DNA repair protein MGMT protects against N-methyl-N-nitrosourea-induced conversion of benign into malignant tumors

Klaus Becker1,4, Cornelia Gregel1, Christa Fricke1, Dymitr Komitowski2, Jörg Dosch3 and Bernd Kaina3,5

1 DNA Repair Group, Institute of Plant Genetics, Corrensstrasse 3, D-06466 Gatersleben
2 German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg
3 Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz
4 Schering AG, Research Laboratories D-13342 Berlin, Germany


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Tumor formation is a multi-step process that can be divided into the stages of tumor initiation, promotion and progression. Previously, we showed that overexpression in skin of mice of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) protects against N-methyl-N-nitrosourea (MNU)-induced tumor initiation without affecting tumor promotion. This indicated that O6-methylguanine, which is specifically repaired by MGMT, is a major tumor-initiating lesion. Here we extended this transgenic approach to the study of tumor progression. Benign papillomas that arose on the skin of CkMGMT transgenic mice upon initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) expressed higher levels of MGMT than papillomas that appeared on DMBA/TPA treated non-transgenic NMRI mice. Treatment of papillomas with MNU resulted in the formation of malignant carcinomas to a significantly lower frequency in CkMGMT mice as compared with the non-transgenic control. The data provide evidence that increased DNA repair protects against the conversion of benign into malignant tumors. They show at the same time that a particular type of damage induced in DNA, namely O6-methylguanine, is decisively involved in triggering tumor progression. This supports the concept that the major cause of both tumor initiation and tumor progression is mutation. Data also indicate that alkylating anti-neoplastic drugs may provoke tumor progression in case of failure of tumor therapy, which is attenuated by DNA repair.

Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; MGMT, O6-methylguanine-DNA methyltransferase; MNU, N-methyl-N-nitrosourea; SCC, squamous cell carcinomas; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
A generally accepted paradigm of tumorigenesis considers tumor formation as a multi-step process that can be divided into the stages of tumor initiation, promotion and progression (1–6). Tumor initiation is related to the hereditary change of a normal cell into a pre-malignant one without clear phenotypic alterations. Tumor promotion is considered to be due to clonal expansion of the initiated cell that results in the formation of a benign tumor, whereas tumor progression denotes the conversion of a benign into a malignant tumor. These mechanistically defined stages of tumor formation are probably best studied in the classical multi-stage skin carcinogenesis model in mice, which offers an excellent experimental tool to quantify and to characterize defined genetic and epigenetic events underlying the particular stages of tumorigenesis, which can experimentally be controlled (7). Currently, it is generally accepted that the induction of point mutations in critical genes, such as the proto-oncogene c-Ha-ras, is the underlying cause of tumor initiation (8,9), whereas tumor promotion brought about by tumor promoters such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is due to stimulation of expression of genes involved in hyperproliferation and/or inflammation (7,10–12) or reduction in the level of apoptosis (13–15). Tumor promotion is generally considered to be an epigenetic phenomenon. Whether genomic changes such as numerical and structural chromosomal aberrations are also involved in tumor promotion is still a matter of debate (16,17).

Tumor progression is a spontaneously rarely occurring process that can be achieved experimentally by treatment of a benign tumor with a carcinogenic DNA-damaging agent but not by treatment with a tumor promoter (18). This indicates that stable genetic changes are involved in tumor progression. However, DNA-damaging agents may also evoke transient cellular responses such as protein modification, activation of growth factor receptors and transcriptional stimulation of genes involved in growth regulation (e.g. c-fos and c-jun) (19–22). Therefore, the involvement of epigenetic changes in tumor progression cannot be excluded. A particular type of DNA damage that triggers tumor progression has not been identified so far.

The concept that point mutations are the underlying cause of tumor initiation has gained substantial support from two-step carcinogenesis studies with transgenic mice that overexpress the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) specifically in skin cells (for review see ref. 23). MGMT is a key suicide enzyme repairing the mispairing base O6-methylguanine, which is induced in DNA as a minor lesion (<8% of total alkylation) by methylating environmental and experimental carcinogens as well as various methylating antineoplastic drugs (e.g. DTIC, procarbazin, streptozotozine and temozolomide). Because O6-methylguanine possess a high potential to mispair with thymine (24) its pre-replicative repair by MGMT prevents the induction of gene mutations and genotoxic effects. This has extensively been proven in several laboratories in studies with isogenic cell lines in vitro differing in MGMT expression (e.g. ref. 25). As overexpression of MGMT also protected against tumor initiation by N-methyl-N-nitrosourea (MNU) evidence was provided that O6-methylguanine-induced point mutations are a major cause of tumor initiation without affecting tumor promotion (26).

Here we addressed the question of whether O6-methylguanine causes tumor progression as well and whether MGMT is able to protect against it. We made again use of the transgenic CkMGMT mouse model and show that the conversion of benign papillomas into malignant carcinomas upon the treatment with MNU is significantly reduced in mice expressing MGMT at a high level in skin, as compared with the non-transgenic control. This clearly indicates that genetic changes brought about by O6-methylguanine trigger the process of tumor progression. At the same time evidence is provided that DNA repair is an effective mechanism of protection against conversion of benign into malignant tumors.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Transgenic mice
Transgenic mice were used for tumor progression experiments, which harbour a transgene construct consisting of two copies of the human MGMT gene (cDNA) directed by the bovine cytokeratin CkIII (BK5) and CkIV (BK6b) promoter, respectively. By virtue of these promoter elements the expression of the MGMT transgene was targeted to the interfollicular epidermis and to the outer cells of hair follicles. The tissue-specific transgene expression resulted in significantly higher cellular MGMT activity in the skin of transgenic as compared with non-transgenic mice (26). The transgenic Gat:NMRI mouse line TgN(CkMGMT)3Bec was kept in our laboratory as a homozygous colony with respect to the transgene.

MGMT analysis
Papillomas were carefully dissected from the surrounding tissue and snap-frozen in liquid nitrogen. MGMT activity in papillomas was assayed essentially as described (26,27).

Multi-stage skin carcinogenesis
Nine to 12 week old transgenic (n = 20) and non-transgenic (n = 23) mice were initiated by application of 50 nmol 7,12-dimethylbenz[a]anthracene (DMBA) dissolved in 100 µl acetone to the shaved back skin (stage I: initiation). Seven days after initiation the animals were treated twice weekly with the tumor promoting phorbol ester TPA (10 nmol in 100 µl acetone) for 10 weeks (stage II: promotion). The number and location of the appearing papillomas was recorded weekly for each mouse. The tumor response was expressed as papilloma rate (number of papilloma-bearing mice/number of survivors) and papilloma yield (number of papillomas/number of surviving mice). At week 12, 1 week after discontinuation of tumor promotion, mice were topically treated by a second initiator, MNU to achieve malignant conversion of benign papillomas to malignant carcinomas (stage III: progression). MNU was recrystallized in methanol, dissolved in acetone at a concentration of 20 µmol/100 µl and immediately applied to the dorsal skin of each papilloma-bearing mouse once a week for 5 weeks. The number and location of emerging carcinomas was recorded weekly for each mouse. The developing skin tumors were classified as carcinomas if the base of the papillomas was enlarged and the surrounding tissue margins showed signs of suspected tumor infiltration. Mice were killed in cases of excessive tumor load or when the size of single carinomas exceeded the diameter of 1 cm. At week 35 after initiation the experiment was terminated and the remaining mice were killed. All mice were also inspected for internal tumors. The incidence of carcinoma-bearing mice in both groups was statistically compared by means of the Kaplan–Meier cancer-free survival probability over 24 weeks. The papilloma–carcinoma conversion ratio in non-transgenic and transgenic mice was statistically compared by calculating the conversion ratio for each papilloma until death of the carcinoma-bearing animal by means of the Wilcoxon two-sample test and the Exact Test.

Histological examination of suspected skin tumors
Suspected skin carcinomas were recorded at the time of appearance. They were characterized by a size of ~1 cm, elevated margins of the tumor and by cratering. During the following 2 weeks, ulceration of the tumor generally proceeded; therefore these mice were killed and the suspected carcinomas (22 tumors of the control and 20 tumors of the transgenic group) were carefully removed from the back of these mice, fixed in Bouins fixative for ~24 h, repeatedly washed in 70% ethanol and processed for histological analysis by a pathologist in order to confirm the gross morphological diagnosis. The principal morphological changes in the investigated mice were epidermal hyperplasia, papillomas and squamous cell carcinomas (SCC). These changes can be related to the stages of experimentally induced carcinogenesis. The hyperplasia with an increase of the epidermal layers from normally 2–3 to 10–20 µm was observed predominantly in association with the papillomas.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Papilloma formation in CkMGMT transgenic and nontransgenic mice (stages I and II)
Benign papillomas were induced in CkMGMT transgenic mice and their non-transgenic Gat:NMRI controls according to the classical skin carcinogenesis protocol: animals were exposed to a subthreshold dose of DMBA followed by TPA treatment for 10 weeks (26). Under these conditions papilloma formation occurred with nearly identical latency period and overall frequency in CkMGMT transgenic and non-transgenic mice. One week after the last TPA treatment, 91% of the non-transgenic controls and 95% of the transgenic mice exhibited at least one papilloma (Figure 1AGo). At week 11 after DMBA initiation, on average 8.2 papillomas were detected per non-transgenic individual and 10.0 papillomas/transgenic mouse (Figure 1BGo). One non-transgenic and one transgenic mouse had to be excluded from further study (progression, phase III), because they did not develop papillomas. The data show that CkMGMT transgenic and non-transgenic control mice did not differ in the DMBA-induced formation of benign papillomas during two-stage skin tumorigenesis. Moreover, there was also no difference in the size and growth of papillomas upon repeated treatment with TPA in both groups of mice.



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Fig. 1. Formation of benign papillomas in CkMGMT transgenic and control mice at stage II of skin carcinogenesis. Twenty transgenic and 23 non-transgenic animals were initiated with a single dose of DMBA and promoted by TPA application for 10 weeks as described (26). The number of cumulative papillomas was registered weekly and the tumor response of CkMGMT transgenic and non-transgenic mice was expressed as (A) papilloma rate (number of papilloma-bearing mice/treated micex100) and (B) papilloma yield (cumulative number of papillomas/treated mouse). {blacksquare} NMRI control; {circ} transgenic mice.

 
Expression of MGMT in papillomas
To examine whether papillomas that arose on two-stage skin carcinogenesis continue to express the transgenic DNA repair protein MGMT, as shown previously for the normal epidermal cells of CkMGMT transgenic mice (26), papillomas were harvested from transgenic and control mice and subjected to MGMT analysis. Measurement of MGMT activity was performed in protein extracts of 12 papillomas that arose on different transgenic animals and six papillomas of control mice. As shown in Figure 2Go, the MGMT level was significantly higher in all papillomas isolated from CkMGMT transgenic individuals than in papillomas of control animals. The MGMT activity ranged in the non-transgenic control group between 14 and 43 fmol/mg protein as compared with 98 and 832 fmol/mg protein, with average expression levels of 21 and 322 fmol/mg protein, respectively. The low and high MGMT level in papillomas of non-transgenic and CkMGMT transgenic mice is in accordance with the MGMT expression level that has been detected in non-treated skin of the same groups of mice (26). Obviously, during tumor initiation and promotion the expression of the MGMT transgene has been maintained in the target cell population. Thus, the CkMGMT mouse model is suitable for examination of a possible role of MGMT in protection against malignant progression.



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Fig. 2. Expression of MGMT in papillomas of CkMGMT-transgenic and control mice. Papillomas were harvested from the back of different transgenic and non-transgenic individuals. Protein extracts were made in order to measure the MGMT activity as described in the Material and methods.

 
Malignant progression of papillomas following MNU treatment (stage III)
In order to induce malignant progression of papillomas that evolved on the back skin of mice upon DMBA initiation and TPA promotion, 19 CkMGMT transgenic and 22 control (non-transgenic) mice were subjected to five topical applications (once weekly for 5 weeks) of 20 µmol of the O6-methylguanine generating agent MNU. Ten weeks after MNU treatment (week 22 after DMBA initiation), the first suspected carcinomas appeared in both groups of mice. Thus, carcinoma latency was not different in transgenic and non-transgenic individuals. Whereas carcinoma formation was accelerated in non-transgenic animals during the following weeks, CkMGMT transgenic mice revealed a significant reduction in the incidence of carcinomas. At week 28 after initiation with DMBA, 72.7% (16/22) of non-transgenic mice developed skin carcinomas as compared with 26.3% (5/19) of the transgenic mice. At week 34, the end of the study, 90.9% (20/22) of non-transgenic and 78.9% (15/19) of the transgenic animals had developed at least one carcinoma (Figure 3AGo). The mean cancer-free survival time was significantly shorter in non-transgenic (26.96 weeks) compared with transgenic (30.81 weeks) mice (P = 0.0134, Kaplan–Meier log-rank test, graphic not shown). Data shown in Figure 3BGo represent the papilloma to carcinoma conversion rate, i.e. the percentage of carcinomas formed during phase III in relation to the total number of papillomas that appeared at the end of phase II. At week 28, non-transgenic controls revealed a conversion frequency of 28.3%, whereas in transgenic mice only six out of 200 papillomas progressed to carcinomas leading to a conversion rate of 3%. The final cumulative papilloma to carcinoma conversion rate in week 34 was 54% in the control as compared with only 14.5% in CkMGMT transgenic mice. The difference in the overall conversion ratio between both groups was statistically significant (one-sided Wilcoxon two-sample test, P = 0.0378; Exact test, P = 0.0345). Overall, overexpression of MGMT in benign papillomas resulted in a significant reduction of malignant progression of papillomas to carcinomas upon MNU treatment.



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Fig. 3. Malignant progression of papillomas to SCC following MNU treatment. Papilloma-bearing CkMGMT transgenic (dark bars) and non-transgenic mice (black bars) were treated with MNU once weekly for 5 weeks. The appearance of carcinomas was recorded weekly after the beginning of treatment and expressed as (A) the cumulative percentage of mice with carcinomas (%) and (B) the papilloma to carcinoma conversion rate (cumulative number of carcinomas on surviving mice per number of papillomas, %).

 
Histological examination of carcinomas
Suspected carcinomas were recorded at the time of appearance. They were characterized by a size of ~1 cm, elevated margins of the tumor and by cratering. During the following 2 weeks, ulceration of the tumor generally proceeded; therefore these mice were killed and the suspected carcinomas (a total of 44 tumors) were histologically examined in order to confirm the gross morphological diagnosis. All non-transgenic and 90% of the transgenic skin tumors turned out to be SCC. Most of these SCC were well differentiated with clearly visible intercellular connections, keratinization and formation of keratin pearls (Figure 4Go). Most of the carcinomas (17 control and 15 transgenic SCC) began to infiltrate the corium. Only one carcinoma of the transgenic group of mice showed undifferentiated areas with signs of transition to a spindle cell carcinoma. The papillomatous appearance of the tumors indicate that the carcinomas induced in stage III originated from benign papillomas that appeared after DMBA/TPA treatment. Some of the lesions showed clearly invasive growth with disruption of the basament membrane.



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Fig. 4. Histology of papillomas and invasive carcinomas. (A) Flat papilloma. The tumor shows broad projections confined to the stroma. On the surface keratinization is clearly detectable (labeled by arrow). (B) Well defined SCC with the formation of keratin pearls (labeled by arrow). (C) SCC with infiltrative growth (arrow) in the subcutaneous muscle tissue.

 

    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Tumor formation is considered to be a multi-step process involving both genetic and epigenetic changes (28,29). It is well accepted that tumor initiation is due to the induction of point mutations since DNA-damaging agents with high mutagenic potential (e.g. MNU) are very efficient initiators (30) whereas agents eliciting predominantly clastogenic effects and bearing low mutagenic potential (e.g. methyl methanesulfonate) are poor initiators (16). Also, the mutation spectrum found in activated oncogenes such as c-Ha-ras or inactivated tumor suppressor genes such as p53 indicates the importance of point mutations for tumor initiation (31–33). One of the most powerful point mutation-inducing DNA lesions is O6-methylguanine, which mispairs with thymine (34). Various transgenic mouse models characterized by overexpressing MGMT (26,35–38) or lack of MGMT due to null mutation (39ndash;41) provided compelling evidence that O6-methylguanine is mainly responsible for tumor formation. In most of these approaches tumor induction occurred by exposing the individuals to a single high dose of an alkylating agent, which does not allow a distinction as to the stages to be involved. By utilizing transgenic mice overexpressing MGMT specifically in skin cells and applying the two-stage protocol of skin carcinogenesis, we were able to show that MGMT specifically protects against tumor initiation provoked by a single low dose of MNU, without affecting tumor promotion (26). Interestingly, initiation was suppressed both for MNU and the chloroethylating agent ACNU (35) demonstrating that not only O6-methylguanine but also larger O6-alkyl-adducts such as O6-chloroethylguanine which is efficiently repaired by MGMT possess tumor-initiating potential.

Here we extended the approach of multi-step skin carcinogenesis by converting benign papillomas into carcinomas, comparing the response of ‘wild-type’ and CkMGMT transgenic mice. Papillomas were induced by DMBA initiation and TPA promotion to nearly equal frequency in control and CkMGMT mice. These tumors clearly expressed higher MGMT levels in transgenic mice than in the wild-type indicating that stimulation of initiated keratinocytes by TPA did not result in loss or down-regulation of MGMT gene expression. Treatment of control and transgenic papillomas expressing low and high MGMT activity, respectively, with MNU caused conversion into carcinomas with significant higher frequency in low than in high MGMT expressing papillomas. This provides evidence that increase of DNA repair capacity of the cell provoked by elevated MGMT protects against alkylation-induced malignant conversion. At the same time the data demonstrate that O6-methylguanine (and presumably also the minor lesion O4-methylthymine) is not only a tumor-initiating lesion but also a lesion causing tumor progression. The protective role of MGMT is of high clinical impact, since the prevention of tumor converting changes is considered to have a profound effect on the long-term survival of tumor patients.

It is generally accepted that conversion of a benign into a malignant tumor requires further accumulation of genetic changes (42). The nature of these alterations and their underlying causes are, however, largely unknown. This is also true for the mouse skin system for which the role of point mutations, recombinations (SCEs) and chromosomal aberrations in the conversion of skin papillomas to carcinomas has not yet been elucidated in detail. Since repair of O6-methylguanine by MGMT reduces both the level of gene mutations, SCEs and chromosomal aberrations (25) the available data do not allow a precise conclusion as to whether point mutations or chromosomal changes would be majorly involved in the process. However, we should stress the finding that aberrations triggered by O6-methylguanine require a much higher number of non-repaired lesions than point mutations (43). Therefore, a relatively high dose of MNU would be required for the induction of aberrations, which exert at the same time strong cytotoxic effects. Upon treatment of papillomas with MNU no inflammation or necrosis of the back skin has been observed. We therefore suppose that the dose applied induced mainly gene mutations, which provoked the process of malignant progression. A preliminary analysis of p53 point mutations in carcinomas revealed base substitution mutations with a frequency of 10–30% (3/10 versus 1/10 mutations in wild-type and CkMGMT transgenic mice, respectively). This is in agreement with the relatively low frequency of p53 point mutations observed in SCC induced either in complete or two-stage skin carcinogenesis protocols in mice upon DMBA and DMBA + TPA treatment (44); for MNU similar data are not available. Further studies are required to elucidate in more detail the role of point mutations versus chromosomal aberrations in tumor progression. The induction of epigenetic changes has also been discussed to be involved in tumor formation (7). According to our knowledge O6-methylguanine does not provoke epigenetic changes. Therefore, the strong tumor suppressive effect of MGMT argues against a significant contribution of epigenetic changes in tumor progression, at least in case of alkylation-induced carcinogenesis.

Overexpression of MGMT reduced tumor conversion ~4-fold. The lack of complete suppression of conversion of benign into malign tumors indicates either saturation of MGMT leaving non-repaired O6-methylguanine lesions in DNA that were fixed into mutations by replication, or that other lesions than O6-methylguanine (such as the less efficiently repaired minor lesion O4-methylthymine and N-alkylations) contribute to skin tumor formation. It would therefore be interesting to elucidate in future work whether especially non-repaired N-alkylation lesions causing predominantly chromosomal aberrations (43) also exert tumor-converting potential thus triggering tumor progression. It should be noted that papillomas consist of hyperproliferating cells. Hyperproliferation and increased DNA replication rate in itself could be a factor contributing to increased carcinogenesis even in case of increased repair capacity for critical lesions because of replication-dependent fixation of lesions.

Simple alkylating agents are not only widely distributed environmental carcinogens (45) but are also endogenously formed by metabolic processes (46). Moreover, they are frequently used in tumor chemotherapy (47). Therefore, various alkylation sources may drive tumor progression once a benign tumor has been formed. MGMT is highly differentially expressed in tumors and normal tissue (48,49). Treatment of tumors expressing low MGMT, such as brain tumors, with methylating agents (e.g. DTIC and temozolomide) yields a comparatively high curative response, which was shown to be related to the low MGMT expression (50,51). But at the same time, in case of failure to achieve complete remission, progression of residual tumor cells could be provoked by non-repaired O6-methylguanine leading finally to a more aggressively growing tumor because of the treatment. Therefore, in the case of methylating agents used for therapy, non-repaired O6-methylguanine lesions may be acting as an initiator for the development of secondary tumors. At the same time they may also be critical as a driving force for conversion of residual tumor tissue into a more aggressively growing type of tumor in the case that complete remission was not achieved by chemotherapy.


    Notes
 
5 To whom correspondence should be addressed Email: kaina{at}mail.uni-mainz.de Back


    Acknowledgments
 
We are grateful to H.F.Ulbrich for statistical analysis. Work was supported by DFG, SFB519, B4 and KA724/4-4.


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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
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Received September 3, 2002; revised November 25, 2002; accepted November 27, 2002.





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