Base Excision Repair Activities Differ in Human Lung Cancer Cells and Corresponding Normal Controls

Abstract

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway for the repair of oxidized modifications both in nuclear and mitochondrial DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three cell lines used. However, the specific activities and cancer versus control comparison differed significantly between the nuclear and mitochondrial compartments. OGG1 activity, as measured by 8-oxodA incision, was up-regulated in cancer cell mitochondria but down-regulated in the nucleus when compared to control cells. Similarly, NTH1 activity was also up-regulated in mitochondrial extracts from cancer cells but did not change significantly in the nucleus. Together, these results support the idea that alterations in BER capacity are associated with carcinogenesis.

Living cells are constantly exposed to environmental agents and endogenous processes that can damage DNA. Among these, reactive oxygen species (ROS) are generated at relatively high rates, and particularly within the mitochondrion, in close proximity to the mitochondrial DNA (mtDNA), which is physically associated with the mitochondrial inner membrane through the nucleoids. Thus, excessive ROS generation is likely to cause genome instability, not only in the mitochondria, but also in the nucleus. Oxidative DNA damage, such as base modifications and strand breaks, accumulates under physiological conditions and in various pathological conditions, such as cancer (1). Carcinogenesis is, indeed, a process of cellular transformation that is initiated by mutations which may arise from DNA damage.

All living organisms have evolved DNA repair pathways which counteract the effects of DNA damage. Several biochemically distinct pathways have evolved to deal with chemically distinct lesions, in particularly excision repair pathways, such as the nucleotide and the base excision repair (BER) pathways (2). The nucleotide excision repair pathway removes bulky adducts and modifications which cause gross alterations of the double-helix. The BER pathway, on the other hand, removes small covalent modifications which do not distort the DNA helix, such as the base modifications generated by ROS and single-strand breaks. The BER pathway is highly conserved in all cellular organisms, from bacteria to man. The repair is carried out in four sequential enzymatic steps catalyzed by the enzymes DNA glycosylase, AP-endonuclease, DNA polymerase and DNA ligase. Although these enzymes do not form proteinb stable complexes, they do interact functionally through an intermediate handling mechanism (3). DNA glycosylases initiate BER by recognizing and excising the modified bases; these enzymes recognize defined sets of substrates, providing the specificity of the pathway. There are two classes of DNA glycosylases, type I or II, depending on their reaction mechanisms, for which the products are an abasic site or a single strand break, respectively. When the product is an abasic site, this is further processed by an abasic site (AP) endonuclease, which then catalysis the hydrolysis of the site generating a single-strand break. The 3′ and 5′ ends around the break can be further processed by AP-endonuclease itself and by the deoxyribose-phosphate (dRP) lyase activity of polymerase β in the nucleus or polymerase γ in the mitochondria, which then also insert a new nucleotide. The gap is then finally sealed by a DNA ligase. This pathway replaces only one nucleotide, and is known as single-patch BER. When the 5′ end is resistant to the dRP lyase activity, DNA polymerase β can perform strand-displacement synthesis generating a flap, which is processed by the structure specific endonuclease FEN-1 and the gap is the also ligated by DNA ligase. This sub-pathway is known as the long-patch BER (4).

Because oxidative damage has been detected at higher levels in cancerous tissues in comparison to normal surrounding tissues, it has long been speculated that alterations in BER activity play a causative role in carcinogenesis, particularly regarding mtDNA, which is more prone to accumulate oxidative DNA damage than is nuclear DNA (5, 6).

However, the only BER gene which has been clearly implicated in carcinogenesis is hMYH, the homologue of the bacterial MutY gene, which codes a DNA glycosylase that removes adenine pared opposite oxidized purines, such as the abundant 8-hydroxyguanine modification (7). The role of other BER activities in cellular transformation has been suggested by association studies, such as the role of the DNA glycosylase OGG1, which removes oxidized purines. Some studies have suggested that mutations in the OGG1 gene predispose towards lung cancer (8–10).

This study measured BER activities in nuclear and mitochondrial extracts of three different lung cancer cell lines and their corresponding control, non-transformed cell lines. The goal was to verify whether cancer cells exhibited a pattern of alterations in BER activities which could be correlated to the cancerous state. Thus, the three major DNA glycosylases were measured: OGG1; NTH1, an glycosylase oxidized pyrimidines; and UDG, which removes uracils in DNA. The activity of the next enzyme in the pathway, AP-endonuclease, which catalyses the hydrolysis of the abasic site, was also measured. While no clear pattern of alterations in BER activities was observed in any cell line, lesion- and compartment-specific changes, which could lead to imbalanced BER and contribute to the genomic instability and mutagenesis leading to cellular transformation, were observed.

Materials and Methods

Materials

HEPES, benzamidine, dithiothreitol (DTT), bovine serum albumin (BSA), and acrylamide/bis-acrylamide (19:1) were from Sigma (St. Louis, MO, USA). Leupeptin was from Roche (Mannheim, Germany). [γ-³²P]ATP was from NEN Life Science Products (Boston, MA, USA), G25 spin columns were from GE Healthcare. T4 polynucleotide kinase was from Stratagene (San Diego, CA, USA). All other reagents were of American Chemical Society grade.

Cell lines and culture conditions

Three different lung cancer cell lines and their corresponding controls were used in the present study (Table I). All cells were obtained from the American Type Culture Collection (ATCC), and were grown under similar conditions except CRL-5983 (NCI-H2107) and CRL-5915 (NCI-H2052). Cancer and normal cell lines were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) with 2 mM L-glutamine, supplemented with 10 mM HEPES and 1 mM sodium pyruvate, plus 10% FBS. The cultures were maintained at 37°C at 5% CO₂/95% relative humidity. CRL-5983 (NCI-H2107) cells were cultured in a mixture of equal parts of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 medium, with the following additions: 0.02 mg/ml insulin, 0.01 mg/ml transferrin, 25 nM sodium selenite, 50 nM hydrocortisone, 1 ng/ml epidermal growth factor (EGF), 0.01 mM ethanolamine, 0.01 mM phosphorylethanolamine, 0.1 mM triodothyronine, 2 mg/ml bovin serum albumin, 10 mM HEPES, 0.5 mM sodium pyruvate and 2 mM L-glutamine. CRL-5915 (NCI-H2052) cells were cultured in RPMI-1640 medium supplemented with 10% FBS. The cells were harvested at 80% confluence with trypsin, washed with PBS, and immediately processed for mitochondria and nuclei isolation.

Table I.

Cell lines used.

Tumor cell lines				Normal cell lines
ATCC number (Designation)	Cancer type	Source	Morphology	ATCC number (Designation)	Source	Morphology
NCI-H2087 (CRL-5922)	Adenocarcinoma; non-small cell lung cancer	Lymphonode	Epithelial	NCI-BL2087 (CRL-5965)	Peripheral blood	Lymphoblast
NCI-H2107 (CRL-5983)	Carcinoma, small cell lung cancer	Bone marrow	Epithelial	NCI-BL2107 (CRL-5966)	Peripheral blood	Lymphoblast
NCI-H2052 (CRL-5915)	Mesothelioma	Pleural effusion	–	NCI-BL2052 (CRL-5963)	Peripheral blood	Lymphoblast

Oligonucleotides

The sequences of all oligonucleotides used in this study are presented in Table II. Control and uracil-containing oligonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX, USA). The 5-OHdC oligonucleotide was kindly provided by Dr. Michelle Ham (MIT, Cambridge, MA, USA). THF-containing oligonucleotides were kindly provided by Dr. David Wilson III (NIA, NIH). Oligonucleotides containing either the DNA lesion or an unmodified base were purified through a 20% PAGE/ 7 M urea gel and 5′-end labeled using T4 polynucleotide kinase and [γ-³²P]ATP. Unincorporated radioactivity was removed with G25 spin columns, and the labeled strand was duplexed with an unlabeled complementary strand by heating at 90°C for 5 min and allowing the samples to cool down slowly.

Table II.

Oligonucleotide substrates used.

Oligonucleotide name	Sequence
C	5′-ATATACCGCGGCCGGCCGATCAAGCTTATT 3′-TATATGGCGCCGGCCGGCCGATCAAGCTTATT
OHC	5′-ATATACCGCGG(OHC)CGGCCGATCAAGCTTATT 3′-TATATGGCGCCGGCCGGCTAGTTGGAAATA
U	5′-ATATACCGCGGCCGGCCGATCAAGCTTATT 3′-TATATGGCGCCCAGCCGGCTAGTTCGAATAA
OA	5′-ATATACCGCGG(oxodA)CGGCCGATCAAGCTTATT 3′-TATATGGCGCCCGCCGGCTTCGAATAA
THF	5′-GAAGACCT(THF)GGCGTCC 3′-CTTCTGGAUCCCGCAGG

C: Control; OHC: 5-OH cytosine; THF: tetrahydrofuran; U: uracil; OA: 8-oxoadenine.

Cellular fractionation

Mitochondria were isolated from cultured human cells using a combination of differential centrifugation and Percoll gradient centrifugation, as described in Souza-Pinto et al. (11). Briefly, cells were harvested, washed with PBS and pelleted at 500 × g. The cell pellet was resuspended in MSHE buffer (0.21 M mannitol, 70 mM sucrose, 10 mM HEPES (pH 7.4), 1 mM EGTA, 2 mM EDTA, 0.15 mM spermine, and 0.75 mM spermidine), with the following protease inhibitors added just before use: 5 mM DTT, 2 μg/ml leupeptin and 2 μM benzamide-HCl. Small aliquots of 5% digitonin (in DMSO) were added and checked for cell integrity using trypan blue. After all cells were stained blue, the cell suspension was homogenized using a glass-to-glass Douncer homogenizer. The suspension was centrifuged at 500 × g and the pellet containing the nuclei was kept −80°C for preparation of nuclear extract. The supernatant was transferred to a new tube and crude mitochondria were pelleted at 10,000 × g. The crude mitochondrial pellet was then suspended in a MSHE:Percoll (1:1) solution and subjected to a Percoll gradient separation at 50,000 × g for 1 h. Purified mitochondria were recovered from the gradient, washed with MSHE and stored as pellets at −80°C.

Preparation of nuclear extracts

The nuclear pellets were centrifuged at 20,000 × g for 20 min, the supernatant was discarded and the pellet was suspended in Buffer A [20 mM HEPES (pH 7.4), 1 mM EDTA, 5% glycerol, 5 mM DTT, 300 mM KCl, protease inhibitors (1 μg/ml aprotinin, pepstatin, chymostatin, 2 μg/ml leupeptin, 2 μM benzamidine, 1 mM PMSF, 0.5 μM E-64)]. After the addition of 0.5% Triton X-100 the samples were incubated for 10 min on ice, followed by a centrifugation at 130,000 × g for 1 h. The supernatants were transferred into Centricon 30 concentrators (Millipore) and the buffer was exchanged for Buffer B (20 mM HEPES (pH 7.4), 1 mM EDTA, 25% glycerol, 5 mM DTT, 100 mM KCl, and 0.015% Triton X-100 and protease inhibitors) and concentrated to 1/5 of the initial volume. The extracts were then aliquoted and stored at −80°C.

Oligonucleotide incision assays

Oligonucleotide incision assay conditions varied for each enzyme being assayed, and are described in detail elsewhere (12). For 5-OH-cytosine and uracil, incision reactions (20 μl) contained 40 mM HEPES-KOH (pH 7.6), 5 mM EDTA, 2 mM DTT, 75 mM KCl, 5% glycerol, ³²P-labeled duplex oligonucleotide, and 25 μg mitochondrial or 50 μg nuclear protein for 5-OHdC incision, or 0.5 μg protein for uracil incision. Reactions were incubated at 37°C for 4h for 5-OHC incision and for 1 h for uracil incision. For 8oxoA incision, reactions contained 20 mM HEPES-KOH (pH 7.6), 5 mM EDTA, 2 mM DTT, 25 mM KCl, 10% glycerol, 0.2 mg/ml BSA, 0.6 mM MgCl₂, 100 fmoles ³²P-labeled duplex oligonucleotide and 10 μg mitochondrial or nuclear extracts, and were incubated at 32°C for 2 h. For THF incision, the reactions contained 40 mM Hepes-KOH (pH 7.6), 5mM EDTA, 1 mM DTT, 50 mM KCl, 10% glycerol, 100 fmoles ³²P-labeled duplex oligonucleotide and 10 μg mitochondrial protein or 1 μg nuclear protein. These were incubated at 37°C for 2 h for mitochondria and 30 min for nuclear extracts.

All reactions were terminated by addition of 1 μl 5 mg/ml Proteinase K and 1 μl 10% SDS and incubated at 55°C for 30 min. The DNA was ethanol-precipitated and analyzed in a denaturing 20% polyacrylamide gel containing 7 M urea. The gels were visualized by PhosphorImager, and quantified using ImageQuant NT software. Incision activity was calculated from the ratio of damage-specific cleavage product to the total product and substrate in the reaction.

Western blot analysis

Mitochondrial protein (20–50 μg) was separated on 12% Tris-glycine gels (Invitrogen). Transfer to PVDF membranes (Invitrogen) was done by electroblotting in transfer buffer (12 mM Tris, pH 8.3; 196 mM glycine, 20% methanol) for 2 h at 30 V. The membrane was blocked for 16 h at 4°C in 5% non-fat dry milk (Bio-Rad, Hercules, CA, USA) in TBST (20 mM Tris-HCl pH 7.2, 137 mM NaCl, 0.1% Tween-20). Fresh milk-TBST was added with the primary antibody, which was either mouse monoclonal anti-lamin B2 (Novocastra, Newcastle upon Tyne, UK) or mouse monoclonal anti-cytochrome oxidase IV (Invitrogen). Detection was performed with ECL+Plus^®(GE Healthcare, Piscataway, NJ, USA).

DNA isolation and PCR-RFLP analysis

Genomic DNA samples were isolated using the Easy-DNA^™ kit (Invitrogen) according to the manufacturer’s recommendations. PCR-RFLP was used to detect the OGG1 Ser326Cys polymorphism. Primers for hOGG1 were obtained from Midland Certified Reagent Co (Midland, TX, USA) and the sequences are presented in Table III. PCR analysis were carried out in 20 μl reactions containing 25 ng of template DNA, 2 mM dNTPs (Stratagene), 15 mM MgCl₂, 60 pM of each primer pair and 0.25 Unit of Taq polymerase (Stratagene). Fifty μg of each PCR sample were digested with 6 units of Fnu4HI (Stratagene) at 37°C overnight and resolved on 2.5% agarose to detect any differences in the RFLP patterns (107 and 100 bp fragments for the 326Cys allele).

Statistical analysis

The results are reported as the mean±standard deviation of three independent experiments, assayed in duplicate. For each experiment, two different gels were run. The differences among cell lines were analyzed by Student’s t-test, and p≤0.05 was considered statistically significant.

Results

Mitochondria and nuclei were isolated from all the cell lines as described. In order to ascertain that the mitochondrial extracts were free of nuclear contamination, mitochondrial extracts were tested for the presence of lamin B2, an abundant nuclear matrix protein, by Western blot. Figure 1 shows a typical blot with mitochondrial extracts from all six cell lines. No lamin B2 signal was detected in any mitochondrial extracts, as opposed to a strong band in a nuclear extract from a normal lymphoblast line (CRL-5963). The blots were co-incubated with an antibody against the mitochondrial protein CoxIV to show the presence of mitochondrial proteins.

Figure 1.

Western blot analysis of mitochondrial and nuclear extracts. 20 μg of mitochondrial (lanes 1–6) or nuclear (lane 7) extracts of the cell lines indicated were analyzed using monoclonal anti-cytochrome oxidase IV (CoxIV) and polyclonal anti-Lamin B2 antibodies.

In order to compare BER efficiency in these cell lines, NTH1, OGG1, UDG and APE-1 activities were measured in nuclear and mitochondrial extracts using an incision assay and DNA oligonucleotide substrates containing single DNA lesions (oligonucleotide substrates are shown in Table II). Initially, experiments were performed with increasing amounts of protein extracts to determine the linear range of the assay for each extract and oligonucleotide (data not shown). All subsequent assays were carried out using protein concentrations and incubation times found to be within the linear range of the incision assay.

DNA glycosylases initiate BER by recognizing specific modifications, and thus provide the substrate specificity of the pathway. This study measured the activities of the three major DNA glycosylases, OGG1, NTH1 and UDG, which recognize and cleave oxidized purines, oxidized pyrimidines and uracil, respectively. Reaction products were separated on denaturing polyacrylamide gels and incision activity was quantified for each gel. Each figure shows a typical gel for the incision reaction and the quantification of at least three independent measurements for each cell line, carried out in duplicates. Each experiment also included negative control reactions, carried out with a lesion-free DNA oligonucleotide, and a positive control reaction, carried out with recombinant enzymes specific for each lesion.

Glycosylase activities in mitochondrial and nuclear extracts of three lung cancer cell lines and their corresponding normal control lines were compared. Figures 2–4 show results for assays using oligonucleotide DNA substrates containing 5-OH-dC (Figure 2), 8-oxoA (Figure 3) and uracil (Figure 4) to assess the enzymes NTH1, OGG1 and UDG, respectively. These results show that the changes in activity were lesion and compartment specific. For nuclear BER, no difference between the cancer and normal cells for 5-OHdC and uracil incision was observed, and only minor differences in 8oxodA incision in two out of the three cancer lines were observed. In mitochondrial extracts, on the other hand, a significant increase in both 5OHdC and 8-oxodA was observed in all cancer lines, except for one pair for 8-oxodA. On the other hand, uracil incision in mitochondrial extracts was unchanged, similarly to the nuclear extracts.

Figure 2.

Incision of a 5-OHdC-containing oligonucleotide was carried out with 25 μg of mitochondrial (A) or 50 μg of nuclear extracts (B), as described in the Materials and Methods. Migration of the substrate (S) and incision products (P) are shown in typical autoradiograms for each extract. The results presented are the mean ± SD of 3 experiments performed in duplicate.

Figure 4.

Incision of a U-containing oligonucleotide was carried out with 0.5 μg (A–B) of mitochondrial and nuclear extracts (A and B). The reactions were incubated and processed as described in the Materials and Methods. Migration of the substrate (S) and incision products (P) are shown in typical autoradiograms for each extract. The results presented are the mean ±SD of 3 experiments performed in duplicate.

Figure 3.

Incision of an 8-oxoA-containing oligonucleotide was carried out with 10 μg (A–B) of mitochondrial and nuclear extracts (A and B). The reactions were incubated and processed as described in the Materials and Methods. Migration of the substrate (S) and incision products (P) are shown in typical autoradiograms for each extract. The results presented are the mean ±SD of 3 experiments performed in duplicate.

Abasic site endonuclease activity, the next step in BER after the DNA glycosylase, was also measured with an incision assay, using oligonucleotides containing the abasic site analogue THF. Both in the nucleus and in mitochondria, APE is the major AP-endonuclease responsible for carrying out this step in BER. While increased APE activity was observed in both nuclear and mitochondrial extracts of one cell line compared to its normal control (Figure 5), the two other pairs evaluated here showed decreased mitochondrial and unchanged nuclear APE activity.

Figure 5.

Incision of a TFH-containing oligonucleotide was carried out with 10 μg of mitochondrial (A) or 1 μg and nuclear extracts (B), as described in the Materials and Methods. The reactions were processed as described. Migration of the substrate (S) and incision products (P) are shown in typical autoradiograms for each extract. The results presented are the mean ±SD of 3 experiments performed in duplicate.

Because changes in 8oxodA incision were observed in both mitochondrial and nuclear extracts it was investigated whether these cell lines carried a polymorphism in the OGG1 site, Ser326Cys, for which the Cys/Cys genotype has been associated with lung cancer in population studies (9, 13, 14). It was found that of the six cell lines, three were homozygous for the wild-type, Ser/Ser genotype, while the other three were heterozygous (Ser/Cys) for the polymorphism (Figure 6). However, there was no obvious association of the genotype with the cancerous or normal state of the cell line, as the polymorphism was found in a normal line and two controls, while one of the cancer lines had the wild-type Ser/Ser genotype.

Figure 6.

Analysis of the hOGG1 Ser326Cys polymorphism. Ser/Ser genotype (wild-type, WT) gives one band of 200 bp. The Cys/Cys genotype (mutant, M) gives two bands at 100 bp and 107 bp and the Ser/Cys genotype (heterozygous, H) gives the composite bands at 100 bp, 107 bp and 207 bp.

Discussion

Cancer is a disease of genomic instability, in which the accumulation of mutations in critical genes leads to cellular transformation (15). These mutations can arise from unrepaired DNA lesions, and much correlative evidence indicates that defects in DNA repair pathways can contribute to mutagenesis and cellular transformation. In fact, several syndromes caused by mutations in DNA repair genes are highly cancer prone (16). Known deficiencies in nucleotide excision repair and mismatch repair activities in human cancers, underlie UV-induced skin cancer and hereditary nonpolyposis colorectal cancer cells, respectively (17–20).

However, a causative role for BER defects in human cancers has still not been demonstrated. This study investigated whether three different lung cancer cell lines had alterations in BER activities relative to their normal, non-transformed, counterpart cell line. Moreover, this study attempted to differentiate alterations in the nuclear and mitochondrial compartments, since mitochondrial dysfunction and oxidative DNA damage accumulation has been proposed to play a causative role in cancer (21) and mutations in the mtDNA have been identified in cancer of the bladder, breast, colon, head, neck, kidney, liver, lung, stomach and in hematologic malignancies such as leukemia and lymphoma (22–24). Thus, the rationale is that a selective loss of mtDNA BER activities could lead to accumulation of oxidative DNA damage in mitochondria, and this would contribute/promote cellular transformation. However, since DNA repair is generally studied in the nuclear DNA, very little is actually known about mitochondrial DNA repair.

The results of this study did not indicate a general deregulation of BER in cancer cells. Rather, lesion- and compartment-specific changes were observed, such as an up-regulation of incision of the oxidative lesions (5-OHdC and 8oxodA) in mitochondria from the cancer lines, but not in the nucleus (Figures 3 and 4). This observation suggests that cancer cell mitochondria are under oxidative stress, and this is in agreement with the observation from other groups that mtDNA from cancer tissue have more mtDNA oxidative damage than normal surrounding tissue (25, 26). The observation that UDG activity, which repairs a product of cytosine deamination and thus is not directly involved in oxidative damage repair, is not altered in either the nucleus or mitochondria from cancer cells also supports the notion that oxidative DNA damage, in particular, may be relevant lesions in carcinogenesis.

In parallel, a decrease in 8oxodA incision in the nucleus was also observed of two out of the three cancer lines. This decrease was not, however, associated with the known evidence exists regarding the repair capacity of the hOGG1 Cys326 variant. Functional evidence is also equivocal, with some in vitro studies showing that hOGG1 Cys326 protein has identical catalytic activity to wild-type Ser326-hOGG1, and others reporting significant differences (27, 28). Janssen et al. performed a study to investigate whether an association exists between OGG1 polymorphism and decreased OGG1 activity in vivo in normal human cells. Their results showed that this polymorphism is not associated with the alteration of OGG1 DNA repair activity in the corresponding lymphocytes (29). In a previous study, a case–control study in the Turkish population, no correlation was found between the Ser326Cys polymorphism and the levels of 8-OHdG, suggesting that this polymorphism is not a genetic risk factor for lung cancer (30). Since oxidized purine incision activity in human mitochondria has been assigned primarily to the α-isoform of hOGG1, the same isoform that is present in the nucleus (31), it is likely that this difference observed in 8oxodA incision activity between the nuclear and mitochondrial extracts is due to changes in protein targeting to each compartment. In this regard, Szcezny et al. have shown that during the aging process, OGG1 targeting to mitochondria is impaired, and although the amount of OGG1 protein which gets to mitochondria is higher in mitochondria from older than in younger animals, the net activity in the matrix is lower because OGG1 becomes stuck in the outer mitochondrial membrane (32).

Abasic site endonuclease activity was also modulated in a compartment-specific manner; increased activity in mitochondria was observed from one cancer cell line and decreased activity seen in the two other lines. Interestingly, the same line which showed increased mitochondrial APE also showed enhanced nuclear APE, while the other two showed no changes in nuclear APE. Thus, it is likely that the cell line which showed increased APE in both compartments had a general up-regulation of this enzyme. Tumor promotion by APE-1 has been previously shown both in in vitro and in vivo, where higher levels of APE-1 expression and altered APE-1 localization have been correlated with tumor progression and poor prognosis for patients with various malignancies (33). Moreover, subcellular localization of APE has also been shown to be altered with age, such that a 2-fold increase in APE levels is observed in the cytoplasmic fraction of the cell, and a 2-fold decrease is observed in the nucleus (32). However, since APE alterations were observed in only one cancer cell line, it is unlikely that this is a general event that causes malignancy in lung tumors.

In conclusion, these results indicate that BER enzymes are altered in the different cancer cell lines when compared to their respective non-transformed controls. However, the alterations in specific enzymes vary from one line to the other, and no specific pattern was observed. However, one must keep in mind that BER is a pathway carried out by four enzymes in a sequential manner, in which the product of one reaction is handed out to the next step, in a manner alike to the ‘passing the baton’ (34). Thus, imbalances in one enzymatic step could result in a deregulation of the pathway as a whole, with accumulation of highly toxic intermediates, such as abasic sites and single-strand breaks. In fact, Rinne et al. demonstrated that over expression of the glycosylase MPG which repairs damage to alkylated bases of DNA increases cellular sensitivity to alkylating agents, likely due to the accumulation of BER intermediates (35). It is now important to measure overall BER activity in lung cancer cell lines in order to investigate whether imbalanced BER leads to genomic instability which could account for cellular transformation.