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Tumor Biology |
Banyu Tsukuba Research Institute in Collaboration with Merck Research Laboratories, Tsukuba, Ibaraki 300-2611 [T. A., V. P. K., K. K., S. N.]; Department of Cell Biology, The Cancer Institute, Japanese Foundation for Cancer Research, Toshima-Ku, Tokyo 170-8455 [T. A., O. M., T. N.]; Mouse Functional Genomics Research Group, RIKEN Genomic Sciences Center, Totsuka-ku, Yokohama, Kanagawa 244-0804 [O. M.]; CREST, Japan Science and Technology Corporation, Kawaguchi 332-0012 [O. M., T. N.]; and Department of Molecular Genetics, Tohoku University School of Medicine, Aoba-Ku, Sendai 980-8575 [T. N.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TA transversions. These results showed that inactivation of the Ogg1 gene leads to a higher risk of cancer because cells with accumulated 8-OH-G still retain the ability to proliferate, leading to an increase in the mutation frequency. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TA transversions as a result of mispairing with an A base (2, 3, 4, 5, 6, 7, 8, 9, 10)
. In the mammalian cell, the MMH/OGG1 gene encodes for a DNA glycosylase/AP lyase that has the capacity to excise 8-OH-G from DNA (11, 12, 13, 14, 15, 16, 17) . The OGG1 protein initiates the base excision repair pathway by recognizing and excising the oxidative DNA lesion. In addition to OGG1, the mammalian cell contains two further enzymes, MYH and MTH, that contribute to the protection from 8-OH-G damage. MYH is a monofunctional glycosylase that removes an A base that has mispaired with 8-OH-G (18) . MTH is an 8-OH-dGTPase that degrades 8-OH-dGTP in the nucleotide pool, thereby preventing its incorporation into DNA (19) . The importance of the 8-OH-G repair systems is reflected in the fact that homologues have been identified in various species from prokaryotes to eukaryotes.
With respect to involvement of the OGG1 gene in human cancer, significant effort has been made to link the inactivation of the OGG1 gene with cancer risk in clinical tumor samples (20, 21, 22, 23, 24) . However, the data, such as somatic mutation, loss of heterozygosity, and polymorphism, are contradictory. Moreover, it remains to be firmly established that the accumulation of 8-OH-G causes carcinogenesis by using experimental animal models. In this study, we showed that 8-OH-G accumulated in liver DNA of Ogg1-/- mouse treated with KBrO3, which is a known carcinogen and oxidative agent for rat kidney (25, 26, 27, 28) , and persisted in the same level after cessation of KBrO3 treatment as in the case of kidney DNA as reported previously (29) . Furthermore, after partial hepatectomy, liver with such oxidative DNA lesions regenerated normally without repair of 8-OH-G, followed by a considerable increase in the mutation frequency. These results indicated that there is no major replication coupled repair of preexisting 8-OH-G. Because induction of carcinogenesis must be coupled with cell proliferation, this system would be a good animal model for long-term carcinogenesis study to elucidate the involvement of 8-OH-G.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Time Course Experiment of KBrO3 Treatment.
Seven to 8-week-old Ogg1+/+, Ogg1+/-, and Ogg1-/- mice were prepared. KBrO3 was administered at a dose of 2 grams/liter in the drinking water to Ogg1 mutant mice. Three male and three female Ogg1 mutant mice of each genotype were killed at each time point of 1, 4, 8, and 12 weeks. Control mice were given water and killed at the same time point. A proportion of the Ogg1 mutant mice was given water 4 weeks after cessation of KBrO3 treatment for 8 weeks and killed. The livers were excised from the sacrificed mice and stored at -80°C before measurement of the 8-OH-G levels.
Partial Hepatectomy Experiment of Mice Treated with KBrO3.
In this experiment, male gpt/Ogg1+/- and gpt/Ogg1-/- mice at the age of 7–8 weeks old were used. KBrO3 was administered at a dose of 2 grams/liter to three mice of each genotype for 12 weeks. The mice were kept for 1 week after treatment with KBrO3 before partial hepatectomy. Partial hepatectomy, involving the removal of the anterior and left lateral hepatic lobes, was performed by the procedure of Higgins and Anderson (31)
. At the time of partial hepatectomy, the resected liver was weighed, and this information was used to calculate the remnant liver weight for each mouse based on the assumption that the remnant liver = (the resected liver) x 3/7. All mice were killed 10 days later. On sacrifice, the remnant liver was weighed, and the proliferation ratio of the remnant liver was calculated. The isolated liver was stored at -80°C before analysis of the 8-OH-G levels and mutation.
Measurement of the 8-OH-G Level.
About 100–200 mg of liver were used for the measurement of 8-OH-G levels using an HPLC-electrochemical detector. Genomic DNA extraction, preparation of the sample, and measurement of 8-OH-G were performed as described previously (30)
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gpt Mutation Assay.
High molecular weight genomic DNA was extracted from 50–80 mg of liver using the RecoverEase DNA Isolation Kit (Stratagene). The gpt mutation assay was performed according to the procedure of Nohmi et al. (32)
. Briefly, extracted genomic DNA was packaged into 
phage. The phage was infected to Escherichia coli YG6020 and plated, and the sequence of the gpt gene from the resulting mutant colonies was obtained. To remove the possibility that mutational events might arise from clonal expansion, identical mutations occurring more than once in the same liver sample were counted as a single, independent mutation. The gpt mutation frequency was calculated by dividing the number of independent colonies resistant to Cm and 6-TG by the number of colonies resistant to Cm alone.
Statistics.
The significance of differences in the measured parameters was evaluated by using the Student t test or ANOVA followed by Scheffe multiple comparison test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 8-OH-G levels of liver DNA from male and female mice within the same group were similar, shown together in Fig. 1
. The amount of 8-OH-G in liver DNA from both control Ogg1+/+ and Ogg1+/- mice was constant at 2.6/106 dG after 12 weeks. In control Ogg1-/- mice, the amount increased proportionally with time, from 8 to 15.3/106 dG. At 12 weeks, equal to 19–20 weeks old, the 8-OH-G level of control Ogg1-/- mice was 5.9 times that of control. These results are in good agreement with those of our previous study (30)
. When Ogg1 mutant mice were treated with KBrO3, the amount of 8-OH-G in liver DNA from Ogg1+/+ and Ogg1+/- mice increased during the first 4 weeks but thereafter remained mainly unchanged from 4 to 12 weeks. The averages were 3.7 and 5/106 dG, respectively. The absence of OGG1 enzyme caused a high accumulation of 8-OH-G in liver DNA from Ogg1-/- mice treated with KBrO3. The amount of 8-OH-G tremendously increased in proportion to time with KBrO3 treatment and reached 96.6/106 dG. This represents a 37.2-fold increase over control Ogg1+/+ mice and 26.1-fold increase compared with KBrO3-treated Ogg1+/+ mice. These results show that, in mice, KBrO3 produces 8-OH-G in liver DNA, in addition to kidney DNA (29)
, and that the 8-OH-G in liver DNA of Ogg1-/- mice accumulates because of chronic oxidative stress.
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. Three male and three female mice were killed at each point. In Ogg1+/+ and Ogg1+/- mice liver DNA, the amount of 8-OH-G decreased to control levels (2.9 and 3.3/106 dG, respectively). By contrast, the accumulated 8-OH-G (68.3/106 dG) in liver DNA from Ogg1-/- mice did not change (65.6/106 dG) after the cessation of KBrO3 treatment. It appears that the 8-OH-G accumulated in liver by chronic oxidative stress cannot be repaired in Ogg1-/- mice. These results show that the major enzyme to repair 8-OH-G in liver is the Ogg1 gene product, which is in good agreement with our previous observations in kidney (29)
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and 2
). Only male mice were used to remove the effect of sex differences on cell proliferation after partial hepatectomy. KBrO3 was administered at a dose of 2 grams/liter in the drinking water to Ogg1+/- and Ogg1-/- mice for 12 weeks. Partial hepatectomy was performed 1 week after discontinuing treatment with KBrO3. All mice were killed 10 days after the partial hepatectomy. We did not find any histological difference in regenerated liver sections stained with H&E from Ogg1+/- and Ogg1-/- mice 10 days after partial hepatectomy (data not shown). The weight of the remnant liver from all mice increased 2.2–2.4 times after 10 days (Fig. 3)
. These results indicate that liver with high amounts of 8-OH-G can proliferate normally.
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). After liver regeneration, there were no differences in the 8-OH-G levels in the mice as compared with before liver regeneration. In control Ogg1-/- mice, the 8-OH-G level, at 16.4/106 dG, decreased to 10.6/106 dG after liver regeneration (P < 0.05). Furthermore, in Ogg1-/- mice treated with KBrO3, the 8-OH-G level, at 113.5/106 dG, decreased to 47.1/106 dG (P < 0.01). The decreases in 8-OH-G levels observed after liver regeneration were 64.6 and 41.5% in control and KBrO3-treated Ogg1-/- mice, respectively. It appears that the decreases are related to the amount of liver proliferation. These results show that, after cell proliferation, the accumulated 8-OH-G levels in liver are diluted by a factor of two, indicating that 8-OH-G is not removed during cell proliferation.
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. In control Ogg1+/- mice, the mutation frequency showed no difference before or after liver regeneration. The mutation frequency of control Ogg1-/- mice seemed higher than that of control Ogg1+/- mice, in agreement with our previous result (30)
, and showed a tendency to increase after liver regeneration. When Ogg1+/- and Ogg1-/- mice were treated with KBrO3, we found no large increase in the mutation frequencies compared with each control. It is likely that KBrO3 produces no mutation in mouse liver during 12 weeks. In liver DNA from Ogg1-/- mice treated with KBrO3, the mutation frequency (10.1 x 10-6) substantially increased, 3.5 times (35.4 x 10-6), after liver regeneration over 10 days (P < 0.03). This mutation frequency was 6.2-fold that of control Ogg1+/- mice (P < 0.01). The results indicate that cell proliferation plays a pivotal role in fixing the mutation in tissue with a high accumulation of 8-OH-G.
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. We found no difference in the mutation frequency and spectrum before and after liver regeneration in Ogg1+/- mice. GCTA transversions were 35.7% before regeneration and 33.3% after. These results show that liver regeneration does not cause DNA mutations in wild-type mouse under normal conditions. GCTA transversions in control Ogg1-/- mice (50% before regeneration and 57.1% after) were more than that of control Ogg1+/- mice. It seems that GCTA transversion increased slightly after liver regeneration in control Ogg1-/- mice. When KBrO3 was administered to Ogg1+/- and Ogg1-/- mice, the number of deletion mutants increased, although we found no deletion in Ogg1-/- mice before liver regeneration. There were no differences in mutations, other than deletions, before and after liver regeneration in KBrO3-treated Ogg1+/- mice compared with control Ogg1+/- mice. In KBrO3-treated Ogg1-/- mice, GCTA transversions before and after liver regeneration were 55 and 81.6%, respectively. Cell proliferation promoted a 5.2-fold increase in mutation frequency of GCTA (5.6 x 10-6 before regeneration and 28.9 x 10-6 after). This GCTA mutation frequency in liver DNA from KBrO3-treated Ogg1-/- mice after liver regeneration was 14.5 times that of control Ogg1+/- mice before regeneration. These results indicate that GCTA transversion accounts for 92.1% of the mutation when 8-OH-G, generated by oxidative stress, is fixed by cell proliferation in vivo, and GCTA transversion is the major mutation caused by 8-OH-G even in an animal model.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To our knowledge, this is the first report to clarify whether a cell having a high accumulation of 8-OH-G possesses the ability to proliferate. Studies in yeast have shown that DNA polymerase 
replicates only 
10% of DNA containing 8-OH-G when compared with the replication of undamaged DNA (4)
. Asagoshi et al. (33)
investigated the translesion synthesis of 8-OH-G using E. Coli DNA polymerase I Klenow fragment. The translesion synthesis of 8-OH-G was slower than that of G because of the inefficiency of nucleotide incorporation opposite 8-OH-G and extension past the lesion. When single-stranded DNA containing 8-OH-G was transfected to COS7 cells, the recovery yield of replicated plasmid was 80% when compared with DNA containing G (7)
. A variety of genes has been identified whose transcription is initiated during the S phase of the cell cycle but whose expression is silent during the G0-G1 phase. Recently Le Page et al. (34)
reported that 8-OH-G blocks transcription by RNA polymerase II. These reports led us to the idea that proliferation may be inhibited in cells having high amounts of 8-OH-G. Such cells, we envisaged, would experience cell cycle arrest or undergo apoptosis as a mechanism to prevent cancer development. Therefore, we investigated the effect of 8-OH-G on cell proliferation by partial hepatectomy of Ogg1-/- mice treated with KBrO3. We showed that liver with a high amount of 8-OH-G can proliferate to the same extent as normal liver. Although it could be argued that rare cells with no accumulation of 8-OH-G were responsible for the proliferated tissue, we found no significant difference in the proliferation index of all liver samples examined 48 h after partial hepatectomy (data not shown). Furthermore, in comparison with liver samples taken at partial hepatectomy, after liver regeneration, significant increases in the mutation frequency were observed. The main mutation was GCTA transversion, believed to be caused mainly by 8-OH-G. Taken together, it suggests that cells having high amounts of 8-OH-G have the ability to proliferate in vivo.
When partial hepatectomy was performed and liver regeneration was induced, the weight of remnant liver from the mice in all groups increased 2.2–2.4 times. After liver regeneration, the amount of 8-OH-G decreased 50% in liver DNA from treated and nontreated Ogg1-/- mice in terms of 8-OH-G residue per guanine residue in DNA. Although the 8-OH-G level did not appear to decrease in Ogg1+/+ mice, a slight dilution in the amount of 8-OH-G at steady-state levels would be difficult to distinguish from background because no technique is currently available to avoid artificial oxidation of a G base during the course of isolation of DNA (35)
. From these results, it is concluded that cell division dilutes the level of the DNA lesion by a factor of two, and no replication-coupled repair of 8-OH-G occurs on the parent strand. Hazra et al. (36
, 37)
discovered two new enzymes, OGG2 and NEH1, capable of removing 8-OH-G from oligonucleotides. Our results propound that these enzymes are responsible for the repair of 8-OH-G when incorporated into the nascent strand by DNA replication.
From experiments performed in vitro, eukaryotic DNA polymerase 
and were shown to incorporate an A base opposite 8-OH-G during DNA synthesis on a oligodeoxynucleotide template, which contained 8-OH-G (3, 4, 5)
. Similarly, transfection of mammalian cells with single-stranded DNA containing 8-OH-G revealed that only GCTA transversion occurs after replication (6
, 7)
. However, Tan et al. (8)
reported that 8-OH-G induced not only GCTA but also GCAT and GCCG using a similar method. Kamiya et al. (9
, 10) analyzed the mutation in foci obtained on transfection of the c-Ha-ras gene containing 8-OH-G to NIH3T3 cells and found GCTA, GCAT, and GCCG. The fact that discrepancies exist in the tissue culture experiments compounds the need for the use of an animal model to investigate the mutation spectrum caused by 8-OH-G. Difficulties in determining the mutation spectrum generated by 8-OH-G in animals have arisen because oxidative stress reagents often produce many kinds of DNA lesions in addition to 8-OH-G. We have succeeded in analyzing the mutation spectrum caused by 8-OH-G, which accumulated in liver DNA after partial hepatectomy of Ogg1 mutant mice treated with KBrO3. Our results indicate that 8-OH-G mainly causes GCTA transversion in an inactive gene after cell proliferation.
The activity of the OGG1 enzyme is inhibited by nitric oxide, which is associated with chronic inflammation (38) . Indeed, it was reported that the 8-OH-G level in liver with chronic hepatitis was higher than that of normal liver (39) . Chronic inflammation predisposes individuals to the development of carcinoma in the esophagus, gastric mucosa, pancreas, colon, and liver (40, 41, 42) . From our results, it is predicted that 8-OH-G could continue to accumulate during inflammation followed by an increase in the mutation frequency after cell proliferation. We hypothesize that inactivation of the OGG1 enzyme by chronic inflammation could contribute to the risk of cancer.
It is likely that the accumulation of 8-OH-G resulting from a deficiency of the Ogg1 gene or inactivation of the OGG1 enzyme could contribute to the initiation and promotion of cancer. For induction of cancer, the cells must be proliferated. To assess this possibility, we are planning to perform a long-term carcinogenesis test using partial hepatectomized Ogg1 mutant mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Banyu Tsukuba Research Institute in Collaboration with Merck Research Laboratories, 3 Okubo, Tsukuba, Ibaraki 300-2611 Japan. Phone: 81-298-77-2000; Fax: 81-298-77-2034; E-mail: nismrasm{at}banyu.co.jp 
2 The abbreviations used are: 8-OH-G, 8-hydroxyguanine; HPLC, high-performance liquid chromatography; 6-TG, 6-thioguanine; Cm, chloramphenicol.
Received 2/16/03. Accepted 4/30/03.
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