Induction of microsatellite mutations by oxidative agents in human lung cancer cell lines

Shanbeh Zienolddiny2, David Ryberg and Aage Haugen1

Department of Toxicology, National Institute of Occupational Health, PO Box 8149 Dep., N-0033 Oslo, Norway


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genomic instability has been associated with cancer development. Oxidative DNA damage seems to contribute to genetic instability observed in cancer. We have used human lung cancer cell lines carrying a plasmid vector containing a (CA)13 microsatellite sequence to study frameshift mutations mediated by ROS-generating chemicals paraquat and hydrogen peroxide. Exposure of the cells to both paraquat and hydrogen peroxide resulted in significantly higher mutation frequencies compared with untreated control cells. Mutation frequencies up to 27-fold higher than the spontaneous mutation frequencies were obtained. The majority of the reversion mutants contained frameshift mutations within the target sequence. However, the pattern of deletions and additions was significantly different in the two cell lines. These results indicate that oxidative damage may play a role in instability of microsatellite sequences in vivo.

Abbreviations: CFE, colony forming efficiency; H2O2, hydrogen peroxide; Hyg, hygromycin; Hyg B, hygromycin phosphotransferase B; MSI, microsatellite instability; neo, neomycin; PBS, phosphate-buffered saline; ROS, reactive oxygen species.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oxidative damage has been implicated in human carcinogenesis (1). Many of the biological effects of oxidative damage is mediated by the highly reactive oxygen species (ROS) (2). A large amount of ROS is generated by endogenous metabolism, indirectly by inflammatory processes and by environmental agents (3). In vitro exposure of normal cells to chemicals producing ROS has been shown to result in genomic alterations similar to those found in common cancers (4,5). ROS can induce DNA base modifications, strand breaks and sister chromatid exchanges which may contribute to genomic instability (6). The high levels of oxidative DNA damage observed in smokers indicate that oxygen radicals are persistently produced due to cigarette smoking (7,8). Several compounds in tobacco smoke may enhance lung carcinogenesis by the radical-mediated reactions and, indirectly, via the action of inflammatory cells and induction of inflammatory processes.

Instability of microsatellite sequences (MSI) is most profound in tumors with deficiencies in DNA mismatch repair genes (9). However, inflammatory conditions may also lead to MSI (10,11). Furthermore, some tumor cell lines produce a high level of ROS, which may contribute to the genetic instability of these cells (12). Studies have also demonstrated an interplay between environmental carcinogens and genomic instability using minisatellites as markers (13,14). The study reported here was designed to investigate possible induction of microsatellite mutations by ROS in vitro in human lung cancer cell lines. The cell lines were transfected with a plasmid vector carrying a hygromycin gene (hyg) disrupted by a (CA)13 microsatellite sequence (15). Mutations within this sequence can restore the reading frame of the hyg gene giving rise to hygr colonies. Hydrogen peroxide and paraquat were selected as ROS-generating chemicals.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Biological reagents
Culture media and chemicals were purchased from Sigma or Gibco BRL unless otherwise stated. Hydrogen peroxide (30% stock) was diluted with ddH2O to working solution just before experiments. Paraquat (methyl viologen) was dissolved in phosphate-buffered saline (PBS) to 1 M solution, filtered through a 0.2 µM filter and stored at –20°C.

Cell lines
Human lung cancer cell lines HCC-15 and NCI-H2009 were donated by Dr Adi F.Gazdar (16) and were maintained in HITES medium with 5% fetal bovine serum.

Plasmid vectors
The mutagenesis system is based on the plasmid vector pMV7pIHyg13 as described previously (15). In pMV7pIHyg13 the hyg gene has been rendered out of frame by insertion of a (CA)13 microsatellite sequence immediately following the ATG start codon. Thus, pMV7pIHyg13-transfected cells are neomycin resistant (neor) but hygromycin sensitive (hygs). Certain spontaneous or chemically induced frameshift mutations can restore the reading frame of the hyg gene, and hygr revertants are selected in the presence of hygromycin phosphotransferase B (hyg B). Changes in the length of the microsatellite are detected by PCR amplification of the DNA fragment that includes the microsatellite sequence. The pMV7pIHyg12 vector is identical to pMV7pIHyg13 except that the microsatellite is a (CA)12 and PMV7pIHyg12-transfected cells become neor and hygromycin resistant (neor-hygr). This vector is used as positive control for hygr and external DNA size standard. Both plasmids become integrated into the genome.

Transfection of recipient cell lines and isolation of neor clones
Transfections were performed as described previously using infectious, replication-deficient viral stocks of the plasmids (15). Briefly, 5x105 cells were incubated with pMV7pIHyg12 or pMV7pIHyg13 in presence of 8 µg/ml polybrene. Forty-eight hours later, 1x105 cells were seeded into 15 cm dishes containing the appropriate amounts of the neomycin analogue G418. After 1–2 weeks selection with G418, neor colonies were trypsinized and single-cell clones were established in 24-well dishes containing medium with 0.1 mg/ml G418. Cells were then trypsinized and transferred to 9 cm dishes and grown until sub-confluent.

PCR analysis of chromosomal integration of vectors
Genomic DNA from each neor clone was isolated by Qiagen Blood and Cell Culture kit as instructed by the manufacturer. PCR reactions containing 100 ng DNA, 2.75 mM MgCl2, 200 µM dNTP, 2.5 U DynaZyme polymerase, 10 pmol each of primers Hyg13 and HygRev in 1x PCR reaction buffer supplied by the manufacturer. Cycling parameters were 5 min at 95°C and 30 cycles of 40 s at 94°C, 40 s at 58°C, 40 s at 72°C and a final incuba- tion of 5min at 72°C. The sequences of primers used were Hyg13, 5'-ACTAGTTGAGATATGCACAC-3' and HygRev, 5'-AAAGCTTCTATT- CCTTTGCC-3' for amplification of the entire fragment of 1087 bp containing the hyg gene. One neor clone from each cell line was chosen for experiments with paraquat and hydrogen peroxide (H2O2).

Paraquat and H2O2 survival assay
Cells (103) were seeded on 6 cm dishes in triplicate. One day later the medium was removed and cells were incubated with paraquat (0, 0.1, 0.2, 0.4, 0.8, 1.0 and 2 mM) for 2 and 24 h, or H2O2 (0, 1, 5, 10, 20, 40 and 100 µM) for 30 min and 24 h. After the end of the incubation periods, the medium was removed, cells were washed twice with PBS and grown in regular medium until macroscopic colonies appeared (10 days). Colonies were fixed and stained in a solution containing 4% formalin and 0.25% crystal violet, and counted. Surviving fractions were determined by relating the number of colonies (>20 cells) in treated dishes to that of the control dishes.

Selection of hygr colonies and determination of mutation frequencies
Cells (1.5x106) were seeded in a series of 9 cm dishes. The next day, cells were washed with PBS and incubated with regular medium (control) or paraquat (0.1, 0.4 and 2 mM) for 2 h, and 0.1 mM paraquat for 24 h. Cells were incubated with 20 µM H2O2 for 2 h. The medium was then removed, the cells were trypsinized and counted. To select for hygr colonies four to eight subcultures (3x105 cells/9 cm dish) were established from each of the control or treated dishes. In order to allow cells to recover and have sufficient time to express hygr phenotype, hyg B (0.2 mg/ml) was added 48 h after subculturing. The cells were grown in hyg B-containing medium for 3–4 weeks. Using cloning cylinders, one or two hygr colonies were isolated from each dish and grown in six-well plates in medium containing hyg B. Remaining colonies were fixed and stained with a solution of 4% formalin and 0.25% crystal violet and counted. In addition, four dishes (103 cells/dish) were seeded for determination of generation numbers and colony forming efficiencies (CFE). The cells in two of these dishes were trypsinized and counted at day 16 and the number of generations was calculated according to the formula N = 3.32 (lnNH lnNI), where NI is the number of cells seeded and NH is the number of cells harvested (17). Colonies in the two other dishes were stained and counted, and CFEs were determined. Mutation frequencies (mutations/cell/generation) were calculated as follows: the number of hygr colonies from each subculture was divided by the number of cells seeded, corrected for mean CFE and then divided by the mean number of generations. Each experiment was performed twice with independent cell populations. Accumulated data from both experiments were used to compare mutation frequencies of control and treated cells using the non-parametric Wilcoxon signed ranks test.

Analysis of (CA)13 length alterations
Genomic DNA from parental neor clones and their hygr derivative colonies was used to PCR amplify the fragment containing integrated (CA)n repeat sequence using primers Hyg1, 5'-CTGCATCAGGTCGGAGACGC-3' and fluorescein-labeled MV7RI5', 5'F-GCGCGTCTTGTCTGCGGAAT-3'. DNA (100 ng) was used in PCR reactions containing 2.0 mM MgCl2, 100 µM dNTP, 0.02% DMSO, 1 U DynaZyme polymerase and 3 pmol each of the primers Hyg1 and MV7RI5' in 1x PCR reaction buffer supplied by the manufacturer. Cycling parameters were 4 min at 95°C and 30 cycles of 30 s at 94°C, 30 s at 58°C, 30 s at 72°C, and a final incubation of 10 min at 72°C. PCR products were analyzed by high resolution capillary electrophoresis (ABI-310 machine) using TAMRA 500 as internal standard. This system had a resolution of better than one base. A 159 bp PCR fragment was detected in genomic DNA isolated from neor-hygr NCI-H2009 cells transfected with pMV7pIHyg12 vector. A 161 bp fragment was detected in neor clones derived from the same cell line but transfected with pMV7pIHyg13 vector. Since pMV7pIHyg12 differs in one CA repeat unit from pMV7Hyg13, we were able to detect alteration of one CA repeat by this method. Therefore, DNA isolated from pMV7pIHyg12-transfected NCI-H2009 cells and the parental neor cells were used as external standards in all PCR reactions.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression vector pMV7pIhyg13 contains a neo gene to select for transfected cells and the reporter out-of-frame hyg gene (Figure 1Go). Human lung cancer cell lines HCC-15 and NCI-H2009 were transfected with a viral stock of pMV7pIHyg13 vector. Independent single-cell neor clones from each cell line were established. The presence of the full-length DNA fragment containing the hyg gene was confirmed by PCR amplification (data not shown). One neor clone from each cell line was chosen for mutagenesis studies with paraquat and H2O2. A neor clone from NCI-H2009 transfected with the pMV7pIHyg12 vector was also established. The cells from this clone were hygr and DNA isolated from these cells was used as a control template in parallel PCR reactions.



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Fig. 1. Schematic representation of the pMV7pIHyg13 vector. A (CA)13 repeat tract has been inserted immediately downstream of the ATG start codon in the hyg gene. The neor gene constitutively driven by the herpes virus thymidine kinase promoter (tkneo) provides a selectable marker.

 
The cell lines showed different survival after treatment with paraquat or H2O2 with NCI-H2009 being more sensitive than HCC-15 (Figure 2Go). Both cell lines treated with H2O2 for 24 h failed to form colonies.



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Fig. 2. Cytotoxicity of paraquat and H2O2 in human lung tumor cell lines. (A) Cells were treated with various concentrations of paraquat for 2 h (closed symbols) or 24 h (open symbols). After 10 days in regular medium colonies >20 cells were counted. Surviving fractions were determined by dividing the number of colonies obtained from treated dishes to the number of colonies in control dishes. (B) Cells were treated with H2O2 for 30 min and surviving fractions were determined as in (A). Shown are mean values from three determinations. Error bars represent standard deviations.

 
The mutagenicity experiments were performed with various concentrations and incubation times. Treatment of the cell lines with paraquat or H2O2 resulted in mutation frequencies significantly higher than the mutation frequencies of the control cells (Tables I and IIGoGo). The mutation frequencies in HCC-15 and NCI-H2009 treated with paraquat were 2–27.3- and 2.4–3.6-fold higher, respectively, than the corresponding control cells (Tables I and IIGoGo). Initially, the mutagenicity of hydrogen peroxide was tested at 20 and 40 µM for 30 min but no changes in the mutation frequencies were observed. However, incubation with 20µM for 2h increased mutation frequencies 2.3-fold in HCC-15 (Table IGo) and 1.7-fold in NCI-H2009 (Table IIGo) as compared with the respective control cells. Concentrations >20 µM and longer incubation times inhibited cell survival. The cloning efficiencies of the treated cells obtained in mutagenicity experiments were different from the initial survival assays. This may be due to difference in the cell density at which cells were treated with the chemicals.


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Table I. Effects of paraquat and H2O2 on HCC-15 cells
 

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Table II. Effects of paraquat and H2O2 on NCI-H2009 cells
 
Changes in the length of the 161 bp fragment including the target (CA)13 sequence were determined. Examples of the PCR products are shown in Figure 3Go. A sample of 98 hygr colonies isolated from control and treated dishes was analyzed. In the HCC-15 cell line, 18/18 (100%) colonies from control dishes and 25/31(81%) colonies from treated dishes had changes within the target sequence (Table IIIGo). The majority of colonies derived from control (14/15, 93%) or treated NCI-H2009 cells (29/34, 85%) also had changes within the target sequence (Table IIIGo). The distribution of frameshift mutations was different in the two cell lines. Both deletions and insertions were common in HCC-15, whereas 88% deletions and only 12% insertions were observed in NCI-H2009 (Figure 4Go). This difference between the two cell lines in mutation types was statistically significant (P = 0.006, Fisher's exact test). Deletion of two bases was common (25/43, 58%) in HCC-15, whereas eight-base deletion was most frequent (37/43, 86%) in NCI-H2009 cells (Table IIIGo).



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Fig. 3. PCR amplification of the DNA fragments of the transduced hyg gene containing (CA)n repeat from the genomic DNA of human lung tumor cell lines. (A) PCR products using DNA from NCI-H2009 cells transduced with pMV7pIHyg12 (solid line) or pMV7pIHyg13 (dotted line) as template. (B) Examples of insertion (+4 bp) and deletions (–2, –8 bp) mutations obtained from PCR reactions using genomic DNA isolated from some of the hygr colonies.

 

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Table III. Changes in the lengths of PCR products in hygr revertants as compared with parental cells
 


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Fig. 4 Distribution of frameshift mutations in NCI-H2009 and HCC-15.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oxidative damage could be an important factor in enhancing the mutation rate leading to cancer. There is evidence that large amounts of oxygen radicals are persistently produced due to cigarette smoking (7). Normal cellular metabolism also generates a variety of the radical species capable of causing oxidative damage to DNA (18,19). Possible overloading in the lung is associated with inflammatory responses, fibrosis and tumor development. ROS may create a transient hypermutable state which may lead to genomic instability during the process of carcinogenesis. For the purpose of studying whether oxidative damage is associated with MSI, lung tumor cell lines were treated with paraquat and H2O2 as sources of oxidative stress. The results indicate that oxygen-induced DNA damage can affect the stability of microsatellite sequences.

It is assumed that mutations induced by oxidative damage contribute significantly to genetic instability in tumors (1). ROS induces DNA base alterations, abasic sites and strand breaks that may contribute to the instability of repetitive sequences. However, the mechanism(s) by which oxygen radicals induce MSI are not fully understood. Altered bases may mispair or cause pausing of DNA polymerase at the damaged site resulting in strand displacement and misalignment. The fidelity of DNA polymerases may also be altered by free radicals (20). ROS-generated free DNA ends are highly recombinogenic and have been shown to increase instability of minisatellite sequences in yeast (21). Moreover, studies have shown that ROS may have suppressive effect on DNA repair enzymes (22). There is also some evidence that repetitive sequences may adopt a non-B DNA structure which may make them less efficient substrates for DNA repair enzymes (23). ROS may also alter activity of some transcription and growth regulatory factors such as NF-{kappa}B and protein kinases, respectively (24).

Various vector systems have been used to determine mutation frequency of microsatellite sequences in cultured cells (2527). Different spontaneous mutation frequencies were obtained in the neor clones from HCC-15 (~0.4x10–5) and NCI-H2009 (~2.0x10–4). However, since only one-third of frameshift mutations result in hygr the actual mutation rate in each cell line is probably higher. Mutation frequency for the neor clones may depend upon the context in the genome within which the vector has been integrated. Chromosomally integrated transgenes are known to be subject to chromatin structure and/or DNA methylation changes (28). This may result in differential expression of the hyg gene depending on the site of integration and chromosomal environment. In addition, other factors (see below) may also contribute to the observed variation in the mutation frequencies.

The mutagenicity experiments were performed with concentrations yielding various cellular toxicity. Treatment with paraquat increased the mutation frequencies in a dose- and time-dependent manner. For both cell lines, the level of induction was inversely correlated with the degree of cellular toxicity. In HCC-15, the most toxic and growth inhibitory dose was 0.1 mM for 24 h which gave the highest increase (27-fold) in mutation frequency. The induction of mutation frequencies by H2O2 was significant in both cell lines. The 2.3-fold induction obtained in our experiments is similar to the reported mutagenicity for H2O2 in other mammalian cells (2931).

The cell lines showed different cellular toxicity and mutagenicity to H2O2 and paraquat. Variation in the cellular sensitivity of mammalian cells to oxidizing agents has been shown to depend upon extent of free radical generation, inherent DNA repair rate and fidelity, antioxidative enzyme activities and cell growth (29). Type of oxidizing agent, concentration and exposure time may also affect the cellular toxicity; high concentrations or long incubation periods will saturate DNA repair and detoxifying activities leading to higher cytotoxicity and mutagenicity (29,30). Paraquat was more effective in inducing mutation frequencies than H2O2. The paraquat concentrations used were several-fold higher than H2O2. Paraquat is a redox cycling chemical that produces superoxide anions which can damage DNA directly or may undergo spontaneous or dismutase catalyzed dismutation to form H2O2 (32). H2O2, via Fenton-type reactions, generates hydroxyl radicals which may cause DNA damage (2). Addition of transition metals to the medium such as iron may further increase the effects of oxidative damage caused by H2O2. Some of the mutagenic effects observed in the treated cells may be due to inhibitory effects of ROS on cellular proteins such as DNA repair enzymes and DNA polymerases.

Frameshift mutations in microsatellite sequences commonly result from additions and deletions of intact repeat units (33). The length changes in the 161 bp fragment in the majority of hygr colonies were alterations of two bases or a multiple of two bases indicating gain or loss of intact repeat units. Because the hyg gene is in –1 reading frame, di-nucleotide mutations that may restore hygr phenotype include deletions of two, eight, 14, 20 or 26 bases and insertions such as four and 10 bases. Eighty-one of 86 insertions and deletions found in both cell lines will restore the reading frame of the hygr phenotype. In the case of the five colonies with two base insertions, additional mutations might have occurred in order to establish hygr phenotypes in these colonies. The observation that 11 of the 12 colonies without length changes and four of five colonies with two base insertions were recovered from H2O2 or paraquat treated dishes indicates that the entire hyg gene may be a target of mutagenesis by ROS.

There was a significant difference between deletion and insertion mutations in the two cell lines. In NCI-H2009, the frameshift mutations were dominated by deletions (mainly loss of eight bases) in comparison with HCC-15 where the majority of changes were two base deletions and only a small fraction (2.3%) of colonies had deletions of eight bases. Several factors may affect the pattern of mutations. The site of integration in the genome is an important factor. The stability of the human minisatellite CEB1 in yeast has been correlated with the chromosomal region at which it is inserted (34). The context of the neighboring sequences has also been shown to affect mutation patterns in mammalian cells (31). Microsatellite sequences may form DNA loops in vivo which are processed by the complexes of DNA mismatch repair proteins (35). The cellular extracts prepared from both cell lines were shown to be proficient in binding to the various DNA mismatches (36). Characterization of the exact integration site of the target microsatellite and surrounding sequences may add new knowledge in understanding the reported `position effect' phenomenon (34).

Studies indicate that small insertions and deletions within microsatellite sequences may be predominant in mammalian cells (25,26). The type of mutations observed in the hygr colonies from the cell lines indicate that insertion and deletion mutations involving the smallest number of repeat units are also common in this target sequence. If mutations were evenly distributed we should have observed deletions of up to 26 bases and insertions larger than 10 bases. This observation is consistent with results from other studies using similar vector systems (25,27).

In summary, ROS have been implicated in a wide range of disorders, including carcinogenesis. Here we have presented evidence that oxidative stress may be an important factor contributing to genetic instability and thereby promote a higher propensity for tumor progression.


    Notes
 
1 To whom correspondence should be addressed Email: age.haugen{at}stami.no Back

2 Present address: Unit of Carcinogen Identification and Evaluation, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, France Back


    Acknowledgments
 
We acknowledge the helpful technical assistance of Tove Andreassen. We thank Dr Adi F.Gazdar for providing the cell lines. This work was supported by the Norwegian Research Council, the Norwegian Cancer Society and EU grant ENV4-CT97-0469.


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 Introduction
 Materials and methods
 Results
 Discussion
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Received February 15, 2000; revised February 15, 2000; accepted April 12, 2000.