Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M311132200 on December 19, 2003

J. Biol. Chem., Vol. 279, Issue 11, 9857-9866, March 12, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/11/9857    most recent
M311132200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, M.-R.
Right arrow Articles by You, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, M.-R.
Right arrow Articles by You, H. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Transcription Factors NF-YA Regulate the Induction of Human OGG1 Following DNA-alkylating Agent Methylmethane Sulfonate (MMS) Treatment*

Mi-Rha Lee{ddagger}, Soo-Hyun Kim{ddagger}, Hyun-Ju Cho{ddagger}, Kun-Yeong Lee{ddagger}, Ae Ran Moon{ddagger}, Hye Gwang Jeong{ddagger}, Jung-Sup Lee{ddagger}, Jin-Won Hyun§, Myung-Hee Chung¶, and Ho Jin You{ddagger}||**

From the {ddagger}Research Center for Proteineous Materials, Chosun University, 375 Seosuk-dong, Gwangju 501-759, Korea, the ||Department of Pharmacology, School of Medicine, Chosun University, 375 Seosuk-dong, Gwangju 501-759, Korea, the §Department of Biochemistry, College of Medicine Cheju National University, Jeju, Jeju-do, Korea, and the Department of Pharmacology, Seoul National University College of Medicine, 28 Yongon-dong, Seoul 110-799, Korea

Received for publication, October 9, 2003 , and in revised form, November 24, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A human 8-oxoguanine-DNA glycosylase (hOGG1) is the main enzyme that repairs 8-oxoG, which is a critical mutagenic lesion. There is a great deal of interest in the up- or down-regulation of OGG1 expression after DNA damage. In this study, we investigated the effect of a DNA-alkylating agent, methylmethane sulfonate (MMS), on hOGG1 expression level and found that MMS treatment resulted in an increase in the functional hOGG1 expression in HCT116 cells. A region between –121 and –61 of the hOGG1 promoter was found to be crucial for this induction by MMS. Site-directed mutations of the two inverted CCAAT motifs substantially abrogated the induction of the hOGG1 promoter as a result of MMS treatment. In addition, the NF-YA protein (binding to the inverted CCAAT box) was induced after exposing cells to MMS. Moreover, gel shift and supershift analyses with the nuclear extracts prepared from HCT116 cells identified NF-YA as the transcription factor interacting with the inverted CCAAT box. Mutations of the inverted CCAAT box either prevented the binding of this factor or abolished its activation as a result of MMS treatment. Finally, this study showed that hOGG1-expressing HCT116 cells exhibited increased hOGG1 repair activity and resistance to MMS. Overall, these results demonstrate that MMS can up-regulate hOGG1 expression through the induction of the transcription factor, NF-YA, and increased transcription level of the hOGG1 gene correlates with an increase in enzyme activity providing functional protection from MMS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS)1 causes a variety of damage to DNA, including oxidized bases, abasic (AP) sites, strand breaks, and DNA-protein cross-links (1–6). Among oxidative lesions, 8-oxoguanine (8-oxoG) is one of the major base lesions formed after oxidative attack to DNA (7). Relatively large quantities of 8-oxoG are produced in mammalian cells, either as a byproduct of the normal oxidative metabolism, or as a result of exogenous sources of ROS, such as ionizing radiation, single oxygen sensitizer dyes, and redox-active organic molecules (8). 8-oxoG preferentially mispairs with adenosine during replication and thereby gives rise to G:C -> T:A transversion mutations (9–12). Because of its persistent generation, relative abundance, and potent mutagenicity, 8-oxoG is believed to represent a major source of spontaneous mutagenesis in all aerobic cells. In order to prevent the mutagenic effect of 8-oxoG, the bacterium, Escherichia coli, contains a GO system (13–15). The bacteria GO system consists of three proteins: MutM (also known as the Fpg protein), a DNA glycosylase/lyase that recognizes 8-oxoG:C and catalyzes the excision of 8-oxoG (16, 17); MutY, which is a DNA glycosylase that recognizes 8-oxoG:A and catalyzes the excision of A (18); and MutT, specific phosphatase that cleaves 8-oxo-dGTP (19). In mammalian cells, the main defense enzyme against the mutagenic effects of 8-oxoG in cellular DNA is 8-oxoguanine-DNA glycosylase (OGG1), which is structurally unrelated but functionally similar to formamidopyrimidine glycosylase (Fpg) in E. coli (20–27). A human DNA glycosylase/AP lyase encoded by the OGG1 gene has an activity that removes 8-oxoG from the DNA and suppresses the mutagenic effect of 8-oxoG (28). The inactivation of the OGG1 gene in yeast and mice leads to an increase in the spontaneous mutation frequency in cells (29–31). The human OGG1 (hOGG1) gene is found on the chromosome 3p26.2, and allelic deletions of this region occur frequently in a variety of human cancers (32–36). In addition, the hOGG1 gene is somatically mutated in some cancer cells and is highly polymorphic among human populations (37–40).

In human tissues, the level of OGG1 expression is much higher in the thymus, testis, kidney, intestine, brain, cerebrum, and the germinal center of B cells than in other tissues (25, 41, 42). Moreover, a specific increase in 8-oxoG incision activity occurs in the mitochondria with age (43), suggesting that OGG1 expression may be modulated by a variety of stimuli. There is mixed evidence for the role of oxidative stress in influencing OGG1 expression. For example, three reports have suggested that the induction of hOGG1 expression is associated with oxidative stress (44–46). However, other studies have failed to observe the induction of OGG1 by ROS (47, 48). Therefore, in this study, we investigated whether or not a ROS producing agent, H2O2, and a DNA-alkylating agent, methylmethane sulfonate (MMS), can modulate the mRNA and protein expression of hOGG1 in HCT116 cells. The results showed that MMS could effectively induce functional hOGG1 expression but not H2O2. A series of experiments were performed to identify the regulatory elements of the hOGG1 promoter. Two inverted CCAAT motifs were identified as MMS regulatory elements necessary for MMS activation of the hOGG1 promoter. A disruption of the inverted CCAAT motif abolished activation of the hOGG1 promoter by MMS. In support of these findings, it was also demonstrated that the nuclear factor-YA (NF-YA) protein was induced as a result of MMS treatment. In addition, the binding affinity of NF-YA to its consensus sequences was also enhanced after MMS treatment. Finally, it was shown that an increase in the functional hOGG1 expression level could protect cells against MMS. This is the first report showing the characterization of an important molecular mechanism by which MMS regulates hOGG1 as well as its biological significance.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell and Reagents—The human colorectal carcinoma HCT 116 cells were a generous gift from Bert Vogelstein (Johns Hopkins University, Baltimore), and cultured in McCoy's 5A-modified medium supplemented with 10% fetal bovine serum, 2 mm L-glutamine, 100 units of penicillin/ml, and 100 µg of streptomycin/ml (Invitrogen, Life Technologies, Inc.). MMS and H2O2 were purchased from Sigma Chemical Co. MMS was dissolved in dimethyl sulfoxide (Me2SO, Sigma) in a stock solution.

Plasmid Constructs—The human OGG1 cDNA was amplified by RT-PCR using the hOGG1 oligo primer (5'-ATGCCTGCCCGCGCGCTTCTGCC-3', 5'-CTAGCCTTCCGGCCCTTTGGAAC-3') from human fibroblast GM00637 cells. After confirming the DNA sequence, the hOGG1 cDNA was cloned into a pcDNA3 mammalian expression vector driven by the CMV promoter (Invitrogen). In order to clone the hOGG1 promoter, genomic DNA from the human fibroblast GM00637 cell line was prepared according to standard protocols and a hOGG1 promoter region search at the NCBI site using GenBankTM accession number AF521807 [GenBank] . The hOGG1 promoter fragment –1980/+93 bp, p(–1980)-hogg1luc was amplified from the genomic DNA of the human fibroblast GM00637 cell line by PCR using a sense primer (5'-ATA AAC TGT CAA TTT AGA ATT CTT TGT TCT-3') and an antisense primer (5'-TTT CCT GAG GTG TAG GTC CC-3'). This fragment was then cloned into SacI and XhoI sites of a promoterless luciferase vector pGL3-Basic (Promega, Madison, WI) and was used to generate nested deletions. PCR was performed to prepare deletion mutants. Constructed plasmids and sense primers are as follows: p(–1060)hogg1luc: sense primer 5'-TGA CCA ACA TGG TGA AAC CC-3'; p(–700)hogg1luc: sense primer 5'-TCC AGG CGA CTA GAA GCC AG-3'; p(–590)hogg1luc: sense primer 5'-ATG AGC AGA TGG AGT ATG GA-3'; p(-390)hogg1luc: sense primer 5'-AGC TGT GTT CGC TCC GGT TC-3'; p(–240)hogg1luc: sense primer 5'-GGA ACC GCG GGG CGC CCA GAT-3'; p(–20)-hogg1luc: sense primer 5'-TGC TGT GGT CTG CCC CTG GAG-3'. The serial mutagenesis of the region from –240 to –20 was generated using an Erase-a-BaseR system (Promega). Briefly, an Erase-a-BaseR system was designed for the rapid construction of the 5'-flanking region subclones containing the progressive unidirectional deletions of any inserted DNA. The Erase-a-BaseR method was used according to manufacturer's instructions. To insert point mutations in one inverted Sp1, proximal-inverted CCAAT, or a distal-inverted CCAAT motif, various mutant constructs were generated by PCR using the p(–240)hogg1luc promoter. The constructs and primers used are as follows: mSp1 (5'-CGCCTCAGAACGGCCCtaCCACCTGATTTCAT GGGCC-3'); mP-CCAAT (5'-ACCCTGATTTCTCtTTGGCGCTCCTACTCCTCTCGG-3'); mD-CCAAT (5'-ACCTCCTCCTCGGtTTGGCTACCTCTAGGTGAAATGAGCGG-3'). The lowercase, underlined characters indicate the nucleotide substitutions in insert mutations. The nucleotide sequence of each construct was confirmed by cycle sequencing using an ABI PRISM 310 genetic analyzer (PerkinElmer Life Science).

Promoter Luciferase Activity Assays—The reporter plasmids were transiently transfected into cells using the LipofectAMINE method according to the manufacturer's protocol (Invitrogen, Life Technologies, Inc.). The transfection efficiency was determined with the Renilla luciferase gene-containing pRL-CMV plasmid (Promega). HCT116 cells were transiently transfected with reporter plasmid and then treated with 200 µM MMS. 24 h after treatment, transfected cells were washed twice with the phosphate-buffered saline (PBS) and lysed in a lysis buffer (5x PLBR, Promega) with gentle shaking at room temperature for 20 min. The cell lysate was centrifuged at 13,000 rpm for 2 min to pellet the cell debris. The supernatants were transferred to a fresh tube, and the dual luciferase activity in cell extracts was determined according to the manufacturer's protocol (Promega). Briefly, each assay mixture contained 2 µl of the cell lysates and 10 µl of a firefly luciferase-measuring buffer (LAR ll R, Promega). The firefly luciferase activity was measured by a luminometer (the luminometer was programmed to perform a 2-s premeasurement delay, followed by a 10-s measurement period for each reporter assay). After measuring the firefly luciferase activity (Stop & GloR, Promega), a Renilla luciferase-measuring buffer was added, and the Renilla luciferase activity was then measured. Each transfection was performed in duplicate, and all were repeated at least three times.

Western Blotting—Cells in the medium were exposed to 200 µM MMS or 100 µM H2O2 for 0, 6, 12, 24, and 48 h. Cells were then washed with PBS, and lysed at 0 °C for 30 min in a lysis buffer (20 mM Hepes, PH 7.4, 2 mM EGTA, 50 mM glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µ/ml aprotinin, 1 mM Na3VO4, and 5 mM NaF). The protein content was determined using the Bio-Rad dye-binding microassay, and 20 µg of protein per lane were electrophoresed on 10% SDS-polyacrylamide gels after boiling for 5 min in a Laemmli sample buffer. The proteins were blotted onto the Hybon ECL membranes (Amersham Biosciences). The markers (MBI) were used as size standards. After electroblotting, the membranes were blocked with Tris-buffered saline with Tween-20 (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% milk, and were incubated with the anti-hOGG1, APE, NF-YA, NF-YB, NF-YC, and {alpha}-tubulin antibodies (Santa Cruz Biotechnology) diluted in blocking buffer for 4 h. The primary antibody dilutions were those recommended by the manufacturer. The membranes were the washed, incubated with the appropriate secondary antibodies (1:4,000) in a blocking buffer for 2 h, and repeatedly washed. The blotted proteins were detected using an enhanced chemiluminescence system (iNtRON, Biotech, Seoul, Korea).

Semiquantitative Reverse Transcriptase Polymerase Chain Reaction—RNA extraction from the human colorectal carcinoma HCT116 cell line was conducted using the RNA-STAT-60 according to the manufacturer's instructions (TEL-TEST, Inc., Friendswood, TX). Briefly, cells in the medium were exposed to 200 µM MMS or 100 µM H2O2 and collected at the indicated time points. After homogenizing cells in the RNA STAT-60, the homogenate was mixed with chloroform (5:1; v/v), shaken vigorously for 15 s, and then centrifuged at 13,000 rpm for 15 min at 4 °C. The RNA presented in the upper colorless aqueous phase was precipitated by adding isopropyl alchohol, washed twice with 70% ethanol, and air-dried for 10 min. The RNA was then resuspended in DEPC. 10-µl RNA aliquots were prepared and stored at –70 °C until needed. 2 µg of the total RNA were reverse-transcribed using a M-MLV cDNA synthesis system (Promega), and the reverse-transcribed DNA was subjected to PCR. The profile of the replication cycles was denaturation at 94 °C for 50 s, annealing at 58 °C for 50 s, and polymerization at 72 °C, for 1 min. In each reaction, the same amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers used for PCR are as follows: hOGG1 forward, 5'-CTG CCT TCT GGA CAA TCT TT-3'; hOGG1 reverse, 5'-TAG CCC GCC CTG TTC TTC-3' designed to amplify a 551-bp region; hAPE forward, 5'-GGA AGA AGC CC CAG ATA TAC-3'; hAPE reverse, 5'-GTG TCA CAG TGC TAG GTA TA-3' designed to amplify a 705-bp region; GAPDH forward, 5'-CCA TGG AGA AGG CTG GGG-3'; and GAPDH reverse 5'-CAA AGT TGT CAT GGA TGA CC-3' designed to amplify a 194-bp region (total number of cycles: 26). The PCR products were resolved on 1% agarose gels, stained with ethidium bromide, and then photographed.

Electrophoretic Mobility Shift Assay—The nuclear protein was extracted from HCT116 cell line cultures as described previously (49). Briefly, 2 x 106 cells were treated with 0, 100, or 200 µM MMS for 12 h, cells were then washed with PBS and pelleted at 4 °C for 5 min at 2,500 rpm. The upper supernatant was carefully removed, and the pellet was resuspended in 1 ml of an ice-cold cell lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, 1 mM leupeptin). The suspension was incubated on ice for 15 min with occasional mixing by inversion. Subsequently, 24 µl of 10% Nonidet P-40 was then added, cells were mixed by inversion, and nuclei were immediately pelleted at 4 °C for 5 min at 3,000 rpm. The supernatant was discarded, and the pellet containing the nuclei was resuspended by adding 50 µl of an ice-cold nuclear extraction buffer (30 mM HEPES, 0.3 mM EDTA, 1.5 mM MgCl2, 0.2 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, 1 mM leupeptin, 450 mM KCl) dropwise. The mixture was then incubated on ice for 30 min, and the nuclear debris were pelleted at 15,000 rpm for 20 min. The protein concentration of the nuclear extracts was determined using a BCA protein assay kit (Pierce) according to the manufacturer's instructions. The nuclear extracts were stored at –70 °C until needed. EMSA binding reactions were incubated in a 20-µl final volume for 20 min at room temperature containing 5 µg of the nuclear extract, 20 fmol of the end-labeled double-stranded oligonucleotide, and 1 µg of the poly(dI-dC)·(dI-dC). The binding buffer contained 30 mM HEPES, 0.3 mM EDTA, 1.5 mM MgCl2, 0.2 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, 1 mM leupeptin. The supershift assays were performed by adding 1 µl of polyclonal antibodies against NF-YA (Santa Cruz Biotechnology). The reactions were incubated at room temperature for 30 min prior to adding the radiolabeled probe. Competition was performed in the presence of a 10- or 20-fold excess of the unlabeled oligonucleotide cassette and incubating for 20 min at room temperature prior to adding the labeled probe. The complexes were resolved using a 5% non-denaturing polyacrylamide gel in 0.5x Tris borate/EDTA and electrophoresis at 200 volt for 1 h. The gels were dried and exposed overnight at –70 °C to x-ray film.

Endonuclease Nicking Assay—Cells at the exponential phase were treated with 0–200 µM MMS for 24 h, and cell pellets (106 cells per each assay) were then suspended in 2 volumes of homogenation buffer (50 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, 5% glycerol, and 0.05% 2-mercaptoethanol, pH 7.5) and homogenized. The homogenates were mixed with streptomycin (final concentration 1.5%) to remove nucleic acids. Supernatants obtained by centrifugation were dialyzed extensively against the homogenation buffer and used as cell extracts for endonuclease nicking assay. 8-oxoguanine (8-oxoG)-containing 21-mer with the sequence 5'-CAGCCAATCAGTXCACCATTC-3' (X = 8-oxoG), and its complementary strand were chemically synthesized (The Midland Certified Reagent Co., Midland, TX). Oligonucleotides were 3'-end-labeled using terminal transferase and [{alpha}-32P]ddATP (Amersham Biosciences, 3000 Ci/mmol). The end-labeled oligomer was annealed with its complementary oligonucleotide, and the resulting duplex DNA was used as the assay substrate. The duplex substrate DNA (20 pmol) was incubated with the cell extracts (10 µg of protein) at 37 °C for 1 h in 1 ml of reaction mixture (50 mM Tris-HCl, 50 mM KCl, and 1 mM EDTA, pH 7.5). The reaction was terminated by heating at 90 °C for 3 min. Reaction products were electrophoresed on 20% denaturing (7 M urea) polyacrylamide gels (DNA sequencing gel). After electrophoresis, the gels were wrapped in Saran Wrap and the amounts of the cleaved substrate were determined by autoradiography and Quantity One software (Bio-Rad Laboratories). Incision activity was calculated as the amount of radioactivity in the band corresponding to the damage-specific cleavage product over the total radioactivity in the lane in the reaction.

Clonogenic Cell Survival Assay—Clonogenic survival assay was measured as described previously (50), Briefly, cells were seeded at 4 x 105 cells per 25 cm culture flask and incubated at 37 °C in a 5% CO2 atmosphere. Cells were then treated with different doses of MMS for 12 h, washed twice with PBS, trypsinized, and resuspended in fresh medium. They were counted with a Coulter counter, and the number of cells required for plating was obtained by successive dilutions in fresh complete medium. Cells were then allowed to grow at 37 °Cina5%CO2 atmosphere for 14 days. Fresh medium was added on the fifth day. Two weeks after drug treatment, cultures were fixed by methanol and stained with Giemsa. The number of colonies exceeding 50 cells was counted with a binocular lens. The surviving fraction was determined as the ratio of the number of colonies observed after treatment to the number of cells seeded, adjusted to the plating efficiency.

Measurement of N7-Methylguanine—N7-methylguanine (N7-meG) was measured as described by van Delft et al. (51). Briefly, cells were treated with different doses of MMS for 6 h, and DNA was extracted from cells by multiple treatments with phenol-chloroform, RNase, and proteinase K. The DNA was heated for 1 h at 100 °C and cooled at 0 °C followed by addition of 1 M HCl, after 10 min on ice, the DNA precipitate was collected by centrifugation at 12,000 rpm at 4 °C for 10 min. Supernatant was removed, neutralized with 1 M K2HPO4, and analyzed by HPLC (elution buffer; 25 mM H3PO4-KOH, pH 6.0, 5% methanol; flow rate, 0.5 ml/min) with electrochemical detection (coulometric array detector, ESA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MMS Treatment Induced Human OGG1 mRNA and Protein Expression—In order to determine if H2O2 and MMS could induce human OGG1 expression in cultured cells, HCT116 cells were treated with either 200 µM MMS or 100 µM H2O2, and hOGG1 mRNA levels were determined in attached cells after 0, 6, 12, 24 h of MMS or H2O2 treatment. The PCR exponential phase was obtained and the optimal number of PCR cycles, which was 26 cycles (Fig. 1A), was determined. Semiquantitative RT-PCR analysis showed that hOGG1 mRNA accumulation was increased by more than 2.5-fold as early as 24 h after incubating cells with 200 µM MMS (Fig. 1B). However, no effect on the hOGG1 mRNA expression level was observed after treating cells with 100 µM H2O2 at any incubation time. Western blotting was performed to determine if this increase in the hOGG1 mRNA level corresponds to an increase in the hOGG1 protein level (Fig. 2). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the whole cell extracts of the protein from untreated and MMS-treated cells. As shown in Fig. 2A, Western blotting with a specific antibody against hOGG1 showed that a small quantity of the hOGG1 protein was present in the untreated HCT116 cells. However, treating cells with MMS caused an increase in hOGG1 protein accumulation within 48 h of post-treatment with MMS. Dose-response experiments showed that the effect of MMS reached a maximum at 200 µM MMS with approximately a 4-fold increase in the hOGG1 protein level (Fig. 2B). The specificity of MMS-induced hOGG1 expression was demonstrated by the lack of an effect on the expression of the human apurinic endonuclease (hAPE) or the housekeeping gene {alpha}-tubulin at any of the incubation times or MMS concentrations investigated (Fig. 2, A and B). This study also investigated the effect of H2O2 on hOGG1 expression in HCT116 cells. Cells were treated with 100 µM H2O2 for 0, 12, 24, and 48 h, and the hOGG1 and hAPE expression levels were measured. As shown in Fig. 2C, treating cells with H2O2 resulted in no detectable effect on the hOGG1 protein level, whereas increases in the hAPE protein level were observed within 24–48 h after adding H2O2 to the medium. These results suggest that hOGG1 expression is effectively induced in response to the DNA-alkylating agent, MMS, but not to H2O2. In order to determine the functional significance of the MMS-mediated increase in hOGG1 expression, nuclear extracts from the MMS-treated cells were prepared and examined for their ability to cleave 8-oxoG using a 21-mer oligonucleotide containing a single 8-oxoG at nucleotide 13. As shown in Fig. 3, pretreating cells with MMS (0–200 µM) for 24 h resulted in a concentration-dependent increase in the ability of the nuclear extracts to cleave the 8-oxoG:C substrate. These results suggest that MMS treatment results in the stimulation of hOGG1 expression and leads to an increase in hOGG1 repair activity.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 1.
Induction of hOGG1 mRNA after treating HCT116 cells with MMS. A, hOGG1 mRNA levels were analyzed by semiquantitative RT-PCR. The PCR exponential phase was 21–29 cycles, and the optimal number of PCR cycles was determined to be 26. B, increased expression levels of hOGG1 mRNA as a result of MMS. Human colorectal carcinoma HCT116 cells were cultured in media containing MMS (200 µM) or H2O2 (100 µM) for 0, 6, 12, 24 h, and the hOGG1 mRNA level was evaluated by semiquantitative RT-PCR using the hOGG1-specific primers on 26 cycles. The relative ratio of the hOGG1/glyceraldehyde-3-phosphate dehydrogenase expression level in each cell to that in MMS or H2O2 untreated control cells is shown.

 



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 2.
Western blot analysis of hOGG1 protein levels in the MMS-treated HCT116 cells. A, HCT116 cells were treated with 200 µM MMS and lysed at the indicated times. The total protein was extracted and quantified as described in the "Experimental Procedures." Anti-hOGG1 and anti-APE antibodies were used to evaluate the hOGG1 and APE levels after the MMS treatment. {alpha}-Tubulin was used as the loading control. B, cells were treated with the various doses of MMS and harvested 48 h later. Antibodies were used as described in A. C, cells were treated 100 µM H2O2, and the cell protein was sequentially extracted at the indicated times thereafter. Antibodies were used as described in A. Fold inductions were quantified as the relative response to the respective MMS or H2O2 untreated levels using Quantity One software.

 



View larger version (75K):
[in this window]
[in a new window]
  		FIG. 3.
Effect of MMS on 8-oxoG repair activity. A 21-mer containing an 8-oxoG lesion was incubated with the cell extracts from the 0, 100, and 200 µM MMS-treated HCT116 cells for 24 h, and oligonucleotide cleavage products (lanes 3–5) were analyzed on DNA sequencing gels and subjected to autoradiography as described under "Experimental Procedures." Human OGG1 (E, lane 2) and buffer alone (NT, lane 1) serve as positive and negative controls, respectively. The arrow indicates the DNA cleavage products (13-mer).

 
A MMS Responsive Element Is Located between Nucleotides –121 and –61 of the Human OGG1 Gene—A reporter plasmid p(–1980)hogg1luc, was constructed by inserting the 2,073-kb (1980/+93 bp) SacI-XhoI genomic fragment containing the hOGG1 promoter upstream of the firefly luciferase cDNA in pGL3-Basic vector to identify putative MMS response elements in the hOGG1 promoter in HCT116 cells. The luciferase activity was detectable in the absence of MMS but was increased ~7-fold in its presence (Fig. 4A). Therefore, elements within p(–1980)hogg1luc directed both the basal hOGG1 transcription and induction by MMS. In order to determine the region within the hOGG1 promoter responsible for the transcriptional activation effect of MMS, a series of 5'-deletion constructs of p(–1980)hogg1luc were made using progressively smaller fragments of the 5'-flanking sequence, and these constructs were cloned into the pGL3-Basic vector. Following the transfection of these reporter constructs into HCT116 cells, cells were treated with MMS, and promoter activities were measured. The results indicated that five additional reporter plasmids containing promoters of various lengths (p(–1060)hogg1luc, p(–700)hogg1luc, p(–590)hogg1luc, p(–390)hogg1luc, and p(–240)hogg1luc) were still highly induced by MMS, whereas the MMS-induced promoter activity was significantly reduced in the p(–20)hogg1luc, lacking the upstream of position –20 (Fig. 4A), indicating that the region between –240 and –20 contains the important controlling elements necessary for the responsiveness of the hOGG1 promoter to MMS treatment. In order to determine the region responding to MMS treatment, further deletion analysis of the promoter region between –240 and –20 bp was performed. A construct containing the region between –240 and –121 [p(–211)hogg1luc, p(–175)hogg1luc, p(–151)hogg1luc, and p(–121)hogg1luc] maintained significant induction after MMS treatment, whereas a construct containing the region, –61/+93 bp, p(–61)hhogg1luc, completely lost its responsiveness (Fig. 4B). These observations suggest that a 61-bp region from –121 to –61 contains the major MMS responsive element.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4.
Mapping of hOGG1 promoter regions required for MMS-mediated expression. The HCT116 cells were transiently transfected with a series of reporter plasmids containing the hOGG1 promoter sequences that drive the expression of the luciferase gene, and then treated with MMS at a dose of 200 µM. Cells were harvested 24 h after treatment, and a luciferase assay was performed as described under "Experimental Procedures." In order to determine the transfection efficiency, the renilla luciferase expression vector (pRL-CMV) was co-transfected with each of the plasmids tested, and the Renilla luciferase activity level was detected as the internal control of transfection. The values are reported as mean ± S.D. from six separate experiments. ** denotes p < 0.01.

 
Two Inverted CCAAT Motifs Contribute to the Induction of the Human OGG1 Promoter Activity by MMS—DNA sequence analysis revealed one inverted Sp1 motif (CGCC, –125 to –122) and two inverted CCAAT boxes (proximal position –97 to –93; distal position –71 to –67) located in this region of the hOGG1 promoter (Fig. 5A). In order to determine if these inverted Sp1 or inverted CCAAT motifs indeed participate in the MMS-mediated induction of hOGG1, a variety of mutants of hOGG1 promoter reporters were constructed, where the inverted Sp1 or inverted CCAAT motifs were mutated in different combinations, using PCR-based mutagenesis. A mutation in the Sp1 binding site (mSp1) did not lead to a reduction in hOGG1 promoter activation following a treatment with MMS. However, mutations in either the proximal inverted CCAAT (mP-CCAAT) or the distal inverted CCAAT (mD-CCAAT) box was shown to significantly affect the activation of the hOGG1 promoter caused by the MMS treatment. Induction of these reporters was reduced by 44 ± 6% and 39 ± 4%, respectively (Fig. 5B). A double mutation in the proximal and distal CCAAT motifs (mP&D-CCAAT) resulted in a marked suppression of the MMS-mediated increase in hOGG1 promoter activity (82 ± 7% decrease by luciferase analysis). Therefore, the presence of these two inverted CCAAT boxes is essential for MMS-mediated activation of the hOGG1 promoter in HCT116 cells.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5.
Two inverted CCAAT motifs are essential for the MMS-induced activation of the hOGG1 promoter. A, DNA sequence analysis indicates that there is one Sp1 site and two inverted CCAAT boxes located in the region of the hOGG1 promoter from –121 to –61. B, site-directed mutagenesis of the three potentially important cis-acting elements demonstrated that the two inverted CCAAT boxes were essential for the MMS induction of the hOGG1 promoter activity. In particular, a mutation of the two inverted CCAAT boxes completely abolished MMS-induced activities. A mutation of the inverted Sp1 site did not affect the MMS induction of the hOGG1 promoter activity. The values are reported as mean ± S.D. from six separate experiments. ** denotes p < 0.01.

 
The NF-YA Proteins Are Induced Following MMS Treatment—Further experiments were carried out to determine if the protein bound to the inverted CCAAT motifs are induced by subsequent MMS treatment. CCAAT boxes have been demonstrated to bind a number of transcription factors including NF-Y, CP2, NF-1, and C/EBP (52). In addition, the NF-Y protein levels were found to increase following MMS treatment (53). Therefore, this study first verified that MMS treatment led to an increase in the NF-Y expression level in our system. In order to investigate the effect of MMS on the NF-Y expression level, the HCT116 cells were treated with 200 µM MMS and analyzed by Western blotting at the indicated times. Mammalian NF-Y comprises three subunits, NF-YA, NF-YB, and NF-YC, all of which are highly conserved throughout evolution (52, 54). Two isoforms of the NF-YA protein, 46 and 42 kDa, were detected by Western analysis. The larger form (46 kDa) of NF-YA exhibited a dramatic induction but the small form (42 kDa) did not show significant elevation (Fig. 6). However, the NF-YB and NF-YC protein, both of which are subunits of the NF-Y factor, did not show any induction following MMS treatment. As a negative control, the detection of the {alpha}-tubulin protein was included, and its expression level remained constant after treatment. Therefore, the NF-YA proteins are induced in response to MMS, and their induction may play a role in the activation of the hOGG1 promoter.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 6.
Cellular NF-YA proteins are induced in response to MMS treatment. The human colorectal carcinoma HCT116 cells were exposed to 200 µM MMS. Cells were then harvested at the indicated time points, and the total protein was extracted and quantified, as described under "Experimental Procedures." 100 µg of the total protein was loaded onto an SDS-polyacrylamide gel for Western blot analysis. Antibodies against NF-YA, NF-YB, and NF-YC were used. The detection bands and their respective size are shown. {alpha}-Tubulin was used as the loading control.

 
NF-YA Interacts with the CCAAT Box of the Human OGG1 Promoter—In order to determine whether or not the NF-YA protein binds to the hOGG1 promoter, and to determine if the MMS-induced NF-YA expression contributes to the enhancement of hOGG1 promoter activity by MMS, the NF-YA binding activity in HCT116 cells following MMS treatment was analyzed by EMSA using oligonucleotides corresponding to the hOGG1 promoter region between –110 and –61, which contain two inverted CCAAT boxes. When the labeled oligonucleotide (Intact) was incubated with the nuclear extracts, a prominent slowly migrating band (I) was observed. The density of this band increased substantially with the nuclear protein in cells treated with MMS (Fig. 7A). However, this band disappeared when two inverted CCAAT motifs were mutated (Mutant). In the competition experiments, this prominent band was efficiently removed with an excess of the unlabeled intact sequence (Self) but the oligonucleotide with mutated inverted CCAAT motifs (Mutant) was not (Fig. 7B). In order to confirm the composition of the NF-YA complexes formed at the proximal and distal inverted CCAAT sites, specific antibodies against the NF-YA transcription factor were added to the DNA-protein binding assay. As shown in Fig. 7B, supershifted protein-DNA complexes (II) were observed after adding the NF-YA antibodies to the DNA binding reaction performed with the intact oligonucleotide. In parallel, the density of the original band (I) was greatly diminished. These results suggest that DNA-alkylating agent MMS-induced NF-YA protein can bind to inverted CCAAT boxes in the hOGG1 promoter.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 7.
EMSA with the hOGG1 promoter region containing the inverted CCAAT motifs. A, the 48-bp (converting the region of the hOGG1 promoter from –110 to –61) oligo probes containing the intact inverted CCAAT motifs (Intact) or mutated inverted CCAAT motifs (Mutant) were incubated with the nuclear extracts isolated from human HCT116 cells. Nuclear extracts were prepared from cells treated with 0, 100, or 200 µM MMS. The DNA-protein complexes were run on neutral polyacrylamide gels and visualized by autoradiography. B, EMSA was performed in the presence of the indicated amounts (fold) of the unlabeled oligo containing the intact inverted CCAAT consensus sequence (Self) or the oligo containing the mutated inverted CCAAT sites (Mutant). For supershifts, specific antibodies to NF-YA were added to the reaction mixtures and incubated for 30 min prior to separating the DNA-protein complexes. The results shown are representative of at least three independent experiments.

 
hOGG1 Expression Protects Cells from MMS-induced Cytotoxicity—The human OGG1 was subcloned into the vector pcDNA3, to form pcDNA3-hOGG1. This construct was transfected into the human colorectal carcinoma HCT116 cells. Nine stable transfected cell lines were established after selection using G418 for 5 weeks. The 8-oxoG cleavage assay revealed that these clones had a significantly higher 8-oxoG cleavage activity (Fig. 8A). To test the functional significance of hOGG1 expression, cells were subjected to the DNA-alkylating agent, MMS, over a range of concentrations. We first measured the level of methylation adduct in cells after MMS treatment. Control and hOGG1-expressing cells were treated with various doses of MMS for 6 h, and then the N7-meG were measured by HPLC with an electrochemical detector. As shown in Fig. 8B, following incubation with MMS, the levels of N7-meG in control and hOGG1-expressing cells were increased in a dose-dependent manner. We next investigated the effect of hOGG1 on MMS-induced cytotoxicity. Following treatment of control and hOGG1-expressing cells with various doses of MMS for 12 h, the cellular sensitivity was determined by clonogenic survival assay. Those cells transfected with hOGG1 were significantly more resistant to MMS when compared with the empty vector (pcDNA3)-transfected cells (Fig. 8C), which suggests that the enhanced hOGG1 activity contributes to cell viability in response to MMS in HCT116 cells.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 8.
hOGG1-expressing cells are protected from MMS. A, cleavage activity on 8-oxoG-21mer by hOGG1-expressing cells. A 21-mer containing an 8-oxoG lesion was incubated with the cell extracts from either the hOGG1-(OG1, lane 4; OG2, lane 6; and OG3, lane 8) or pcDNA3-(C1, lane 3; C2, lane 5; and C3, lane 7) transfected cells. 8-oxoG cleavage activity was measured as described under "Experimental Procedures." Human OGG1 (E, lane 2) and buffer alone (NT, lane 1) serve as positive and negative controls, respectively. The arrow indicates the DNA cleavage products (13-mer). The incision activity was calculated as the ratio of cleavage product to the total product in the lane. The values are presented as a mean ± S.D. from two experiments performed in duplicate. B, levels of N7-meG in pcDNA3-expressing clone-1 (C1) and hOGG1-expressing clone-3 (OG3). Cells were treated with indicated concentration of MMS for 6 h, and the formation of N7-meG was measured by HPLC in combination with electrochemical detector. The values are presented as a mean ± S.D. from three separate experiments. C, increasing hOGG1 expression results in an increase in MMS resistance. GM00627 cells (None), three individual HCT116 + hOGG1 clones (clone-1, OG1; clone-2, OG2; and clone-3, OG3), and three individual HCT116 + pcDNA3 clones (clone-1, C1; clone-2, C2; and clone-3, C3) were treated with different doses of MMS, and the cell viability was determined by a clonogenic survival assay. The hOGG1-expressing clones demonstrated increased protection compared with HCT116 cells containing only the pcDNA3 vector. The values are presented as a mean ± S.D. from four separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study analyzed the effect of the alkylating agent, MMS, and the ROS-generating agent, H2O2 on expression of human OGG1 in HCT116 cell lines. It was found that MMS treatment stimulated hOGG1 expression, which resulted in an increase in the 8-oxoG cleavage activity, while the H2O2 incubation failed to induce hOGG1 expression. Using 5'-deletion analyses, the region of the hOGG1 promoter between –121 and –61 was found to be associated with the MMS regulatory elements. Further analyses indicated that the two inverted CCAAT motifs located in this region were essential for inducing hOGG1 promoter in response to MMS. Mutations made in these DNA binding sites abrogated the induction of hOGG1 promoter by MMS. Importantly, the NF-YA protein levels were observed to increase after MMS treatment, and specific complexes formed on the hOGG1 promoter DNA probe were supershifted by anti-NF-YA antibodies. Finally, it was shown that hOGG1-expressing HCT116 cells exhibited an increase in the hOGG1 repair activity as well as an increased resistance to MMS. Therefore, these findings demonstrate that MMS induces hOGG1 expression via the transactivation of the hOGG1 promoter, and the NF-YA protein induced by MMS plays a key role in activating the hOGG1 promoter in response to MMS. Furthermore, an increased functional hOGG1 expression level via this pathway may contribute to functional protection from MMS.

Recently, the hOGG1 promoter region was cloned (48), and sequence analysis of the promoter region revealed two CpG islands and no TATA or CAAT boxes, suggesting the hOGG1 gene is essentially a housekeeping gene, which is believed to be expressed in a constitutive manner. However, the activity of the housekeeping gene can be modulated under certain conditions, such as oxidative stress (55). The presence of a Nrf2 binding site, which contributes to regulating the antioxidant responses and coordinating the induction of genes coding the detoxifying enzymes (56), within the hOGG1 promoter region suggests that hOGG1 expression is modulated by oxidative stress. In support of this hypothesis, several groups have demonstrated that hOGG1 is inducible by ROS at the mRNA level in several experimental systems. For example, one of the environmental pollutants, diesel exhaust particles (DEP), is known to generate ROS and induce lung cancer in experimental animals. The treatment of rats with DEP resulted in dose- and time-dependent changes in the levels of 8-oxo-G as well as the induction of hOGG1 mRNA (44). In addition, the hOGG1 gene expression level was elevated after rats were exposed to crocidolite asbestos, which is associated with epithelial cell injury in the process of carcinogenesis via oxidative stress (45). Moreover, the OGG1 mRNA expression level in the rat kidney after ischemia-reperfusion injury was higher than that in a normal kidney (46). However, other studies have failed to observe the induction of hOGG1 by ROS. For example, the treatment of HeLa cells with oxidative generating agents as H2O2 or LPS did not modify the hOGG1 expression level (47), and treating the human lung carcinoma A549 cells with H2O2 has no detectable effect on the hOGG1 mRNA or protein expression levels (48), suggesting that the modulation of hOGG1 expression by ROS is dependent on the experimental model chosen. In our system, treating HCT116 cells with H2O2 had no effect on the hOGG1 mRNA levels as assessed by semi-quantitative RT-PCR or the protein levels as assessed by Western blotting (Fig. 1B). Surprisingly, it was found that treating the HCT116 cells with 200 µM MMS leads to a time-dependent increase in the hOGG1 mRNA expression level as measured by semi-quantitative RT-PCR (Fig. 1B), which leads to a time-and concentration-dependent increase in hOGG1 protein expression level as measured by Western blotting (Fig. 2, A and B). The induction of hOGG1 expression in response to MMS was observed in the absence of any changes in the hAPE and {alpha}-tubulin expression level. Therefore, the induction of hOGG1 caused by MMS is not secondary to cytotoxicity. In addition, treating cells with MMS resulted in a concentration-dependent increase in the cellular capacity to cleave the 8-oxoG:C substrate (Fig. 3), indicating that the enhancement of hOGG1 expression by MMS is functionally important in HCT116 cells.

The DNA-alkylating agent, MMS, which caused DNA damage that can be repaired by the base excision repair (BER) pathway, is known to activate several transcription factors such as activator protein 1 (AP1) and NF-{kappa}B via the c-Jun NH2 terminal kinase/stress-activated protein kinase (JNK/SAPK) or NF-{kappa}B pathway, and alter expression levels of a group of genes (56, 57). In this study, we found that the region between –121 and –61 of the hOGG1 promoter contains the important controlling elements necessary for responsiveness to MMS treatment (Fig. 4). The activity of fragments spanning –121 to –61 of the hOGG1 promoter were analyzed using Transfac software; this segment contains one inverted Sp1 site and two inverted CCAAT motifs (Fig. 5A). A single mutation in the Sp1 site did not substantially abrogate the induction of the hOGG1 promoter following MMS treatment (Fig. 5B). In contrast, mutations in either of the two inverted CCAAT motifs can abrogate the hOGG1 induction by MMS. Mutations in both inverted CCAAT motifs can completely eliminate the induction of the hOGG1 promoter by MMS. Therefore, the presence of these two inverted CCAAT boxes is essential for the MMS-mediated activation of the hOGG1 promoter in the HCT116 cells. A number of factors interact with the inverted CCAAT boxes, including NF-Y, YB-1, and c/EBP (52). Their binding sites are related but do not generally share a common consensus sequence. Data obtained in this study suggest that the inverted CCAAT box factor regulating hOGG1 expression is NF-YA. NF-Y is a ubiquitous transcription factor consisting of three subunits A, B, and C. NF-Y specifically recognizes a CCAAT box motif. NF-Y regulates the transcription of a variety of genes, including some that are constitutive, tissue-specific, or developmentally or cellcycle-regulated (52, 54). Recently, it was reported that NF-YA, but not NF-YB and NF-YC, was induced after cells were exposed to MMS (53). The induction of the NF-YA protein is mediated through a post-transcriptional mechanism and does not require a normal cellular p53 function. These observations suggest that the NF-YA proteins can participate in the cellular response to genotoxic stress, possibly via the regulation of their downstream genes. This study demonstrated that the NF-YA protein is induced subsequent to MMS treatment (Fig. 6), and the specific complexes formed on the hOGG1 promoter DNA probe are supershifted by anti-NF-YA antibodies (Fig. 7). These results suggested that the MMS-mediated increase in hOGG1 transcription results from the increased binding of the transcription factor NF-YA to the two inverted CCAAT motifs of the hOGG1 promoter. However, the mechanism underlying this MMS-mediated increase in NF-YA-DNA binding is not known. Further studies will be needed to determine the mechanism by which MMS promotes NF-YA-DNA binding and to answer the question of how many genes are regulated by this mechanism.

The important question that remains to be answered is the biological role of the increased hOGG1 expression level in response to MMS. One hypothesis is that a high hOGG1 expression level after an MMS treatment may confer protection against MMS-induced cytotoxicity. Monofunctional alkylating agents, such as MMS and N-methyl-N'-nitro-N''-nitrosoguanine (MNNG), are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing (59). These alkylating agents are generating in DNA primarily N7-meG, O6-methylguanine (O6-meG) as well as a number of other minor base modifications, which are removed by DNA repair enzymes including O6-methylguanine DNA methyltransferase (MGMT) or 3-methyladenine DNA glycosylase (MPG) (60). Recently, several lines of evidence have indicated that cellular DNA repair systems are important in the sensitivity of cells to alkylating agents. For example, expression of MGMT confers increased chemoresistance to chloroethylnitrosourea, one class of alkylating agents. Conversely, O6-meG induced by methylating carcinogen is the predominant cytotoxic lesion in cells lacking MGMT (61–65). In addition, cells deficient in DNA polymerase {beta} are impaired in base excision repair and hypersensitivity to DNA-alkylating agents, such as MMS (66–68). Thiotepa (N,N',N''-triethylenethiophosphoramide), aziridine, and 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), alkylating chemotherapeutic agents, induce the formation of N7-aminoethyl adducts of guanine and adenine, and these adducts are unstable, further degrading into imidazole ring opening lesions (69–71). These ring-opened damaged bases, formamidopyrimidines (Fapy), prevent the movement of the replication fork and block DNA synthesis (69, 72). Therefore, incompletely repaired Fapy may induce apoptosis in a DNA replication-dependent way. E. coli Fpg (Mut M) and hOGG1 are well known repair enzymes, which are able to excise several oxidized bases including 8-oxoG (28). Additionally, these enzymes have shown to release alkylated ringopened guanine adducts, such as Fapy, C8 guanine aminofluorene, N7 guanine aflatoxin-B1, or N7 hydroxyethylthioethlyguanine (73–75). Furthermore, the expression of the Fpg or hOGG1 protein in mammalian cells protects cells from alkylating agents, such as thiotepa, aziridine, and BCNU (74, 76–78). These observations suggest that the Fpg/hOGG1 repair activity contributes to the protection against MMS. The data represented in this report show that increased hOGG1 activity, by stable transfection with hOGG1-expressing vectors, results in protection against MMS in HCT116 cells (Fig. 8), suggesting that an enhancement in the functional OGG1 expression level as a result of MMS treatment may, at least in part, be involved in the cell survival against MMS in these cell lines. Accordingly, investigations aimed at determining the detailed mechanism of the protective effect of hOGG1 on MMS-induced cytotoxicity as well as the biological significance of MMS-mediated increase in hOGG1 expression are currently under way.


   FOOTNOTES
 
* This work was supported by the Ministry of Science and Technology of Korea and the KOSEF through the Research Center for Proteineous Materials, and by research funds from Chosun University, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed. Tel.: 82-62-230-6337; Fax: 82-62-233-3720; E-mail: hjyou{at}mail.chosun.ac.kr.

1 The abbreviations used are: ROS, reactive oxygen species; 8-oxoG, 8-oxoguanine; AP sites, abasic sites; Fpg, formamidopyrimidine glycosylase; OGG1, 8-oxoguanine-DNA glycosylase; MMS, methylmethane sulfonate; MNNG, N-methyl-N'-nitro-N''-nitrosoguanine; NF-YA, nuclear factor-YA; EMSA, electrophoretic mobility shift assay; DEP, diesel exhaust particle; Thiotepa, N,N',N''-triethylenethiophosphoramide; Fapy, formamidopyrimidines; BCNU, 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea; O6-meG, O6-methylguanine; N7-meG, N7-methylguanine; PBS, phosphate-buffered saline. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nakayama, T., Kaneko, M., Kodama, M., and Nagata, C. (1985) Nature 314, 462–464[CrossRef][Medline] [Order article via Infotrieve]
  2. Halliwell, B., and Aruoma, O. I. (1991) FEBS Lett. 281, 9–19[CrossRef][Medline] [Order article via Infotrieve]
  3. Breimer, L. H. (1990) Mol. Carcinog. 3, 188–197[Medline] [Order article via Infotrieve]
  4. Dizdaroglu, M. (1991) Free Radic. Biol. Med. 10, 225–242[CrossRef][Medline] [Order article via Infotrieve]
  5. Feig, D. I., Reid, T. M., and Loeb, L. A. (1994) Cancer Res. 54, 1890–1894
  6. Wiseman, H., and Halliwell, B. (1996) Biochem. J. 313, 17–29[Medline] [Order article via Infotrieve]
  7. Cadet, J., Berger, M., Douki, T., and Ravanat, J. L. (1997) Rev. Physiol. Biochem. Pharmacol. 131, 1–87[Medline] [Order article via Infotrieve]
  8. Wood, M. L., Dizdaroglu, M., Gajewski, E., and Essigmann, J. M. (1990) Biochemistry 29, 7024–7032[CrossRef][Medline] [Order article via Infotrieve]
  9. Shibutani, S., Takeshita, M., and Grollman, A. P. (1991) Nature 349, 431–434[CrossRef][Medline] [Order article via Infotrieve]
  10. Cheng, K. C., Cahill, D. S., Kasai, H., Nishimura, S., and Loeb, L. A. (1992) J. Biol. Chem. 267, 166–172[Abstract/Free Full Text]
  11. Grollman, A. P., and Moriya, M. (1993) Trends Genet. 9, 246–249[CrossRef][Medline] [Order article via Infotrieve]
  12. Bjoras, M., Luna, L., Johnsen, B., Hoff, E., Haug, T., Rognes, T., and Seeberg, E. (1997) EMBO J. 16, 6314–6322[CrossRef][Medline] [Order article via Infotrieve]
  13. Michaels, M. L., and Miller, J. H. (1992) J. Bacteriol. 174, 6321–6325[Free Full Text]
  14. Tchou, J., and Grollman, A. P. (1993) Mutation Res. 299, 277–287[Medline] [Order article via Infotrieve]
  15. Tajiri, T., Maki, H., and Sekiguchi, M. (1995) Mutat. Res. 336, 257–267[Medline] [Order article via Infotrieve]
  16. Boiteux, S., O'Connor, T. R., and Laval, J. (1987) EMBO J. 6, 3177–3183[Medline] [Order article via Infotrieve]
  17. Tchou, J., Kasai, H., Shibutani, S., Chung, M. H., Laval, J., Grollman, A. P., and Nishimura, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4690–4694[Abstract/Free Full Text]
  18. Michaels, M. L., Pham, L., Nghiem, Y., Cruz, C., and Miller, J. H. (1990) Nucleic Acids Res. 18, 3841–3845[Abstract/Free Full Text]
  19. Maki, H., and Sekiguchi, M. (1992) Nature 355, 273–275[CrossRef][Medline] [Order article via Infotrieve]
  20. van der Kemp, P. A., Thomas, D., Barbey, R., de Oliveira, R., and Boiteux, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5197–5202[Abstract/Free Full Text]
  21. Nash, H. M., Bruner, S. D., Scharer, O. D., Kawate, T., Addona, T. A., Spooner, E., Lane, W. S., and Verdine, G. L. (1996) Curr. Biol. 6, 968–980[CrossRef][Medline] [Order article via Infotrieve]
  22. Aburatani, H., Hippo, Y., Ishida, T., Takashima, R., Matsuba, C., Kodama, T., Takao, M., Yasui, A., Yamamoto, K., and Asano, M. (1997) Cancer Res. 57, 2151–2156[Abstract/Free Full Text]
  23. Arai, K., Morishita, K., Shinmura, K., Kohno, T., Kim, S. R., Nohmi, T., Taniwaki, M., Ohwada, S., and Yokota, J. (1997) Oncogene 14, 2857–2861[CrossRef][Medline] [Order article via Infotrieve]
  24. Lu, R., Nash, H. M., and Verdine, G. L. (1997) Curr. Biol. 7, 397–407[CrossRef][Medline] [Order article via Infotrieve]
  25. Radicella, J. P., Dherin, C., Desmaze, C., Fox, M. S., and Boiteux, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8010–8015[Abstract/Free Full Text]
  26. Roldan-Arjona, T., Wei, Y. F., Carter, K. C., Klungland, A., Anselmino, C., Wang, R. P., Augustus, M., and Lindahl, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8016–8020[Abstract/Free Full Text]
  27. Rosenquist, T. A., Zharkov, D. O., and Grollman, A. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7429–7434[Abstract/Free Full Text]
  28. Boiteux, S., and Radicella, J. P. (2000) Arch. Biochem. Biophys. 377, 1–8[CrossRef][Medline] [Order article via Infotrieve]
  29. Thomas, D., Scot, A. D., Barbey, R., Padula, M., and Boiteux, S. (1997) Mol. Gen. Genet. 254, 171–178[CrossRef][Medline] [Order article via Infotrieve]
  30. Minowa, O., Arai, T., Hirano, M., Monden, Y., Nakai, S., Fukuda, M., Itoh, M., Takano, H., Hippou, Y., Aburatani, H., Masumura, K., Nohmi, T., Nishimura, S., and Noda, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4156–4161[Abstract/Free Full Text]
  31. Klungland, A., Rosewell, I., Hollenbach, S., Larsen, E., Daly, G., Epe, B., Seeberg, E., Lindahl, T., and Barnes, D. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13300–13305[Abstract/Free Full Text]
  32. Naylor, S. L., Johnson, B. E., Minna, J. D., and Sakaguchi, A. Y. (1987) Nature 329, 451–454[CrossRef][Medline] [Order article via Infotrieve]
  33. Hibi, K., Takahashi, T., Yamakawa, K., Ueda, R., Sekido, Y., Ariyoshi, Y., Suyama, M., Takagi, H., and Nakamura, Y. (1992) Oncogene 7, 445–449[Medline] [Order article via Infotrieve]
  34. Yokota, J., and Sugimura, T. (1993) FASEB J. 7, 920–925[Abstract]
  35. Hosoe, S., Shigedo, Y., Ueno, K., Tachibana, I., Osaki, T., Tanio, Y., Kawase, I., Yamakawa, K., Nakamura, Y., and Kishimoto, T. (1994) Lung Cancer 10, 297–305[CrossRef][Medline] [Order article via Infotrieve]
  36. Ishida, T., Hippo, Y., Nakahori, Y., Matsushita, I., Kodama, T., Nishimura, S., and Aburatani, H. (1999) Cell Genet. 85, 232–236
  37. Chevillard, S., Radicella, J. P., Levalois, C., Lebeau, J., Poupon, M. F., Oudard, S., Dutrillaux, B., and Boiteux, S. (1998) Oncogene 16, 3083–3086[CrossRef][Medline] [Order article via Infotrieve]
  38. Kohno, T., Shinmura, K., Tosaka, M., Tani, M., Kim, S. R., Sugimura, H., Nohmi, T., Kasai, H., and Yokota, J. (1998) Oncogene 16, 3219–3225[CrossRef][Medline] [Order article via Infotrieve]
  39. Blons, H., Radicella, J. P., Laccourreye, O., Brasnu, D., Beaune, P., Boiteux, S., and Laurent-Puig, P. (1999) Mol. Carcinog. 26, 254–260[CrossRef][Medline] [Order article via Infotrieve]
  40. Ishida, T., Takashima, R., Fukayama, M., Hamada, C., Hippo, Y., Fujii, T., Moriyama, S., Matsuba, C., Nakahori, Y., Morita, H., Yazaki, Y., Kodama, T., Nishimura, S., and Aburatani, H. (1999) Int. J. Cancer 80, 18–21[CrossRef][Medline] [Order article via Infotrieve]
  41. Kuo, F. C., and Sklar, J. (1997) J. Exp. Med. 186, 1547–1556[Abstract/Free Full Text]
  42. Nishioka, K., Ohtsubo, T., Oda, H., Fujiwara, T., Kang, D., Sugimachi, K., and Nakabeppu, Y. (1999) Mol. Biol. Cel. 10, 1637–1652[Abstract/Free Full Text]
  43. Souza-Pinto, N. C., Croteau, D. L., Hudson, E. K., Hansford, R. G., and Bohr, V. A. (1999) Nucleic Acids Res. 27, 1935–1942[Abstract/Free Full Text]
  44. Tsurudome, Y., Hirano, T., Yamato, H., Tanaka, I., Sagai, M., Hirano, H., Nagata, N., Itoh, H., and Kasai, H. (1999) Carcinogenesis 20, 1573–1576[Abstract/Free Full Text]
  45. Kim, H. N., Morimoto, Y., Tsuda, T., Ootsuyama, Y., Hirohashi, M., Hirano, T., Tanaka, I., Lim, Y., Yun, I. G., and Kasai, H. (2001) Carcinogenesis 22, 265–269[Abstract/Free Full Text]
  46. Tsuruya, K., Furuichi, M., Tominaga, Y., Shinozaki, M., Tokumoto, M., Yoshimitsu, T., Fukuda, K., Kanai, H., Hirakata, H., Iida, M., and Nakabeppu, Y. (2003) DNA Repair (Amst.) 2, 211–229[Medline] [Order article via Infotrieve]
  47. Dhenaut, A., Boiteux, S., and Radicella, J. P. (2000) Mutat. Res. 461, 109–118[Medline] [Order article via Infotrieve]
  48. Hodges, N. J., and Chipman, J. K. (2002) Carcinogenesis 23, 55–60[Abstract/Free Full Text]
  49. Schreiber, E., Matthias, P., Muller, M. M., and Schaffiner, W. (1989) Nucleic Acids Res. 17, 6419[Free Full Text]
  50. Cho, H. J., Jeong, H. G., Lee, J. S., Woo, E. R., Hyun, J. W., Chung, M. H., and You, H. J. (2002) J. Biol. Chem. 277, 19358–19366[Abstract/Free Full Text]
  51. van Delft, J. H., van Winden, M. J., van den Ende, A. M., and Baan, R. A. (1993) Environ. Health Perspect. 99, 25–32[Medline] [Order article via Infotrieve]
  52. Mantovani, R. (1999) Gene (Amst.) 239, 15–27[CrossRef][Medline] [Order article via Infotrieve]
  53. Jin, S., Fan, F., Fan, W., Zhao, H., Tong, T., Blanck, P., Alomo, I., Rajasekaran, B., and Zhan, Q. (2001) Oncogene 20, 2683–2690[CrossRef][Medline] [Order article via Infotrieve]
  54. Matuoka, K., and Yu Chen, K. (1999) Exp. Cell Res. 253, 365–371[CrossRef][Medline] [Order article via Infotrieve]
  55. Kletzien, R. F., Harris, P. K., and Foellmi, L. A. (1994) FASEB J. 8, 174–181[Abstract]
  56. Venugopal, R., and Jaiswal, A. K. (1998) Oncogene 17, 3145–3156[CrossRef][Medline] [Order article via Infotrieve]
  57. Dosch, J., and Kaina, B. (1996) Oncogene 13, 1927–1935[Medline] [Order article via Infotrieve]
  58. Fritz, G., and Kaina, B. (2001) J. Biol. Chem. 276, 3115–3122[Abstract/Free Full Text]
  59. Singer, B. (1975) Prog. Nucleic Acids Res. Mol. Biol. 15, 219–284[Medline] [Order article via Infotrieve]
  60. Hansen, W. K., and Kelley, M. R. (2000) J. Pharmacol. Exp. Ther. 295, 1–9[Abstract/Free Full Text]
  61. Kaina, B., Fritz, G., and Coquerelle, T. (1993) Environ. Mol. Mutagen. 22, 283–292[Medline] [Order article via Infotrieve]
  62. Davis, B. M., Reese, J. S., Koc, O. N., Lee, K., Schupp, J. E., and Gerson, S. L. (1997) Cancer Res. 57, 5093–5099[Abstract/Free Full Text]
  63. Koc, O. N., Reese, J. S., Szekely, E. M., and Gerson, S. L. (1999) Cancer Gene Ther. 6, 340–348[CrossRef][Medline] [Order article via Infotrieve]
  64. Maze, R., Carney, J. P., Kelley, M. R., Glassner, B. J., Williams, D. A., and Samson, L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 206–210[Abstract/Free Full Text]
  65. Phillips, W. P. Jr., Willson, J. K., Markowitz, S. D., Zborowska, E., Zaidi, N. H., Liu, L., Gordon, N. H., and Gerson, S. L. (1997) Cancer Res. 57, 4817–4823[Abstract/Free Full Text]
  66. Sobol, R. W., Horton, J. K., Kuhn, R., Gu, H., Singhal, R. K., Prasad, R., Rajewsky, K., and Wilson, S. H. (1996) Nature 379, 183–186[CrossRef][Medline] [Order article via Infotrieve]
  67. Ochs, K., Sobol, R. W., Wilson, S. H., and Kaina, B. (1999) Cancer Res. 59, 1544–1551[Abstract/Free Full Text]
  68. Ochs, K., Lips, J., Profittlich, S., and Kaina, B. (2002) Cancer Res. 62, 1524–1530[Abstract/Free Full Text]
  69. Gombar, C. T., Tong, W. P., and Ludlum, D. B. (1980) Biochem. Pharmacol. 29, 2639–2643[CrossRef][Medline] [Order article via Infotrieve]
  70. Muller, N., and Eisenbrand, G. (1985) Chem. Biol. Interact. 53, 173–181[CrossRef][Medline] [Order article via Infotrieve]
  71. Chetsanga, C. J., and Lindahl, T. (1979) Nucleic Acids Res. 6, 3673–3684[Abstract/Free Full Text]
  72. O'Connor, T. R., Boiteux, S., and Laval, J. (1988) Nucleic Acids Res. 16, 5879–5894[Abstract/Free Full Text]
  73. Karahalil, B., Girard, P. M., Boiteux, S., and Dizdaroglu, M. (1998) Nucleic Acids Res. 26, 1228–1233[Abstract/Free Full Text]
  74. Gill, R. D., Cussac, C., Souhami, R. L., and Laval, F. (1996) Cancer Res. 56, 3721–3724[Abstract/Free Full Text]
  75. Deutsch, W. A., Yacoub, A., Jaruga, P., Zastawny, T. H., and Dizdaroglu, M. (1997) J. Biol. Chem. 272, 32857–32860[Abstract/Free Full Text]
  76. Dobson, A. W., Grishko, V., LeDoux, S. P., Kelley, M. R., Wilson, G. L., and (2002) Am. J. Physiol. Lung Cell. Mol. Physiol. 283, L205-L210[Abstract/Free Full Text]
  77. Xu, Y., Hansen, W. K., Rosenquist, T. A., Williams, D. A., Limp-Foster, M., and Kelley, M. R. (2001) J. Pharmacol. Exp. Ther. 296, 825–831[Abstract/Free Full Text]
  78. Kobune, M., Xu, Y., Baum, C., Kelley, M. R., and Williams, D. A. (2001) Cancer Res. 61, 5116–5125[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
M.-H. Kim, H.-B. Kim, S. Acharya, H.-M. Sohn, J. Y. Jun, I.-Y. Chang, and H. J. You
Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor {alpha}1 Expression
Mol. Cell. Biol., April 15, 2009; 29(8): 2264 - 2277.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
I.-Y. Chang, M. Jin, S. P. Yoon, C.-K. Youn, Y. Yoon, S.-P. Moon, J.-W. Hyun, J. Y. Jun, and H. J. You
Senescence-Dependent MutS{alpha} Dysfunction Attenuates Mismatch Repair
Mol. Cancer Res., June 1, 2008; 6(6): 978 - 989.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. L. Habib, D. J. Riley, L. Mahimainathan, B. Bhandari, G. G. Choudhury, and H. E. Abboud
Tuberin regulates the DNA repair enzyme OGG1
Am J Physiol Renal Physiol, January 1, 2008; 294(1): F281 - F290.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
C.-K. Youn, P. I. Song, M.-H. Kim, J. S. Kim, J.-W. Hyun, S.-J. Choi, S. P. Yoon, M. H. Chung, I.-Y. Chang, and H. J. You
Human 8-Oxoguanine DNA Glycosylase Suppresses the Oxidative Stress Induced Apoptosis through a p53-Mediated Signaling Pathway in Human Fibroblasts
Mol. Cancer Res., October 1, 2007; 5(10): 1083 - 1098.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Das, T. K. Hazra, I. Boldogh, S. Mitra, and K. K. Bhakat
Induction of the Human Oxidized Base-specific DNA Glycosylase NEIL1 by Reactive Oxygen Species
J. Biol. Chem., October 21, 2005; 280(42): 35272 - 35280.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C.-K. Youn, S.-H. Kim, D. Y. Lee, S. H. Song, I.-Y. Chang, J.-W. Hyun, M.-H. Chung, and H. J. You
Cadmium Down-regulates Human OGG1 through Suppression of Sp1 Activity
J. Biol. Chem., July 1, 2005; 280(26): 25185 - 25195.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. Hu, S. Z. Imam, K. Hashiguchi, N. C. de Souza-Pinto, and V. A. Bohr
Phosphorylation of human oxoguanine DNA glycosylase ({alpha}-OGG1) modulates its function
Nucleic Acids Res., June 7, 2005; 33(10): 3271 - 3282.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
V. I. Grishko, L. I. Rachek, D. R. Spitz, G. L. Wilson, and S. P. LeDoux
Contribution of Mitochondrial DNA Repair to Cell Resistance from Oxidative Stress
J. Biol. Chem., March 11, 2005; 280(10): 8901 - 8905.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. G. Jeong, C.-K. Youn, H.-J. Cho, S.-H. Kim, M.-H. Kim, H.-B. Kim, I.-Y. Chang, Y.-S. Lee, M.-H. Chung, and H. J. You
Metallothionein-III Prevents {gamma}-Ray-induced 8-Oxoguanine Accumulation in Normal and hOGG1-depleted Cells
J. Biol. Chem., August 13, 2004; 279(33): 34138 - 34149.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C.-K. Youn, M.-H. Kim, H.-J. Cho, H.-B. Kim, I.-Y. Chang, M.-H. Chung, and H. J. You
Oncogenic H-Ras Up-Regulates Expression of ERCC1 to Protect Cells from Platinum-Based Anticancer Agents
Cancer Res., July 15, 2004; 64(14): 4849 - 4857.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/11/9857    most recent
M311132200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, M.-R.
Right arrow Articles by You, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, M.-R.
Right arrow Articles by You, H. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement