Transgenic expression of human MGMT blocks the hypersensitivity of PMS2-deficient mice to low dose MNU thymic lymphomagenesis
Xiusheng Qin,
Hang Zhou,
Lili Liu and
Stanton L. Gerson1
Division of Hematology/Oncology and the Ireland Cancer Center, Case Western Reserve University, Cleveland, OH 44106-4937, USA
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Abstract
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Mice deficient in the DNA mismatch repair (MMR) gene, PMS2, develop spontaneous thymic lymphomas and sarcomas. We have previously shown that PMS2/ mice were hypersensitive to a single i.p. injection of 50 mg/kg of N-methyl-N-nitrosourea (MNU) for thymic lymphoma induction. We postulated that MNU sensitivity was due to formation of O6-methylguanine (O6-mG), which, if unrepaired by O6-alkylguanine DNA alkyltransferase (AGT), leads to apoptosis in MMR competent cells and O6-mG:T mismatches in MMR deficient cells. Tumor induction is less in MMR+/+ mice because cells with residual DNA adducts die, whereas mutagenized cells survive in MMR/ mice. Overexpression of AGT (encoded by the methylguanine DNA methyltransferaseMGMTgene) is known to block MNU induced tumorigenesis in mice with functional MMR. To further determine the sensitivity of PMS2/ mice to MNU and the protective effect of hAGT overexpression, a low dose of MNU (25 mg/kg) was studied in PMS2/ mice and PMS2//hMGMT+ mice. No thymic lymphomas were found in MNU-treated PMS2+/+ and PMS2+/ mice. At 1 year, 46% of the MNU-treated PMS2/ mice developed thymic lymphoma, compared with an incidence of 25% in both untreated PMS2/ mice and MNU treated PMS2//hMGMT+ mice. In addition, a significantly shorter latency in the onset of thymic lymphomas was seen in MNU-treated PMS2/ mice. K-ras mutations were detected almost equally in the thymic lymphomas induced by MNU in both PMS2/ and PMS2//hMGMT+ mice, but not in the spontaneous lymphomas. These data suggest that PMS/ mice are hypersensitive to MNU, that there are different pathways responsible for spontaneous and MNU induced thymic lymphomas in PMS2/ mice, and that overexpression of hMGMT protects the mice by blocking non-K-ras pathways.
Abbreviations: AGT, O6-alkylguanine DNA alkyltransferase protein; MGMT, methylguanine DNA methyltransferase gene; MMR, mismatch repair; MNU, N-methyl-N-nitrosourea.
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Introduction
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Environmental, endogenous and inherited factors affect cellular maintenance of the genome and play important roles in tumorigenesis. Alkylating agents are well known environmental carcinogens (1). N-methyl-N-nitrosourea (MNU), a methylating agent, induces various tumors in mice including thymic lymphomas, breast tumors and GI tumors based on the production of carcinogenic O6-methylguanine (O6-mG) lesions in DNA (210). Unless repaired by O6-alkylguanine DNA alkyltransferase (AGT), O6-mG preferentially pairs with thymine (T) instead of cytosine (C) during DNA replication, resulting in a O6-mG:T mismatch and, after another round of DNA replication, to a G:C
A:T transition mutation (1113). AGT is the product of the O6-methylguanine DNA methyltransferase gene (MGMT) and specifically repairs O6-mG (14) by directly transferring the methyl group from DNA to its cysteine residue at position 145 (15). Generally, carcinogenic N-alkyl-N-nitrosoureas are known to induce tumors preferentially in tissues with low AGT activity (1619). Overexpression of MGMT prevents G
A mutations, protects cells from malignant transformation and protects animals from tumorigenesis following treatment with alkylating agent (16,20).
The O6-mG:T mismatch, whether it persists for minutes or days (based on AGT levels), will be recognized by the DNA mismatch repair (MMR) system. Normally, MMR is triggered by single mismatched bases and slippage at base repeats and initiates long patch removal and repair. Repair is initiated from the newly synthesized strand and resynthesizes the patch using the parent strand template (21). However, with the O6-mG:T mismatch, the newly synthesized strand contains the T, and during DNA repair synthesis, a T will again be inserted opposite the O6-mG, leading to repetitive aberrant cycles of repair, consumption of ATP and apoptosis (22). Further, the long single strand patches are prone to homologous recombination and chromosomal aberrations (23). MMR deficient cells, on the other hand, cannot recognize these mismatches and are resistant to apoptosis. This results in greater cell survival coupled with numerous O6-mG:T mismatches, which leads to G:C
A:T transition mutations (24,25). Some of these mutations may result in activation of proto-oncogenes and cause malignant transformation, since G:C
A:T transition mutations have typically been detected at the second guanine of codon 12 (GGT) of the K-ras or H-ras oncogenes in rodent tumors induced by methylating carcinogens (2628).
Defects in mismatch repair are causative of human hereditary non-polyposis colorectal cancer (HNPCC) (21). Mice defective in mismatch repair genes, PMS2, MSH2 or MLH1, develop spontaneous thymic lymphoma, skin tumor and intestinal tumors (2932). We have recently shown that these MMR deficient mice are hypersensitive to MNU carcinogenesis. A dose of 50 mg/kg MNU induced a 100% incidence of thymic lymphomas by 181 days (33). Surprisingly, while overexpression of the hMGMT transgene completely blocked 50 mg/kg MNU carcinogenesis in PMS2+/+ mice (2), hMGMT overexpression only partially protected PMS2/ mice from induction of lymphomas by this comparatively high dose of MNU (33).
Why does overexpression of hMGMT fail to completely protect against MNU? We proposed that other DNA adducts besides O6-mG might play a role in MNU tumorigenesis in PMS2/ mice or that the survival of PMS2/ cells after MNU without cell cycle arrest would result in rapid conversion of O6-mG to G
A mutations in critical oncogenes even before these lesions could be repaired. To further assess this issue, we treated PMS2/ mice with a low dose of MNU. By using a dose that is not carcinogenic in normal mice, we approached an exposure that is more indicative of endogenous methylation. We evaluated the incidence of thymic lymphoma in the presence and absence of hMGMT overexpression. We found that PMS2/ mice were hypersensitive to thymic lymphomagenesis even at this low dose of MNU, but were completely protected from MNU by hMGMT overexpression. From this, we conclude that hMGMT overexpression not only efficiently protects genetically normal mice, but also mice that are carcinogen-hypersensitive due to loss of MMR and genomic instability.
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Materials and methods
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Chemicals
Taq DNA polymerase, deoxynucleotide triphosphate, proteinase K and agarose were purchased from Gibco BRL (Gaithersburg, MD). BstNI was obtained from New England Biolabs (Beverly, MA) and FokI was purchased from Boehringer Mannheim (Indianapolis, IN). MNU was obtained from Sigma (St Louis, MO).
hMGMT transgenic mice and PMS2 knockout mice
The construct of hMGMT-CD2 transgenic mice has been described (2). The PMS2 knockout mice were generated by Baker et al. (29) in 129 ES cells, chimeric mice propagated in C57BL/6 for more than eight passages and offspring crossed to generate the line. As described previously (33), heterozygous PMS2 knockout mice (PMS2+/) were crossmated with hMGMT transgenic mice (hMGMT+) to generate F1 PMS2+//hMGMT+ mice which were backcrossed to PMS2+/ mice and the resulting offspring were used for these experiments. The genotypes of all the mice were determined by PCR amplification of tail DNA.
Animal treatment
Twenty-six PMS2/ mice, 18 PMS2//hMGMT+ mice, 19 PMS2+/mice, 12 PMS2+//hMGMT+ mice, 28 PMS2+/+ mice and four PMS2+/+/hMGMT+ mice were given a single i.p. injection of MNU at a dose of 25 mg/kg body wt at 5 weeks of age. Forty-four PMS2/ and 51 PMS2//hMGMT+ additional mice were observed for spontaneous onset of lymphomas. The mice were carefully observed at least three times a week. Mice found short of breath were killed under carbon dioxide (CO2) anesthesia and an immediate necropsy was done on all of the dead mice. Some of the mice developed palpable splenomegaly. Tumor and organ sections were frozen in liquid nitrogen and stored at 70°C for analysis of K-ras mutations and AGT activity. The remaining sections were fixed in 10% buffered formalin, embedded in paraffin and 3 µm sections were cut and stained with H&E.
Thymic lymphoma-free survival curves were plotted by KaplanMeier Cumulative Survival Curves and compared by standard statistical techniques using Statview software with different Chi-square tests. The cumulative hazard was also analyzed using KaplanMeier Cumulative Hazard Curves. The survival curve describes the relationship between time and the survival probability, which is simply the probability that an individual survives or is disease-free from one time to another. Hazard function is another frequently used measure of risk, sometimes called the force of mortality, failure rate, or instantaneous death rate. A hazard rate measures interval or instant risk of death at a specific time. Hazard analysis is more useful as a measure of prognosis than is the survival curve (34,35). In this study, we used hazard analysis to evaluate the potential of thymic lymphoma development.
PCR-RFLP for K-ras
Activating G
A point mutations in codon 12 of K-ras were identified as previously described (10,33).
Alkyltransferase assay
These assays were performed as described earlier (16). The overexpression of human MGMT in PMS2//hMGMT+ mice was confirmed and reported previously (33) (data not shown).
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Results
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Spontaneous thymic lymphoma development
As reported previously, PMS2/ mice develop thymic lymphoma spontaneously (29,33). Lymphoma incidence in the PMS2/ and PMS2//hMGMT+ mouse crosses were similar in both the groups with a lymphoma incidence at 1 year of ~25% (Figure 1A
). The cumulative hazard analysis shown in Figure 1B
demonstrates the identical results. No obvious protective effect of hMGMT overexpression on spontaneous thymic lymphoma development was noted in 51 PMS2//hMGMT+ mice.

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Fig. 1. Spontaneous thymic lymphoma development in PMS2/ mice with or without human MGMT transgenes. The data were from 51 transgenic (PMS2//hMGMT+; closed circle) and 44 non-transgenic (PMS2/; open circle) PMS2 deficient mice. No tumors were found in any of the normal (PMS2+/+) or heterozygous (PMS2+/) mice (data not shown). About 25% tumor incidence was similarly observed in both the hMGMT transgenic and non-transgenic PMS2 deficient mice (A). The cumulative hazard curves are parallel to each other (B). There was no effect of high hMGMT expression on spontaneous thymic lymphoma development in PMS2/ mice.
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Thymic lymphoma induction by MNU
The 25 mg/kg dose of MNU did not induce any thymic lymphomas in PMS2+/+ or PMS2+/ mice (data not shown). In contrast, 46% of the MNU-treated PMS2/ mice developed thymic lymphomas. Compared with untreated PMS2/ mice, MNU-treated PMS2/ mice showed significantly higher incidence of thymic lymphomas (Figure 2A
, P < 0.05). In addition, significantly shorter latency of thymic lymphoma development was seen in the MNU-treated PMS2/ mice. The cumulative hazard curve plateaus for the untreated PMS2/ mice, but continues to increase for the MNU-treated PMS2/ mice (Figure 2B
). This confirms the hypersensitivity to MNU in these mice and the synergy (since this dose of MNU does not induce tumors in normal mice) between MNU and MMR deficiency.

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Fig. 2. Thymic lymphoma development in MNU-treated PMS2/ and non-treated PMS2/ mice. Twenty-six MNU-treated PMS2/ mice (closed triangle) were compared with 44 non-treated PMS2/ mice (spontaneous, open circle) in the KaplanMeier Cumulative Survival Curve. 42% thymic lymphoma-free survival was found in PMS2/ mice (A), although the actual number of thymic lymphoma carrying mice was 12 out of 26 (46%). The difference was significant (P < 0.05). The KaplanMeier Cumulative Hazard Curve for MNU-treated PMS2/ mice clearly shows an increasing tendency while spontaneous thymic lymphoma development levels off (B).
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The incidence of lymphomas in MNU-treated PMS2//hMGMT+ mice was the same as that of untreated PMS2//hMGMT+ mice (Figure 3A
). The cumulative hazard curves for these two groups of mice are parallel to each other (Figure 3B
). Since the hMGMT transgene had no effect on the incidence of spontaneous thymic lymphomas, the data indicate that hMGMT overexpression prevents MNU carcinogenesis in PMS2/ mice.

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Fig. 3. The thymic lymphoma induction in MNU-treated PMS2//hMGMT+ mice was compared with non-treated PMS2//hMGMT+ mice. Eighteen MNU-treated PMS2//hMGMT+ mice (open triangle) had a similar incidence, compared with the non-treated PMS2//hMGMT+ mice (spontaneous; closed circle) (A). An identical tendency was also demonstrated in the KaplanMeier Cumulative Hazard Curves (B). The effect of 25 mg/kg MNU was not demonstrated in PMS2//hMGMT+ mice.
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Median latency of thymic lymphomas in MNU-treated PMS2//hMGMT+ mice was 181 days, which was longer than the median latency of 150 days in MNU-treated PMS2/ mice (Figure 4A
). The cumulative hazard continuously increased for MNU-treated PMS2/ mice, but not for MNU-treated PMS2//hMGMT+ mice (Figure 4B
). To distinguish the incidence and latency of MNU-induced lymphomas compared with spontaneous lymphomas, survival curves were calculated separately for PMS2/ and PMS2//hMGMT+ mice, as shown in Figure 5
. A significant difference (P < 0.05) in the MNU-specific lymphoma incidence was demonstrated between PMS2/ and PMS2//hMGMT+ mice.

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Fig. 4. Effects of overexpression of hMGMT on thymic lymphoma induction by MNU in PMS2/ mice. The data from the 26 MNU-treated PMS2/ mice (closed triangle) and 18 MNU-treated PMS2//hMGMT+ mice (open triangle) were plotted. The median latency of thymic lymphomas in MNU-treated PMS2//hMGMT+ mice was 181 days, which was longer than the median latency of 150 days in MNU-treated PMS2/ mice (A). A clear difference in cumulative hazard risk was shown in the KaplanMeier Cumulative Survival Curves (B).
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Fig. 5. The MNU-specific lymphoma incidence (A) and cumulative hazard risk (B) in MNU-treated PMS2/ and PMS2//hMGMT+ mice was calculated based on the data from MNU-treated and non-treated spontaneous incidence of each group. A significant difference (P < 0.05) was clearly demonstrated, confirming the blocking effects of overexpression of hMGMT on 25 mg/kg MNU carcinogenesis in PMS2//hMGMT+ mice.
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K-ras mutation analysis
Six out of 12 lymphomas from PMS2/ mice, three out of five lymphomas from PMS2//hMGMT+ mice and five samples from spontaneous tumors including both PMS2/ and PMS2//hMGMT+ mice were analyzed for a mutation in codon 12 (GGT) of the K-ras oncogene (the most common site of activated ras mutations in MNU-induced lymphomas; 2628). A similar mutation rate of K-ras codon 12 was found in MNU-induced lymphomas of PMS2/ mice (3/6) and PMS2//hMGMT+ mice (2/3), whereas no mutations were detected from the spontaneous tumor samples (0/5). As expected, all of the K-ras mutations detected in the present study were G
A transition point mutations leading to a GGT
GAT transition mutation at K-ras codon 12, consistent with the specificity of DNA alkylation and mutation by MNU (2628).
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Discussion
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MMR plays an important role in stabilizing the genome. Defects in MMR are causative in human hereditary nonpolyposis colorectal cancer (21). Mice defective in MMR genes, PMS2, MSH2 or MLH1, show increased mutation rates and develop lymphomas, sarcoma and intestinal tumors (2932) even in the absence of any mutagenic treatment. Tumors in MMR-deficient mice have microsatellite DNA instability (29).
MMR-defective cells are resistant to the killing effect of methylating agents due to the failure to initiate the MMR system. As a result, these cells may suffer from a large load of DNA adducts, which lead to mutations and high tumor induction. Therefore, we hypothesized that the PMS2/ mice are hypersensitive to environmental methylating carcinogens and that overexpression of the hMGMT transgene should protect these mice from carcinogenesis of methylating agents by highly efficient repair of O6-mG lesions. Our studies have shown that PMS2/ mice are hypersensitive to a single i.p. injection of 50 mg/kg MNU (33). MNU-treated PMS2/ mice demonstrated a 100% incidence of thymic lymphoma induction, compared with an incidence of 52% in genetically normal mice, and had a shorter mean latency (81 versus 102 days). However, the protection of overexpression of hMGMT following 50 mg/kg MNU was only partially demonstrated in our previous study (33). Even though overexpression of the hMGMT transgene results in a 40-fold increase in AGT, and thus a 40-fold increase in capacity to repair O6-mG, and is able to completely protect normal mice from 50 mg/kg MNU (2,5), PMS2//MGMT+ mice remain hypersensitive to 50 mg/kg MNU. The explanation may be that O6-mG adducts not repaired by MGMT are carcinogenic. This is possible even though our recent studies in genetically normal mice suggest that carcinogenic O6-mG lesions are removed in hMGMT+ mice (36). A more interesting possibility is that there were sufficient O6-mG adducts to be rapidly converted to G
A mutations, because cell cycle arrest is not present in the absence of MMR (3739). Thus, even with rapid removal of most O6-mG lesions by hAGT, the lack of a cell cycle check point may have `locked in' sufficient lesions to be carcinogenic.
To further evaluate the hypersensitivity of PMS2/ mice to MNU and to evaluate the protection of the hMGMT transgene, we designed the present `low dose' MNU carcinogenesis experiment. This study demonstrated that MNU (25 mg/kg body wt) does not induce thymic lymphomas in PMS2+/+ or PMS2+/ mice, indicating that this dose is a subthreshold, non-carcinogenic dose. MNU-treated PMS2/ mice developed a higher incidence of thymic lymphomas compared with spontaneous thymic lymphomas (P < 0.05), at a significantly shorter latency (P < 0.05). G
A point mutations in the second position of K-ras codon 12 were detected in 50% (3/6) of tumors examined compared with none of the spontaneous tumors, suggesting that the increased incidence is due to MNU. Overexpression of hMGMT transgene significantly reduced the MNU effect; therefore, most, if not all, of the thymic lymphomas found in the MNU-treated PMS2//hMGMT+ mice appear to be spontaneous.
The K-ras mutation rate in tumors from PMS2/ mice is similar to the rate observed at the high dose (50 mg/kg) of MNU (51.6% in PMS2//hMGMT+ mice versus 26% in PMS2/ mice, respectively) (33). K-ras mutations are often observed in lymphomas induced by MNU at an incidence that is dose dependent and affected by genetic background. In normal mice, K-ras mutations are seen in 1030% of lymphomas (33,40,41). Of interest, Newcomb et al. reported no clear relation between elevated levels of ras proteins and K-ras mutations and shorter latency, rate of metastasis, or proportion presenting with a leukemic phase (42). Thus, while K-ras mutations are observed in some MNU-induced lymphomas, other oncogenes and/or inactivation of unidentified tumor suppressor gene(s) may also be involved, and may complement the proliferative advantage that K-ras mutations may provide. The role of K-ras in animals with genomic instability is also unclear.
It appears that overexpression of the hMGMT gene does not impact on the development of spontaneous thymic lymphomas in MMR deficient mice (33,46, this study). Moreover, mice deficient in MGMT expression, produced by gene targeting technology, do not develop spontaneous lymphomas (4345). In addition, no K-ras mutations were detected in the spontaneous tumors (33, this study), indicating that endogenous methylation does not appear to be important in spontaneous thymic lymphoma development. The spontaneous thymic lymphomas may be due to abnormalities in thymocyte growth, lack of cell cycle regulation or other consequences of genomic instability (47). It is also possible that other lesions, such as oxidative DNA damage, are responsible. Oxidative damage is the most common form of spontaneous DNA damage. In steady-state rat cells there are ~24 000 8-oxo-dG lesions per cell (48). Transcription-coupled repair of thymine glycol, one of the major products of oxidative damage, depends on the MMR system in yeast (49), suggesting that MMR can recognize and repair oxidative damage. The mutation rate of MMR-defective yeast strains can be dramatically lowered under anaerobic conditions, which are associated with lower levels of oxidative damage, further supporting the role of oxidative damage repair by MMR (50).
In summary, hMGMT overexpression protects PMS2-deficient mice from hypersensitivity to MNU-induced lymphomagenesis. These data also indicate that methylation damage to DNA is significantly more carcinogenic in the absence of mismatch repair.
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Acknowledgments
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We would like to thank Dr R.M.Liskay, Department of Molecular/Medical Genetics, Oregon Health Sciences University, Portland, OR, for providing us with PMS2 gene knockout mice. We thank Ms Alana Hart, the Grants/Publications Specialist, for word processing. This work was supported in part by USPHS grants P30CA43703, RO1CA63193, RO1ES06288, RO1CA73062 and UO1CA75525.
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Notes
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1 To whom correspondence should be addressed Email: slg5{at}po.cwru.edu.. 
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Received March 12, 1999;
revised May 27, 1999;
accepted June 9, 1999.