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Carcinogenesis Advance Access originally published online on March 26, 2007
Carcinogenesis 2007 28(8):1629-1637; doi:10.1093/carcin/bgm072
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Targeting human 8-oxoguanine DNA glycosylase (hOGG1) to mitochondria enhances cisplatin cytotoxicity in hepatoma cells

Haihong Zhang1,{dagger}, Takatsugu Mizumachi2,{dagger}, Jaime Carcel-Trullols2, Liwen Li1, Akihiro Naito2, Horace J. Spencer3, Paul M. Spring4, Bruce R. Smoller1, Amanda J. Watson6, Geoffrey P. Margison6, Masahiro Higuchi2 and Chun-Yang Fan1,5,*

1 Department of Pathology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA
2 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA
3 Department of Biostatistics, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA
4 Department of Otolaryngology—Head and Neck Surgery, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA
5 John L. McClellan Memorial Veterans Hospital, 4300 West 7th Street, Little Rock, AR 72205, USA
6 Division of Carcinogenesis, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK

* To whom correspondence should be addressed. Tel: +501 257 6469; Fax: +501 257 6430;Email: fanchunyang{at}uams.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many chemoradiation therapies cause DNA damage through oxidative stress. An important cellular mechanism that protects cells against oxidative stress involves DNA repair. One of the primary DNA repair mechanisms for oxidative DNA damage is base excision repair (BER). BER involves the tightly coordinated function of four enzymes (glycosylase, apurinic/apyrimidinic endonuclease, polymerase and ligase), in which 8-oxoguanine DNA glycosylase 1 initiates the cycle. An imbalance in the production of any one of these enzymes may result in the generation of more DNA damage and increased cell killing. In this study, we targeted mitochondrial DNA to enhance cancer chemotherapy by over-expressing a human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene in the mitochondria of human hepatoma cells. Increased hOGG1 transgene expression was achieved at RNA, protein and enzyme activity levels. In parallel, we observed enhanced mitochondrial DNA damage, increased mitochondrial respiration rate, increased membrane potential and elevated free radical production. A greater proportion of the hOGG1-over-expressing hepatoma cells experienced apoptosis. Following exposure to a commonly used chemotherapeutic agent, cisplatin, cancer cells over-expressing hOGG1 displayed much shortened long-term survival when compared with control cells. Our results suggest that over-expression of hOGG1 in mitochondria may promote mitochondrial DNA damage by creating an imbalance in the BER pathway and sensitize cancer cells to cisplatin. These findings support further evaluation of hOGG1 over-expression strategies for cancer therapy.

Abbreviations: AP, apurinic/apyrimidinic; APE, AP endonuclease; BER, base excision repair; HE, dihydroethidium; hOGG1, human 8-oxoguanine DNA glycosylase 1; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ROS, reactive oxygen species


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Nuclear and mitochondrial DNAs are constantly exposed to damaging agents from endogenous and exogenous sources, which can generate DNA base lesions or base loss. These DNA base lesions may eventually become mutagenic, carcinogenic or lethal to cells if not removed through the base excision repair (BER) pathway to restore the DNA's chemical integrity (1,2). BER is a multi-step process, which typically starts with a DNA glycosylase that cleaves the N-glycosyl bond between the deoxyribose sugar moiety and the damaged DNA base, generating an apurinic/apyrimidinic (AP) site (3). Resultant AP sites are the substrate of AP endonuclease (APE1/HAP1), which cleaves the sugar–phosphate backbone 5' to the AP site, leaving a 3'-hydroxyl group and a 5'-deoxyribose phosphate group flanking the nucleotide gap (4,5). In the next repair step, the DNA polymerase incorporates the undamaged nucleotides and then removes the abasic sugar–phosphate (dRP) group, producing a nicked duplex DNA molecule (6). The resultant gap is subsequently sealed by DNA ligase (7). The specificity of BER is provided by DNA glycosylases that act on specific substrates. Four major pathway components (DNA glycosylase, AP endonuclease, DNA polymerase and ligase) have to be precisely regulated and highly coordinated for error-free and efficient DNA repair.

Overproduction of a single DNA glycosylase is detrimental rather than beneficial (8–14). Enhanced repair capacities of hNTH1 and human 8-oxoguanine DNA glycosylase 1 (hOGG1) genes in TK6 human lymphoblast cells significantly increase the number of double-strand breaks in DNA and elevate sensitivity to the cytotoxic and mutagenic effects of ionizing radiation (8). Similar results have been obtained in Escherichia coli, in which over-expression of the gene FPG, a bacterial homolog of hOGG1, resulted in these outcomes (9). Extensive investigations performed in MPG, a DNA glycosylase gene, showed that over-expression of MPG rendered cells more sensitive to various alkylating agents (10,11). Similar results have been achieved using human cancer cell lines (12–14).

Cisplatin is one of the most active cytotoxic agents in the treatment of cancer. Many studies suggest that cisplatin mediates its anti-neoplastic effects via DNA adduct formation and generation of reactive oxygen species (ROS) (15–17). Previous study has showed that increased oxidative stress induced by inhibiting mitochondrial respiration could significantly enhance the tumor-killing effects of cisplatin in leukemic cells(18).

Mitochondria are dynamic intracellular organelles that play central roles in energy metabolism (ATP production), production of ROS such as superoxide radicals and hydrogen peroxide and regulation of apoptosis (19). Mitochondrial genomes consist of circular DNA molecules ~16 kb in length, encoding 13 polypeptides primarily involved in mitochondrial oxidative phosphorylation (19). Because of its close proximity to the electron transport chain and lack of protective histone protein, mitochondrial DNA is highly susceptible to ROS damage produced during oxidative phosphorylation. The most important DNA repair mechanism for oxidative damage in mitochondria is BER, and the detection of various BER enzymes in mitochondria underscores the significance of maintaining mitochondrial DNA integrity for normal cellular functioning (20,21). Because of its critical role in cellular functions, mitochondrial DNA has been previously targeted for enhanced cancer chemotherapy by over-expressing E.coli exonuclease III (a bacteria homolog of APE/HAP1) or MPG in the mitochondria of human breast cancer cells (12,21).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
The hOGG1 construct
The MT-hOGG1 mammalian expression vector (Figure 1A) that over-expresses hOGG1 in mitochondria was constructed using a 1.5 kb segment containing the full-length hOGG1 (mitochondrial isoform type 2a) cDNA (22), which was removed from the pSPORT1-hOGG1 plasmid (a gracious gift from Dr D.Barnes and T.Lindahl, Imperial Cancer Research Fund, UK) and inserted into the Bam HI cloning site of MThGH vector (a gracious gift from Dr Geoffrey P.Margison, CRC Paterson Institute for Cancer Research, Manchester, UK) (22). This vector contains a 1.7 kb mouse metallothionein-I regulatory element at 5' and a 1.8 kb transcription unit of human growth hormone gene and was used successfully to generate transgenic mice over-expressing the human O6-alkylguanine-DNA alkyltransferase gene (23). The hOGG1 type 2a cDNA insert was sequenced to confirm the integrity of the open reading frame prior to cell transfection.


Figure 1
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  		Fig. 1. Construction, stable transfection, and over-expression of MT-hOGG1 mammalian gene expression vector. (A) Schematic representation of the MT-hOGG1 construct (H; Hind III, B; Bam HI, S; Sca I). (B) PCR identification for MT-hOGG1 transgene integration in 10 G418-resistant HepG2 cell clones following transfection with MT-hOGG1 and pCDNA3.1 plasmids. The MT-hOGG1 transgene was integrated only in clones 7, 9 and 10. (C) Over-expression of MT-hOGG1 mRNA in cell clones harboring the MT-hOGG1 transgene. While all cell lines expressed similar levels of human ß-actin (middle panel), only clones 7, 9 and 10 expressed MT-hOGG1 hybrid transcript (upper panel). Total RNA samples were also subjected to DNA Taq polymerase reaction (lower panel) without prior reverse transcription reaction. Negative PCR amplicons indicate that total RNA preparations are devoid of genomic DNA contamination.

 
Cell culture and stable transfection
The human hepatoma cell line HepG2 was obtained from American Type Culture Collection. Cells were transfected with either the MT-hOGG1 vector using FuGENE 6 reagent (Roche Diagnostics Corporation, Indianapolis, IN) according to the manufacturer's instructions or with MThGH vector alone. For selection, plasmid pcDNA 3 containing a neomycin-resistant gene (Clontech, Mountain View, CA) was co-transfected with MT-hOGG1 or MThGH at a 3:5 ratio (3 pcDNA 3:5 MT-hOGG1 or MThGH). Stable transfectants were selected in Dulbecco's modified Eagle's medium supplemented with 950 µg/ml G418 (Gibco, Grand Island, NY) for 2 weeks. Integration of the MT-hOGG1 transgene in G418-resistant clones was confirmed by polymerase chain reaction (PCR) using a transgene-specific primer set that only amplified the integrated MT-hOGG1 transgene but not the endogenous hOGG1. The transgene-specific primer sequences are as follows: 5'-ACT ATG CGT GGG CTG GA-3' (sense), derived from mouse metallothionein promoter, and 5'-CTA GCC TGG CTC TTG TCT-3' (anti-sense), derived from exon 2 of the hOGG1 gene. The size of the resultant PCR product was 487 bp extending from the mouse MT promoter into exon 2 of the hOGG1 gene. The integration of parental vector MThGH without the hOGG1 insert in G418-resistant clones was identified by PCR using primers designed to amplify a 290 bp fragment within the mouse MT promoter. These primers will not recognize the endogenous human MT promoter sequence. The primer sequences are as follows: 5'-TCC AGG AAA GGA GAA GCT GA-3' (sense) and 5'-TCC AGC CCA CGC ATA GT-3' (anti-sense).

Immunofluorescent staining for hOGG1 protein
To verify that hOGG1 protein is successfully over-expressed, HepG2 cells transfected with MT-hOGG1 or MThGH were cultured with coverslips. The coverslips were fixed with methanol/acetone (1:1) for 10 min at –20°C. After fixation, they were washed with Tris-buffered saline and auto fluorescence was quenched by 0.1% sodium borohydride in Tris-buffered saline for 5 min. The coverslips were blocked with phosphate-buffered saline (PBS) containing 10% normal goat serum and 1% bovine serum albumin for 1 h and stained with rabbit polyclonal anti-hOGG1 antibody (kindly provided by Dr Sankar Miltra, University of Texas Medical Branch, Galveston, TX) at 1:100 dilutions for 2 h at room temperature followed by reaction with green fluorescent Alexa488-conjugated anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA) at 1:1000 dilution. After washing, cells were counterstained with DAPI to visualize the nuclei. Cells were then examined under a confocal fluorescence microscope equipped with a photographic system.

RT–PCR analysis of hOGG1 mRNA expression
Total cellular RNA was isolated using TRIzol reagent/chloroform (Gibco Life Technologies, Paisley, UK) from a confluent plate of cells transfected with MT-hOGG1 or a control vector, MThGH, followed by DNase I treatment (Invitrogen) to remove contaminated DNA. MT-hOGG1 transgene mRNA expression was analyzed by conventional RT–PCR using the SuperScrip One-Step RT–PCR Kit (Invitrogen) with a transgene-specific primer set (see Cell culture and stable transfection). RT–PCR for human ß-actin mRNA was performed in parallel as an internal control. Primer sequences for the human ß-actin gene were as follows: 5'-GCT CGT CGT CGA CAA CGG CTC-3' (sense) and 5'-CAA ACA TGA TCT GGG TCA TCT TCT C-3' (anti-sense), with an expected PCR amplicon of 353 bp.

Oligonucleotide incision assay for hOGG1 activity
Specific hOGG1 activity was analyzed as described previously (24). Briefly, a 25mer oligonucleotide with 8-oxoguanine at the seventh position (5-CCG CTA [8-oxo]CG GGT ACC GAG CTC GAA T-3') was end labeled with 32[P]-ATP (Amersham Biosciences, Piscataway, NJ 10 mCi/ml) by T4 polynucleotide kinase (Roche) as substrate. Ms Amanda J.Watson and Dr Geoffrey P.Margison of Paterson Institute for Cancer Research and Christie Hospital, Manchester, UK, provided assistance with the hOGG1 enzyme activity assay.

Long-range PCR for detection of mitochondrial genome
Long-range PCR was employed to amplify the entire 16 kb mitochondrial genome as described previously (25). For each sample, DNA (1 µg) was used for long-range PCR using AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen) with the following primers: MITO F 5'-TGA GGC CAA ATA TCA TTC TGA GGG GC-3' (sense) and MITO R 5'-TTT CAT CAT GCG GAG ATG TTG GAT GG-3' (anti-sense).

Measurement of mitochondrial respiration activity
Oxygen consumption by intact cells, an indicator of mitochondrial respiration, was measured as described previously (26). Oxygen consumption was measured with Oxytherm (Hansatech Instrument, King's Lynn, UK) in a sealed chamber at 37°C. Cultured cells stably transfected with MT-hOGG1 or control vector were harvested and 1 x 106 cells were suspended in 1 ml fresh culture media pre-equilibrated with 21% oxygen. The oxygen contents in the starting medium were normalized assuming an O2 concentration of 220 µM in air-saturated medium at 37°C. The two cell lines were initially measured for oxygen consumption for 10 min without any drug treatment. Following that, a mitochondrial respiratory chain complex III inhibitor, antimycin A (400 ng/ml, Sigma, St Louis, MO), or a mitochondrial uncoupler, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP, 100 µM; Sigma), were added to cell suspensions. The cell suspensions were then monitored for an additional 10 min for changes in oxygen consumption.

Measurement of intracellular free radical
Intracellular hydrogen peroxide and superoxide were determined in cells with or without cisplatin treatment by flow cytometry, as described previously (27). The intracellular generation of ROS was measured using 5 µM dihydroethidium (HE) for superoxide radical and 10 µM DHR123 for hydrogen peroxide. Cultured cells stably transfected with MT-hOGG1 or control vector were first treated overnight with 1 µM cisplatin. As a control, cells derived from these two lines were treated with PBS overnight. The cells were then trypsinized, re-suspended with medium and stained with the two fluorescent dyes, HE or DHR123, for 30 min at 37°C. After that, the samples were cooled on ice and evaluated on a FACS Calibur Flow Cytometer (FACSCalibur, Becton Dickinson, Mountain View, CA). FL1 fluorescence for hydrogen peroxide measurement or FL2 fluorescence for superoxide measurement of 10 000 cells was collected in each experiment by gating and acquiring the cell population.

Detection of mitochondrial membrane potential
To visualize mitochondrial membrane potential in situ, cells were seeded overnight at a concentration of 1 x 105 cells per ml in a Lab-Tek Chamber (Nunc International Corp., Naperville, IL). Cells were then incubated with 4 mM of JC-1 (Invitrogen) in complete medium for 30 min and observed under a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) using 488 nm excitation and a 600/20 nm bandpass emission filter for JC-1 aggregates (red fluorescence) and a 525/50 nm (green fluorescence) emission filter for JC-1 monomer. JC-1 forms "J aggregates" that appear red at higher mitochondrial membrane potential but remain as a monomer that appears green at low mitochondrial membrane potential. Mitochondrial membrane potential was also assessed quantitatively by flow cytometry using the mitochondria-specific fluorescent dye 5,5',6,6',-tetrachloro1,1',3,3',-tetraethylbenzimidazolocarbocyanine iodide (JC-1) (Molecular Probes, Eugene, OR), as described previously with slight modifications (28). Three separate measurements were performed for each cell line and the mean red fluorescence intensities of 10 000 cells per sample were presented and analyzed.

Analysis of apoptosis by annexin V–fluorescein isothiocyanate/propidium iodide staining
Apoptosis was analyzed using the Annexin V–fluorescein isothiocyanate Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA). Cells (1 x 106) transfected with MT-hOGG1 or vector only (MThGH) were plated and allowed to attach overnight. Cells were then treated with 10 µM cisplatin (Sigma) in the media for 24 h and then harvested for apoptosis analysis. Cells were stained with annexin V–FITC and propidium iodide, respectively. Stained cells (1 x 104) were analyzed by flow cytometry.

Determination of cell survival using colony formation assay
The transfected cells were allowed to grow to confluence and then trypsinized. Transfected cells (1 x 103) of hOGG1 or vector only were plated in triplicate on 6 cm cell culture dishes and allowed to attach overnight. The cells were then treated with cisplatin at specified doses (0.01, 0.1, 0.5, 1.0 and 5.0 µM) overnight. Control cells were treated with PBS. Following drug treatment, cells were rinsed twice with PBS and allowed to grow in fresh medium. After 10 days, cells were rinsed twice with PBS, followed by fixation with methanol/acetic acid in a 3:1 ratio for 10 min at room temperature and air-dried. Cells were then stained with hematoxylin 2 (Richard-Allan Scientific, Kalamazoo, MI) and the colonies were scored. HepG2 cells with the hOGG1 transgene were compared with those without hOGG1 with regard to cell survival based on plating efficiency and their ability to form a colony after treatment with various doses of cisplatin.

Statistical analysis
Analysis of variance models were used to evaluate the main effects of cell type (MT-hOGG1 or vector only) on membrane potential, free radical production and oxygen consumption and the main effects of both cell type and drug (PBS or cisplatin) on apoptosis. For each outcome, the interaction between cell type and drug was tested. In this case, two-sample t-tests were used to evaluate main effects. P values were generated using the permutation distributions of the test statistics of interest. When feasible, the entire permutation distribution was enumerated and used in the calculation of P values; otherwise, 2000 random permutations of the data were used to provide an estimate of the permutation distribution. A linear mixed-effect model was used to analyze the effect of cisplatin on cell survival. In particular, cell types were compared with respect to the slope of the line estimating cell survival in response to increasing doses of cisplatin. P values equal to or less than the 0.05 levels were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Increased mRNA levels in cells harboring the MT-hOGG1 transgene
Following stable transfection with the MT-hOGG1 construct (Figure 1A) or the control vector, MThGH, DNA samples were isolated from G418-resistant clones and subjected to PCR amplification to confirm transgene integration using primers specific for MT-hOGG1 or MThGH constructs. Among 10 G418-resistant clones transfected with MT-hOGG1 construct, three (clones 7, 9 and 10) were positively identified as having integrated the hOGG1 transgene in their genome (Figure 1B). Seven G418-resistant clones transfected with control vector, MThGH, were all positive for the mouse MT sequence, indicating stable integration of the MThGH vector DNA (data not shown).

To investigate whether the integrated MT-hOGG1 transgene was over-expressed in HepG2 cells, total RNA (0.2 µg) samples from 10 G418-resistant clones were subjected to RT–PCR using the primers specific for the MT-hOGG1 construct. As shown in , 3 out of 10 G418-resistant clones positive for MT-hOGG1 (clones 7, 9 and 10 in Figure 1B) expressed the MT-hOGG1 transgene mRNA. No MT-hOGG1 mRNA was detected in G418-resistant clones transfected with the MThGH control vector (data not shown).

Increased hOGG1 enzyme activity in cells harboring the MT-hOGG1 transgene
Whole-cell extracts from two MT-hOGG1-transfected cell clones (clones 7 and 9) and a vector-only-transfected cell clone (clone 2) were subjected to a specific hOGG1 enzyme activity assay. hOGG1-specific enzyme activities for the MT-hOGG1-transfected cell clones were 15.2 and 9.7 fmol (femtomoles)/µg DNA per h, respectively, whereas the vector-only-transfected cell clone was 4.4 fmol/µg DNA per h. These results show that the over-expressed hOGG1 protein was functional in MT-hOGG1-transfected cells. The following data were derived from a single positive clone (clone 7). In parallel, the vector-only-transfected clone (clone 2) was used as a control.

Increased hOGG1 protein expression in cells harboring the MT-hOGG1 transgene
To confirm that increased hOGG1 protein was over-expressed, MT-hOGG1 and vector-only-transfected HepG2 cells were stained with red-fluorescent rhodamine dye (MitoTracker Red CMXRos, Invitrogen) to localize the mitochondria. Cells were also stained with anti-hOGG1 polyclonal antibody (a gracious gift from Dr Sankar Mitra, University of Texas Medical Branch, Galveston, TX), which was subsequently linked to a green fluorescent Alexa488-conjugated anti-rabbit secondary antibody to visualize the over-expressed hOGG1 protein. Under a confocal fluorescence microscope, increased hOGG1 protein (green fluorescence) is represented as fine cytosol granules, consistent with mitochondrial targeting of the over-expressed hOGG1 protein (Figure 2B). In contrast, control cells transfected by vector only showed only faint cytoplasmic staining for the hOGG1 protein (Figure 2A). Our results are consistent with those obtained by Takao et al. (29) who demonstrated that the type 2 mitochondrial isoform of the hOGG1 gene is exclusively expressed in the mitochondria.


Figure 2
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  		Fig. 2. Increased hOGG1 protein expression and mitochondrial DNA deletion in hOGG1-over-expressing cells. HepG2 hepatoma cells transfected with MT-hOGG1 transgene (A) or vector only (B) were stained with anti-hOGG1 antibody and labeled with green fluorescing FITC to visualize the hOGG1 protein. There is much more intense granular, green fluorescence in the cytoplasm of cancer cells harboring the MT-hOGG1 transgene (A) than in vector-only-transfected cells (B), consistent with targeted expressed hOGG1 protein in the mitochondria. The nuclei were counterstained with DAPI. Increased mitochondrial DNA deletions are detected in cells over-expressing MT-hOGG1 (C). Long-range PCR using mitochondria-specific primers was performed on MT-hOGG1- (lane 2) or vector-only (lane 1)-transfected HepG2 cells (clone 7). There were more abundant mtDNA deletions in cells harboring the MT-hOGG1 transgene (lane 2) as demonstrated by additional PCR bands of low molecular weights when compared with the vector-only transfectants (lane 1). (Magnification for A and B: 400x.)

 
Enhanced mitochondrial DNA deletions in cells over-expressing MT-hOGG1
Total DNA was isolated from MT-hOGG1 or vector-only-transfected cells and subjected to long-range PCR using mitochondria-specific primers to amplify the entire 16 kb human mitochondrial genome. Whereas the majority of MT-hOGG1 or vector-only transfectants contained an intact 16 kb mitochondrial genome (Figure 2C), MT-hOGG1 transfectants (lane 2) contained more mitochondrial DNA deletions (i.e. DNA bands of lower molecular weight) as compared with vector-only transfectants (lane 1). This indicates that targeted over-expression of hOGG1 in mitochondria causes increased mitochondrial DNA damage.

Increased mitochondrial respiration in cells over-expressing MT-hOGG1
To determine how mitochondrial respiration and membrane potential may be affected in hOGG1-over-expressing cells that show enhanced mitochondrial DNA damage, oxygen consumption by intact cells was measured as an indicator of mitochondrial respiratory activity, as described previously (18). As illustrated in Figure 3, without any drug treatment, oxygen consumption rates in hOGG1-over-expressing cells were much higher (1792 and 1877 nmol/min/106 cells, respectively, in two separate assays; Figure 3C and D) than those in vector-only-transfected cells (719 and 796 nmol/min/106 cells, respectively, in two separate assays; Figure 3A and B). These results indicate that over-expression of hOGG1 mitochondrial isoform protein is correlated with enhanced mitochondrial respiration activity.


Figure 3
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  		Fig. 3. Increased oxygen consumption in cells over-expressing MT-hOGG1. Mitochondrial respiration activity was determined by measuring oxygen consumption rates in intact cells transfected with vector only (A, B) or MT-hOGG1 (C, D). Both cell lines were first analyzed for oxygen consumption rates for 10 min. This was followed by adding the mitochondrial respiration complex III inhibitor, antimycin A (A, C), or mitochondrial uncoupler, FCCP (B, D), in cell suspension and then by an additional period (10 min) of measuring oxygen consumption. Average rate of oxygen consumption over 10 min (nmol/min) was placed above each individual oxygen consumption curve. A single measurement was made for each cell type and treatment group.

 
To determine whether or how much the oxygen is actually consumed in the mitochondria, oxygen consumption was also measured in the presence of a mitochondrial respiratory chain complex III inhibitor, antimycin A. Following treatment with antimycin, the rates of oxygen consumption dropped from 719 to 197 nmol/min (73% reduction) in vector-only-transfected cells (Figure 3A) and from 1792 to 256 nmol/min (86% reduction) in hOGG1-over-expressing cells (Figure 3C). These results indicate that most oxygen is consumed by the mitochondria in both control and hOGG1-over-expressing cells.

To analyze the maximal oxygen consumption capacity in mitochondria, we also measured oxygen consumption in the presence of a mitochondrial uncoupler, FCCP. Following FCCP treatment, oxygen consumption rates increased from 796 to 1366 nmol/min (1.7-fold increase) in vector-only-transfected cells (Figure 3B) and from 1877 to 10925 nmol/min (5.8-fold increase) in hOGG1-over-expressing cells (Figure 3D). These results indicate that the mitochondrial coupling mechanism is intact in both control and hOGG1-over-expressing cells and that the maximal oxygen consumption capacity in mitochondria is much greater in hOGG1-over-expressing cells (Figure 3D) than in control cells (Figure 3B).

Increased membrane potential in cells over-expressing MT-hOGG1
We also analyzed mitochondrial membrane potential in cells transfected with an MT-hOGG1 construct or with vector. Cells were first stained with JC-1 (Invitrogen) and observed under a confocal laser scanning microscope. The red fluorescence (JC-1 aggregates) intensity in individual cells, indicative of mitochondrial membrane potential, was much more intense in MT-hOGG1-transfected cells (Figure 4C) than in vector-only-transfected cells (Figure 4A). In contrast, the green fluorescence (JC-1 monomer) intensity in individual cells, reflective of mitochondrial number, was similar between MT-hOGG1-transfected cells (Figure 4D) and vector-only-transfected cells (Figure 4B). To further confirm that mitochondrial membrane potential indeed increases in MT-hOGG1-transfected cells, both MT-hOGG1- and vector-only-transfected cells were stained with JC-1 dye followed by quantitative flow cytometric analyses. Three independent measurements were made on each cell lines. The average fluorescence intensity derived from 3 separate measurements was 255 [standard deviation (SD) = 4.4] in MT-hOGG1-transfected cells and 134 (SD = 19.3) in vector-only-transfected cells. The difference in mean fluorescent intensity in these two cell lines is statistically significant (P = 0.05). As presented in Figure 4E, the mean fluorescence (JC-1 aggregates) intensity was about twice as much in MT-hOGG1-transfected cells (255) as in the vector-only-transfected cells (134) using JC-1 fluorescent dye.


Figure 4
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  		Fig. 4. Increased membrane potential in cells over-expressing MT-hOGG1.Mitochondrial membrane potential was first analyzed in situ in individual cells transfected with MT-hOGG1 (C, D) or vector only (A, B). Cells were stained with JC-1 fluorescent dye and subjected to confocal laser scanning microscope examination for the presence of J-aggregates (red fluorescence), indicative of increased membrane potential (A, C), or a JC-1 monomer (green fluorescence) (B, D), reflective of mitochondrial number. Membrane potential was also quantitatively measured by flow cytometry using JC-1 fluorescent dye in cells transfected with vector only or MT-hOGG1 (E). Three separate quantitative measurements for membrane potential were made with an averaged mean fluorescence intensity presented in Figure 4E.

 
Increased production of free radicals in cells over-expressing MT-hOGG1
The amount of intracellular superoxide radicals or hydrogen peroxide was analyzed in cells without or with cisplatin treatment by flow cytometry using fluorescent dyes HE or DHR123, respectively. The measurements were first made in both MT-hOGG1- and vector-only-transfected cell lines in the absence of cisplatin. The average mean fluorescence intensities, derived from 3 separate measurements, for HE, reflective of the amount of superoxide radicals, were 10.4 (SD = 0.56) in MT-hOGG1- and 8.1 (SD = 0.3) in vector-only-transfected cells. The difference in superoxide production was statistically significant (P = 0.05). The average mean fluorescence intensities, derived from 3 separate measurements, for DHR123, reflective of the amount of hydrogen peroxide, were 562.9 (SD = 39) in MT-hOGG1- and 273.4 (SD = 32) in vector-only-transfected cells. The difference in hydrogen peroxide production was statistically significant (P = 0.05). In order to determine how free radical production was affected by the treatment of cisplatin, the amount of free radicals was measured in both MT-hOGG1- and vector-only-transfected cell lines following treatment with 1 µM cisplatin. A single measurement was made in this experiment for each treatment group. The mean HE fluorescence intensities, reflective of the amount of superoxide, were 242 and 126 in MT-hOGG1- and vector-only-transfected cells, respectively (Figure 5A). These increased to 292 and 140, respectively, in the presence of 1 µM cisplatin (Figure 5C). In the absence of cisplatin, the mean DHR123 fluorescence intensities, reflective of the amount of hydrogen peroxide in MT-hOGG1- or vector-only-transfected cells, were 163 and 87, respectively (Figure 5B). These increased to 186 and 101, respectively, in the presence of 1 µM cisplatin (Figure 5D). The results demonstrate that more free radicals are produced in hOGG1-over-expressing cells as compared with those in control cells and that treating cells with cisplatin enhances the production of free radicals in both control and hOGG1-over-expressing cells.


Figure 5
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  		Fig. 5. Increased intracellular free radicals in cells over-expressing MT-hOGG1. Intracellular superoxide radical and hydrogen peroxide were analyzed in cells (1 x 106) transfected with vector only or MT-hOGG1 by flow cytometry using fluorescing dyes, HE (A, C) and DHR123 (B, D), respectively, in the absence (A, B) or presence (C, D) of cisplatin. Fluorescence intensities, reflective of total amount of free radicals, were listed within parentheses underneath the identity of the cell type. A single measurement was made for each cell type and treatment group.

 
Enhanced sensitivity of MT-hOGG1 cells to cisplatin
To determine whether an increase in intracellular free radicals could trigger more active apoptosis, we characterized apoptosis in MT-hOGG1- or vector-only-transfected cells with or without cisplatin treatment by flow cytometric analysis. Three separate measurements were made and the average percentage of apoptotic cells was displayed and analyzed. The proportions of apoptotic cells were 23 and 15.6%, respectively, in MT-hOGG1- and vector-only-transfected cells prior to cisplatin treatment; these increased to 31.5 and 18.5%, respectively, following drug treatment (Figure 6A). The overall interaction is statistically significant (P = 0.0018) between MT-hOGG1- and vector-only-transfected cells with regard to different treatment plans (PBS versus cisplatin) and between PBS and cisplatin-treated cells with regard to transgene status (MT-hOGG1 versus vector). The significant difference on the test of overall interaction indicates that apoptosis in MT-hOGG1-transfected cells is significantly more active than that in vector-only-transfected cells regardless of whether the cells are treated with cisplatin or not and that apoptosis in cisplatin-treated cells is significantly more active than that in PBS-treated cells regardless of whether the cells are transfected with MT-hOGG1 or not.


Figure 6
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  		Fig. 6. Enhanced sensitivity of MT-hOGG1 cells to cisplatin. Proportions of cells undergoing apoptosis were analyzed by annexin V–FITC/propidium iodide staining followed by flow cytometry in MT-hOGG1- or vector-only-transfected cells with or without cisplatin (A). Long-term survival of MT-hOGG1- or vector only-transfected cells following cisplatin treatment was also analyzed using colony formation assay (B). Three independent experiments are represented for apoptosis study and five independent experiments are represented for long-term survival study.

 
To determine whether hOGG1 over-expression with resultant increased free radical production will enhance the cytotoxicity of cisplatin, we characterized the long-term survival of MT-hOGG1- and vector-only-transfected cells following cisplatin treatment at specified doses (0.01, 0.1, 0.5, 1 and 5 µM) using colony formation assays. The average numbers of colonies for MT-hOGG1- and vector-only-transfected cells without cisplatin treatment were 167.4 and 160.5, respectively; the difference was not statistically significant. Following treatment with cisplatin at specified doses of 0.01, 0.1, 0.5, 1 and 5 µM, the average numbers of colonies for MT-hOGG1-transfected cells were 134.8, 109.6, 66.4, 16.4 and 2, respectively, whereas those for vector-only-transfected cells were 158.8, 147, 116, 78.6 and 2.4, respectively. Five separate experiments were performed and the average number of colonies were displayed and analyzed. In MT-hOGG1-transfected cells treated with cisplatin, colony formation was significantly decreased at all doses except for the highest dose (5 µM) as compared with that of vector-only-transfected cells (Figure 6B). Overall, the difference in colony formation between MT-hOGG1- and vector-only-transfected cells following cisplatin treatment was statistically significant (P = 0.048).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, we targeted foreign gene expression to the mitochondria using a mitochondrial isoform of the hOGG1 gene, type 2a. It has been shown previously that the hOGG1 protein encoded by mitochondrial isoforms, hOGG1 types 1b, 1c and 2, is expressed exclusively in the mitochondria, because these isoforms contain only mitochondrial targeting signals in the final protein product (29).

We demonstrated that over-expression of the hOGG1 gene in the mitochondria of hepatoma cells is correlated with more extensive mitochondrial DNA damage, increased mitochondrial respiration, increased membrane potential, increased intracellular production of free radicals, enhanced apoptosis and ultimately, enhanced sensitivity of tumor cells to cisplatin. The results presented here were obtained with a single clone (clone 7) that integrated and over-expressed the hOGG1 transgene. Similar phenotypes, particularly on gene expression, free radical production, membrane potential and apoptosis, were observed in another positive cell clone (clone 9). Thus, it is highly likely that the observed biological and cellular changes are due to over-expressed hOGG1 in the mitochondria rather than incidental disruption of an important nuclear gene in the process of transgene integration.

Our results are consistent with those obtained by others who demonstrated that over-expression of DNA glycosylases impaired DNA repair and increased cellular sensitivity to irradiation or chemotherapeutic agents (8–14). We postulate that over-expression of a single DNA glycosylase gene will disrupt the BER pathway and lead, paradoxically, to more DNA damage through the following mechanisms: (i) the non-lethal, readily repairable clustered DNA base lesions or multiple damaged sites that are produced in close proximity to two opposite strands and are subsequently converted by over-expressed DNA glycosylases into lethal double-strand breaks (8,9) or (ii) the number of toxic BER intermediates, such as AP sites generated by over-expressed DNA glycosylases, exceed the incision capacity of other downstream BER enzymes, such as APE, resulting in accumulation and persistence of these toxic AP sites with a resultant increase in mutation frequency (12–14,30).

It is noteworthy that some investigators have shown that over-expression of oxidative DNA glycosylases (hOGG1 or FPG) is beneficial and protects cells against oxidative DNA damage (31–33).The impact of over-expressed oxidative DNA glycosylases (hOGG1 or FPG) on cellular response to oxidative stress is likely to be influenced by factors such as the levels of DNA repair gene expression and the extent of oxidative DNA damage. Improved DNA repair with prolonged cell survival under oxidative stress would be expected if there is only a mild increase in DNA glycosylase activity and/or in the production of free radicals, resulting in a slight increase in AP sites that can be easily repaired by the downstream BER enzymes. In contrast, impaired DNA repair with shortened cell survival under oxidative stress would occur if there is a dramatic increase in DNA glycosylase activity and/or in the production of free radicals, generating large numbers of toxic AP sites that overwhelm the downstream BER enzymes.

Our results endorse the theory put forward by many investigators that efficient BER relies on coordinated regulation of all components of the pathway and that over-expression of a single enzyme in the pathway is more likely to be detrimental rather than beneficial due to an imbalance in BER. This conclusion is supported by the studies involving over-expression of DNA glycosylases (8–14,34), APE (21,35,36) and DNA polymerase (37–40).

We also observed a series of molecular events associated with over-expressed hOGG1 genes in the mitochondria; these included increased mitochondrial DNA damage, elevated cellular respiration, increased mitochondrial membrane potential, increased ROS production, enhanced apoptosis and decreased long-term survival in the presence of cisplatin.

While the mechanisms and exact sequence by which the over-expressed hOGG1 protein induces the above molecular events, and the cause–effect relationships among them, remain unknown and beyond the scope of our current investigation, we hypothesize that over-expression of hOGG1 in the mitochondria would disrupt the balance of BER, resulting in enhanced mitochondrial DNA damage and decreased ATP synthesis. This would bring about a series of molecular changes, such as elevated cellular respiration, increased mitochondrial membrane potential and increased ROS production, by an adaptive mechanism to compensate for loss of ATP and cell function. In our study, the amount of damaged mitochondrial DNA in hOGG1-over-expressing cells is significantly more than that seen in control cells (Figure 2C).

Because mitochondrial DNA contains important genes involved in the oxidative phosphorylation chain, the primary function of which is to turn oxygen and substrate into ATP and water, the damage to mitochondrial DNA would jeopardize the mitochondrial oxidative phosphorylation function, ultimately resulting in reduced synthesis of ATP, which is required by many important cellular functions. This would, in turn, set in motion an adaptive mechanism to compensate for the loss of ATP, leading to more active oxidative phosphorylation with enhanced mitochondrial respiration, increased membrane potential and increased free radical production. This adaptive mechanism has been noted in patients with the MELAS (myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome, a maternally inherited mitochondrial disease in which expression of mitochondrial and nuclear genes involved in oxidative phosphorylation are increased in cells with mitochondrial DNA mutations and defective mitochondrial function (41). Similarly, an adaptive mechanism is demonstrated in the brain of aged mice, which sustained more extensive oxidative DNA damage, but expressed higher levels of mitochondrial genes involved in oxidative phosphorylation (42).

Mitochondria play a crucial role in regulating apoptosis. Upon receiving an apoptotic signal, mitochondria will either release cytochrome c into the cytosol to initiate a caspase-dependent apoptosis pathway and/or release an apoptosis-inducing factor to the nuclei to launch a caspase-independent apoptosis pathway (43,44). It has been shown that oxidative stress in cells with subsequent ROS-induced DNA damages is primarily involved in a caspase-independent apoptosis pathway by triggering the activation of poly (ADP-ribose) polymerase-1, required for the translocation of apoptosis-inducing factor from mitochondria to the nuclei, and subsequent cell death (45,46). Our results showed that hOGG1-over-expressing cells generated more free radicals (Figure 5A and B) as compared with vector-only-transfected cells and that the production of free radicals was further enhanced when these cancer cells were treated with a chemotherapeutic agent, cisplatin (Figure 5C and D). The above results may help explain the fact that cells over-expressing hOGG1 were more sensitive to the cytotoxic effects of cisplatin in terms of apoptosis and long-term survival (Figure 6A and B).

The use of an oxidative DNA glycosylase, such as hOGG1, may have significant clinical implications, particularly when combined with targeted expression of the hOGG1 gene to cancer tissue by gene therapy. The enzyme, hOGG1, specifically removes 8-oxoguanine, an oxidative DNA base lesion generated by a wide range of antitumor agents such as ionizing radiation and chemotherapy. By inducing an imbalanced BER, hOGG1 over-expression can simultaneously sensitize cancer cells to a wide variety of antitumor regimens that generate ROS and 8-oxoguanine. The possibility of combining targeted hOGG1 over-expression in conjunction with combined chemoradiation therapy may yield more potent antitumor activity with fewer clinical side effects because enhanced antitumor effects will reduce the need for large doses of cytotoxic agents and the associated side effects.


   Footnotes
 
{dagger} These two authors contributed equally to this work. Back


   Acknowledgments
 
The authors would like to thank Mr James P.Morgan, Office of Grants and Scientific Publications at University of Arkansas for Medical Sciences for his careful editing of the manuscript. This work was supported in part by a grant from the National Institutes of Health (5K08CA089029 to C.Y.F.) and in part by research funds from the Department of Pathology and Otolaryngology—Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas. Findings were presented at the 95th Annual Meeting of United States and Canadian Academy of Pathology, February 11–17, 2006, Atlanta, Georgia.

Conflict of Interest Statement: None declared.


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 28, 2007; revised March 1, 2007; accepted March 19, 2007.


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