Carcinogenesis

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Carcinogenesis, Vol. 20, No. 2, 279-284, February 1999
© 1999 Oxford University Press

Reduced lung tumorigenesis in human methylguanine DNA—methyltransferase transgenic mice achieved by expression of transgene within the target cell

Lili Liu, Xiusheng Qin and Stanton L. Gerson¹

Division of Hematology and Oncology, Department of Medicine and the University/Ireland Cancer Research Center, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, BRB-3, 10900 Euclid Avenue, Cleveland, OH 44106-4937, USA

	Abstract

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Human methylguanine–DNA methyltransferase (MGMT) transgenic mice expressing high levels of O⁶-alkylguanine-DNA alkyltransferase (AGT) in lung were crossbred to A/J mice that are susceptible to pulmonary adenoma to study the impact of O⁶-methylguanine (O⁶mG)–DNA adduct repair on NNK-induced lung tumorigenesis. Expression of the chimeric human MGMT transgene in lung was identified by northern and western blot analysis, immunohistochemistry assay and enzymatic assay. AGT activity was 17.6 ± 3.2 versus 1.2 ± 0.4 fmol/µg DNA in lung of MGMT transgenic mice compared with non-transgenic mice. Immunohistochemical staining with anti-human AGT antibody showed that human AGT was expressed throughout the lung. However, some epithelial cells of bronchi and alveoli did not stain for human AGT, suggesting that the human MGMT transgene expression was heterogeneous. After 100 mg/kg NNK i.p. injection in MGMT transgenic mice, lung AGT activity remained much higher and levels of lung O⁶mG–DNA adducts in MGMT transgenic mice were lower than those of non-transgenic mice. In the tumorigenesis study, mice received 100 mg/kg NNK at 6 weeks of age and were killed 44 weeks later. Ten of 17 MGMT transgenic mice compared with 16 of 17 non-transgenic mice had lung tumors, P < 0.05. MGMT transgenic mice had lower multiplicity and smaller sized lung tumors than non-transgenic mice. Moreover, a reduction in the frequency of K-ras mutations in lung tumors was found in MGMT transgenic mice (6.7 versus 50% in non-transgenic mice). These results indicate that high levels of AGT expressed in mouse lung reduce lung tissue susceptibility to NNK-induced tumorigenesis due to increased repair capacity for O⁶mG, subsequently, decreased mutational activation of K-ras oncogene. Heterogeneity in the level of AGT expressed in different lung cell populations or other forms of carcinogenic DNA damage caused by NNK may explain the residual incidence of lung tumors in MGMT transgenic mice.

Abbreviations: AGT, alkyltransferase; ECL, enhanced chemiluminescence; hAGT, human AGT; hMGMT, human MGMT; MGMT, O⁶-methylguanine–DNA methyltransferase; MNU, N-methylnitrosourea; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; O⁶mG, O⁶-methylguanine; PBS, phosphate-buffered saline; TBS, Tris-buffered saline.

	Introduction

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Using transgenic mice, it has been possible to access the role of a single gene in malignant transformation induced by chemical carcinogens in a target tissue. Transgenic mice expressing the human MGMT gene which encodes the DNA repair protein, AGT, are a suitable system, because AGT specifically repairs O⁶mG–DNA adducts formed by methylating agents without involving other proteins (1–4). Furthermore, both the mutagenic and carcinogenic consequence of unrepaired O⁶mG have been well characterized (5–8). Persistent unrepaired O⁶mG behaves like adenine during DNA replication, resulting in GCAT transition in the ras gene family. The mutational activation of ras participates in the initiation of tumor development (9,10). Thus, the ability of cellular AGT to remove the O⁶mG lesion is critical to prevent carcinogenesis initiated by methylating agents.

Earlier work from our laboratory showed marked protection of MGMT transgenic mice from N-methylnitrosourea (MNU)-induced thymic lymphoma (11). In this transgenic model, thymus expression of human MGMT was specifically targeted by constructing a chimeric gene containing the ß-actin promoter, human MGMT and the locus control region from the CD2 gene (11). Thymic protection was achieved by uniform MGMT expression and rapid repair of O⁶mG–DNA adducts in thymus (12,13). From this study, we concluded that a single DNA repair gene expressed in the target cell would block MNU carcinogenicity. Based on these results, another strain of transgenic mice with the same MGMT–CD2 gene construction, which were found highly expressing human AGT in lung, were crossbred with A/J mice. The A/J mouse is a sensitive strain to spontaneous and chemically induced lung tumors. Treatment of A/J mice with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) increased the multiplicity and reduced the latency of pulmonary tumor formation (14). Moreover, NNK-induced lung tumors in this strain had high frequency of GCAT transition in the K-ras oncogene at codon 12 (15,16), which appears to be a major contributor to lung tumor susceptibilities in this strain.

NNK is a nitrosamine present in tobacco and tobacco smoke. It has been shown to be a powerful and organ-specific carcinogen (17,18). The potent carcinogenicity of NNK is due to metabolic activation via both methylene hydroxylation to produce a methylating species and methyl hydroxylation to produce a pyridyloxobutylating species (17,18). The hallmark of these active species is their reaction with a number of sites in DNA to form N⁷- and O⁶-methylguanine or pyridyloxobutylated DNA. Organotropism studies of lung carcinogenesis induced by NNK carried out in A/J mice and rats have shown a strong correlation between lung tumor formation and levels of O⁶mG adducts (19–22). Other studies in A/J mice focused on NNK-induced pyridyloxobutylated DNA and found that this bulky DNA adduct inhibits repair of O⁶mG and induces GCAT transitions and GCTA transversions in codon 12 of the K-ras gene (23). Pyridyloxobutylation also appears important in rat lung carcinogenesis induced by NNK (24). Therefore there is a potential role for methyl adducts as well as pyridyloxobutyl adducts in the tumorigenesis of NNK.

The objective of the present study was to generate transgenic mice expressing human MGMT in lung. We determined whether repair of NNK-formed O⁶mG–DNA adducts by increased AGT in lung would prevent mutations in the K-ras oncogene and block the tumorigenicity of NNK.

	Materials and methods

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Chemicals
NNK was purchased from Chemsyn Science Laboratories (Lenexa, KS). Mouse anti-human alkyltransferase monoclonal antibody mT3.1 was provided kindly by Drs T.Brent (St Judes Children Hospital, Memphis, TN) and D.Bigner (Duke University, Durham, NC).

Transgenic mice
Mice harboring the human MGMT (hMGMT) cDNA were generated as previously described (11). Briefly, a chimeric gene was constructed consisting of the ß-actin promoter, hMGMT cDNA, the poly-A region from bovine growth hormone and the CD2 locus control region (11). Two founder mice derived from C57BL/6xSJL mice (B6SJL) expressed high levels of hMGMT in multiple organs of which one was used to study MNU-induced lymphomas (11) and the other, reported here, was used to study azoxymethane-induced aberrant crypt foci in mouse colon (28). This founder expresses hMGMT in lung parenchyma. In present experiments, B6SJL hMGMT^+/– mice were bred to A/J mice to generate hMGMT⁺ and hMGMT^– F1 B6A/J mice.

Alkyltransferase assay
AGT activity was measured by the amount of [³H]methyl group removed from O⁶ [³H]mG present in calf thymus DNA alkylated with N-[³H]methyl-N-nitrosourea. The alkylated [³H-methyl]O⁶mG and N⁷mG bases were separated by HPLC and quantified by liquid scintillation. AGT activity was expressed as fmol O⁶mG removed/µg DNA (25).

DNA isolation and adduct determination
Briefly, DNA was isolated from lung by digestion with proteinase K and RNase, two extractions in chloroform/isoamyl alcohol and 2-ethoxyethanol precipitation. Quantitation of DNA adducts was performed with a Rainin HPLC equipped with a double SCX-5 ion exchange column coupled to a Hitachi F-1000 fluorescent detector set to excitation of 290 nm and an emission of 360 nm, using Dynamax chromatogram analysis software from Rainin Instruments (Bedford, MA). O⁶mG and N⁷mG and guanine were separated by isocratic elution in 100 mM NH₄PO₄, pH 2.5, 10% methanol at 1.0 ml/min (13).

Northern analysis
Total cellular RNA was prepared from various tissues, separated by formaldehyde–agarose gel electrophoresis, and subjected to northern blot analysis with a ³²P-labeled MGMT cDNA probe.

Western blotting
Cell extracts were resolved by SDS–PAGE gels (12% acrylamide) and the gels were performed in a Bio-Rad (Hercules, CA) minigel apparatus at 150 V for 1 h. Proteins were transferred onto PVDF membranes, using a Bio-Rad mini Trans-Blot cell for 1 h at 100 V. The blotted membranes were blocked with 5% dry milk in Tris-buffered saline (TBS) buffer and then probed for 2 h with a mouse monoclonal antibody, which is specific for human cellular MGMT. After three 5 min washes with TBS–TW20, the blots were incubated with secondary antibody, anti-mouse HRPO-anti IgG for 1h. Antibody binding was visualized by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (26).

Alkyltransferase immunohistochemistry
Tissues were fixed in Carnoy (60% ethanol, 30% chloroform and 10% acetone) for 90 min and then transferred to 70% ethanol. Paraffin-embedded sections were brought to water, and endogenous peroxidase activity was blocked by 0.3% H₂O₂ in methanol. Sections were treated with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min and incubated with the mouse monoclonal anti-human AGT antibody overnight at 4°C. Following PBS washes, sections were incubated with peroxidase-labeled goat anti-mouse antibodies for 1 h. The peroxidase reaction was developed with diaminobenzidine for 10 min. Omission of the primary antibody was performed as a control (27).

PCR–RFLP for K-ras
An aliquot of 10 µl of DNA extracted from lung tumor was used as substrate for the first PCR amplification of the first exon of the K-ras oncogene (codons 4–40) using the following primers: P#1: 5'-AACTTGTGGTGGTTGGACCTG-3' (the underlined C indicates the site of a point mutation in the primer); P#2: 5'-AGCGTTACCTCTATCGTAGG-3'. The PCR product of 106 bp resulted in creation of a BstN1 restriction site (BstN-1; CCTGG) by primer #1 in the wild-type sequence but not in the sequence with a mutation in the second position of codon 12. The amplification product was subjected to digestion by BstN-1, which cuts the wild-type K-ras sequence to 86 and 20 bp fragments and leaves the mutated sequence intact. The BstN-1 digested product was diluted (1:100) and 10 µl was used for second PCR amplification by the primers, P#3: 5'-AACTTGTGGTGGTTGGAGGTG-3'; P#4: 5'-CTCATCCACAAAGTGATTCT-3'. The primer #3 created an Hph-1 restriction site (GGTGA) in amplicons with a GGTGAT mutation at codon 12, whereas the wild-type sequence and other mutations at this site do not restrict. Fragments were separated by a 4% agarose gel (28). The wild-type sequences remain intact whereas the K-ras mutation with GAT at codon 12 was cut into 50 and 30 base fragments.

Statistical analysis
The unpaired Student's t-test and ² test were used.

	Results

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Expression of human MGMT in lung of transgenic mice
hMGMT transgenic mice bred to A/J mice to generate B6A/J F1 mice expressed 14-fold higher levels of lung AGT than that of non-transgenic mice (Figure 1A). mRNA transcripts of the chimeric human MGMT gene were identified in lung of the B6A/J hybrids (Figure 1B). Human AGT protein was detectable by western blot using a mouse monoclonal antibody mT3.1, which specifically recognizes the human AGT. As shown in Figure 1C, human cellular AGT was not detected in lung of non-transgenic littermates.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Identification of human MGMT gene expression in lung of transgenic mice. (A) AGT activity in lung of MGMT transgenic mice versus non-transgenic mice. (B) Northern blot analysis of the chimeric human MGMT gene in transgenic mice. 1, thymus; 2, spleen; 3, lung. (C) Western blot assay for human AGT protein. 1, positive control (extraction of K562 cells transfected with human MGMT cDNA); 2, lung of transgenic mice; 3, lung of non-transgenic mice.

 
Human AGT expression in lung was also determined by immunohistochemistry. The AGT staining (which showed a brown color) was observed throughout the lung (Figure 2A). Transgenic human AGT was predominantly expressed either in the nuclei or cytoplasm of cells in the mucosal lining of the bronchiolus and alveolar epithelium (Figure 2B). However, it was notable that at least 10% cells of bronchiolus and 20% alveolar cells did not have detectable staining for MGMT antibody. Non-transgenic lung showed no staining for MGMT (Figure 2C and D).

View larger version (150K): [in this window] [in a new window]   Fig. 2. Immunohistochemical staining for human AGT expression in lung of MGMT transgenic and non-transgenic mice. (A, B) Human AGT expressed in lung cells. (C, D) AGT staining in lung of non-transgenic mice.

 
Formation of O⁶-methylguanine–DNA adducts and depletion of lung AGT activity following NNK
NNK at 100 mg/kg was used to evaluate the level of O⁶mG–DNA adducts formed and the degree to which these adducts depleted AGT. Repair of O⁶mG adducts utilizes AGT such that residual AGT is a measurement of the remaining capacity for DNA repair. After NNK, levels of O⁶mG–DNA adducts were detected in lung of transgenic mice that were one-third of that in non-transgenic lung. However, detectable O⁶mG adducts persisted for >72 h (Figure 3A). As expected, levels of N⁷mG were similar, suggesting identical bioavailability of the metabolically activated NNK in the two groups of mice (Figure 3B). AGT activity in human MGMT-transgenic lung was 17.6 ± 3.2 fmol/µg DNA compared with 1.2 ± 0.4 fmol/µg DNA in non-transgenic littermates. At 6 h after NNK injection, AGT decreased to 15.1 fmol/µg DNA in MGMT transgenic lung compared with a drop to 0.2 fmol/µg DNA (limit of detection is 0.1 fmol/µg DNA) in non-transgenic lung. Residual AGT activity in non-transgenic mice remained close to the limit of detection over the next 72 h (Figure 4). These results indicate that there were sufficient O⁶mG adducts formed after a carcinogenic dose (100 mg/kg) of NNK to inactivate lung AGT activity in non-transgenic mice but not in transgenic mice. Because in hMGMT⁺ mice, there were substantial residual O⁶mG adducts seen in lung tissue with adequate hAGT, this suggests that there was heterogeneity in expression of human MGMT and thus in O⁶mG repair throughout the lung, as noted by immunohistochemistry analysis.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Levels of methylating DNA adducts in lung at intervals after injection of mice with 100 mg/kg NNK. (A) Persistence of O6mG–DNA adduct in lung after NNK treatment. (B) Formation of N7mG–DNA adduct in lung after NNK treatment.

 

View larger version (47K): [in this window] [in a new window]   Fig. 4. Depletion of AGT activity in lung of MGMT transgenic mice versus non-transgenic mice after NNK (100 mg/kg) treatment.

 
Lung tumorigenesis by NNK
Mice were treated with NNK at 6 weeks of age and killed 44 weeks later. The results of lung tumor induction are summarized in Table I. After a single 100 mg/kg dose of NNK, 16 of 17 mice had lung tumors in the non-transgenic mice cohort compared with 10 of 17 mice in the hMGMT transgenic cohort (P < 0.05). A single dose of NNK induced a high rate of tumor-bearing mice but a low tumor multiplicity (assayed by number of tumors per whole lung). Fewer tumors were observed in the hMGMT transgenic group compared with non-transgenics. Moreover, in non-transgenic group, the number of lung tumors sized >2 mm was more than that observed in the hMGMT transgenic group. Therefore, it appears that protection of the hMGMT transgene from lung tumor induced by NNK can be presented by the lesser number of small sized tumors in MGMT transgenic mice.

View this table: [in this window] [in a new window]   Table I. Lung tumors induced by NNK in MGMT transgenic and non-transgenic mice

 
Identification of activating mutations in K-ras
NNK-induced lung tumors from transgenic and non-transgenic mice were analyzed for K-ras mutation at codon 12 by PCR–RFLP (Figure 5A). As shown in Figure 5B, a significantly higher rate of activating GCAT transition in codon 12 of the K-ras gene was observed in eight of 16 tumors from non-transgenic mice compared with one of 17 tumors from the transgenic group (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Frequency of K-ras mutation in lung tumors arose from NNK-treated MGMT transgenic mice versus non-transgenic mice. (A) K-ras mutation at codon 12 by PCR–RFLP. (B) Comparison of frequency of K-ras mutation between lung tumors induced by NNK in MGMT transgenic and non-transgenic mice.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
This study provides direct linkage between NNK-induced formation and repair of O⁶mG–DNA adducts and lung carcinogenesis using human MGMT transgenic mice. We found that overexpression of the human MGMT gene in transgenic mouse lung reduced the incidence of NNK-induced lung tumors. In addition, the frequency of K-ras oncogene activation due to GCAT transitions at codon 12 was significantly reduced in the tumors that arose from NNK-treated MGMT transgenic mice.

Numerous previous studies have linked O⁶mG–DNA adducts to NNK carcinogenesis in the mouse and rat (29,30). Intracellular metabolic activation of NNK by P450 enzymes in particular cell types leads to differential levels of DNA adduct formation (29,31). Thus, lung cell types thought to be targets for NNK include those with high potential for bioactivation and those with high levels of persistent O⁶mG–DNA adducts. The studies of NNK in rat lung demonstrated that the highest levels of methyl adducts were detected in Clara cells due to P450 enzyme activity and/or lack of AGT (29,32) compared with that in other alveolar cells. A strong correlation was obtained between the level of O⁶mG in Clara cells and the incidence of lung tumors in NNK-exposed rats (29). Therefore, Clara cells have been identified as the target cell for the formation of O⁶mG–DNA adduct and a contributor to the carcinogenesis of NNK in the rat (29). However, in the mouse, both alveolar type II and Clara cells have the highest levels of persistent O⁶mG–DNA adducts after NNK treatment, whereas the alveolar type II cell is thought to be the origin of mouse lung adenomas (30). These previous data have provided indirect evidence linking carcinogenic risk with metabolic activation and DNA adduct persistence but have not been able to show the specific nature of this by altering the level of persistent adducts in the cells at risk for tumorigenesis.

The MGMT transgene we studied was expressed throughout the lung, resulting in high levels of AGT activity. Immunohistochemical studies reveal AGT expression in the nuclei of mucosal cells of the bronchi, alveolar epithelium, as well as in vascular epithelium. However, AGT expression was not uniform. The explanation for this heterogeneity is presumably due to variable degrees of the hMGMT transgene expression, perhaps in a cell-specific fashion. This appears to explain why despite AGT activity that was 14-fold higher in the hMGMT mouse lung, the levels of O⁶mG persistent in the transgenic lung were low, but still detectable in 30% of the non-transgenic lung, despite high levels of AGT in most lung cells of the transgenic mice. Another aspect of this discrepancy may be the heterogenous formation of O⁶mG in different cell types due to metabolic activation of NNK. As a result, even though we saw a protective effect of hMGMT on NNK-induced lung tumors, variable levels of AGT activity in some cells may have led to NNK-tumorigenicity in the MGMT transgenic mice. This could explain why NNK-induced lung adenomas were reduced but not eliminated in the MGMT transgenic mice.

Since we have previously noted a very homogenous pattern of expression of MGMT in the thymus of transgenic mice and the virtual complete elimination of MNU-induced lymphomagenesis along with rapid removal of O⁶mG–DNA adducts, we conclude that when the MGMT transgene is expressed uniformly and O⁶mG–DNA adducts are formed to a similar extent in all cells of the target tissue, the MGMT transgene is able to confer significant protection from MNU-induced carcinogenesis (11–13). It is clear that in the lung, both the pattern of MGMT transgene expression and the pattern of NNK activation and O⁶mG formation are much more complex. Nonetheless, our data clearly show the importance of MGMT in protecting the lung from NNK-induced carcinogenesis.

Investigators have recently provided evidence of the importance of the NNK-pyridyloxobutylation pathway in lung carcinogenesis (22,23,33,34). The O⁶ of guanine is one of the potential sites of DNA alkylation by the pyridyloxobutyl group, and this adduct can compete with O⁶mG for repair by AGT. Thus, either this adduct directly or through depletion of AGT can result in direct mutagenic damage or increase mutagenesis due to increased persistence of O⁶mG–DNA adducts. In either case, this second alkylation product of NNK can contribute to carcinogenesis, whereas its potency should be reduced by MGMT transgene expression. The exact link between NNK-induced by these two O⁶ adducts and their repair by human AGT in MGMT transgenic mice remains to be determined. It is likely that repair of both contributed to the decreased incidence of tumor formation by NNK.

The importance of K-ras mutation in carcinogenesis has been well documented in tumors induced by methylating agents (35). The studies on K-ras gene from NNK-induced lung tumors provide evidence that this oncogene is a potential target for activation by NNK (36). It has been shown that virtually all K-ras mutations detected from NNK-induced lung tumors in A/J mice have a GCAT transition mutation in the second base of codon 12 (16). The predominance of this mutation is consistent with the formation and persistence of O⁶mG–DNA adducts at this position in the target cell after NNK treatment. Since the K-ras gene has been shown to have a susceptible allele in A/J mouse (37), the mutation in this gene appears to particularly sensitize the mouse to NNK (38). The markedly reduced frequency of K-ras mutations in MGMT transgenic mice suggests that efficient repair of O⁶mG reduces the contribution of the K-ras pathway to NNK tumorigenesis. Moreover, these results implicated strongly that other types of DNA alkylating damage (24) may be responsible for mutagenesis/carcinogenesis in MGMT transgenic mice. However, identification of a second oncogene involved in this malignant transformation is no easy task. For instance, it is of interest that p53 has rarely been found to be the target for methylating agents in carcinogenesis in the mouse nor have other ras genes been linked in the mouse lung model (16).

In summary, these data indicate that expression of the human MGMT transgene in the mouse lung decreases susceptibility to NNK tumorigenesis and markedly reduces the frequency of K-ras activation in developing tumors. Heterogeneity of DNA alkylation and DNA repair results in an imbalance favoring persistence of DNA adducts in some cells and may explain the residual tumorigenesis occurring in the lung of mice expressing high levels of the MGMT transgene. These studies further provide evidence of protection achieved by expression of DNA repair transgene within the target cell.

	Acknowledgments

 
This work was supported by Public Health Service grants RO1CA73062, RO1ES06288, UO1CA75525 and P30CA43703 from the USPHS.

	Notes

 
¹ To whom correspondence should be addressed Email: slg5{at}po.cwru.edu

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Received June 29, 1998; revised September 16, 1998; accepted October 2, 1998.

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