1 Division of Pulmonary, Allergy, Critical Care, and Occupational Medicine, Departments of Medicine, 2 Section of Hematology/Oncology, Herman B Wells Center for Pediatric Research, Pediatrics, and 3 Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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The pulmonary complications of 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) are among the most important dose-limiting factors of BCNU-containing cancer chemotherapeutic regimens. BCNU damages DNA of both cancer cells and normal cells. To increase the resistance of lung cells to BCNU, we employed gene transfer of Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) and human 8-oxoguanine-DNA glycosylase (hOGG1) to A549 cells, a lung epithelial cell line, using a bicistronic retroviral vector, pSF91-RE, that encoded both FPG/hOGG1 and an enhanced green fluorescent protein. The transduced epithelial cells were sorted by flow cytometry, and expression of FPG/hOGG1 protein was determined by the level of FPG/hOGG1 RNA and enzyme activity. The single-cell gel electrophoresis (comet assay) measured DNA damage induced by BCNU. FPG/hOGG1-expressing A549 cells incubated with 40-500 µg/ml BCNU exhibited significantly less DNA damage than vector-transduced cells. In addition, FPG- and/or hOGG1-expressing cells incubated with 10-40 µg/ml BCNU showed at least a 25% increase in cell survival. Gene transfer of FPG/hOGG1 reduced BCNU-induced DNA damage and cytotoxicity of cultured lung cells and may suggest a new mechanism to reduce BCNU pulmonary toxicity.
deoxyribonucleic acid repair; pulmonary toxicity; N7-alkylated guanine; formamidopyrimidine-DNA glycosylase; human 8-oxoguanine-DNA glycosylase; 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea
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INTRODUCTION |
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PULMONARY COMPLICATIONS of cancer chemotherapy are common (36). Although many chemotherapeutic drugs damage DNA in neoplastic cells, these drugs can also damage DNA in normal lung cells, resulting in both early-onset and delayed-onset pulmonary disorders (36). Because pulmonary toxicity remains one of the major adverse reactions limiting the use of more aggressive anticancer regimens (36), correcting or preventing the genotoxic and/or cytotoxic effects to lung cells would likely improve the therapeutic efficacy of these regimens.
1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) is a chemotherapeutic drug commonly used for human intracranial tumors, Hodgkin's disease, non-Hodgkin's lymphomas, melanoma, and gastrointestinal cancer (34, 36). In addition to bone marrow suppression and gastrointestinal toxicity, pulmonary toxicity by BCNU is a major fatal complication and occurs in as high as 20-30% of the patients treated by BCNU (34, 35). Proposed mechanisms of BCNU-induced pulmonary toxicity include DNA damage, glutathione depletion, or adverse inflammation and an immune response in the lung (6, 22). The pathological changes of BCNU-induced pulmonary toxicity include alveolar edema and protein accumulation, diffuse hyperplasia, and hypertrophy of alveolar epithelial cells, metaplasia of alveolar epithelium, fibroblastic proliferation in the alveolar septae, and alveolar septal fibrosis (23, 35).
As an alkylating agent, BCNU causes DNA damage by modifying bases, cross-linking, and inducing DNA strand breaks (37). BCNU alkylates DNA predominantly (>90%) at N7 positions of guanine and to a less extent, at the O6 position of guanine (32). O6 adducts result in miscoding and subsequently cause interstrand cross-linking as well, so they account for both mutagenic and cytotoxic activities of BCNU (37). The O6 guanine adducts generated by BCNU may be excised by O6-methylguanine-DNA methyltransferase (MGMT) and repaired through a direct-reversal DNA repair pathway (9, 39).
Although N7 guanine adducts can exist in cells for a long time without lethal effect (19), they are prone to imidazole ring rupture (24). Decomposition of these adducts through the rupture of the imidazole ring will give rise to an altered base, a formamidopyrimidine (Fapy), which can block DNA synthesis (24, 26). Thus Fapy lesions are lethal if not repaired. Also, N7-alkylated guanine is a major adduct of various DNA-alkylating agents (24). Repair of this lesion is important for cell survival. Studies have shown that base excision repair is involved in repairing various alkylated DNA adducts, including Fapy lesions (30).
Base excision repair is a multienzymatic process against various lethal or mutagenic DNA adducts generated by irradiation and chemotherapeutic drugs (11, 30). The initial step of this process is carried out by a DNA glycosylase that recognizes certain adducts specifically and releases the modified base from the sugar molecule of a nucleotide leaving an apurinic/apyrimidinic (AP) site in the DNA sequence (11, 30). Next, an AP endonuclease or a DNA lyase nicks the DNA backbone next to the AP site. The following steps include a DNA polymerase filling in the gap with appropriate nucleotides and a DNA ligase finally sealing the gap (11, 30).
Escherichia coli formamidopyrimidine-DNA glycosylase (FPG), a 30-kDa globular monomer, is a combined DNA glycosylase-AP lyase that removes the damaged bases and cleaves phosphodiester bonds in the DNA backbone next to AP sites (3, 27). FPG has a broad range of substrates, such as ring-opened guanine or adenine, oxidized guanine, cytidine, or uridine, and ring-opened and oxidized thymidine. Most of these substrates are adducts derived from oxidative and alkylating agents that damage DNA (2, 11, 12, 15, 30). A recently identified human functional homologue of FPG, 8-oxoguanine-DNA glycosylase (hOGG1), possesses similar enzymatic activities to FPG (1, 28, 29).
Previous studies have shown that overexpression of FPG and/or hOGG1 in mammalian cells reduces the toxicity of thiotepa and aziridine (5, 8, 16). Both thiotepa and aziridine alkylate guanine at the N7 position (25). Another study has shown that the expression of FPG and hOGG1 protects murine fibroblasts from the lethal effect of BCNU (40). We hypothesized that FPG and hOGG1 would increase resistance of lung cells against BCNU toxicity. Therefore, we introduced FPG and hOGG1 into A549 cells, a human alveolar epithelial cell line. Using a modified bicistronic retroviral vector, pSF91-RE, we established stable transduced cells and investigated the protective effects of FPG and hOGG1 on BCNU-induced DNA damage and cell death.
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METHODS |
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Cell culture. The human alveolar epithelial cell line A549 (ATCC no. CCL-185; American Type Culture Collection, Manassas, VA) was grown in DMEM (BioWhittaker, Walkersville, MA) with 10% FBS (Hyclone, Logan, UT) containing 1,000 U/ml penicillin and 100 U/ml streptomycin (Biosource International, Rockville, MD) at 37°C in 5% CO2.
Retroviral vector constructions.
The retrovirus vector pSF91N was a gift from Dr. Christopher Baum of
the University of Hamburg (Hamburg, Germany; see Ref. 13).
It was reconstructed to a bicistronic retroviral vector, pSF91-RE, that
contained an internal ribosome entry site (IRES) upstream to a gene
expressing an enhanced green fluorescence protein (EGFP). This was
achieved by replacing the IRES element of pSF91N with the IRES-EGFP
fragment (between EcoR I and Not I restriction sites) obtained from the vector pIRES-EGFP (Clontech, Palo Alto, CA).
After transforming DH5 competent cells (Life Technologies, Grand
Island, NY) with the new vector, pSF91-RE, colonies containing pSF91-RE
were confirmed by restriction endonuclease digestion and
electrophoresis. DNA sequencing confirmed the integrity of pSF91-RE.
Finally, a large amount of vector was produced using the Plasmid Maxi
Kit (QIAGEN, Chatsworth, CA).
Retroviral transduction. A549 cells (1 × 105) were plated in a six-well culture dish (Falcon, Franklin Lakes, NJ) and cultured overnight. The next day, 2 ml of supernatant of viral vector were added to the cell culture. After a 4-h incubation at 37°C, 2 ml of 10% DMEM were added, and the infection was continued overnight. Two days after infection, transduced cells were sorted using FACStarPlus (Becton-Dickinson, San Jose, CA) as EGFP-expressing cells. Transduced A549 cells were maintained in 10% DMEM and subcultured two times per week. EGFP expression levels in transduced cell populations were determined using FACSCalibur (Becton-Dickinson) by measuring fluorescence intensities of 10,000 cells. Flow cytometry data were analyzed by Cell Quest 3.3 software (Becton-Dickinson). EGFP expression levels were also visualized by fluorescent microscopy using an Olympus BX60 system microscope (Olympus, Tokyo, Japan).
Northern blot analysis. Total RNA was isolated from transduced cells (~5 × 106) by an RNeasy Mini Kit (QIAGEN). Total RNA (10 µg) was resolved on 1% agarose gel containing 18% formaldehyde. Subsequently, separated RNA was transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). FPG, hOGG1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA probes were made by random oligonucleotide priming using the Megaprime DNA Labeling System (Amersham Pharmacia Biotech, Piscataway, NJ). The activity of the probes was >1 × 108 dpm/µg. Hybridization was performed overnight at 60°C. After low-, medium-, and high-stringency washes, the hybridized membrane was visualized by autoradiography for 3 days.
Enzyme activity assay.
A 26-mer 8-oxoguanine-containing oligonucleotide,
5'-AATTCACCGGTACCOTCTAGAATTCG-3', where O is 8-oxoguanine, was used as
the substrate of FPG and hOGG1. The oligonucleotide was labeled with [-32P]ATP at the 5'-end with polynucleotide kinase.
The labeled oligonucleotide was then hybridized to its complementary
strand by heating the mixture to 95°C and then gradually cooling to
room temperature. To examine the enzyme activity, 10 µg protein from
cell-free extract were allowed to react with 2.5 pmol labeled substrate
in 70 mM HEPES-KOH, pH 7.6, 100 mM KCl, and 10 mM EDTA for 60 min at
37°C. Reaction products were separated by 20% PAGE containing 7 M
urea. The autoradiography was directly derived from PAGE. A unit is defined as the amount that excises 1 pmol of 8-oxoguanine-containing oligonucleotide in 60 min at 37°C (18).
Single cell gel electrophoresis/comet assay. BCNU (Carmustine; Sigma, St. Louis, MO) solutions were freshly prepared for each experiment. BCNU was first dissolved in 30 µl of 100% ethanol and then diluted to desired concentrations with 10% DMEM. The final concentration of ethanol in each solution was 0.03% (vol/vol). Transduced A549 cells were seeded on six-well cell culture dishes (Falcon) at a density of 5 × 105 cells/well. The next day, cells were treated with BCNU at concentrations of 0, 40, 100, 200, and 500 µg/ml for 1 h at 37°C. The CometAssay kit (Trevigen, Gaithersburg, MD) was used to assess DNA damage. Following a manufacturer-recommended protocol, cells were first detached by scraping and resuspended in Ca2+- and Mg2+-free PBS (Trevigen) at a concentration of 3 × 105 cells/ml. The cell suspension was then mixed with liquefied agarose at a 1:10 ratio. A small aliquot of the mixture was immediately transferred to a provided slide. After solidification and cell lysis at 4°C, all slides were treated with alkali solution (0.3 M NaOH, 1 mM EDTA) for 20 min to unwind the double-stranded DNA. Subsequently, the slides were electrophoresed at 20 V for 10 min to show the comet tails. After being stained with provided fluorescent staining solution, samples were examined and photographed by fluorescent microscopy using an Olympus BX60 system microscope (Olympus) and Paxit software (MIS, Franklin Park, IL). During data analysis, tail length was defined as the distance between the leading edge of the nucleus and the end of the tail. In each experiment, ~80 determinations were made for each sample using Adobe Photoshop software (Adobe Systems, San Jose, CA). Data represent a minimum of three separate experiments.
Colony-forming assay. Transduced A549 cells were seeded on six-well cell culture dishes (Falcon) at a density of 2 × 105 cells/well. The next day, cells were treated with BCNU at 0, 10, 20, and 40 µg/ml for 1 h at 37°C. After being washed and diluted, cells were subcultured in triplicate in 100-mm cell culture dishes (Falcon) until the colonies were visualized easily. The survival of BCNU-treated cells was expressed as a percentage of the number of colonies formed by the untreated cell population. The final result was a conclusion of three separate experiments.
Statistics. The Student's t-test was applied in the comet assay and the colony-forming assay at each BCNU concentration point. Significance was accepted as P < 0.05.
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RESULTS |
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Expression of EGFP.
The expression of EGFP in transduced A549 cells was confirmed by
multiple FACS analyses. After sorting, the EGFP-positive cells among
pSF91-RE-, pSF91-FPG-, and pSF91-hOGG1-transduced A549 cells were
90.3 ± 2.2, 92.9 ± 3.7, and 94.9 ± 1.6%,
respectively, whereas only 1.3 ± 1.0% of the untransduced A549
cells were positive. The mean fluorescent intensities were 42.9 ± 3.1, 94.1 ± 11.5, and 211.2 ± 13.2 for pSF91-RE-,
pSF91-FPG-, and pSF91-hOGG1-transduced A549 cells, respectively,
whereas the background reading of untransduced A549 cells was 3.9 ± 0.4. The EGFP expression of these cells was followed for up to 25 cell passages. There was no discernible decrease in the fluorescent
intensity. The fluorescence among these cells was also visualized using
microscopy (Fig. 1). Also, retroviral
transduction did not alter the rate of replication of A549 cells (data
not shown).
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Expression and activity of FPG/hOGG1.
The expression of FPG mRNA was confirmed by Northern blot analysis
(Fig. 2A).
pSF91-FPG-transduced A549 cells expressed FPG mRNA at an amount
comparable to GAPDH mRNA, whereas untransduced and pSF91-RE-transduced
cells did not show any FPG expression. Similarly, the enzymatic
activity of FPG revealed that 10 µg of protein from the
pSF91-FPG-transduced cell-free extract completely cleaved 2.5 pmol
labeled 26-mer substrate, revealing an activity of at least 250 units
in each milligram of cell-free extract (Fig. 2B); in
contrast, untransduced and pSF91-RE-transduced cell extract did not
show any activity. Similar findings were obtained when detecting hOGG1
mRNA and activity in pSF91-hOGG1-transduced cells using the same assays
(data not shown). Although A549 cells potentially express hOGG1, we did
not detect any hOGG1 activity in untransduced or pSF91-RE-transduced
cells (Fig. 2B). Thus endogenous repair activity of
N7-alkylated guanines is at
undetectable background levels in A549 cells.
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FPG/hOGG1 and BCNU-induced DNA damage.
The single-cell gel electrophoresis or comet assay was employed to
detect DNA strand breaks induced by BCNU in transduced A549 cells. BCNU
caused a concentration-dependent increase of DNA damage in pSF91-RE-,
pSF91-FPG-, and pSF91-hOGG1-transduced cells (Fig. 3). In untreated
cells, the comet tail length reflecting background DNA damage was
minimal. pSF91-FPG-transduced cells showed significantly reduced DNA
damage at all concentrations of BCNU, i.e., 40, 100, 200, and 500 µg/ml (Fig. 3A,
P < 0.001). pSF91-hOGG1-transduced cells also
exhibited significantly reduced tail lengths at the same BCNU
concentrations except 500 µg/ml (Fig. 3B,
P < 0.01).
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FPG/hOGG1 and BCNU-induced cytotoxicity.
The protective effect of FPG and hOGG1 on cell survival was evaluated
by a series of colony-forming assays. BCNU caused a decreased survival
of transduced A549 cells in a concentration-dependent manner.
pSF91-FPG-transduced A549 cells revealed significantly increased
survival in the presence of 20 and 40 µg/ml BCNU (Fig. 4A, P < 0.05), whereas pSF91-hOGG1-transduced A549 cells showed reduced cell
death at only 20 µg/ml BCNU (Fig. 4B, P < 0.05).
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DISCUSSION |
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Previous studies have indicated two cellular mechanisms of reducing BCNU-induced toxicity, glutathione-S-transferase (GST)-mediated detoxification of BCNU (31) and MGMT-mediated direct reversal DNA repair of BCNU-induced O6-methylguanine (9, 39). In the current study, retroviral vector-mediated gene transfer of FPG and hOGG1 to human lung cells resulted in a stable and functional expression of the DNA repair proteins. This expression led to a significant reduction in both BCNU-induced DNA damage and cytotoxicity in human lung cells. Combined with a previous study in murine fibroblasts (40), FPG/hOGG1-mediated DNA base excision repair may represent another mechanism to reduce BCNU toxicity.
The reduced DNA damage in FPG/hOGG1-expressing A549 cells is likely the result of FPG/hOGG1 releasing the ring-opened structures of N7-alkylated guanine adducts and initiating a base excision repair process. It is known that FPG can release ring-opened N7-alkylguanines, such as N7-methylguanine and N7-hydroxyethylguanine, in a cell-free system (4, 20, 33). Furthermore, FPG reduces cytotoxicity of thiotepa and aziridine, both of which form N7-aminoethylguanine adducts (5, 8, 16). The structures of these adducts are similar to BCNU-modified N7 guanines, i.e., N7-chloroethylguanine (37). Our results are consistent with FPG excising various ring-ruptured, N7-alkylated purine adducts (4, 20, 33).
It is likely that after FPG/hOGG1 releases the damaged bases and cuts
the DNA backbone next to AP sites, human AP endonucleases (such as
Ape1), DNA polymerase-, and DNA ligase are necessary to finish the
subsequent repair steps (11, 30). The protective effect of
FPG/hOGG1 on BCNU-induced DNA damage in our study suggests that
FPG/hOGG1 at the initial step of this particular base excision repair
process might be rate limiting. The less protection from BCNU-induced
DNA damage observed at higher concentrations of BCNU may be
1) the result of enzyme saturation of either FPG/hOGG1 or
any of its downstream repair enzymes or 2) because
non-FPG/hOGG1-repairable DNA damage, such as
O6-alkylguanine, contributes more to the
damaging effect of BCNU than
N7-alkylguanine.
Although FPG and hOGG1 are functional homologues, in the current study, FPG exhibited a stronger protective effect against BCNU toxicity than hOGG1. This finding is consistent with a previous study that shows hOGG1 only partially suppresses the spontaneous mutator phenotype of FPG-deficient E. coli strains (28). A possible explanation comes from a recent study that shows that an increased amount of Ape1 stimulates the activity of hOGG1 through enhancing hOGG1 turnover (14). In this case, solely overexpressing hOGG1 without upregulating Ape1 may not increase this specific DNA repair activity. On the other hand, there is no similar report on FPG. In addition, FPG and hOGG1 may have different substrate specificities, and their interaction with potential regulators or their metabolic pathways in the lung cells may be different.
FPG and OGG1 are multifunctional DNA repair enzymes. In addition to DNA glycosylase/lyase activity, they possess deoxyribose phosphotase (dRPase) activity (10, 29). In an alternative base excision repair pathway initiated by a DNA glycosylase that does not simultaneously function as a DNA lyase (11), an AP endonuclease nicks the DNA backbone, leaving a 5'-deoxyribose phosphate (dRP) group flanking the 3'-side of the nicking site (10, 11, 29). dRPase activity is required to hydrolyze the dRP for the subsequent enzyme activities (11). Thus FPG and OGG1 are capable of participating in different parts of base excision repair. A high level of FPG or OGG1 protein in the transduced A549 cells might not only enhance the release of BCNU-modified bases but may also facilitate DNA repair triggered by other DNA glycosylases.
It is noteworthy that FPG/hOGG1-mediated base excision repair is not the only way of protecting lung cells from BCNU toxicity. Endogenous mechanisms such as GST-mediated detoxification of BCNU- and MGMT-mediated direct reversal of DNA damage may also function in these lung cells (9, 31, 39). In the current study, a high level of FPG/hOGG1 did not reduce the BCNU-induced DNA damage and cytotoxicity to the background levels exhibited by the untreated cells. This suggested that, if combined with other approaches to increase intracellular levels of GST, glutathione, and/or MGMT activity, introducing FPG/hOGG1 may provide an optimal protection from BCNU toxicity to lung cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. David A. Williams for review and valuable suggestions. We also thank Renee L. Tritt for providing technical support.
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FOOTNOTES |
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These studies were supported by National Cancer Institute Grant PO1-CA75426 (W. J. Martin II and M. R. Kelley).
Address for reprint requests and other correspondence: W. J. Martin II, Indiana Univ. School of Medicine, Div. of Pulmonary, Allergy, Critical Care, and Occupational Medicine, 1110 W. Michigan St. LO 401, Indianapolis, IN 46202.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 28, 2001; 10.1152/ajplung.00316.2001
Received 9 August 2001; accepted in final form 19 September 2001.
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