[CANCER RESEARCH 46, 5701-5705, November 1986)
Enhancement of 06-Methylguanine-DNA-Methyltransferase
Various Treatments in Mammalian Cells1
Activity Induced by
Patricia Lefebvre and FrançoiseLaval2
Groupe "Radiochimie de l'ADN, " Institut Gustave-Roussy, 94805 Villejuif Cedex, France
mutagenic effects of Ar-methyl-/V"-nitro-yV-nitrosoguanidine and
ABSTRACT
that this adaptive response was related to an increase in the
number of methyltransferase molecules per cell (15).
activity was determined in a rat hepatoma cell line after treatment with
In rats, the methyltransferase activity can be increased by
ultraviolet or 7-irradiation, heat treatment, or incubation with c/'.vpretreatments
with a variety of agents including partial hepadiamminedichloroplatinum(ll), 2-methyI-9-hydroxyeIIipticinium, or bleotectomy (16,17), aflatoxin B, (18), 2-acetylaminofluorene (19),
mycin. The assay measured the removal of 0*-methylguanine from 'IIdimethylnitrosamine (20, 21), or other carcinogens (22). An
alkylated DNA by cellular extracts. The results show that 48 h after the
increase of 06-methylguanine repair was also observed after
various treatments, the methyltransferase activity is increased by 2- to
whole-body
7-irradiation of rats (23); 2 days after a 10-Gy dose,
5-fold. This increase is due to de novo specific protein synthesis. It is not
the activity increases about 5-fold in liver extracts and about 4related to a modification of the cell cycle parameters, as a similar
enhancement is observed in plateau-phase cells treated with ionizing
and 2-fold in lung and kidney, respectively.
radiations or m-dichlorodiammineplatinum(II).
We have studied whether the number of methyltransferase
The increase of the methyltransferase activity measured using an
molecules could be modified in cells treated in vitro with various
alkylated substrate represents an actual increase of the active molecules
physical or chemical agents. The results show that, when the
in the cells, as the mutation frequency is much lower in cells treated with
A'-methyl-/V'-nitro-/V-nitrosoguanidine 48 h after an irradiation (3 Gy) cells are treated with either UV or ionizing radiations, or with
different chemical compounds, the number of methyltransferase
than in nonirradiated cells.
molecules is 2- to 5-fold higher than in control cells.
This induction of the methyltransferase was not observed in Chinese
The 06-methylguanine-DNA-methyltransferase
(methyltransferase)
hamster ovary cells after 7-irradiation, and therefore it does not seem to
occur in cells which have a low constitutive level of O'-methylguanine
repair.
MATERIALS
AND METHODS
Cell Culture. H4 cells (epithelial cells derived from a rat hepatoma)
were grown in Dulbecco's medium supplemented with 5% fetal calf
INTRODUCTION
Among the different lesions produced by alkylating agents
(1), O6-methylguanine is one of the most important because of
its mutagenic (2) and potentially carcinogenic (3) properties. In
bacteria (4) as well as in mammalian cells (5), this lesion is
repaired by a methyltransferase,3 which transfers the alkyl
group from DNA to one of its own cysteine residues. The
properties of this protein, which can act only once, have been
extensively studied (6).
The methyltransferase is implicated in the sensitivity to al
kylating agents; Mer~ (7) or Mex" (8) cells, which do not carry
methyltransferase molecules, are more sensitive to these com
pounds than the proficient Mer+ or Mex+ cells. This protein is
thought to prevent alkylation mutagenesis, as Mer" cells have
a higher mutation frequency than Mer+ cells (reviewed in Ref.
9), and it seems also implicated in the level of cytogenetic
damage (10). Therefore, the number of methyltransferase mol
ecules per cell seems to be a determinant for the cellular
resistance to mutagenesis mediated by alkylating agents.
The number of methyltransferase molecules can be increased
during the adaptive treatment of Escherichia coli with alkylating
agents (reviewed in Ref. 11). Although it seems to vary from
cell line to cell line, an adaptive response has also been described
in mammalian cells (12, 13). We have previously shown (14)
that a rat hepatoma cell line could be adapted to the toxic and
Received 3/11/86; revised 6/17/86; accepted 6/18/86.
The costs of publication of this article were defrayed in pan by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the Centre National de la Recherche
Scientifique, Institut National de la Santéet de la Recherche Médicale,and
Association pour la Recherche sur le Cancer (Villejuif).
2 To whom requests for reprints should be addressed.
'The abbreviations used are: methyltransferase, O'-methylguanine-DNAmethyltransferase; O'-MeGua, O'-methylguanine; MNU, N-methyl-A'-nitrosourea; MNNG, jV-methyl-W-nitro-A'-nitrosoguanidine;
cis-DDP, r/i-diamminedichloroplatinum(II); CHO, Chinese hamster ovary.
serum, 5% horse serum, penicillin (50 units/ml), and streptomycin (50
¿¿g/ml)
in a humidified 5% CÛ2atmosphere. The doubling time was
about 15 h (14). Plateau-phase cells were obtained by daily feeding the
cells until they attained density inhibition of growth. CHO cells (ob
tained from the American Type Culture Collection) were grown in the
same conditions.
Irradiations and Drug Treatment. 7-Rays were delivered by a *°Co7ray source operating at room temperature at a dose rate of 1.0 Gy/min.
Ultraviolet irradiation was carried out with a General Electric 254-nm
germicidal lamp, at a fluence rate of 10 J/m2/s. Cis-DDP (Roger Bellon,
Paris, France) and 2-methyl-9-hydroxyellipticinium (Institut Pasteur,
Paris, France) were dissolved in water; bleomycin (Roger Bellon, Paris,
France) was dissolved in 0.9% NaCl solution, then the solutions were
diluted to the appropriate concentrations in culture medium. Incuba
tions with these compounds were run for l h in complete medium at
37"C. These various treatments were carried out 24 h after seeding 3.5
x IO6cells in 75-cm2 flasks. Cycloheximide (Sigma Chemical Company)
was added in the culture medium (1.5 fig/ml) immediately after -,
irradiation for a period of 18 h.
Survival and Mutation Frequency Determinations. For survival mea
surement, cells were subcultured in an appropriate number in order to
yield about 50 viable colonies per dish, and they were grown for 14
days until the appearance of clones (14). Mutation frequency experi
ments were carried out as previously described (14). Briefly, cells were
incubated for l h in complete medium containing MNNG (10 ><M).
washed, trypsinized, and subcultured in fresh medium for 6 days. This
time period was already found to be the optimal expression time for
MNNG-treated H4 cells (14). They were then either plated for survival
or plated (2.10s cells/dish) in Dulbecco's medium supplemented with
10% dialyzed calf serum and 6-thioguanine (2.5 ¿tg/ml).
Determination of 0*-Methylguanme-DNA-Methyltransferase
Activ
ity. The cells were trypsinized 48 h after irradiation or drug treatment,
washed twice in Earle's balanced salt solution, and then resuspended in
a buffer containing 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.6, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol,
and 10% glycerol (IO7 cells/100 M')- About 2.5 mg of proteins were
contained in 100 n\ of cell suspension. Protease inhibitors were added
to the cell suspension (antipain, leupeptin, and aprotinin, 2 Mg
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ENHANCEMENT
OF METHYLTRANSFERASE
each), and the cells were disrupted by adding Triton VI00 (linai
concentration, 0.1%) at 0°C.The transferase activity was measured by
incubating cell extracts with [3H]MNU-treated DNA, prepared as al
ready described (24), and measuring the disappearance of O'-MeGua
from this substrate. The reaction mixture contained, in a final volume
of 100 n\, the incubation buffer [70 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.6, 1 mM EDTA, and 1 mM dithiothreitol], 30 nmol of [3H]MNU-DNA (about 60 pmol of O'-MeGua),
and increasing amounts of cell extracts. After incubation at 37°Cfor
20 min, the substrate was hydrolyzed, and the remaining O6-MeGua,
was measured by high-pressure liquid chromatography, as already de
scribed (24).
Miscellaneous Determinations. To determine DNA and protein syn
thesis, the cells were incubated for l h in growth medium containing
the appropriate precursor: [3H]thymidine (0.5 //Ci/ml) or [14C]protein
hydrolysate (0.3 iiCi/ml). Radioactivity and DNA or protein concentra
tions were measured in the acid-insoluble material, as previously de
scribed (25). Measurement of total acid-soluble thiols was done as
described by Sedgwick and Robins (26).
RESULTS
Methyltransferase Activity after ^-Irradiation. H4 cells were
7-irradiated (3 Gy) and grown for 48 h, and the methyltransferase activity was determined. Fig. 1 represents the activity
measured in extracts corresponding to the same number of
control or irradiated cells. The methyltransferase activity is
enhanced in irradiated cells: 26 /¿g
of protein removed 9 and 45
fmol of O6-MeGua in control and irradiated cells, respectively.
The same experiment was run with different 7-ray doses, less
than 3 Gy, in order to avoid high cellular toxicity. For each 7ray dose, the methyltransferase activity was measured using
increasing amounts of cell extracts, and the number of meth
yltransferase molecules was calculated from the linear part of
each curve. The methyltransferase increase is detectable after a
dose of 0.5 Gy (Table 1). The maximum increase (5-fold) is
observed in cells irradiated with 3 Gy. The irradiated population
(3 Gy) contains 94% viable cells (measured by trypan blue
exclusion) at the time of the methyltransferase activity deter
mination and 30% surviving cells (measured by cloning effi
ciency) (Table 1).
In order to determine the changes of the methyltransferase
Ü
60
Ë
§30
50
PROTEIN
150
(
ACTIVITY
activity in irradiated cells with time, H4 cells were irradiated (3
Gy), and the activity was measured at different times after
irradiation. The cells were maintained in exponential growth
during the experiment. The number of methyltransferase mol
ecules per cell was calculated from the linear part of each curve
obtained with increasing amounts of cell extracts. Immediately
after irradiation, there is no change in the number of methyl
transferase molecules per cell (Fig. 2). This number begins to
increase 7 h after the irradiation, reaches a maximum value
after 48 h, and then decreases to reach the control value after
120h.
The influence of sequential 7-ray doses on the methyltrans
ferase activity was also determined. They were administered
every 48 h up to a total of 3 irradiations, and the cells were
maintained in exponential growth during this period of time.
The number of methyltransferase
molecules per cell was
285,000, 316,000, and 320,000 after 1, 2, or 3 irradiations,
respectively, and therefore this number is not significantly
different after a single or repeated 7-ray doses.
Methyltransferase Activity in CHO Cells. The methyltrans
ferase activity was measured in CHO cell extracts prepared 48
h after 7-irradiation of an exponentially growing population.
Doses between 1 and 3 Gy were delivered to these cells, as they
show the same radiosensitivity as H4 cells (data not shown).
The number of methyltransferase molecules per cell (less than
2500) was not changed after irradiation, suggesting that the
enhancement of activity does not occur in cells which have a
low constitutive level of this protein.
Macromolecular Synthesis in Irradiated Cells. In order to
determine whether this methyltransferase increase was due to
de novo protein synthesis, the cells were 7-irradiated (3 Gy) and
then cultured in the presence or absence of cycloheximide (1.5
Mg/ml) for 18 h. This incubation with cycloheximide decreased
the plating efficiency from 95 to 85%. Results (Fig. 3) show
that, in cycloheximide-treated cells, the methyltransferase ac
tivity is not enhanced by the irradiation, suggesting that the
increase of activity is due to de novo protein synthesis. It should
be noted that, in nonirradiated cells, the presence of cyclohex
imide for 18 h in the culture medium decreases the methyltrans
ferase activity to about 70% of the control value.
This enhancement of the methyltransferase activity might be
due to an increase of the overall protein synthesis in the
irradiated cells. Therefore, the protein synthesis was measured
at different times after irradiation (Table 2). The results show
a decrease of the protein synthesis with the culture time length,
as the cells become more confluent, but for each time period
studied, there is no significant difference between control and
250
Fig. 1. Removal of O'-MeGua from alkylated DNA by H4 cell extracts.
[3H]MNU-treated DNA was incubated with extracts from control (O) or yirradiated (3 Gy) cells (•).The transferase activity was measured 48 h after
irradiation.
Table 1 Number of methyltransferase molecules in -^-irradiated H4 cells
Exponentially growing H4 cells were irradiated and then either immediately
plated for survival or grown for 48 h prior to the methyltransferase activity
measurement. For details, see "Materials and Methods."
•y-ray
dose
(Gy)0
0.51.0
9280
3.0Survival(%)100
35No.
' Mean ±SD of three separate experiments.
of transferase
molecules/cell54,000
±6,600°
95,500 ±8,400
161,600 ±12,300
285,000 ±27,600
24
48
72
96
120
POST IRRADIATION TIME (hours)
Fig. 2. Number of methyltransferase molecules in H4 cells at different times
after -y-irradiation. The cells received a dose of 3 Gy at time 0 and then were
grown in fresh medium for various time lengths prior to the determination of the
transferase activity.
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ENHANCEMENT
OF METHYLTRANSFERASE
ACTIVITY
dose, is much lower in irradiated than in control cells, suggest
ing that the results described above represent a real increase of
the active methyltransferase molecules in the irradiated cells. It
should be noted that, in cells irradiated and treated with
MNNG, the number of mutants does not increase even after
expression times longer than 6 days (data not shown). The
levels of total acid-soluble thiols in control and irradiated cells
were 18.1 ±1.5 and 20.9 ±2.4 ¿ig/106cells, respectively. This
50
PROTEIN
250
150
(
Fig. 3. Influence of cycloheximide on the transferase activity in irradiated
cells. The removal of O'-MeGua from |'H]MNU-treated DNA was measured
using extracts of control cells (CD),control cells incubated for 18 h with cyclohex
imide (1.5 im in11(A), irradiated cells grown for 18 h in either control medium
(•)or the presence of cycloheximide (1.5 >ig/ml) (A).
Table 2 Protein and DNA synthesis in y-irradiated H4 cells
H4 cells were irradiated (3 Gy) or not and then grown in fresh medium for 7,
24, or 48 h. They were either incubated with [MC]protein hydrolysate or [3H]thymidine for 30 min. The incorporated radioactivity was measured as described
in "Materials and Methods."
ofTime
Specific activity
after irra
(h)7diation
ofcontrol10096.790.2
Control cells
cells24
Irradiated
8,9408,365
Control cells
cells48
Irradiated
7,0005,237
7656.5
ofcontrol100
11,1948,668
91.271.3
8,1585,061
67.141.6
Control cells
6,148%
5,880%
63DNAcpm/Mg12,152
Irradiated cellsProteinscpm/mg9,215
50.6
Table 3 Mutation frequency in H4 cells
Exponentially growing H4 cells were irradiated or not and then 48 h later
incubated for 1 h with MNNG (10 UM).The number of 6-thioguanine mutants
was determined as described in "Materials and Methods."
PretreatmentNone
treatmentNone
of mutants/105
survivors0.9
±0.2"
36.6
3.23.4
±
None1.5
MNNGNone
Gy
Gy3.0
1.5
±0.8
MNNGNoneMNNGNo. 15.5
±2.58.0
Gy
3.0 GyChallenge
" Mean ±SD of two separate experiments.
rules out the influence of sulfhydryl groups on the mutation
frequency.
Methyltransferase Activity after Various Treatments. In order
to determine whether the methyltransferase activity increased
only after -y-irradiation or also after various cell treatments, H4
cells were treated with different agents known to induce differ
ent types of damage in the cellular DNA. They were either U V
irradiated, or heated (41 or 42°C),or incubated for 1 h with
different chemical compounds; then they were grown for 48 h
in fresh medium. The methyltransferase activity was then mea
sured and compared to that of control cells. Each treatment
was delivered in order to result in the same range of survival.
The results (Table 4) show that the different treatments tested
increase the methyltransferase activity in H4 cells.
The modulation of the methyltransferase activity was also
investigated in plateau-phase H4 cells during the stationary
phase of growth. Confluent cultures were either 7-irradiated (3
Gy) or incubated for 1 h with cis-DDP (5 UM).The transferase
activity was measured 48 h after the treatments, using increas
ing amounts of cell extracts. Fig. 4 shows that the results are
similar to those obtained with exponentially growing cells. The
number of methyltransferase per cell is 46,000, 125,000, and
210,000 for control, cis-DDP-treated, and 7-irradiated cells,
respectively. The surviving fractions are 30 and 80% for irra
diated and cis-DDP-treated cells, respectively, the cis-DDP
being less toxic in plateau than in exponential cultures (27).
Therefore a similar increase of the methyltransferase activity is
observed in exponential and plateau-phase cells treated with
equitoxic doses of 7-rays or cis-DDP.
Table 4 Number of methyltransferase molecules in H4 cells 48 h after various
treatments
The cells were either UV irradiated, or heated (41 or 42'C), or incubated for
±1.5
10.3 ±2.0
1 h with the different compounds, rinsed, and grown for 48 h in fresh medium.
The number of methyltransferase molecules was then calculated from the linear
part of the curves obtained with increasing amounts of cell extracts.
irradiated cells, ruling out the role of a nonspecific increase of
the protein synthesis.
As the variations of the methyltransferase activity could be
the reflection of a cell cycle modification after irradiation, DNA
synthesis and cell growth were measured at different times after
irradiation. The DNA synthesis is slightly lower in irradiated
than in control cells (Table 2), but the variations are not in the
same range as those detected in the methyltransferase activity.
Furthermore, the doubling time of H4 cells, measured during
72 h after irradiation, was 16 and 16.5 h for control and
irradiated cells, respectively.
Mutation Frequency in MNNG-treated Cells. The mutation
frequency in MNNG-treated H4 cells, irradiated or not, was
measured in order to ascertain whether the increased methyl
transferase activity, measured in vitro using an alkylated sub
strate, represented the presence of physiologically active mole
cules in the cells. H4 cells were 7-irradiated (1.5 or 3 Gy) and
48 h later incubated for 1 h with MNNG (10 ¿¿M);
then the
mutation frequency was determined. Results (Table 3) show
that the number of mutants, obtained with the same MNNG
of methyl
transferase mole
cules/cell54,000
6,600°88.
±
treatmentNoneUV
Cell
irradiation
2.5 J/m2
5 J/m2
J/m2Heating
10
90
4096
41'C, 2h
hcis-DDP2.5
42-C, 1
32,300 ±15,600
90805555 116,100
13,4501 ±
MM
MM2-Methyl-9-hydroxyellipticinium
5.0
M
1 g/ml2.5
mlBleomycin
ni:
10 Mg/ml
25 Mg/mlSurvival(%)10098
" Mean ±SD of three separate experiments.
100 ±8,900
108,000 ±11,050
135,600
12,8801 ±
64,300 ±18,500
12,27020 ±
207,300
1,600 ±7,450
5.90089,500
±
306030No.252,000
±6,650
120,400 ±11,200
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ENHANCEMENT
OF METHYLTRANSFERASE
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DNA, did not induce the adaptive response to mutagenicity, we
proposed that the increase of the methyltransferase during
adaptation was related to the amount of O6-MeGua residues
50
150
PROTEIN
(
250
Mg
)
Fig. 4. Methyltransferase activity in plateau-phase H4 cells. [3H]MNU-treated
DNA was incubated with extracts of control (O), 7-irradiated (3 Gy) (•),or cisDOP (5 ,.\ii iriMti'd cells (A). The cells were treated during plateau phase and
used 48 h after the treatment.
DISCUSSION
The ability of H4 cells to remove O6-MeGua from alkylated
DNA is greatly increased when the cells have been previously
7-irradiated. This enhancement is time and dose dependent and
is due to de novo protein synthesis, as it is not observed when
cycloheximide is added in the culture medium. When the cells
are grown in the presence of cycloheximide, the constitutive
activity is reduced by about 30%, suggesting that a proteolytic
activity degrades 30% of the molecules. The discrepancy be
tween this degradation rate (30%) and the enhancement after
irradiation (500%) suggests that the latter process is not due to
a reduced proteolytic activity in the irradiated cells. However,
the possibility that this enhancement is due to a methyltransferase activity different from the constitutive one cannot be
ruled out. The increase of the methyltransferase activity on an
alkylated substrate in vitro correlates with a greater resistance
of the irradiated cells to the mutagenic effect of MNNG.
Induction of the methyltransferase activity does not occur in
CHO cells which have a low constitutive activity.
It has been shown that the regulation of base excision repair
was related to the proliferarne rate of a cell population (28)
and that the removal of methylated bases from DNA was cycle
dependent (29). However, the increase of methyltransferase
described in this paper is not due to a modification of the cell
cycle, as the doubling time and the DNA synthesis are not
significantly different in control and irradiated cells during the
48 h which follow the irradiation. These values are in agreement
with radiobiological data, as it has been shown (30) that irra
diated and control cells divide with apparently the same dou
bling time, the difference being that nonsurviving cells will only
carry out a limited number of divisions. Furthermore, the
increase of methyltransferase activity in stationary cultures
rules out the influence of cell cycle parameters on this process.
It also suggests that the increase observed in animals after
various treatments is not simply due to cell proliferation (16,
22).
Treatment of cells with alkylating agents results in depletion
of the number of methyltransferase molecules per cell and is
followed by a repopulation (31, 32). In irradiated cells, no
decrease of methyltransferase activity is observed after irradia
tion. Therefore, the enhancement observed cannot be explained
by a repopulation of the methyltransferase molecules whose
number could fluctuate before reaching the control value.
We have previously shown that the pretreatment of H4 cells
with repeated nontoxic doses of MNNG increased the resist
ance of these cells towards the toxic and mutagenic effects of
high MNNG doses (14) and increased the number of methyl
transferase molecules per cell (15) by about 3-fold. As other
alkylating agents, which produce less O6-MeGua in the cellular
produced in the cellular DNA during the adaptive treatment
(14). However, the increase described in this paper seems in
duced by another mechanism. In order to know whether the
methyltransferase enhancement was related to the single strand
breaks produced by T-rays, the cells were treated with agents
known to induce various DNA lesions and at doses resulting in
comparable survival. As in each case the methyltransferase
activity was increased, there is no apparent relationship between
the type of initial DNA damage and the enhancement of the
methyltransferase activity, although we cannot rule out the
implication of the DNA breaks enzymatically produced during
the repair of these lesions (33). It has also been shown that,
when the cells are confronted with adverse changes, they utilize
a defense mechanism termed heat-shock response. This re
sponse is characterized by the synthesis of specific proteins and
is accompanied by transcription and translation of genes which
were active before the environmental insult (for review, see Ref.
34). That this type of cellular response modifies the methyl
transferase activity cannot be excluded. The possibility that
other DNA repair activities are also increased is under investi
gation.
ACKNOWLEDGMENTS
The authors thank Dr. G. P. Margison for kindly communicating
his results prior to publication. The skillful technical assistance of M.
Menage was greatly appreciated.
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ENHANCEMENT
OF METHYLTRANSFERASE
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.
Enhancement of O6-Methylguanine-DNA-Methyltransferase
Activity Induced by Various Treatments in Mammalian Cells
Patricia Lefebvre and Françoise Laval
Cancer Res 1986;46:5701-5705.
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