Carcinogenesis vol.24 no.3 pp.541–546, 2003
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
1DNA
Repair Group, Institute of Plant Genetics, Corrensstrasse 3, D-06466
Gatersleben, 2German Cancer Research Center, Im Neuenheimer Feld 280,
D-69120 Heidelberg, 3Division of Applied Toxicology, Institute of
Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131
Mainz and 4Schering AG, Research Laboratories,
D-13342 Berlin, Germany
whom correspondence should be addressed
Email: [email protected]
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-methylguanineDNA methyltransferase (MGMT) protects against Nmethyl-N-nitrosourea (MNU)-induced tumor initiation
without affecting tumor promotion. This indicated that O6methylguanine, 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.
Introduction
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 mechanAbbreviations: 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.
© All rights reserved. Oxford University Press 2003
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5To
istically 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-Otetradecanoylphorbol-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 twostep 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
K.Becker et al.
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 nontransgenic 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
Transgenic mice
MGMT analysis
Papillomas were carefully dissected from the surrounding tissue and snapfrozen 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.
542
Results
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 nontransgenic controls and 95% of the transgenic mice exhibited
at least one papilloma (Figure 1A). At week 11 after DMBA
initiation, on average 8.2 papillomas were detected per nontransgenic individual and 10.0 papillomas/transgenic mouse
(Figure 1B). 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.
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 2, 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
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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.
Fig. 1. Formation of benign papillomas in CkMGMT transgenic and control
mice at stage II of skin carcinogenesis. Twenty transgenic and 23 nontransgenic 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 mice⫻100) and
(B) papilloma yield (cumulative number of papillomas/treated mouse).
j NMRI control; s transgenic mice.
MGMT and conversion of benign into malignant tumors
Fig. 3. Malignant progression of papillomas to SCC following MNU
treatment. Papilloma-bearing CkMGMT transgenic (dark bars) and nontransgenic 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, %).
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.
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 (nontransgenic) 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 3A). 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
Discussion
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 O6methylguanine, which mispairs with thymine (34). Various
transgenic mouse models characterized by overexpressing
MGMT (26,35–38) or lack of MGMT due to null mutation
(39–41) 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). Interes543
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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.
in Figure 3B 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.
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 4). 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.
K.Becker et al.
tingly, 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 O6chloroethylguanine 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
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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.
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 alkylationinduced 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 nonrepaired 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 alkylationinduced carcinogenesis.
Overexpression of MGMT reduced tumor conversion ~4fold. 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 Nalkylation lesions causing predominantly chromosomal aberrations (43) also exert tumor-converting potential thus triggering
MGMT and conversion of benign into malignant tumors
Acknowledgements
We are grateful to H.F.Ulbrich for statistical analysis. Work was supported by
DFG, SFB519, B4 and KA724/4-4.
References
1. Berenblum,I. (1941) The cocarcinogenic action of croton resin. Cancer
Res., 1, 44–48.
2. Boutwell,R.K. (1978) Biochemical mechanism of tumor promotion.
Carcinogenesis, 2, 49–57.
3. Slaga,T.J., Fischer,S.M., Nelson,K. and Gleason,G.L. (1980) Studies on
the mechanism of skin tumor promotion: evidence for several stages in
promotion. Proc. Natl Acad. Sci. USA, 77, 3659–3663.
4. Marks,F. and Fürstenberger,G. (1990) The conversion stage of skin
carcinogenesis. Carcinogenesis, 11, 2085–2092.
5. Fürstenberger,G., Berry,D.L., Sorg,B. and Marks,F. (1981) Skin tumor
promotion by phorbol esters is a two-stage process. Proc. Natl Acad. Sci.
USA, 78, 7722–7726.
6. Nowell,P.C. (1986) Mechanisms of tumor progression. Cancer Res., 46,
2203–2207.
7. Slaga,T.J., Budunova,I.V., Gimenez-Conti,I.B. and Aldaz,C.M. (1996) The
mouse skin carcinogenesis model. J. Invest. Dermatol. Symp. Proc., 1,
151–156.
8. Brown,K., Quintanilla,M., Ramsden,M., Kerr,I.B., Young,S. and
Balmain,A. (1986) v-ras genes from harvey and BALB murine sarcoma
viruses can act as initiators of two-stage mouse skin carcinogenesis. Cell,
46, 447–456.
9. Bailleul,B., Surani,M.A., White,S., Barton,S.C., Brown,K., Blessing,M.
and Balmain,A. (1990) Skin hyperkeratosis and papilloma formation in
transgenic mice expressing a ras oncogene from a subrabasal keratin
promotor. Cell, 62, 697–708.
10. Chen,N., Ma,W.Y., She,Q.-B., Wu,E., Liu,G., Bode,A.M. and Dong,Z.
(2001) Transactivation of the epidermal growth factor receptor is involved
in 12-O-tretradecanoylphorbol-13-acetate-induced signal transduction.
J. Biol. Chem., 276, 46722–46728.
11. Angel,J.M. and DiGiovanni,J. (1999) Genetics of skin tumor promotion.
Progr. Exp. Tumor Res., 35, 143–157.
12. Megosh,L., Gilmour,S.K., Rosson,D., Soler,A.P., Blessing,M., Sawicki,J.A.
and O’Brien,T.G. (1995) Increased frequency of spontaneous skin tumors
in transgenic mice which overexpress ornithine decarboxylase. Cancer
Res., 55, 4205–4209.
13. Wach,S., Schirmacher,P., Protschka,M. and Blessing,M. (2001) Overexpression of bone morphogenic protein-6 (BMP-6) in murine epidermis
suppresses skin tumor formation by induction of apoptosis and
downregulation of fos/jun family members. Oncogene, 20, 7761–7769.
14. Rodriguez-Villanueva,J., Greenhalgh,D., Wang,X.J., Bundman,D., Cho,S.,
Delehedde,M., Roop,D. and McDonnell,T.J. (1998) Human keratin-1.bcl2 transgenic mice aberrantly express keratin 6, exhibit reduced sensitivity
to keratinocyte cell death induction and are susceptible to skin tumor
formation. Oncogene, 16, 853–863.
15. Cho,S.H., Delehedde,M., Rodriguez-Villanueva,J., Brisbay,S. and
McDonnell,T.J. (2001) Bax gene disruption alters the epidermal response
to ultraviolet irradiation and in vivo induced skin carcinogenesis. Int. J.
Mol. Med., 7, 235–241.
16. Fürstenberger,G., Schurich,B., Kaina,B., Petrusevska,R.T., Fusenig,N.E.
and Marks,F. (1989) Tumor induction in initiated mouse skin by phorbol
esters and methyl methanesulfonate: correlation between chromosomal
damage and conversion (stage I of tumor promotion) in vivo. Carcinogenesis, 10, 749–752.
17. Kaina,B. (1989) Chromosomal aberrations as a contributing factor for
tumor promotion in the mouse skin. Teratogen. Carcinogen. Mutagen., 9,
331–348.
18. Hennings,H., Shores,R., Wenk,M.L., Spangler,E.F., Tarone,R. and
Yuspa,S.H. (1983) Malignant concersion of mouse skin tumors is increased
by tumor initiators and unaffected by tumor promoters. Nature, 304, 69–71.
19. Dosch,J. and Kaina,B. (1996) Induction of c-fos, c-jun and junD mRNA
and AP-1 by alkylating mutagens in cells deficient and proficient for the
DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT)
and its relationship to cell death, mutation induction and chromosomal
instability. Oncogene, 13, 1927–1935.
20. Bender,K., Blattner,C., Knebel,A., Iordanov,M., Herrlich,P. and
Rahmsdorf,H.J. (1997) UV-induced signal transduction. J. Photochem.
Photobiol., 37, 1–17.
21. Norbury,C.J. and Hickson,I.D. (2001) Cellular responses to DNA damage.
Ann. Rev. Pharmacol. Toxicol., 41, 367–401.
22. Wang,Y.J. (1998) Cellular responses to DNA damage. Curr. Opin. Cell
Biol., 10, 240–247.
23. Kaina,B., Fritz,G., Ochs,K., Haas,S., Grombacher,T., Dosch,J.,
Christmann,M., Lund,P., Gregel,C.M. and Becker,K. (1998) Transgenic
systems in studies on genotoxicity of alkylating agents: critical lesions,
thresholds and defense mechanisms. Mutat. Res., 405, 179–191.
24. Abbott,P.J. and Saffhill,R. (1979) DNA synthesis with methylated poly
(dC-dG) templates. Evidence for a competitive nature to miscoding by
O6-methylguanine. Biochim. Biophys. Acta, 562, 51–61.
25. Kaina,B., Fritz,G., Mitra,S. and Coquerelle,T. (1991) Transfection and
expression of human O6-methylguanine-DNA methyltransferase (MGMT)
cDNA in Chinese hamster cells: the role of MGMT in protection against
the genotoxic effects of alkylating agents. Carcinogenesis, 12, 1857–1867.
26. Becker,K., Dosch,J., Gregel,C.M., Martin,B.M. and Kaina,B. (1996)
Targeted expression of human O6-methylguanine-DNA methyltransferase
(MGMT) in transgenic mice protects against tumor initiation in two-stage
skin carcinogenesis. Cancer Res., 56, 3244–3249.
27. Preuss,I., Eberhagen,I., Haas,S., Eibl,R.H., Kaufmann,M., von Minckwitz,G.
and Kaina,B. (1995) O6-methylguanine-DNA methyltransferase activity
in breast and brain tumors. Int. J. Cancer, 61, 321–326.
28. DiGiovanni,J. (1992) Multistage carcinogenesis in mouse skin. Pharmacol.
Ther., 54, 63–128.
29. Fearon,E.R. and Vogelstein,B. (1990) A genetic model for colorectal
tumorigenesis. Cell, 61, 759–767.
30. Pegg,A.E. and Singer,B. (1984) Is O6-alkylguanine necessary for initiation
of carcinogenesis by alkylating agents? Cancer Invest., 2, 221–238.
31. Balmain,A., Ramsden,M., Bowden,G.T. and Smith,J. (1984) Activation of
the mouse cellular Harvey-ras gene in chemically induced benign skin
papillomas. Nature, 307, 658–660.
32. Hollstein,M., Sidransky,D., Vogelstein,B. and Harris,C.C. (1991) p53
mutations in human cancer. Science, 253, 49–53.
33. Hussain,S.P., Hollstein,M.H. and Harris,C.C. (2000) p53 tumor suppressor
gene: at the crossroad of molecular carcinogenesis, molecular epidemiology
and human risk assessment. Ann. N.Y. Acad. Sci., 919, 79–85.
34. Eadie,J.S., Conrad,M., Toorchen,D. and Topal,M.D. (1984) Mechanism of
mutagenesis by O6-methylguanine. Nature, 308, 201–203.
35. Becker,K., Gregel,C.M. and Kaina,B. (1997) The DNA repair protein
O6-methylguanine-DNA methyltransferase protects against skin tumor
formation induced by antineoplastic chloroethylnitrosourea. Cancer Res.,
57, 3335–3338.
545
Downloaded from http://carcin.oxfordjournals.org/ at oxford university press on November 23, 2016
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 nonrepaired 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.
K.Becker et al.
546
44. Ruggeri,B., DiRado,M., Zhang,S.Y., Bauer,B., Goodrow,T. and KleinSzanto,A.L. (1993) Benzo(a)pyrene-induced murine skin tumors exhibit
frequent and characteristic G to T mutations in the p53 gene. Proc. Natl
Acad. Sci. USA, 90, 1013–1017.
45. Bartsch,H. and Spiegelhalder,B. (1996) Environmental exposure to Nnitroso compounds (NNOC) and precursors: an overview. Eur. J. Cancer
Prev., 5 (suppl. 1), 11–17.
46. Marnett,L.J. and Burcham,P.C. (1993) Endogenous DNA adducts: potential
and paradox. Chem. Res. Toxicol., 6, 771–785.
47. Tew,K.D., Colvin,M. and Chabner,B.A. (1996) Alkylating agents. In
Chabner,B.A. and Longo,D. (eds), Cancer Chemotherapy: Principles and
Practice, 2nd edn. J.B. Lippincott Co., Philadelphia, pp. 297–332.
48. Preuss,I., Haas,S., Eichhorn,U. et al. (1996) Activity of the DNA repair
protein O6-methylguanine-DNA methyltransferase in human tumor and
corresponding normal tissue. Cancer Detect. Prev., 20, 130–136.
49. Pegg,A.E. (1990) Mammalian O6-alkylguanine-DNA alkyltransferase:
Regulation and importance in response to alkylating carcinogenic and
therapeutic agents. Cancer Res., 50, 6119–6129.
50. Belanich,M., Pastor,M., Randall,T. et al. (1996) Retrospective study of the
correlation between the DNA repair protein alkyltransferase and survival
of brain tumor patients treated with carmustine. Cancer Res., 56, 783–788.
51. Jaeckle,K.A., Eyre,H.J., Townsend,J.J., Schulman,S., Knodson,H.M.,
Belanich,M., Yarosh,D.B., Bearman,S.I., Giroux,D.J. and Schold,S.C.
(1998) Correlation of tumor O6-methylguanine-DNA methyltransferase
levels with survival of malignant astrocytoma patients treated with bischloroethylnitrosourea: a southwest oncology group study. J. Clin. Oncol.,
16, 3310–3315
Received September 3, 2002; revised November 25, 2002;
accepted November 27, 2002
Downloaded from http://carcin.oxfordjournals.org/ at oxford university press on November 23, 2016
36. Dumenco,L.L., Allay,E., Norton,K. and Gerson,S.L. (1993) The prevention
of thymic lymphomas in transgenic mice by human O6-alkylguanine-DNA
alkyltransferase. Science, 259, 219–222.
37. Zaidi,N.H., Pretlow,T.P., O’Riordan,M.A., Dumenco,L.L., Allay,E. and
Gerson,S.L. (1995) Transgenic expression of human MGMT protects
against azomethane-induced aberrant crypt foci and G to A mutations in
the Ka-ras oncogene of mouse colon. Carcinogenesis, 16, 451–456.
38. Nakatsuru,Y., Matsukuma,S., Nemoto,N., Sugano,H., Sekiguchi,M. and
Ishikawa,T. (1993) O6-methylguanine-DNA methyltransferase protects
against nitrosamine-induced hepatocarcinogenesis. Proc. Natl. Acad. Sci.
USA, 90, 6468–6472.
39. Tsuzuki,T., Sakumi,K., Shiraishi,A. et al. (1996) Targeted disruption of
the DNA repair methyltransferase gene renders mice hypersensitive to
alkylating agent. Carcinogenesis, 17, 1215–1220.
40. Iwakuma,T., Sakumi,K., Nakatsuru,Y., Kawate,H., Igarashi,H.,
Shiraishi,A., Tsuzuki,T., Ishikawa,T. and Sekiguchi,M. (1997) High
incidence of nitrosamine-induced tumorigenesis in mice lacking DNA
repair methyltransferase. Carcinogenesis, 18, 1631–1635.
41. Liu,L., Qin,X. and Gerson,S.L. (1999) Reduced lung tumorigenesis in
human methylguanine DNA-methyltransferase transgenic mice achieved
by expression of transgene within the target cell. Carcinogenesis, 20,
279–284.
42. Aaltonen,L.A., Peltomäki,P., Leach,F.S. et al. (1994) Clues to the pathogenesis of familial colorectal cancer. Science, 260, 812–816.
43. Kaina,B., Fritz,G. and Coquerelle,T. (1993) Contribution of O6-alkylguanine and N-alkylpurines to the formation of sister chromatid exchanges,
chromosomal aberrations and gene mutations: new insights gained from
studies of genetically engineered mammalian cell lines. Environ. Mol.
Mutagen., 22, 283–292.