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ORIGINAL ARTICLE
Apoptosis in malignant glioma cells triggered by the temozolomide-induced
DNA lesion O
6
-methylguanine
WP Roos
1
, LFZ Batista
2
, SC Naumann
1
, W Wick
3
, M Weller
3
, CFM Menck
2
and B Kaina
1
1
Department of Toxicology, University of Mainz, Mainz, Germany;
2
Department of Microbiology, Institute of Biomedical Sciences,
University of Sao Paulo, Sao Paulo, SP, Brazil and
3
Department of General Neurology, Hertie Institute for Clinical Brain Research,
University of Tu
¨bingen, School of Medicine, Tu
¨bingen, Germany
Methylating drugs such as temozolomide (TMZ) are
widely used in the treatment of brain tumours (malignant
gliomas). The mechanism of TMZ-induced glioma cell
death is unknown. Here, we show that malignant glioma
cells undergo apoptosis following treatment with the
methylating agents N-methyl-N0-nitro-N-nitrosoguanidine
(MNNG) and TMZ. Cell death determined by colony
formation and apoptosis following methylation is greatly
stimulated by p53. Transfection experiments with O
6
-
methylguanine-DNA methyltransferase (MGMT) and
depletion of MGMT by O
6
-benzylguanine showed that, in
gliomas, the apoptotic signal originates from O
6
-methyl-
guanine (O
6
MeG) and that repair of O
6
MeG by MGMT
prevents apoptosis. We further demonstrate that O
6
MeG-
triggered apoptosis requires Fas/CD95/Apo-1 receptor
activation in p53 non-mutated glioma cells, whereas in
p53 mutated gliomas the same DNA lesion triggers the
mitochondrial apoptotic pathway. This occurs less effec-
tively via Bcl-2 degradation and caspase-9, -2, -7 and -3
activation. O
6
MeG-triggered apoptosis in gliomas is a late
response (occurring >120 h after treatment) that requires
extensive cell proliferation. Stimulation of cell cycle
progression by the Pasteurella multocida toxin promoted
apoptosis whereas serum starvation attenuated it. O
6
MeG-
induced apoptosis in glioma cells was preceded by the
formation of DNA double-strand breaks (DSBs), as
measured by cH2AX formation. Glioma cells mutated in
DNA-PK
cs
, which is involved in non-homologous end-
joining, were more sensitive to TMZ-induced apoptosis,
supporting the involvement of DSBs as a downstream
apoptosis triggering lesion. Overall, the data demonstrate
that cell death induced by TMZ in gliomas is due to
apoptosis and that determinants of sensitivity of gliomas to
TMZ are MGMT, p53, proliferation rate and DSB repair.
Oncogene (2007) 26, 186–197. doi:10.1038/sj.onc.1209785;
published online 3 July 2006
Keywords: apoptosis; DNA damage; DNA repair;
MGMT; glioblastoma; Fas; p53
Introduction
During the last years, anticancer drugs with methylating
properties (such as temozolomide [TMZ], procarbazine,
dacarbazine, streptozotocine) have received much atten-
tion, notably in the therapy of malignant gliomas.
These drugs target DNA, inducing about a dozen DNA
methylation products (Beranek, 1990). Studies with O
6
-
methylguanine-DNA methyltransferase (MGMT)-defi-
cient cells (Sklar and Strauss, 1981; Scudiero et al., 1984;
Preuss et al., 1996) and MGMT-transfected isogenic cell
lines (Kaina et al., 1991) unequivocally revealed that
one of the lesions, the minor alkylation product O
6
-
methylguanine (O
6
MeG), is a most potent killing lesion.
It acts as a powerful trigger of apoptosis (Kaina et al.,
1997; Tominaga et al., 1997; Meikrantz et al., 1998). In
the cell killing process driven by O
6
MeG mismatch
repair (MMR) is essentially involved (Kat et al., 1993;
Kaina et al., 1997; Hickman and Samson, 1999; Pepponi
et al., 2003). If O
6
MeG is repaired, cells are either
resistant to O
6
MeG-triggered apoptosis or die because
of harmful N-alkylation lesions such as 3-methyladenine,
3-methylguanine and apurinic sites that have arisen
from N-methylpurine hydrolysis (Lindahl, 2000). The
contribution of O
6
MeG to cell death depends on
the ratio of O
6
MeG to N-alkylations in the DNA, the
capacity of the cell to repair O
6
MeG and the rate of
repair of N-alkylations (Kaina et al., 1997). The repair
of O
6
MeG also provokes protection against the
mutagenic, clastogenic and carcinogenic effects of
O
6
-alkylating agents, suggesting that O
6
MeG is not only
an apoptotic but also a genotoxic (for a review see
Margison and Santibanez-Koref, 2002), tumour-initiat-
ing (Dumenco et al., 1993; Becker et al., 1996) and
tumour-converting (Becker et al., 2003) lesion.
Repair of O
6
MeG is accomplished by the suicide repair
protein MGMT via direct methyl group transfer from the
oxygen in guanine to a cystein residue (Cys145) in the
MGMT molecule. Guanine in DNA is thereby restored and
MGMT gets inactivated (for a review see Pegg, 2000).
Because of the stochiometry of the reaction the repair
capacity strictly depends on the amount of pre-existing
MGMT molecules in the cell. The level of MGMT in
tumours is highly variable. Quite low amounts are expressed
in brain tumours (Chen et al., 1992; Preuss et al., 1995;
Silber et al., 1999; Bobola et al., 2001), presumably due to
Received 7 March 2006; revised 22 May 2006; accepted 23 May 2006;
published online 3 July 2006
Correspondence: Professor B Kaina, Department of Toxicology,
University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz,
Germany.
E-mail: kaina@uni-mainz.de
Oncogene (2007) 26, 186–197
&
2007 Nature Publishing Group
All rights reserved 0950-9232/07 $30.00
www.nature.com/onc
MGMT promoter methylation (Esteller et al., 1999, 2001).
MGMT expression level (Chen et al., 1999; Anda et al.,
2003) and, as recently shown, MGMT promoter methyla-
tion (Esteller et al., 2000; Hegi et al., 2004, 2005; Paz et al.,
2004) were predictive for the clinical response to chemother-
apy, stressing the critical role of MGMT in determining
alkylating drug resistance (for a review see Gerson, 2004).
Brain tumours, notably malignant gliomas, are highly
therapy-refractory tumours. Therapy of gliomas includes
tumour resection, followed by radiotherapy and chemother-
apy that usually involves O
6
-alkylating agents. The
methylating agent TMZ was shown to prolong survival of
patients when administered during and after radiotherapy
as part of the first-line treatment (Stupp et al., 2005).
Despite the usefulness of O
6
-methylating agents in
glioma therapy, the median survival times of patients
suffering from the most severe form glioblastoma multi-
forme are still remarkably low (12–14 months). Ob-
viously, there is an urgent need for improving glioma
therapy. One goal that needs to be reached in achieving
this would be to improve our knowledge on the
mechanism of alkylating agent-induced death in glioma
cells. Whereas the mechanism of apoptosis induced by
O
6
-methylating agents has been elucidated in great detail
in various experimental systems such as rodent cell lines
(Ochs and Kaina, 2000), lymphoblastoid cells (Dunkern
et al., 2003; Hickman and Samson, 2004) and peripheral
human lymphocytes (Roos et al., 2004), it remains
enigmatic in malignant gliomas and other tumour types.
Thus, for human glioma cells, TMZ was reported to
induce either a p53-associated G2/M arrest followed
by senescence or p53-independent mitotic catastrophe
(Hirose et al., 2001). In another study, TMZ was
proposed to induce autophagy but failed to induce
apoptosis in malignant glioma cells (Kanzawa et al.,
2004), whereas glioma cells grown as spheriods have been
shown to be able to undergo apoptosis following
alkylating agent treatment (Gunther et al., 2003). These
conflicting data prompted us to study the mechanism of
glioma cell death upon treatment with methylating agents
in detail. Here, we show that human malignant glioma
cells undergo apoptosis dose and time dependently upon
methylating agent treatment for which the specific DNA
lesion O
6
MeG is the decisive trigger. We also demonstrate
that MGMT protects against TMZ-induced apoptosis in
gliomas. O
6
MeG-triggered apoptosis in glioma cells is
dependent on the p53 status that determines whether cells
undergo death receptor or mitochondrial apoptosis.
Furthermore, we show the importance of DNA double-
strand break (DSB) formation and cell proliferation in
O
6
MeG-triggered apoptosis of glioma cells. Clinical
implications will also be discussed.
Results
Methylating agents reduce colony formation of
glioma cells
The cytotoxic effect of the powerful O
6
-methylating
agent N-methyl-N0-nitro-N-nitrosoguanidine (MNNG)
on U87MG (p53wt) and U138MG (p53mt) glioma cells
was examined in colony-forming survival assays. As
shown in Figure 1, U87MG (p53wt) cells are clearly
more sensitive to the killing effect of MNNG than
U138MG (p53mt) cells.
Induction of apoptosis following MNNG or TMZ
treatment and influence of the p53 status on apoptosis
To show that the killing effect observed after methylat-
ing agent treatment in p53 wild-type and p53 mutant
glioma cells is related to the induction of apoptosis, the
apoptotic response was measured as a function of drug
concentration. MNNG and TMZ induced apoptosis in a
concentration dependent manner (Figure 2a and b).
Importantly, the onset of apoptosis was observed at
very late time points in cultures that were growing
exponentially throughout the whole time of the experi-
ment, starting at 96 h after MNNG treatment and
increasing in frequency up to 144 h (Figure 2f). p53 wild-
type glioma cells (U87MG) were clearly more sensitive
than p53 mutated cells (U138MG) to the induction of
apoptosis by MNNG and TMZ through the whole
concentration range tested (Figure 2a and b), indicating
p53 to be involved in triggering apoptosis upon methy-
lation. To substantiate the role of p53 in methylating
agent-induced apoptosis, p53 was inhibited in both cell
lines using the specific inhibitor pifithrin-a. In U87MG
(p53wt) cells, inhibition of p53 caused a significant
decrease in the apoptotic response following MNNG
and TMZ treatment whereas pifithrin-ahad no effect in
p53 mutant cells (Figure 2c and d). To determine
whether MNNG can cause the stabilization and nuclear
localization of p53 in glioma cells, we treated U87MG
Figure 1 Colony formation after genotoxic treatment. Dose–
response curves after MNNG treatment of U87MG (p53 wild-type
’) and U138MG (p53 mutant m) glioma cells. Data are the mean
of three independent experiments.
Alkylating agent-induced apoptosis in glioma cells
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(p53wt) cells with 5 mMMNNG and determined the
p53 protein levels in nuclear extracts using Western blot
analysis. The p53 level increased in the nucleus of p53
wild-type glioma cells after MNNG treatment. This
increase was observed at late time points (Figure 2e),
corresponding to the induction of apoptosis (Figure 2f).
Alkylating agent-induced apoptosis in glioma cells
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In time course experiments, an extra p53 mutant glioma
cell line was included, namely LN308 (p53mt). Both
p53 mutant cell lines, U138MG and LN308, showed a
clearly lower apoptotic response than the p53 wild-type
cell line U87MG (Figure 2f). An additional experiment
supporting the influence of p53 on the induction of
apoptosis was performed by using U87MG glioma
cells stably transfected with siRNA targeted to p53
(Figure 2g). In these cells, p53 was clearly less expressed
than in the parental line (Figure 2g, inset). With a
clinically relevant concentration of TMZ (100 mM), the
knockdown of p53 in U87MG cells significantly reduced
apoptosis. Collectively, the data demonstrate that, upon
methylation, p53 is involved in determining the cyto-
toxic and apoptotic response of glioma cells.
Influence of MGMT on O
6
MeG-triggered apoptosis
The question of whether O
6
MeG lesions are responsible
for apoptosis upon DNA methylation in glioma cells
had to be answered next. To this end, U87MG (p53wt)
and U138MG (p53mt) cells were stably transfected with
MGMT in order to compare the apoptotic response
in cells not expressing and expressing MGMT (see
Figure 3a and b for protein levels). Whereas U87MG
(p53wt) and U138MG (p53mt) cells showed no MGMT
activity, U87MGMT and U138MGMT showed
MGMT activities of B600 and B800 fmol/mg protein,
respectively (Figure 3c and d). MGMT overexpression
decreased apoptosis in U87MG (p53wt) and U138MG
(p53mt) cells by approximately 90% after MNNG (1 or
5mM) treatment (Figure 3e and f). When inhibiting
MGMT in U87MG and U138MG cells that were stably
transfected with MGMT (designated as U87MGMT
and U138MGMT respectively) with O
6
-benzylguanine
(O
6
BG), the cells showed a dramatic increase in apop-
tosis upon treatment with MNNG (Figure 3e and f). As
MGMT specifically repairs O
6
-methylations (the minor
lesion O
4
-methylthymine can be neglected), the strong
apoptotic response in MGMT deficient gliomas and the
inhibition of apoptosis in MGMT overexpressing
glioma cells clearly show that at least 90% of the
apoptotic signalling upon TMZ treatment originates
from O
6
MeG lesions.
Replication dependence of O
6
MeG-triggered apoptosis in
glioma cells
The late onset of apoptosis upon pulse methylation of
proliferating cells indicates that apoptosis occurs not
in the treatment but in one of the post-treatment cell
cycles. To elucidate the dependence of apoptosis on
cell cycle progression following O
6
MeG induction, two
strategies were employed: (1) inhibiting cells from
progressing through the cell cycle and (2) by pushing
cells to divide faster. Firstly, slowing or stopping the
proliferation of U87MG (p53wt) cells by serum starva-
tion provoked the apoptotic response to decrease by
approximately 55% (Figure 4a). Secondly, we proved
the opposite argument that forcing glioma cells to
progress through the cell cycle will give rise to a stronger
apoptotic response. For this reason, U87MG (p53wt)
cells were treated with the powerful mitogen PMT,
thereby increasing the proliferation rate of the cells
(Figure 4b, left panel). Treatment with PMT leads to
more than double the apoptotic frequency (Figure 4b,
right panel). Thus, manipulation of cell cycle progres-
sion after methylating agent exposure demonstrates that
glioma cells require cellular proliferation in order for
O
6
MeG to be able to trigger the apoptotic response. It is
obvious that extensive proliferation stimulates the
apoptotic response provoked by O
6
MeG lesions.
Importance of the formation of DNA DSBs on apoptosis
following O
6
-methylguanine induction
It has been shown previously that unrepaired O
6
MeG
lesions give rise to DSBs (Ochs and Kaina, 2000; Roos
et al., 2004). It has also been shown that ‘clean’ DSBs
are a powerful trigger of apoptosis (Lips and Kaina,
2001). To substantiate a role of DSBs in methylating
agent-induced apoptosis for glioma cells, the formation
of DSBs were assayed following O
6
-methylating agent
treatment. In U87MG (p53wt) cells, MNNG treat-
ment led to the formation of DSBs as visualized by an
increase in the presence of phosphorylated H2AX
(Figure 5a). This was again observed at late time points
(48–96 h), preceding the onset of apoptosis. Next, we
determined whether methylation-induced DSBs are also
related to apoptosis in glioma cells. To this end, MO59K
glioma DNA-PK wild-type and MO59J glioma DNA-
PK-deficient cells were compared following MNNG
(Figure 5b) and TMZ (Figure 5c) treatment. To deplete
MGMT completely, cells were pretreated with O
6
BG.
In both cases, the DNA-PK-deficient cells were more
sensitive to apoptosis induction than wild-type cells
(Figure 5b and c). As DNA-PK is involved in the repair
of DSBs by non-homologous end joining (NHEJ), these
data support the hypothesis that in glioma cells DSBs
are critically involved in O
6
MeG-triggered apoptosis.
It has been suggested that inhibition of transcription
may trigger apoptosis (Ljungman and Zhang, 1996).
Therefore, we checked the level of transcriptional
Figure 2 Apoptosis induced by MNNG and TMZ in p53 wild-type and p53 mutant glioma cells. (a) Dose–response curve of MNNG-
treated U87MG (J) and U138MG () glioma cells after 144 h incubation time. (b) Dose–response curve of TMZ-treated U87MG (J)
and U138MG () glioma cells after 144 h incubation time. (c) Apoptosis of U87MG and U138MG cells after 10 mMMNNG treatment
at 144 h in the presence and absence of the p53 inhibitor pifithrin-a(induced apoptosis was corrected for control). (d) Apoptosis of
U87MG and U138MG cells after 0.1 mMTMZ treatment at 144 h in the presence and absence of the p53 inhibitor pifithrin-a. Data are
the mean of at least three independent experiments (induced apoptosis was corrected for control). (e) p53 nuclear localization in
U87MG p53 wild-type glioma cells after 5mMMNNG treatments at indicated time points (the level of induced apoptosis is shown that
is corrected for control). (f) Time response after 10 mMMNNG treatment in U87MG (’) p53 wild-type, U138MG (m) and LN-308
(E) p53 mutant glioma cells. (g) Apoptosis in U87MG (p53wt) cells transfected with siRNA targeting p53 following treatment with
100 mMTMZ (induced apoptosis means corrected for untreated cells). The appropriate controls are also shown.
Alkylating agent-induced apoptosis in glioma cells
WP Roos et al
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Oncogene
inhibition by TMZ. As shown in Figure 5d, TMZ
(100 mM) did not attenuate the rate of transcription as
measured immediately (2 h) and late (48 h) after treat-
ment. It rather stimulated transcription at late post-
exposure times in U87MG (p53wt) cells. Therefore,
transcriptional inhibition can be excluded as a mechan-
ism of TMZ-induced apoptosis.
Death receptor signalling in O
6
MeG-triggered apoptosis
in glioma cells
To determine whether glioma cells utilize the death
receptor mediated apoptotic pathway triggered by
O
6
MeG, the expression of the Fas receptor (Fas,
CD95, Apo-1) was assayed. In membrane extracts
of U87MG (p53wt) cells, a clear induction of Fas was
observed following MNNG treatment (Figure 6a). This
increase in Fas protein was again observed at late times
(Figure 6a). U138MG (p53mt) cells did not express Fas
(Figure 6a). By using a specific antagonizing antibody
against Fas, thereby inactivating receptor signalling, a
decrease in TMZ-induced apoptosis of approximately
55% was observed in U87MG (p53wt) cells, whereas no
Figure 4 Replication dependence of O
6
MeG-triggered apoptosis.
(a) Cell cycle distribution in the presence and absence of FCS (left
panel). Relative apoptotic response in U87MG (p53wt) cells in the
presence and absence of FCS after 0.1 mMTMZ treatment at 144 h
(right panel). (b) Relative cell number after treatment with the
wild-type (PMTwt) and mutant (PMTC1165s) mitogen PMT in
U87MG (p53wt) cells at 120 h (left panel). Relative apoptosis
induced with 5 mMMNNG in the presence of either PMT
wt
or
PMTC1165s at 120 h (right panel). Data are the mean of three
experiments. The histogram is a representative of one experiment.
Figure 3 Influence of MGMT on O
6
MeG-triggered apoptosis.
(a) Western blot analysis of U87MG MGMT-transfected glioma
cells. (b) Western blot analysis of U138MG MGMT-transfected
glioma cells. (c) MGMT activity in U87MG MGMT-transfected
cells. (d) MGMT activity in U138MG MGMT-transfected cells.
(e) Relative apoptotic response of U87MG MGMT transfected
cells after 1 and 5 mMMNNG treatment at 144 h in the presence
and absence of the specific MGMT inhibitor O
6
BG. (f) Relative
apoptotic response of U138MG MGMT-transfected cells after 1
and 5 mMMNNG treatment at 144 h in the presence and absence of
the specific MGMT inhibitor O
6
BG. Representative Western blots
are shown. Quantitative measurements and apoptosis experiments
were repeated at least three times.
Alkylating agent-induced apoptosis in glioma cells
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effect was seen in U138MG (p53mt) cells (Figure 6b). A
second functional assay to demonstrate the contribution
of death receptor signalling in glioma p53 wild-type
cells was by generating U87MG (p53wt) and U138MG
(p53mt) dominant-negative-FADD (DN-FADD) stable
transfectants (for expression level see Figure 6c
and d). Transfection of U87MG (p53wt) with
DN-FADD protected cells against O
6
MeG-triggered
apoptosis throughout the whole concentration range
tested (Figure 6c). No protective effect was observed for
U138MG (p53mt) cells (Figure 6d).
Mitochondrial signalling in O
6
MeG-triggered apoptosis in
U138MG (p53mt) cells
It has been shown that in p53-mutated rodent cells,
O
6
MeG triggers the mitochondrial apoptotic pathway
characterized by a decline in Bcl-2 protein level (Ochs and
Kaina, 2000). Therefore, we followed the hypothesis that
in p53 mutant glioma cells the mitochondrial pathway is
activated in response to O
6
MeG lesions. Indeed, as shown
in Figure 7a, after MNNG treatment U138MG (p53mt)
cells exhibit the characteristic decline in Bcl-2. This was
not observed in p53wt glioma cells (data not shown).
Bcl-2 decline was accompanied by caspase-9 activation
as well as the activation of the executive caspases-3
and -7 (Figure 7b–d). Also caspase-2 became activated
(Figure 7e) whereas no caspase-8 activation was observed
in U138MG (p53mt) cells (Figure 7f). These data are in
agreement with the view that in p53-mutated glioma cells,
O
6
MeG triggers the mitochondrial-mediated apoptotic
pathway whereas in p53 wild-type cells the same lesion
triggers the death receptor pathway.
Caspase inhibition attenuates O
6
MeG-triggered apoptosis
The activation of multiple caspases, as shown in
Figure 7, prompted us to determine whether apoptosis
Figure 5 DNA DSBs and transcription in O
6
MeG-triggered apoptosis. (a) Phosphorylation of histone H2AX in U87MG (p53wt)
cells after 5 mMMNNG treatment at indicated time points. (b) Time response of induced apoptosis in MO59K DNA-PK wild-type and
MO59J DNA-PK mutant glioma cells after 5 mMMNNG and 10 mMO
6
BG treatment. (c) Time response of induced apoptosis in
MO59K DNA-PK wild-type and MO59J DNA-PK mutant glioma cells after 0.5 mMTMZ and 10 mMO
6
BG treatment. (d) Stimulation
of transcription after TMZ treatment. Data of at least three experiments were pooled.
Alkylating agent-induced apoptosis in glioma cells
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Oncogene
Figure 6 Influence of death receptor signalling on O
6
MeG-triggered apoptosis. (a) Western blot analysis of Fas receptor induction
after 5 mMMNNG treatment in U87MG (p53wt) and U138MG (p53mt) cells. ERK2 was used as the loading control. (b) Apoptosis in
U87MG and U138MG cells after 0.1 mMTMZ treatment in the presence and absence of Fas neutralizing antibody at 144h. (c) Dose–
response curves of apoptosis in U87MG (p53wt) (’) and DN-FADD-transfected cells (m) at 144 h (experiments were performed at
least 3 times). Western blot analysis for positive clone is included where ERK2 was used as the loading control. (d) Apoptosis response
in U138MG (p53mt) (’) and DN-FADD-transfected cells (m) at 144 h. Western blot analysis for positive clone is included where
ERK2 was used as the loading control.
Figure 7 Expression of Bcl-2 and caspases in U138MG cells treated with MNNG. (a) Western blot analysis of Bcl-2 and (b–f)
caspases. For caspase-8, -2, -7 and -3 the antibody was directed against the active fragment. For caspase-9 both the inactive and the
active protein is recognized. The activated form is indicated by arrow. Exponentially growing cells were treated with 5 mMMNNG and
cells were harvested at indicated time points.
Alkylating agent-induced apoptosis in glioma cells
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triggered by O
6
MeG lesions can be blocked by caspase
inhibition. It should be kept in mind that the triggering
of apoptosis by O
6
MeG in glioma cells is dependent on
cellular proliferation (Figure 4) and occurs at very late
times following exposure (Figure 2f). Therefore, an
inhibitor approach might exhibit only low effectiveness
particularly if the inhibitor is unstable and the substrate
gets re-synthesized. In this approach, we applied
the broad-spectrum caspase inhibitor Boc-D-fmk. In
U87MG (p53wt) and U138MG (p53mt) cells, the
caspase inhibitor was able to decrease the apoptotic
response by approximately 30% (Figure 8). This
inhibition of apoptosis was statistically significant in
both cell lines (Po0.001). Together with the demon-
stration of caspase activation (Figure 7 and data not
shown), the attenuation of apoptosis by Boc-D-fmk
demonstrates that caspases are involved in O
6
MeG-
triggered apoptosis in glioma cells.
Discussion
Patients suffering from malignant glioma, in particular
glioblastoma multiforme, have a very poor prognosis.
Standard therapy today, in addition to surgery and
radiotherapy, includes treatment with alkylating agents,
specifically TMZ and the chloroethylating drugs car-
mustine and nimustine. Currently and in the future, the
use of TMZ and presumably also other methylating
drugs will become more dominant than chloroethylating
agents because of less severe side effects. The common
use of O
6
-methylating agents in glioma therapy pro-
vokes the question of how these agents exert their killing
effects and, based on the data obtained, is it possible to
improve glioma chemotherapy? As the molecular action
of O
6
-alkylating agents in gliomas (and other tumour
types) is largely unknown, this study was aimed at
elucidating the mode of death of glioma cells upon
treatment with O
6
-methylating agents.
Here, we show that glioma cells undergo apoptosis
following treatment with TMZ or MNNG (that acts in
the same way as TMZ upon decomposition into reactive
carbenium ions). Apoptosis in glioma cell lines is a very
late response, occurring no earlier than 4 days after
pulse treatment of exponentially growing cultures. This
late response might explain the failure of detection of
apoptosis in previous studies with glioma cells. Modula-
tion of MGMT activity by pharmacological inhibition
with O
6
BG (Dolan et al., 1990) or MGMT cDNA
transfection (Kaina et al., 1991) revealed that O
6
MeG is
the major proapoptotic DNA lesion in malignant
glioma cells upon O
6
-methylating agent treatment. As
MGMT strongly protects against O
6
MeG-triggered
apoptosis, the data also confirm that MGMT is a
decisive determinant of glioma resistance to O
6
-methy-
lating agents.
An intriguing observation was that p53 wild-type-
expressing glioma cells were more sensitive than their
p53 mutant counterparts, and that pharmacological
inhibition of p53 by pifithrin-aor knockdown of p53 by
siRNA provoked O
6
-methylating agent resistance. This
clearly illustrates that p53 is involved in O
6
MeG-
triggered apoptosis in gliomas. As both p53 wild-type
and p53 mutant glioma cells were able to undergo
apoptosis in response to TMZ, we conclude that p53 is
not absolutely required, but stimulates the apoptotic
process. The p53 gene is often mutated in human
cancers. Secondary glioblastomas that develop from
grade II or III astrocytomas exhibit a p53 mutation
frequency of 65%, whereas primary glioblastomas
exhibit a quite low p53 mutation frequency of 10%.
As p53 wild-type exerted a strong positive effect on
O
6
-methylating agent sensitivity, p53 must be considered
as a predictive factor in glioma therapy. As p53 mutant
glioma cells are more resistant to TMZ, the data also
implicate that p53-mutated tumour cells may be selected
after treatment with TMZ or other methylating agents,
resulting in recurrent gliomas that would not respond to
the same treatment.
How does p53 stimulate apoptosis triggered by
O
6
MeG in glioma cells? In p53 wild-type, but not in
p53 mutant glioma cells, O
6
-methylating agents pro-
voked the induction of Fas (CD95, Apo-1) receptor
expression and activation of the Fas-dependent apopto-
tic pathway (Figure 6), including activation of caspase-8
(data not shown). Transfection with DN-FADD atte-
nuated apoptosis, which suggests that Fas is involved
in O
6
MeG-triggered apoptosis in p53 wild-type glioma
cells. p53 is a transcriptional activator of Fas (Muller
et al., 1998; Pohl et al., 1999), which explains its
upregulation upon treatment of glioma cells with O
6
-
methylating agents. Interestingly, in p53 mutant glioma
cells Fas and caspase-8 were not induced, whereas Bcl-2
declined and caspase-9 and -3 were activated in response
to MNNG. The decline of Bcl-2 is a hallmark of
O
6
MeG-triggered apoptosis in p53-mutated cells (Ochs
and Kaina, 2000). The data therefore show that in p53
mutant glioma cells, O
6
-methylating agents are able to
trigger the intrinsic mitochondrial apoptotic pathway. It
is important to note that the mitochondrial pathway is
Figure 8 Influence of caspase inhibition on O
6
MeG-triggered
apoptosis. U87MG (p53wt) and U138MG (p53mt) cells were
treated with 5 mMMNNG and 5 mMO
6
BG in the presence and
absence of the broad-spectrum caspase inhibitor Boc-D-fmk. Boc-
D-fmk (50 mM) was added 72 h after MNNG treatment, before the
onset of apoptosis, and then 25 mMevery 24 h until samples were
stopped. Data are the mean of three experiments7s.d.
Alkylating agent-induced apoptosis in glioma cells
WP Roos et al
193
Oncogene
less efficiently evoked than the death receptor pathway.
Consequently, at equimolar doses of TMZ p53 wild-
type glioma cells deficient in MGMT exhibited a much
greater response than p53 mutant cells. In both
p53 wild-type and mutant glioma cells, transfection
with MGMT blocked apoptosis almost to completion.
This shows that O
6
MeG is the main trigger of apoptosis
irrespective of the p53 status of cells. Given the data,
p53 can be considered as a decision maker of whether
the same DNA lesion, O
6
MeG, triggers the death
receptor or the mitochondrial apoptotic pathway (see
Figure 9). The potency of the lesion to trigger, in the
absence of p53, the mitochondrial pathway is, however,
clearly lower than to trigger the death receptor pathway.
How does p53 get activated in response to O
6
MeG?
The current model supported by a bulk of data is
outlined in Figure 9. It proposes that O
6
MeG does
not trigger apoptosis on its own. It rather becomes
converted into a critical downstream lesion that is
supposed to be DSBs. This occurs, according to a
reasonable model, by mispairing of O
6
MeG lesions with
thymine, which in turn is processed by MutSa-depen-
dant mismatch repair provoking a futile MMR cycle
(Karran and Bignami, 1994; Hampson et al., 1997).
Secondary lesions will be formed that cause replication
interference in the subsequent DNA replication cycle
leading ultimately to DSBs (Ochs and Kaina, 2000). As
shown recently, the glioma cells used in this study
express similar levels of the MMR proteins MSH2
and MSH6 (Hermisson et al., 2006). They show DSB
formation, as demonstrated by gH2AX induction that
preceded apoptosis. A key repair pathway for DSBs is
NHEJ that involves DNA-PKcs. Indeed, apoptosis
induced by MNNG and TMZ was enhanced in DNA-
PKcs mutated glioma cells, which supports the view that
DSBs are formed in response to O
6
MeG, contributing to
apoptosis. The apoptotic response may occur by
stabilization of p53 via the ATM/ATR-Chk1 pathway
(O’Connell and Cimprich, 2005). Interestingly, however,
cells deficient in ATM are hypersensitive to methy-
lating agents (Debiak et al., 2004). Similarly, cells
lacking XRCC2 are hypersensitive to TMZ and MNNG
(Tsaryk et al., 2005). This supports a role of ATM and
XRCC2 in the repair of DSBs originating from O
6
MeG
lesions rather than apoptotic signalling. The apoptotic
response of gliomas upon TMZ treatment can also be
provoked by a p53-independent activity that degrades
Bcl-2. The signalling involved in the Bcl-2-driven path-
way still remains enigmatic.
The model described above implicates DNA replica-
tion and cell proliferation as essential elements in
O
6
MeG-triggered apoptosis in glioma cells. This is
indeed the case, as substantiated by experiments in
which proliferation was either blocked or stimulated.
Inhibition of glioma cell proliferation clearly reduced
the frequency of apoptosis upon O
6
-methylating
agent treatment, whereas stimulation of proliferation
by treatment with the powerful mitogen, Pasteurella
multocida toxin (PMT) (Orth et al., 2003) significantly
enhanced apoptosis. Therefore, proliferation rate ap-
pears to be an important predictive factor in O
6
MeG-
driven apoptosis in gliomas and very likely also in other
tumour types. These data also show that any prolifera-
tion blocking treatment applied together or after
TMZ might attenuate the therapeutic effect of TMZ.
We should note that the concentration range of the
methylating agents used throughout this work was
relatively low, allowing full recovery and propagation
of cells. For TMZ, the concentration range corresponds
to serum levels achieved during treatment, namely
100 mM(Hammond et al., 2004).
In summary, the data reported here show that glioma
cells die after methylating agent treatment owing to the
induction of apoptosis. This apoptotic response is
primarily due to O
6
MeG lesions formed in the DNA.
The apoptotic pathway employed by glioma cells
depends on their p53 status. In p53 wild-type gliomas,
O
6
MeG lesions trigger the death receptor pathway
whereas in p53 mutant cells the same lesion activates
Figure 9 A model of O
6
MeG-triggered apoptosis in p53 wild-type
and p53 mutated glioma cells. For explanation see Discussion.
Alkylating agent-induced apoptosis in glioma cells
WP Roos et al
194
Oncogene
the mitochondrial pathway. The modulation of cell
proliferation and DSB repair had a significant impact on
O
6
MeG-triggered apoptosis. The data obtained suggest
that predictive markers of TMZ sensitivity of gliomas
are MGMT activity, proliferation rate, p53 status and
the efficiency of DSB repair.
Materials and methods
Cell lines and culture conditions
The glioma cell lines U87MG, U138MG, LN-308, M059K and
M059J were used in this study. U87MG is p53 wild-type
whereas U138MG and LN-308 are p53 mutant (Wischhusen
et al., 2003). M059K is DNA-PK wild type and M059J lack the
p350 component of DNA-PK (Allalunis-Turner et al., 1995).
All cell lines were cultured in Dulbecco’s modified Eagle’s
medium containing 10% fetal bovine serum.
Clonogenic survival assays
Colony-forming assays were performed as previously
described (Roos et al., 2000). Shortly, U87MG (p53wt) and
U138MG (p53mt) glioma cells, growing in log phase were
used. Cells were seeded in triplicate at appropriate cell
numbers in 60 mm Petri dishes to yield approximately 100
surviving colonies after MNNG treatment. Cells were allowed
to attach and then exposed to increasing concentrations of
MNNG. After 2–3 weeks colonies were fixed (in acetic
acid:methanol:H
2
O 1:1:8), stained (in 0.01% amido black)
and colonies containing 50–100 cells were counted. The
surviving fraction was plotted on a log scale and fitted to the
linear-quadratic equation.
Apoptosis determined by flow cytometry
The apoptotic response after genotoxic drug treatment was
measured using the flow cytometric method of sub-G1
determination. Treated and untreated cells were harvested,
washed once with phosphate-buffered saline (PBS) and fixed in
70% ethanol. Ethanol-fixed cells were stained with propidium
iodide (16.5 mg/ml) in PBS after RNase (0.03 mg/ml) digestion.
Samples were analysed in a FACS Calibur (Becton Dickinson,
Heidelberg, Germany). For each sample 10 000 cells were
analysed. The number of apoptotic cells was calculated using
the Cell Quest software (Heidelberg, Germany).
Drugs and drug treatment
MNNG (Sigma, Munich, Germany) stock solution was
prepared by dissolving MNNG in dimethyl sulfoxide and then
diluting it with sterile dH
2
O. TMZ (Schering-Plough,
Kenilworth, NJ, USA) stocks were prepared by dissolving
the drug in ethanol and diluting it with sterile dH
2
O. Stock
solutions were filtered, aliquoted and stored at 801C. Glioma
cell lines were treated with increasing concentrations of either
MNNG or TMZ and then harvested after 144 h to determine
apoptosis. For the time responses, the glioma cells were treated
with MNNG and then harvested at the appropriated times.
The p53 inhibitor pifithrin-a, which reversibly blocks p53
dependent transcriptional activation (Komarova and Gudkov,
2000), was added 72 h after treatment with either 10 mM
MNNG or 100 mMTMZ and apoptosis was determined at
time point 144 h. The specific MGMT inhibitor O
6
BG (Pegg
et al., 1993) was added 1 h before MNNG treatment at a
concentration of 10 mM. The powerful mitogen, Pasteurella
multocida toxin (PMT
wt
), and the inactive mutant PMT
C1165S
(Orth et al., 2003) were kept in a 1.1 mg/ml stock solution
(50 mMTris-HCl, pH 7.5, 50 mMNaCl, 2.5 mMCaCl
2
)at
201C and used at a concentration of 10 ng/ml (the mitogen
was a kind gift of Dr Klaus Aktories, Freiburg). Caspases were
inhibited using the broad-spectrum caspase inhibitor Boc-D-
FMK (Calbiochem, San Diego, USA). After treating U87MG
p53 wild-type and U138MG p53 mutant glioma cells with 5 mM
MNNG and O
6
BG, caspases were inhibited with adding 50 mM
Boc-D-FMK 72 h after methylation and then 25 mMevery 24 h
until the samples were harvested at 144 h.
Preparation of protein extracts
Fractionated cell extracts Cell pellets of treated and untreated
samples were suspended in fractionation buffer A (10 mM
HEPES–KOH, pH 7.4, 0.1 mMethylene diaminetetraacetic
acid (EDTA), 1 mMethylene glycol-bis (b-aminoethyl ether),
250 mMsucrose, 1 mMNa
3
VO
4
, 0.5 mMphenylmethylsulfonyl
fluoride (PMSF) and 10 mMdithiothreitol (DTT)). The cells
were lysed by freeze/thaw/vortexing. The lysate was then
centrifuged at 10 000 r.p.m. for 10 min and the supernatant
containing the cytoplasmic proteins was isolated. The pellet,
containing the nuclei, organelles and membranes, were then
suspended in fractionation buffer B (20 mMTris, 1 mMEDTA,
1m
Mb-mercaptoethanol, 5% glycerine, 1 mMNa
3
VO
4
, 0.5 mM
PMSF, 10 mMDTT, pH 8.5). This suspension was homo-
genized by sonication. After centrifugation at 10 000 r.p.m.
for 10 min the supernatant contains the nuclear proteins
and the pellet the membrane fragments. This membrane pellet
was suspended in fractionation buffer B containing 1% Triton
X-100. The protein concentration was determined by the
method of Bradford (Bradford, 1976).
Cell extracts for MGMT activity assay Cells were harvested
and homogenized by sonication in buffer containing 20 mM
Tris-HCl, pH 8.5, 1 mMEDTA, 1 mMb-mercaptoethanol,
5% glycerol and the protease inhibitor PMSF (0.1 mM).
The extract was centrifuged at 10 000 r.p.m. (10 min) in the
cold in order to remove debris and the supernatant was
snap-frozen in aliquots using liquid nitrogen and stored at
801C until use.
Western blot analysis
The method used here is based on the method described by
Renart et al. (1979). Protein (30 mg) of cell extracts was
separated in a 12% SDS polyacrylamide gel. Thereafter,
proteins were blotted onto a nitrocellulose transfer membrane
(Protran; Schleicher & Schuell, Dassel, Germany) for 3 h.
Membranes were blocked for 2 h at room temperature in 5%
(wt/vol) fat-free milk powder in TBS containing 0.1% Tween
20, incubated overnight at 41C with the primary antibody
(1:500–1000 dilution), washed three times with 0.1% Tween 20
in TBS, and incubated for 2 h with a horseradish peroxidase-
coupled secondary antibody 1:3000 (Amersham Biosciences
AB, Uppsala, Sweden). Antibodies used were anti-p53 (BD
PharMingen, San Diego, USA), anti-Fas (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), anti-Bcl-2 and
anti-Bax (Santa Cruz Biotechnology Inc.), anti-GAPDH
(Ambion Inc.), anti-MGMT (Chemicon International Inc.),
anti-ERK2 (Santa Cruz Biotechnology Inc.), anti-gH2AX
(Upstate, Dundee, Scotland), anti-caspase-8 (Cell Signalling,
Danvers, USA), anti-caspase-9 (Cell Signalling), anti-caspase-
2 (Neomarkers, Fremont, USA), anti-caspase-7 (Cell signal-
ling) and anti-caspase-3 (Cell Signalling). After final washing
with 0.1% Tween 20 in TBS (3 times for 10 min each) blots
were developed by using a chemiluminescence detection system
(Amersham Biosciences AB).
Alkylating agent-induced apoptosis in glioma cells
WP Roos et al
195
Oncogene
Transfection of glioma cells with MGMT, siRNA (p53)
and DN-FADD
MGMT transfectants were generated by co-transfection of
U87MG (p53wt) and U138MG (p53mt) cells with the
mammalian expression vector (pSV2MGMT) harbouring
the MGMT gene described previously (Kaina et al., 1991)
and the pSV2neo plasmid for selection. The transfection
method employed was the Effectene transfection kit (Qiagen,
Hilden, Germany). In brief, B1.5 mg of pSV2MGMT and
0.2 mg pSV2neo were transfected and then the transfected cells
were selected for with firstly 1.5 mg/ml G418 for 72 h and then
0.5 mg/ml until clones formed. G418-resistant clones were
picked in 24-well plates and tested for MGMT expression
using Western blot and MGMT activity assay. Transfectant
clones were routinely cultured in a medium containing 0.5 mg/ml
G418 (Sigma-Aldrich, Munich, Germany) that was omitted
during the experiments. DN-FADD transfectants were generated
in U87MG (p53wt) and U138MG (p53mt) cells by transfecting
B1.5 mg pcDNA3-FADD-DN (Tewari and Dixit, 1995) in the
same way as MGMT except that this plasmid also contained the
neo gene so co-transfection was unnecessary. FADD-DN-
positive clones were determined by Western blotting. siRNA
transfectants with siRNA targeted towards p53 have already
been described (Wischhusen et al., 2003).
RNA synthesis
RNA synthesis was determined based on a method described
previously (Balajee et al., 1997). Approximately 4.0 10
4
cells
were plated in 35 mm Petri dishes and 24 h after plating they
were treated with TMZ (0.1 mM) and maintained with the drug
for the indicated times. After this period, cells were incubated
in a medium containing 3% dialysed FCS and (5-
3
H)-uridine
(
3
H-Udi 4.0 mCi/ml, Amersham-Pharmacia Biotech, Uppsala,
Sweden) for 30 min. Cells were then harvested and separated
into two samples. In one of the samples, cells were lysed
(NaCl 0.3 M; Tris-HCl pH 8.0 20 mM; EDTA 2 mM; SDS 1%
and K proteinase 200 mg/ml) and then transferred to Whatman
17 paper and washed twice with 15% trichloroacetic acid
and hydrated ethanol for 30 min, for radioactivity measure-
ment. The second sample was used to determine the
absorbance at 260 nm, for data normalization. The ratio
between radioactivity and absorbance expresses the RNA
synthesis in these cells.
Fas receptor neutralization
Fas receptor (CD95/Apo1) was inhibited by adding 1 mg/ml
anti-Fas neutralizing antibody (ZB4) (Biozol Diagnostica
Vertrieb GmbH, Eching, Germany) 72 h after MNNG
treatment and then every 24 h till samples were harvested.
The percentage of population undergoing apoptosis was
determined after 144 h.
MGMT activity assay
The MGMT activity in U87MG (p53wt), U138MG (p53mt)
and MGMT co-transfected cells were determined using a
method based on the radioactive assay where tritium-labelled
methyl groups are transferred from the O
6
-position of guanine
to protein in the cell extract (Preuss et al., 1995). HeLa S3 cells
expressing MGMT (588786 fmol/mg protein) and HeLa MR
cells deficient in MGMT served as positive and negative
controls, respectively. The radioactivity of the protein was then
measured. For each assay cell extracts containing 200 mg
protein, as determined by the method of Bradford (Bradford,
1976), was incubated with [
3
H]methyl-nitrosourea-labelled calf
thymus DNA containing O
6
-MeG (total 80 000 c.p.m./sample)
in 700 mMHEPES–KOH (pH 7.8), 10 mMDTT, 50 mM
EDTA for 90 min. Data are expressed as femtomoles of
radioactivity transferred from
3
H-labelled DNA to protein per
milligram of protein within the sample.
Acknowledgements
This Work was supported by Deutsche Forschungsge-
meinschaft, DFG KA 724/13-1 and 13-2 and SFB 432/B7
(Mainz), NGFN-2 (Tu
¨bingen) as well as FAPESP (Sao Paulo,
Brazil). We gratefully acknowledge Georg Nagel and Andrea
Piee-Staffa for technical assistance. We also acknowledge a
generous gift of PMT from Professor Klaus Aktories, Freiburg
and TMZ from Schering-Plough.
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