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8-Hydroxyguanine (8-OHG) is an oxidatively damaged mutagenic base which causes G:C->T:A transversions in DNA. OGG1 was cloned as a human gene encoding a DNA glycosylase that specifically excises 8-OHG from DNA in vitro. However, it was not clear whether OGG1 protein suppresses G:C->T:A transversions caused by 8-OHG in human cells in vivo. In the present study we have examined the ability of OGG1 protein to suppress G:C->T:A transversions caused by 8-OHG in human cells by bacterial suppressor tRNA (supF) forward mutation assay using a shuttle vector DNA, pMY189. Introduction of a single 8-OHG residue at position 159 of the supF gene in plasmid pMY189 resulted in a 130-fold increase in mutation frequency compared with untreated plasmid pMY189 after replication in the NCI-H1299 human lung cancer cell line. G:C->T:A transversions at position 159 were detected in >90% of the supF mutants from the 8-OHG-containing plasmid. The mutation frequency of the 8-OHG-containing plasmid was significantly reduced by overexpression of OGG1 protein in NCI-H1299 cells and, in particular, the occurrence of G:C->T:A transversion at position 159 in the supF gene was suppressed. Furthermore, frequencies and spectra of mutations of the untreated pMY189 plasmid did not differ significantly with overexpression of OGG1 protein. These results indicate that OGG1 protein has the ability to suppress G:C->T:A transversions caused by 8-OHG in human cells in vivo.

Abbreviations: AP, apurinic/apyrimidinic; APEX, AP endonuclease; dG, deoxyguanosine; HPLC–ECD, high performance liquid chromatography with electrochemical detection; IPTG, isopropyl-ß-D-thiogalactopyranoside; LB, Luria–Bertani; 8-OHdG, 8-hydroxydeoxyguanosine; 8-OHG, 8-hydroxyguanine; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Some oxidative DNA lesions are highly mutagenic and thus are believed to be involved in human carcinogenesis (1,2). 8-Hydroxyguanine (8-OHG) is a major form of oxidative DNA lesion produced by reactive free radicals (3,4). The presence of 8-OHG in DNA causes G:C->T:A transversions, since 8-OHG allows the incorporation of cytosine and adenine nucleotides opposite the lesion during DNA replication (5). Thus, 8-OHG is a highly mutagenic DNA lesion unless repaired prior to DNA replication.

Genes coding for DNA glycosylases, which excise 8-OHG paired with C from DNA, have been cloned in bacteria (MutM) and yeast (OGG1) (69). The Escherichia coli mutM mutant is deficient in 8-OHG repair and shows a 10-fold increase in spontaneously occurring G:C->T:A transversions compared with the wild-type strain (10). Similarly, an OGG1-disrupted yeast strain exhibits a mutator phenotype with a 50-fold increase in spontaneously occurring G:C->T:A transversions compared with the wild-type strain (11). Recently we and others cloned structural and functional human and mouse homologs of the yeast OGG1 gene, OGG1 and Ogg1, which encode polypeptides with an ability to suppress the mutator phenotype of E.coli mutants deficient in 8-OHG repair (1219). OGG1/Ogg1 protein, expressed in and purified from bacterial cells, catalyzes the DNA glycosylase and apurinic/apyrimidinic (AP) lyase reactions for 8-OHG:C pairs in double-stranded DNA, leaving the AP site with a 3'-blocking end that is thought to be further processed by the AP endonuclease (APEX), DNA polymerase ß and ligase III proteins (12, 1423). In Ogg1 null mice the spontaneous mutation frequency in their liver tissues was shown to be two to three times higher than that in wild-type mice and >50% of base substitutions observed in the liver tissue of Ogg1 null mice consisted of G:C->T:A transversions (24,25). However, a recent study indicated that the spontaneous mutation rates in Chinese hamster ovary cells were not significantly changed by overexpression of OGG1 protein (26). Therefore, it was still obscure whether OGG1 has the ability to suppress G:C->T:A transversions caused by 8-OHG in vivo.

In order to clarify this issue, we have investigated the ability of OGG1 protein to suppress G:C->T:A transversions caused by 8-OHG in human cells by bacterial suppressor tRNA (supF) forward mutation assay using a shuttle vector DNA (pMY189) containing the supF gene of E.coli. We first prepared three types of pMY189 plasmids containing 8-OHG for this purpose. Two of them were treated with riboflavin plus visible light and CuCl2 plus H2O2, respectively, both of which are known to efficiently produce 8-OHG in DNA (27,28). The third one was a modified pMY189 plasmid in which 8-OHG was directly incorporated instead of G at position 159 of the supF gene. These pMY189 plasmids were replicated in a recipient human lung cancer cell line (NCI-H1299) and were recovered to analyze the frequencies and spectra of mutations that occurred in the supF gene. It was found that G:C->T:A transversion was most specifically induced in the plasmid in which 8-OHG was directly incorporated to pair with C in the supF gene among the three types of plasmids examined. Thus, the 8-OHG-containing plasmid was further introduced into NCI-H1299 cells in which OGG1 protein was overexpressed by stable transfection of an OGG1 expression plasmid. The frequency of G:C->T:A transversion at position 159 of the supF gene in the transfectants was significantly lower than that in the parental NCI-H1299 cells. The results indicate that OGG1 has the ability to suppress G:C->T:A transversions caused by 8-OHG in human cells in vivo.


    Materials and methods
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 Materials and methods
 Results
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Cells, bacterial strains, helper phage and plasmids
A human lung cancer cell line, NCI-H1299, was used as the recipient in the supF forward mutation assay. NCI-H1299 is wild-type for the OGG1 gene (data not shown). It is homozygous for the Arg46, Ser326 and Glu308 alleles at three non-synonymous polymorphic sites and is homozygous for major alleles at other synonymous polymorphic sites. The expression level of OGG1 type 1a protein in H1299 cells was lower than the level detectable by western blot analysis using a monoclonal antibody, mAb-7E2 (29). NCI-H1299 has a homozygous deletion of the p53 gene (30). An indicator E.coli strain (KS40/pKY241) and a shuttle vector plasmid (pMY189) were also used in the supF forward mutation assay (31). 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, whereas cells carrying the mutated supF form colonies on plates containing nalidixic acid, chloramphenicol and ampicillin. Escherichia coli cells carrying the active supF gene produce blue colonies, whereas cells carrying the mutated supF produce white colonies on selection plates. Escherichia coli strain XL1-Blue MRF' (Stratagene, Tokyo, Japan), with genotype {Delta} (mcrA)183 {Delta} (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac[F' proAB laclqZDM15 Tn10 (Tetr)], and R408 Interference-Resistant Helper Phage (Stratagene) were used for preparation of single-stranded pMY189 DNA.

Media
Luria–Bertani (LB) medium and LB plates were prepared as described (31). Chemicals listed below were used for the LB medium and LB plates in the supF forward mutation assay as indicated: 50 µg/ml nalidixic acid, 30 µg/ml chloramphenicol, 150 µg/ml ampicillin, 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) and 0.008% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) (Calbiochem, Tokyo, Japan). All chemicals not specifically mentioned above were purchased from Sigma-Aldrich Japan.

Treatment of plasmids with riboflavin plus visible light or CuCl plus H2O2
The pMY189 double-stranded plasmid DNA was prepared as described (31). Briefly, E.coli JM109 cells containing pMY189 were grown in LB medium with ampicillin for 12–14 h, with shaking at 250 r.p.m. at 37°C. The plasmid was purified using the Qiagen Plasmid Purification Kit (Qiagen, Tokyo, Japan), followed by the purification of closed circular DNA using cesium chloride/ethidium bromide density gradient centrifugation. The pMY189 double-stranded plasmid DNA was treated with riboflavin plus visible light as described by Tano et al. (28). A solution containing plasmid DNA (1.6 µg) and riboflavin (5 µg/ml) in 10 mM phosphate buffer in a total volume of 64 µl was irradiated at room temperature with white light (150 W tungsten lamp) for 5 min at a distance of 21 cm from the surface of the sample. The pMY189 double-stranded plasmid DNA was also treated with CuCl2 plus H2O2 as described by Sagripanti and Kraemer (32). A solution of plasmid DNA (1.6 µg) in 10 mM phosphate buffer was incubated for 30 min at 20°C with 0.1 mM CuCl2 plus 1 mM H2O2 in a total volume of 15 µl. The reaction was stopped by addition of 1 µl of 0.2 M EDTA. The two treated plasmid DNAs were ethanol precipitated and re-dissolved in TE buffer, respectively.

Quantification of 8-OHG
The content of 8-OHG in DNA was determined by the methods described by Tsurudome et al. (33), with some modifications. Plasmid DNA was denatured at 100°C for 3 min, digested with nuclease P1 (Yamasa, Tokyo, Japan) at 37°C for 30 min and treated with bacterial alkaline phosphatase at 37°C for 1 h. The resulting deoxynucleoside mixtures were analyzed with a high performance liquid chromatography with electrochemical detection (HPLC–ECD) system. The molar ratio of 8-hydroxydeoxyguanosine (8-OHdG) to deoxyguanosine (dG) was determined from the profile of authentic 8-OHG with an EC detector (ESA Inc., USA) and UV absorbance detector (Tosoh, Tokyo, Japan).

Construction of closed circular double-stranded plasmid DNA containing a single 8-OHG residue
The pMY189 single-stranded DNA was prepared in an E.coli strain (XL1-Blue MRF') as described (34) using the R408 helper phage. The 5'-phosphorylated 24mer oligonucleotide containing a single 8-OHG at nucleotide position 159 of supF (5'-CGACTTCGAA-8-OHG-GTTCGAATCCTTC-3') was synthesized and purified as described (20). Position 159 was chosen for the 8-OHG incorporation site because a single G->T mutation at position 159 led to inactivation of supF in our previous supF forward mutation assay using the pMY189 plasmid. Closed circular double-stranded plasmid DNA containing a single 8-OHG was synthesized by the methods described by Moggs et al. (35), with some modifications. Thirty micrograms of plus single-stranded plasmid DNA were annealed with a 5-fold molar excess of the 5'-phosphorylated 24mer oligonucleotide in a 140 µl reaction mixture containing 10 mM Tris–HCl, pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT at 65°C for 15 min. Then the mixture was cooled to room temperature. After the annealing reaction, 600 µM deoxynucleotide triphosphate, 40 U T4 DNA polymerase (New England Biolabs, Tokyo, Japan), 36 Weiss units T4 DNA ligase (New England Biolabs) and 1 mM ATP were added and primer extension was allowed to proceed at 37°C for 4 h. Closed circular DNA was isolated by cesium chloride/ethidium bromide density gradient centrifugation, then purified by consecutive isoamyl alcohol extraction and ethanol precipitation and re-dissolved in TE buffer.

Construction of OGG1 stable transfectants
The type 1a/pcDNA3 expression plasmid, which contains a full-length cDNA fragment for the OGG1 type 1a isoform, was used (29). This construct allowed constitutive expression of OGG1 type 1a driven by the cytomegalovirus promoter in mammalian cells. A total of 1x106 cells were plated onto a 100 mm dish and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Tokyo, Japan) and antibiotics at 37°C in a 5% CO2 atmosphere. After 16 h cell culture, the type 1a/pcDNA3 plasmid (2.0 µg) was transfected into the cells using DMRIE-C reagent (Life Technologies) according to the supplier's recommendations. After 48 h, 1% of the cells were plated onto a 100 mm culture dish. Geneticin sulfate (600 µg/ml; Life Technologies) was added after 24 h. After 14 days, single clones were isolated and propagated in 24-well plates. The expression status of OGG1 protein was examined by western blot analysis as described below.

Western blot analysis
Cellular proteins were extracted with lysis buffer containing 40 mM HEPES–KOH, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin and 10 µg/ml aprotinin, fractionated by SDS–PAGE and transferred to Hybond-P membrane (Amersham Pharmacia Biotech, Tokyo, Japan). After blocking with 5% non-fat dry milk and 0.1% Tween 20 in Tris-buffered saline, membranes were incubated at 37°C for 1 h with antibody mAb-7E2 (29). The membranes were developed after incubation with goat anti-mouse IgG (H + L)–horseradish peroxidase conjugate (Life Technologies) with the ECL western blotting detection system (Amersham Pharmacia Biotech) according to the supplier's recommendations. Equal loading of protein was confirmed by staining the membrane after detection.

SupF forward mutation assay
A total of 6x105 cells in 10 ml of culture medium were plated onto a 100 mm dish. After 16 h cell culture, plasmid pMY189 (1.0 µg) was transfected into the cells using DMRIE-C reagent (Life Technologies) according to the supplier's recommendations. After 72 h, propagated plasmids were extracted from the cells using a QIAprep Spin Miniprep Kit (Qiagen). The extracted plasmids were digested with DpnI (New England Biolabs) to eliminate unreplicated plasmids, which retained a bacterial methylation pattern. After removal of proteins by passage through a Micropure-EZ filter unit (Millipore, Tokyo, Japan), DNA was purified with a Microcon (Millipore).

The plasmid DNAs recovered were introduced into the KS40/pKY241 indicator bacteria with a Gene Pulser II electroporation apparatus (Bio-Rad, Tokyo, Japan). To select E.coli with a mutated supF gene, the transformed cells were plated onto a LB plate containing nalidixic acid, ampicillin, chloramphenicol, IPTG and X-gal and were cultured at 37°C for 24 h. A white colony on the plate indicated a supF mutant. To determine the total number of transformants, a portion of the transformed cells was plated onto a LB plate containing ampicillin and chloramphenicol. After 24 h cell culture at 37°C the colonies were counted and mutation frequencies, which are defined by the supF mutant fraction divided by the total transformant fraction, were calculated.

Analysis of mutations in the supF gene
A mutant E.coli colony was transferred to 50 µl of LB medium and the mixture was used to amplify DNA fragments containing the supF gene by PCR using forward (5'-TGTAAAACGACGGCCAGT-3', 30–47 bp upstream of the supF gene) and reverse (5'-ATCTCAAGAAGATCCTTTGATC-3', 31–52 bp downstream of the supF gene) primers. One microliter of the mixture was suspended in a total volume of 40 µl of PCR buffer containing 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 300 nM each primer, 200 µM deoxynucleotide triphosphate and 0.5 U Taq DNA polymerase (TaKaRa, Tokyo, Japan). PCR conditions were as follows: 60 s at 95°C, 60 s at 55°C and 60 s at 72°C for 35 cycles, followed by 10 min at 72°C. PCR products amplified from mutant plasmids were first screened by agarose gel electrophoresis to determine the sizes of the products. PCR products without mobility shifts were purified using a QIAquick PCR Purification Kit (Qiagen) and directly sequenced with Big Dye Terminator Cycle Sequencing FS Ready Reaction Kits (PE Applied Biosystems, Tokyo, Japan) and an ABI Prism 310 DNA Sequencer (PE Applied Biosystems).


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Induction of G:C->T:A transversion by 8-OHG
Three types of 8-OHG-containing pMY189 plasmids pMY189-RV, pMY189-CH and pMY189-8-OHG were prepared. pMY189-RV and pMY189-CH were pMY189 plasmids treated with riboflavin plus visible light and CuCl2 plus H2O2, respectively, both of which were shown to induce 8-OHG in double-stranded DNA (27,28). HPLC–ECD analysis showed that the amount of 8-OHG, expressed as the ratio of 8-OHdG to dG, was 12.8x10–5 and 235.7x10–5 in pMY189-RV and pMY189-CH DNA, respectively. Since the amount of 8-OHG in untreated pMY189 DNA was 2.0x10–5, it strongly suggests that 8-OHG was produced in pMY189 DNA by treatment with riboflavin plus visible light and CuCl2 plus H2O2, as reported previously (27,28). We also prepared pMY189-8-OHG, which carried a single 8-OHG residue instead of G at position 159 of the supF gene, by incorporating 8-OHG by in vitro DNA polymerization from an 8-OHdG-containing oligonucleotide on single-stranded pMY189 DNA.

Untreated pMY189, pMY189-RV, pMY189-CH and pMY189-8-OHG were introduced and replicated in NCI-H1299 human lung cancer cells. Plasmids replicated in human cells were recovered and introduced into the indicator E.coli strain KS40/pKY241. The mutation frequency in the supF gene of untreated plasmids was 1.7 ± 0.1x10–4 (Figure 1AGo). In contrast, the mutation frequencies in the supF gene of the pMY189-RV, pMY189-CH and pMY189-8-OHG plasmids were 5.6 ± 0.4x10–4, 8.1 ± 0.8x10–4 and 220.6 ± 6.4x10–4, respectively. Thus, the mutation frequencies in the supF gene were significantly increased in all the pMY189-RV, pMY189-CH and pMY189-8-OHG plasmids compared with the untreated pMY189 plasmid (unpaired t-test) and the most remarkable increase in mutation frequency was observed in pMY189-8-OHG.



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Fig. 1. Mutation frequencies in the supF gene of plasmid pMY189 replicated in human cells. (A) Mutation frequencies in the supF gene of untreated pMY189, pMY189-RV, pMY189-CH and pMY189-8-OHG, which were replicated in NCI-H1299 cells. The mutation frequency in the supF gene of 8-OHG-containing plasmid pMY189 is not shown to scale because of a very elevated frequency. (B) Mutation frequencies in the supF gene of untreated pMY189 and pMY189-8-OHG, which were replicated in parental H1299 and OGG1-transfected NCI-H1299 cells. Columns, mean mutation frequencies of three independent experiments; bars, SE. NS, not significant; ND, not determined.

 
Samples of 385, 364, 190 and 79 supF mutants were randomly selected for untreated pMY189, pMY189-RV, pMY189-CH and pMY189-8-OHG, respectively, to analyze the types of mutations in the supF gene. DNA fragments containing the supF gene were amplified from mutant plasmids by PCR and the sizes of the PCR products were roughly determined by agarose gel electrophoresis. Mutants with apparently normal sized PCR products were sequenced to confirm whether the supF gene was mutated or not. 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. Sequencing of the supF gene revealed that most of the clones from the pMY189-RV and pMY189-CH plasmids carried single or multiple base substitutions in the supF gene, while a small subset of the clones carried small deletions of <20 bp. In contrast, all the sequenced clones from pMY189-8-OHG had single base substitutions at nucleotide position 159, where 8-OHG was incorporated.

The proportion of mutants with base substitutions among all the analyzed mutants for plasmids pMY189-CH and pMY189-8-OHG was significantly higher than, while that for the pMY189-RV plasmid was similar to, that for the untreated plasmid (Table IGo, Fisher's exact probability test). The majority (>90%) of mutations in the supF gene for pMY189-8-OHG were base substitutions, while genetic alterations other than base substitutions, such as deletions and gross rearrangements of the supF gene, were dominant (>65%) for pMY189-RV and pMY189-CH, as in the case of untreated pMY189. The proportion of G:C->T:A transversions among the base substitutions in pMY189-8-OHG was significantly higher than that in untreated pMY189 (Table IIGo, Fisher's exact probability test). In contrast, it was not significantly different between untreated pMY189 and either pMY189-RV or pMY189-CH.


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Table I. Proportion of mutants with base substitution in the supF gene of plasmid pMY189
 

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Table II. Spectrum of base substitutions in the supF gene of plasmid pMY189
 
Among the 75 supF mutants analyzed for pMY189-8-OHG, G:C->T:A transversion at position 159, where 8-OHG was incorporated, was detected in all but one case (Figure 2Go). In contrast, several types of base substitutions were detected at various positions of the supF gene in the mutants for untreated pMY189, pMY189-RV and pMY189-CH. Thus, we conclude that G:C->T:A transversion was specifically induced by 8-OHG after replication of pMY189-8-OHG plasmid DNA in human cells, while it was not specifically induced after replication of either pMY189-RV or pMY189-CH plasmid DNA.



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Fig. 2. The distributions of base substitutions in the supF gene of plasmid pMY189. (A) Base substitutions in the supF gene of (a) untreated pMY189, (b) pMY189-RV, (c) pMY189-CH and (d) pMY189-8-OHG, which were replicated in parental H1299 cells. (B) Base substitutions in the supF gene of (a) untreated pMY189 and (b) pMY189-8-OHG, which were replicated in H1299-OG (–) cells. (C) Base substitutions in the supF gene of (a) untreated pMY189 and (b) pMY189-8-OHG, which were replicated in H1299-OG1 cells. (D) Base substitutions in the supF gene of pMY189-8-OHG, replicated in H1299-OG2 cells. (E) Base substitutions in the supF gene of pMY189-8-OHG, replicated in H1299-OG3 cells.

 
Ability of OGG1 protein to suppress G:C->T:A transversion caused by 8-OHG
OGG1-overexpressing H1299 cells were obtained by selection of stable transfectants with the type 1a/pcDNA3 expression plasmid, which allows constitutive overexpression of OGG1 type 1a protein in human cells (29). Three clones, H1299-OG1, H1299-OG2 and H1299-OG3, in which the OGG1 protein expression levels were more than 10 times higher than that in parental H1299 cells, and one clone, H1299-OG (–), in which OGG1 overexpression was not observed, were obtained (Figure 3Go). Mutation frequencies in the supF gene of the pMY189-8-OHG plasmid after replication in H1299-OG1, H1299-OG2, H1299-OG3 and H1299-OG (–) cells were 33.9 ± 6.6x10–4, 45.3 ± 6.0x10–4, 54.8 ± 12.7x10–4 and 186.2 ± 14.3x10–4, respectively (Figure 1BGo). Therefore, the mutation frequencies in OGG1-overexpressing H1299 cells were significantly lower than that in parental H1299 cells, while the frequency was not significantly different between parental H1299 and H1299-OG (–) cells without OGG1 overexpression (unpaired t-test). Mutation frequencies in the supF gene of the untreated pMY189 plasmid after replication in H1299-OG1 and H1299-OG (–) cells were 2.4 ± 0.8x10–4 and 2.4 ± 1.0x10–4, respectively. Therefore, the mutation frequencies in the supF gene of the untreated pMY189 plasmids were not significantly different among H1299 cells with or without OGG1 overexpression.



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Fig. 3. OGG1-type 1a protein expression in parental and OGG1-transfected H1299 cells. Lane 1, parental H1299; lane 2, H1299-OG (–); lane 3, H1299-OG2; lane 4, H1299-OG3; lane 5, H1299-OG1. The molecular size of the protein is 39 kDa.

 
Samples of 110, 94, 93 and 80 supF mutants for the pMY189-8-OHG plasmids were randomly selected for H1299-OG1, H1299-OG2, H1299-OG3 and H1299-OG (–) cells, respectively, to analyze the types of mutations in the supF gene. The proportion of mutants with base substitutions among all the mutants for pMY189-8-OHG was significantly lower in OGG1-overexpressing cells than that in parental H1299 cells (Table 1, Fisher's exact probability test). In contrast, it was not different between H1299-OG (–)and parental H1299 cells. The proportion of G:C->T:A transversions among the base substitutions detected in OGG1-overexpressing cells was lower than that in parental H1299 cells (Table IIGo). In addition, base substitutions at positions other than position 159, which were not detected for parental and H1299-OG (–) cells, were detected in OGG1-overexpressing cells (Figure 2Go). This result indicated that induction of G:C->T:A transversions by 8-OHG in plasmid pMY189-8-OHG was significantly suppressed by overexpression of the OGG1 gene in H1299 cells.

In contrast, the proportion of mutants with supF base substitutions among all the supF mutants were similar among the parental, H1299-OG (–) and H1299-OG1 cells for untreated plasmid pMY189 (Table IGo). The proportion of G:C->T:A transversions among base substitutions was not reduced in H1299-OG1 cells compared with those in parental H1299 and H1299-OG (–) cells (Table IIGo). Base substitutions were detected not only at position 159 but dispersed at various positions in the H1299-OG (–) and H1299-OG1 cells, as in the case of parental H1299 cells (Figure 2Go).


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In this study it has been shown that the modified pMY189 shuttle plasmid pMY189-8-OHG, in which G at position 159 in the supF gene was replaced by 8-OHG, is useful for assessment of the ability to suppress G:C->T:A transversion caused by 8-OHG. The mutation frequency of plasmid pMY189-8-OHG was 130 times higher than that of untreated plasmid pMY189 due to the exclusive occurrence of G:C->T:A transversions at the position of 8-OHG. This result is consistent with the previous finding that the presence of 8-OHG paired with C in DNA causes G:C->T:A transversion, since 8-OHG allows the incorporation of cytosine and adenine nucleotides opposite the lesion during DNA replication (5,36). In contrast, neither treatment of the pMY189 plasmid with riboflavin plus visible light nor CuCl2 plus H2O2 specifically induced G:C->T:A transversion, although such treatments induced the formation of 8-OHG in plasmid DNA and increased the mutation frequencies, as reported previously (27,28,37). This result is similar to previous studies using E.coli cells as recipients for plasmid or phage DNA (28,37). In these studies the treatment of DNA with riboflavin plus visible light or CuCl2 plus H2O2 led to induction of several types of genetic alteration, including G:C->T:A transversions. Riboflavin plus visible light and CuCl2 plus H2O2 are known to induce several types of base modifications other than 8-OHG (27,38,39). Therefore, base modifications other than 8-OHG, which were formed at various positions of the supF gene in pMY189 plasmid DNA, might have induced the occurrence of several types of base substitutions in the pMY189-RV and pMY189-CH plasmids in H1299 cells. Then the high incidence of genetic alterations caused by DNA lesions other than 8-OHG could mask G:C->T:A transversions induced by 8-OHG in plasmids pMY189-RV and pMY189-CH.

In this study deletions and rearrangements were predominantly found (>65%) in the supF mutants for untreated pMY189, pMY189-RV and pMY189-CH after replication in H1299 cells (Table IGo). In previous studies deletions and rearrangements were also found dominantly (>50%) in plasmid pMY189 and in a similar shuttle vector plasmid, pZ189, which also contains the supF gene, after replication in mammalian cells (40,41). These results were consistent with our findings on the untreated plasmid. Since the shuttle vector is introduced into the cell as naked DNA, it could be easily nicked by cellular processes. In addition, riboflavin plus visible light and CuCl2 plus H2O2 are known to induce single- and double-strand breaks in DNA (32,38,39). Thus, it is possible that the deletions and rearrangements were triggered by strand breaks that occurred in the treated and untreated plasmid DNAs. It is also possible that introduction of the treated and untreated plasmids activated unspecified processes of DNA recombination and other DNA repair pathways in the cells.

We used the pMY189-8-OHG plasmid to investigate the ability of OGG1 protein to suppress G:C->T:A transversion caused by 8-OHG. We isolated several H1299-derived human lung cancer cells which stably overexpressed OGG1 type 1a protein. In human cells multiple isoforms of OGG1 proteins are expressed due to alternative splicing (12,29,42). The OGG1 type 1a protein is predominantly expressed in human cells and is considered to be the major isoform for the repair of chromosomal DNA in the nucleus, since it is a unique isoform carrying both a DNA-binding domain and a nuclear localization signal (29). In OGG1-overexpressing H1299-derived clones the mutation frequency in the supF gene of the replicated pMY189-8-OHG plasmid DNA was significantly decreased compared with parental H1299 cells, in which OGG1 protein is not overexpressed (Figure 1BGo). In addition, the proportion of supF mutants with G:C->T:A transversion at position 159 among all supF mutants was reduced in cells with OGG1 overexpression compared with cells without OGG1 overexpression. These results indicate that OGG1 protein has the ability to suppress G:C->T:A transversion caused by 8-OHG in human cells in vivo. In contrast, frequencies and spectra of mutations of the untreated pMY189 plasmid were not significantly altered by overexpression of OGG1 protein. This result is consistent with the result of a recent study showing that overexpression of human OGG1 type 1a protein has no significant effect on spontaneous mutation rates in the guanine phosphoribosyltransferase locus of Chinese hamster ovary cells (26). Thus, it is possible that, under conditions without severe oxidative stress, G:C->T:A transversion is predominantly induced by DNA lesions other than 8-OHG.

In the present study mutation frequencies of the pMY189-8-OHG plasmid were 20–30 times higher than those of untreated plasmid pMY189 after replication in OGG1- overexpressing cells, in which G:C->T:A transversion caused by 8-OHG was significantly suppressed (Figure 1Go). This may be partially due to the occurrence of G:C->T:A transversion as a replicative consequence of 8-OHG:C before its repair by OGG1 protein. However, if we consider that dominant types of mutations in pMY189-8-OHG were not base substitutions (Table IGo), it is also likely that deletions and gross rearrangements were induced after replication of plasmid pMY189-8-OHG in OGG1-overexpressing cells. It is predicted that a 3'-blocking end generated by the AP lyase activity of OGG1 protein causes a double-strand DNA break, unless removed by APEX protein prior to DNA replication (43). Therefore, it is possible that the 3'-blocking ends generated by overexpression of OGG1 protein led to the formation of DNA strand breaks which initiate DNA recombination. It is also possible that introduction of the 8-OHG-containing plasmid into cells with OGG1 overexpression activated unspecified processes of DNA recombination and other DNA repair pathways, which led to the induction of deletions and gross rearrangements.

The results of the present study strengthen the idea that OGG1 is a critical factor in preventing mutations in human cells. The OGG1 gene is somatically mutated in a small subset of human cancers and is a polymorphic gene, which has several allele variations associated with amino acid substitutions (9,42,4449). Therefore, defects in the 8-OHG-repair pathway due to mutations in the OGG1 gene could be involved in the development and/or progression of cancer by enhancing the mutation rates of defined critical genes, such as oncogenes and tumor suppressor genes. The interindividual differences in 8-OHG repair activity due to OGG1 genetic polymorphisms could also be involved in interindividual differences in cancer susceptibility. Genetic as well as functional analysis of the OGG1 gene should be further performed to elucidate the pathogenetic significance of 8-OHG and 8-OHG repair systems in human carcinogenesis.


    Notes
 
4 To whom correspondence should be addressed Email address: jyokota{at}gan2.ncc.go.jp Back


    Acknowledgments
 
We thank Dr J.D.Minna and A.F.Gazdar of the University of Texas Southwestern Medical Center, USA, for providing us with the NCI-H1299 cell line. We also thank Dr Masatomo Mori of Gunma University School of Medicine for encouragement throughout this study. This work was supported in part by Grants-in-Aid from the Ministry of Health and Welfare for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control and from the Ministry of Education, Science, Sports and Culture of Japan. This work was also supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan. N.Sunaga is a recipient of the Research Resident Fellowship from the Foundation for Promotion of Cancer Research.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 31, 2001; revised April 9, 2001; accepted April 27, 2001.