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Molecular Biology and Genetics |
Division of Applied Toxicology, Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSPreparation of Cell Extracts...AntibodiesMeasurement of Caspase...Caspase InhibitorsNeutral Single-Cell Gel...RESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSPreparation of Cell Extracts...AntibodiesMeasurement of Caspase...Caspase InhibitorsNeutral Single-Cell Gel...RESULTSDISCUSSIONREFERENCES |
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For methylating agents inducing O6 MeG in DNA, evidence is available to show that the lesion pairs with thymine, forming a mispair that is subjected to MMR (13 , 14) . It is believed that, because of faulty repetitive repair cycles, secondary lesions are generated, leading ultimately to reproductive cell death and chromosomal damage (15, 16, 17) . This is in line with the finding that cells that are defective in MMR are more resistant to methylating agents inducing O6 MeG, although these cells do not express MGMT (so-called tolerant cells; Refs.18, 19, 20, 21 ). Overall, as regards O6 MeG as the primary critical DNA damage, both the efficiency of prereplicative repair by MGMT and the postreplicative DNA MMR determine the level of cell killing and chromosomal damage.
Whereas mutations, chromosomal aberrations, and malignant
transformation induced by O6
MeG have
been studied in detail, the molecular mechanism of cell death is only
rudimentarily understood. A deeper insight into cell killing processes
would be especially desirable in view of modifying cell death functions
to optimize tumor therapy. Recently, it has been shown that
MGMT-deficient cells are more sensitive than MGMT-proficient cells in
the induction of apoptosis, indicating that alkylation-induced
cell killing is largely attributable to apoptosis and that
O6
MeG acts as a trigger of this toxic
response (15
, 22
, 23)
. Apoptosis induced by
O6
MeG is likely to be mediated by the
same or a similar pathway acting on damaged DNA, which generates
genotoxic effects, i.e., by the participation of MMR. This
has recently been supported by demonstrating that in hamster and human
cells, MutS
, a mismatch-binding heterodimeric protein complex
composed of MSH2 and MSH6, is required for the initiation of apoptosis
in response to methylation (24)
. The apoptotic signaling,
however, i.e., the activation or inhibition of apoptotic
functions in response to O6
MeG, is
completely unknown. In the present study, we investigated the
expression of relevant apoptotic proteins and their significance for
O6
MeG-triggered apoptosis by comparing
isogenic Chinese hamster cell lines that are MGMT deficient (MGMT-),
MGMT proficient (MGMT+), and that exhibit the tolerance phenotype
(i.e., MGMT-, MMR compromised cells) upon exposure to a
potent DNA-methylating agent.
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MATERIALS AND METHODS |
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Cell Culture
The cell lines neo-C5 (here briefly designated as MGMT-)
and AT17-C3 (designated as MGMT+) are derivatives of CHO-9 and were
generated by transfection with pSV2neo and pSV2neo + pSV2
MGMT, respectively (7)
. MGMT- cells have no detectable
MGMT activity. MGMT+ cells express 750 fmol/mg protein. The line
TK22-cos9-5-1/2-C1-M (here designated as tolerant) is also a derivative
of CHO-9, which was generated as described previously
(18)
. The cells are MGMT deficient and resistant to the
cytotoxic and clastogenic effects of
O6
MeG-generating compounds. They are
cross-resistant to 6-thioguanine and display reduced G-T binding
activity and MSH2 level (18)
. Therefore, they are clearly
representative of the so-called tolerance phenotype. All cell lines
were maintained in DMEM:F-12 (1:1) supplemented with 10% inactivated
FCS containing 1.5 mg/ml G418. The addition of G418 was omitted during
the experiments.
Transfection Experiments
To overexpress Bcl-2, CHO-9 cells were stably transfected with
the expression vector pcDNA3.1(-)Myc-His harboring wild-type
bcl-2 under the transcriptional control of the
cytomegalovirus promoter (25)
. Transfection was performed
using Lipofectamine (Life Technologies, Inc.). Cells were selected with
G418 (1.5 mg/ml), and individual clones were checked for Bcl-2
expression by Western blot analysis.
Cell Death Analysis
MTT Assay.
Cell growth and cytotoxicity after treatment with MNNG was tested using
the MTT assay. Cells (1,500/well), grown in 24-well plates, were
treated with different doses of MNNG 24 h after seeding. After
incubation for 96 h, their potential to metabolize MTT was
quantitated as described (26)
with some modifications. MTT
(50 µl/500 µl of medium) dissolved in PBS (5 mg/ml) was added to
each well and incubated for 3 h at 37°C. After discarding
supernatants, 0.2 ml of 96% ethanol was added to dissolve formazan
crystals, and absorbance (A) was measured at 540 and
690 nm for reference. Results are expressed as the ratio of
A per treatment level/A of untreated control.
Clonogenic Survival Assays.
Reproductive cell death was assayed by measuring colony formation
with and without MNNG treatment as described (7)
.
Apoptosis and Necrosis.
The frequency of apoptosis and necrosis in untreated cell populations
and after their treatment with MNNG was determined using Annexin V and
flow cytometry (27)
. In this assay, unfixed cells were
double-stained with Annexin V and propidium iodide, which allows the
quantitation of both apoptotic and necrotic cells. Exponentially
growing cells were treated with MNNG, harvested by gentle
trypsinization (0.025% trypsin containing EDTA), washed with cold PBS,
subjected to Annexin V and propidium iodide staining (according to the
manufacturer’s protocol; Annexin V-FITC; PharMingen) and to flow
cytometric measurement. Evaluation of cell populations was performed
using a computer-based program (Cell Quest; Becton Dickinson).
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Preparation of Cell Extracts and Western Blotting |
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Antibodies |
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Measurement of Caspase Activities |
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Caspase Inhibitors |
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Neutral Single-Cell Gel Electrophoresis |
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSPreparation of Cell Extracts...AntibodiesMeasurement of Caspase...Caspase InhibitorsNeutral Single-Cell Gel...RESULTSDISCUSSIONREFERENCES |
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. Tolerant cells showed a clearly lower frequency of
apoptosis than MGMT- cells. It was only slightly enhanced above the
level of MGMT+ cells, which did not show significant apoptosis within
the dose range of the mutagen applied (Fig. 1A)
. Time course
experiments revealed that induction of apoptosis is a late event.
Increase of apoptosis was found only at stages later than 48 h
after pulse treatment with MNNG. Thereafter, the frequency of apoptosis
increased gradually with a high yield observed 96 and 120 h after
treatment (Fig. 1B)
. Again, in the time course experiments,
MGMT+ and tolerant cells showed lower levels of apoptosis than MGMT-
cells (Fig. 1B)
. The yield of apoptosis was higher than the
level of necrosis with all doses and times tested (<15%; data not
shown), indicating that apoptosis is the major cause of
O6
MeG-induced cytotoxicity in MGMT-
cells.
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A, MNNG provoked a lower level of expression of Bcl-2 in
MGMT- cells but not in MGMT+ cells, as measured 72 and 96 h after
treatment. Decline in Bcl-2 level was dose dependent (Fig. 2
D,
left panel). Time course analysis revealed that it did not occur
before 24 h after treatment. A decline in Bcl-2 started at 48 h after MNNG exposure and continued (reaching 25% of the control
level) up to 120 h after treatment (Fig. 2, B
and
D, right panel). Interestingly, MGMT+ cells showed a slight
up-regulation of the Bcl-2 protein level (1.8-fold) when treated with a
moderate dose of MNNG (10 µM; Fig. 2, A and D
). Tolerant cells showed nearly no
reduction of Bcl-2 as compared with MGMT- cells upon MNNG treatment
(Fig. 2C)
. The expression levels of proapoptotic Bak (Fig. 2A)
and Bax (Fig. 2B)
remained unchanged in both
MGMT- and MGMT+ cells. Overall, the results show that
O6
MeG-induced apoptosis in MGMT-
cells is associated with a reduced Bcl-2 protein level, whereas the
enhanced apoptotic resistance of MGMT+ and tolerant cells is related to
nearly unchanged Bcl-2 expression.
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. Furthermore, MGMT-depleted cells showed a clearly reduced
level of expression of Bcl-2, as measured 72 h after treatment
with MNNG (Fig. 3B)
. The data support the conclusion that
unrepaired O6
MeG leads to a decline of
Bcl-2 and the induction of apoptosis.
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A, both Bcl-2 overexpressing clones exhibited clearly reduced
frequencies of apoptosis, compared with the control. Bcl-2
overexpression also improved viability of MNNG-treated cells, as
measured by the MTT assay (data not shown) and colony formation (Fig. 4B)
. This indicates that cells overexpressing Bcl-2 were not
only protected against apoptosis but were also able to proliferate,
forming colonies. Western blot analysis revealed that transfection of
Bcl-2 did not block MNNG-induced decline of the endogenous Bcl-2. Thus,
Bcl-2 both in the control and the transfectant wt/20 decreased in
amount after treatment with MNNG (Fig. 4C)
. There was also
reduction of the transfected Myc-tagged Bcl-2, which followed a similar
dose-response as observed for the endogenous protein (Fig. 4C)
. The data indicate that the endogenous and the
transfected Bcl-2 protein are subject to destruction. They also show
that overexpressed Bcl-2 compensates for the degraded endogenous
protein, thus promoting survival.
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illustrates that Bad is present mainly in its hyperphosphorylated form
in untreated MGMT+ and MGMT- cells. MGMT- but not MGMT+ cells treated
with MNNG showed accumulation of hypophosphorylated Bad, as detected 96
and 120 h after treatment. Expression of antiapoptotic Bag-1 and
Bcl-xL remained unaltered. Thus, it appears that
in MGMT- cells treated with MNNG, the decline of Bcl-2 protein is
followed by accumulation of hypophosphorylated Bad.
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. Under the same treatment conditions, MNNG did not provoke
cytochrome c release in MGMT+ cells (Fig. 6A)
.
Regarding caspases, we found an increase in the activity of caspase-9
and caspase-3 in MGMT- cells 48–96 h after treatment with MNNG,
whereas in MGMT+ cells, both caspases remained largely unaffected (Fig. 6B)
. The Mr 85,000
breakdown product of PARP was detectable in MGMT- cells at the
earliest stage 78 h after MNNG treatment. Cleavage culminated at
96 h after treatment (Fig. 6C)
. Collectively, the data
show that CHO cells undergoing MNNG-induced apoptosis exhibit
cytochrome c release from mitochondria, activation of
caspase-9 and caspase-3, and cleavage of PARP.
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A, Fas-L was expressed in untreated CHO cells and remained
unaltered in both cell types after MNNG treatment. Incubation of
untreated and MNNG-treated MGMT- cells with anti-Fas-R-FITC-conjugated
antibody and subsequent flow cytometric measurement revealed that the
same was true for the Fas-R, i.e., the expression of Fas-R
was not changed (not shown). Also, the receptor proximal caspase-8 was
not activated in either MGMT- or MGMT+ cells treated with MNNG (Fig. 7B)
. For control, Jurkat cells were treated with
doxorubicin, which caused a nearly 3-fold induction of caspase-8,
compared with the untreated control, which is in line with a previous
report (38)
. On the basis of this data, we conclude that
MNNG induces apoptosis in MGMT- cells without triggering the
Fas-R/Fas-L system.
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. Z-VAD-FMK, a general caspase inhibitor, showed the most efficient
inhibition of apoptosis (up to 70%). Z-DEVD-FMK and Ac-DEVD-CHO, which
are selective inhibitors of the caspase-3 subfamily, and Z-LEHD-FMK, a
specific inhibitor of caspase-9, also inhibited MNNG-induced apoptosis.
In contrast, Z-IETD-FMK, a specific inhibitor of caspase-8, was
completely ineffective in protecting MGMT- cells against
MNNGinduced apoptosis. Similar results have been obtained when
apoptosis was measured 96 h after MNNG treatment (not shown). The
results support the conclusion drawn above that caspase-9 and
caspase-3, but not caspase-8, are required for
O6
MeG-induced apoptosis in CHO cells.
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, significant induction of DNA dsb was observed in MGMT- cells but not
in MGMT+ and tolerant cells. The frequency of dsb strongly increased
24–48 h after MNNG treatment and thus clearly preceded the induction
of apoptosis (Fig. 10)
. The finding that in tolerant cells dsb were not generated after MNNG
treatment indicates that MMR is involved in the induction of DNA breaks
by unrepaired O6
MeG.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSPreparation of Cell Extracts...AntibodiesMeasurement of Caspase...Caspase InhibitorsNeutral Single-Cell Gel...RESULTSDISCUSSIONREFERENCES |
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to be required for apoptosis in MGMT-deficient cells
(24)
. Overall, the data reported here support previous
findings that O6
MeG is a potent
apoptosis-inducing DNA lesion and that compromised MMR on
O6
MeG-T sites protects cells from
apoptosis (15
, 22
, 23)
.
Mismatch repair at O6
MeG-T sites takes
place both in the first (after DNA replication) and in the second
posttreatment cycle. Abortive MMR, i.e., repetitive
misincorporation of thymine opposite to O6
MeG, may create DNA
single-strand gaps leading to chromosomal breakage in the subsequent
S-phase. This model of formation of MMR-derived secondary lesions
resulting in chromosomal aberrations (15
, 40, 41, 42)
gains
support here by showing that DNA dsb were formed in replicating
MGMT-deficient cells after MNNG treatment. Interestingly, tolerant
cells as well as MGMT+ cells showed no induction of dsb and apoptosis
when treated with MNNG, indicating that formation of dsb is involved in
the initiation of apoptosis. This is further supported by findings that
show: (a) cells out of the exposure cycle do not undergo
apoptosis after MNNG treatment, although O6
MeG-T mismatches were formed in
S-phase; (b) the formation of dsb precedes the appearance of
apoptotic cells (see Fig. 10
); and (c) electroporation of
cells with restriction enzymes specifically provokes the formation of
dsb and induces decline in Bcl-2 and apoptosis as a delayed
response.5
To address the question of the apoptotic pathway that becomes activated in response to O6 MeG, we analyzed various apoptosis-regulating proteins. DNA-damaging agents may induce apoptosis by activating p53-dependent functions via death receptors (such as tumor necrosis factor receptor-1, Fas, or DR5; Refs. 43, 44, 45 ) and/or by mitochondrial damage (46 , 47) . Our data show that expression of Fas (CD95, Apo-1) receptor and Fas ligand is not altered in MGMT- CHO cells upon methylation. More importantly, caspase-8, the most proximal caspase in Fas receptor signaling, was not activated, and inhibition of caspase-8 by Z-IETD-FMK did not block apoptosis. We thus conclude that O6 MeG does not trigger apoptosis via Fas receptor signaling. We should note that CHO cells are mutated for p53 (48 , 49) , which was shown to up-regulate the Fas-R in response to DNA damage (45) . p53 was also reported to up-regulate Bax (50) and to turn on redox-related genes, leading to the generation of reactive oxygen species and mitochondrial damage (51) . Despite the central role p53 is ascribed in apoptosis, its involvement appears to be dependent on cell type and the inducing agent. Clearly, apoptosis in the cell system we were working with does not require p53, and therefore, we can conclude that O6 MeG leads to apoptosis in CHO cells independently of p53. The p53-independent apoptotic pathway seems to play an important role especially in fibroblasts, which is supported by data obtained with p53 knockout fibroblasts from mice. These cells are highly sensitive to the induction of apoptosis by alkylating agents, suggesting that in fibroblasts p53 is not required for apoptosis; it rather protects against it, very likely because of prereplicative DNA repair.6 However, in other cell types, notably lymphoblastoid and liver cells, DNA damage-induced apoptosis is dependent on functional p53 and Fas-R/caspase-8 (52 , 53) . Therefore, in these cell types DNA damage-induced signaling is likely to be different from that in fibroblasts.
We show that a hallmark of O6 MeG-induced apoptosis is the reduction of the expression level of the antiapoptotic protein Bcl-2. Bcl-2 decline was observed prior to and concurrent with the induction of apoptosis in MGMT-deficient cells (and in MGMT- depleted cells upon incubation with the MGMT inhibitor O6 -benzylguanine) but not in MGMT-proficient and -tolerant cells. To our knowledge, this is the first report demonstrating reduced expression of Bcl-2 in response to unrepaired O6 MeG. Furthermore, the results obtained with the tolerant cells demonstrate that protection from induction of apoptosis by defective MMR is associated with steady expression of Bcl-2. To further prove the participation of Bcl-2 in regulation of apoptosis in this cell system, we stably transfected MGMT-deficient cells with wild-type Bcl-2. Overexpression of Bcl-2 blocked MNNG-induced apoptosis by >60% and reduced MNNG cytotoxicity. Overall, the findings strongly indicate that Bcl-2 acts at a proximal step in O6 MeG-triggered apoptosis. The ability of Bcl-2 to protect cells against the induction of apoptosis was also shown for ionizing radiation, alkylating agents, and various chemotherapeutic drugs (22 , 54, 55, 56) . While studying apoptosis in response to the chemotherapeutic agents vincristine and etoposide, Zhang et al. (56) reported a protection by Bcl-2 overexpression but stated that Bcl-2 fails to maintain the ability of the cells to proliferate. In contrast, in our experiments Bcl-2-overexpressing cells deficient for MGMT exhibited a clearly enhanced colony-forming ability upon treatment with MNNG and continued to proliferate. We should note that none of the Bcl-2-overexpressing cell clones we isolated became as resistant as the wild-type when treated with MNNG. This indicates that the signal triggered by O6 MeG-derived lesions must be very strong; it cannot be completely alleviated by Bcl-2 overexpression. It should also be noted that decline of endogenous Bcl-2 still occurred in Bcl-2-overexpressing cells, and that, at the same time, the level of the transfected Bcl-2 was reduced as well. This may be taken to indicate that decline in Bcl-2 level is not attributable to down-regulation of Bcl-2 gene expression but rather is a posttranslational phenomenon. Future work will focus on the mechanism of Bcl-2 down-modulation. It has been shown that inhibitors of microtubuli formation, such as Taxol and vincristine, induce phosphorylation of Bcl-2, which neutralizes the antiapoptotic function of the protein (57) . Whether Bcl-2 phosphorylation provokes Bcl-2 instability is unknown. It will be interesting to see whether a Bcl-2 decline in MGMT- cells upon alkylation is attributable to protein instability or controlled degradation.
Bcl-2 is known to target the protein kinase Raf-1 to mitochondria, allowing the kinase to phosphorylate and thereby inactivate proapoptotic Bad (33 , 58) . Our studies revealed an accumulation of hypophosphorylated Bad after decline of Bcl-2 in apoptotic MGMT-deficient cells. This finding corresponds to the reported ability of hypo- and unphosphorylated Bad to dimerize with Bcl-xL and thereby displace Bax, which then promotes cell death (34) . This is in line with our finding of a reinforcement of apoptosis when the hypophosphorylated form of Bad was dominating (96 h after treatment). From this we conclude that Bad participates in controlling O6 MeG-triggered apoptosis by directly amplifying Bcl-2-mediated signaling.
Damage to mitochondria can result in release of cytochrome c
to the cytosol (59)
. Cytochrome c causes
caspase activation by forming a complex with Apaf-1 and procaspase-9,
leading to activation of caspase-9, which in turn activates
procaspase-3 (60
, 61)
. In this process, Bcl-2 is thought
to be involved by preventing the release of cytochrome c
from mitochondria and thereby the activation of caspases (35
, 62)
. We show that decline of Bcl-2 triggered by
O6
MeG in MGMT- cells is accompanied
by accumulation of cytosolic cytochrome c and activation of
caspase-9 and caspase-3. Caspase-9 and caspase-3 activation clearly
preceded O6
MeG-induced apoptosis (Fig. 10)
, indicating that mitochondria-mediated caspase activation is
decisively involved. Experiments with caspase inhibitors confirmed
this. Both the general caspase inhibitor Z-VAD-FMK and the specific
caspase-9 inhibitor Z-LEHD-FMK, as well as the inhibitors of
caspase-3-like proteases, Z-DEVD-FMK and Ac-DEVD-CHO, significantly
reduced apoptosis in response to
O6
MeG. Contrary to this, no inhibition
of apoptosis was achieved with Z-IETD-FMK, a specific inhibitor of
caspase-8. This supports the conclusion that activation of caspase-9
and caspase-3 is important for
O6
MeG-triggered apoptosis, whereas
caspase-8 is not essential.
We did not observe a change in the level of Bcl-xL and Bax, which heterodimerizes with Bcl-2 and forms dimers with itself (63, 64, 65) . A decline in Bcl-2 level in MGMT- cells after MNNG treatment may result in increased formation of Bax homodimers. Its generation in apoptotic MGMT- cells is likely further enhanced by lack of Bcl-2-mediated hyperphosphorylation of Bad, which binds to Bcl-xL, thus setting Bax free. Overall, our data are consistent with the model that apoptosis induced by O6 MeG is triggered by reduction of the relative ratio of Bcl-2/Bax, causing cytochrome c liberation from mitochondria. It should be noted that lack of cytochrome c in mitochondria may lead to uncoupling of electron chain transport, yielding a burst of reactive oxygen radicals that further damage the cell (64) . Reactive oxygen radicals, however, are not likely to be important for O6 MeG-triggered apoptosis because incubation of MGMT-deficient cells treated with MNNG with various antioxidants completely failed to inhibit apoptosis (data not shown). We should note that only a small proportion of cytochrome c from mitochondria was found to be released. It therefore appears that sufficient cytochrome c still remains within mitochondria, thus maintaining electron transport.
We also show that activation of caspase-9 and caspase-3 after MNNG treatment of MGMT-deficient cells results in cleavage of PARP. Whether PARP cleavage is directly involved in the induction of apoptosis is still unclear. It is unlikely, however, that it is an essential step in O6 MeG-induced apoptosis because it started to take place at a time (>78 h after treatment with MNNG) when apoptotic cells were already appearing. Thus, it seems that PARP cleavage is a late side effect of caspase-9 and caspase-3 activation.
The time sequence of the main apoptotic functions measured in MGMT-
cells upon treatment with MNNG is summarized in Fig. 10
. It is
interesting to note that the formation of dsb precedes the decline of
Bcl-2 and the activation of caspase-9 and caspase-3. Although the data
do not reject the hypothesis that faulty MMR directly activates
apoptotic functions, they support the view that dsb are involved in
triggering the decline of Bcl-2. A model for
O6
MeG-induced apoptosis based on
the available data is shown in Fig. 11
. In summary, O6
MeG induces apoptosis
via mispairs, which are subject to MMR. The repair intermediates
(possibly nonsealed, gapped DNA) may lead to DNA dsb (e.g.,
by nuclease attack at stalled replication forks), which finally
activate functions that down-modulate Bcl-2. This leads to leakiness of
mitochondria, cytochrome c release, and finally activation
of downstream caspase-9 and caspase-3. Decline of Bcl-2 appears to play
a major role in DNA damage-induced, p53/Fas-R/caspase-8-independent
apoptosis in fibroblasts because we observed it not only in
MGMT-deficient cells but also in other hypersensitive cell lines
characterized by defective DNA repair, such as base excision
repair-defective mouse fibroblasts and nucleotide excision
repair-defective Chinese hamster
cells.7
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5% of tumors are
lacking MGMT (65)
; these tumors are likely to be highly
responsive to methylating agents. On the other hand, there is a
tendency toward increase in the MGMT level in tumors with increasing
malignancy (65
, 66) . Interestingly, p53 is involved in
regulation of basal MGMT gene activity (66, 67, 68)
and thus, indirectly, may exert influence on methylating drug
resistance. Nevertheless, because
O6
MeG is a very powerful
apoptosis-inducing lesion, it would be desirable to inhibit its repair
specifically in tumor cells. Selective, highly specific repair
inhibitors of MGMT are available (69)
, and attempts are
currently being made to target them to tumor cells. Because MMR is
essential for O6
MeG-triggered
apoptosis, information on the MMR status of tumors could also be of
predictive value. Also, in view of the decisive role of Bcl-2 in O6
MeG-induced apoptosis, it would
be of interest to develop strategies to inhibit Bcl-2 expression in
tumor cells. A future chemotherapy might thus be based on a combination
of inhibition of DNA repair and amplification of apoptotic signaling in
tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Deutsche
Forschungsgemeinschaft SFB 519/B4 and Stiftung Rheinland-Pfalz. 
2 To whom requests for reprints should be
addressed, at Division of Applied Toxicology, Institute of Toxicology,
University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz,
Germany. Phone: 49-6131-393-3246; Fax: 49-6131-393-3421; E-mail: Kaina{at}mail.uni-mainz.de
3 The abbreviations used are: MGMT,
O6-methylguanine-DNA methyltransferase;
MGMT+, MGMT proficient; MGMT-, MGMT deficient; MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine;
O6 MeG,
O6-methylguanine;
O6-BG,
O6-benzylguanine; dsb, DNA double-strand
breaks; PARP, poly(ADP-ribose) polymerase; Fas-L, Fas ligand; Fas-R,
Fas receptor; MMR, mismatch repair; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; pNA,
paranitroanilide; CHO, Chinese hamster ovary; Z-VAD-FMK,
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; Z-DEVD-FMK,
Z-Asp-Glu-Val-Asp-FMK; Ac-DEVD-CHO,
N-acetyl-Ile-Glu-Thr-Asp-CHO(aldehyde); Z-IETD-FMK,
Z-Ile-Glu(OMe)-Thr-Asp(OMe)-FMK; Z-LEHD-FMK,
Z-Leu-Glu(OMe)-HisAsp(OMe)-FMK.
4 T. Dunkern and B. Kaina, unpublished
data.
5 J. Lips and B. Kaina, unpublished data.
6 D. Lackinger and B. Kaina, Primary mouse
fibroblasts deficient for c-Fos, p53, or for both proteins are
hypersensitive to UV light and alkylating agent-induced chromosomal
breakage and apoptosis, Mutation Res., in press.
Received 1/21/00. Accepted 8/14/00.
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REFERENCES |
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R. Stupp, M. E. Hegi, M. R. Gilbert, and A. Chakravarti Chemoradiotherapy in Malignant Glioma: Standard of Care and Future Directions J. Clin. Oncol., September 10, 2007; 25(26): 4127 - 4136. [Abstract] [Full Text] [PDF]
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 B. Yamini, X. Yu, M. E. Dolan, M. H. Wu, D. W. Kufe, and R. R. Weichselbaum Inhibition of Nuclear Factor-{kappa}B Activity by Temozolomide Involves O6-Methylguanine Induced Inhibition of p65 DNA Binding Cancer Res., July 15, 2007; 67(14): 6889 - 6898. [Abstract] [Full Text] [PDF]
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V. O'Brien and R. Brown Signalling cell cycle arrest and cell death through the MMR System Carcinogenesis, April 1, 2006; 27(4): 682 - 692. [Abstract] [Full Text] [PDF]
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 E. J. Fox, D. T. Leahy, R. Geraghty, H. E. Mulcahy, D. Fennelly, J. M. Hyland, D. P. O'Donoghue, and K. Sheahan Mutually Exclusive Promoter Hypermethylation Patterns of hMLH1 and O6-Methylguanine DNA Methyltransferase in Colorectal Cancer J. Mol. Diagn., February 1, 2006; 8(1): 68 - 75. [Abstract] [Full Text] [PDF]
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 M. Hidaka, Y. Takagi, T. Y. Takano, and M. Sekiguchi PCNA-MutS{alpha}-mediated binding of MutL{alpha} to replicative DNA with mismatched bases to induce apoptosis in human cells Nucleic Acids Res., October 4, 2005; 33(17): 5703 - 5712. [Abstract] [Full Text] [PDF]
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 E. Peterson-Roth, M. Reynolds, G. Quievryn, and A. Zhitkovich Mismatch Repair Proteins Are Activators of Toxic Responses to Chromium-DNA Damage Mol. Cell. Biol., May 1, 2005; 25(9): 3596 - 3607. [Abstract] [Full Text] [PDF]
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 M. E. Hegi, A.-C. Diserens, T. Gorlia, M.-F. Hamou, N. de Tribolet, M. Weller, J. M. Kros, J. A. Hainfellner, W. Mason, L. Mariani, et al. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma N. Engl. J. Med., March 10, 2005; 352(10): 997 - 1003. [Abstract] [Full Text] [PDF]
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 V. A. Barvaux, P. Lorigan, M. Ranson, A. M. Gillum, R. S. McElhinney, T. B. H. McMurry, and G. P. Margison Sensitization of a human ovarian cancer cell line to temozolomide by simultaneous attenuation of the Bcl-2 antiapoptotic protein and DNA repair by O6-alkylguanine-DNA alkyltransferase Mol. Cancer Ther., October 1, 2004; 3(10): 1215 - 1220. [Abstract] [Full Text] [PDF]
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E. Gonzalez-Suarez, F. A. Goytisolo, J. M. Flores, and M. A. Blasco Telomere Dysfunction Results in Enhanced Organismal Sensitivity to the Alkylating Agent N-Methyl-N-Nitrosourea Cancer Res., November 1, 2003; 63(21): 7047 - 7050. [Abstract] [Full Text] [PDF]
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 D. Petrin, A. Baker, S. G. Coupland, P. Liston, M. Narang, K. Damji, B. Leonard, V. A. Chiodo, A. Timmers, W. Hauswirth, et al. Structural and Functional Protection of Photoreceptors from MNU-Induced Retinal Degeneration by the X-Linked Inhibitor of Apoptosis Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2757 - 2763. [Abstract] [Full Text] [PDF]
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 M. T. Tomicic, C. Friedrichs, M. Christmann, P. Wutzler, R. Thust, and B. Kaina Apoptosis Induced by (E)-5-(2-Bromovinyl)-2'-deoxyuridine in Varicella Zoster Virus Thymidine Kinase-Expressing Cells Is Driven by Activation of c-Jun/Activator Protein-1 and Fas Ligand/Caspase-8 Mol. Pharmacol., February 1, 2003; 63(2): 439 - 449. [Abstract] [Full Text] [PDF]
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A. W. Adamson, W.-J. Kim, S. Shangary, R. Baskaran, and K. D. Brown ATM Is Activated in Response to N-Methyl-N'-nitro- N-nitrosoguanidine-induced DNA Alkylation J. Biol. Chem., October 4, 2002; 277(41): 38222 - 38229. [Abstract] [Full Text] [PDF]
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T. J. Page, S. O'Brien, C. R. Jefcoate, and C. J. Czuprynski 7,12-Dimethylbenz[a]anthracene Induces Apoptosis in Murine Pre-B Cells through a Caspase-8-Dependent Pathway Mol. Pharmacol., August 1, 2002; 62(2): 313 - 319. [Abstract] [Full Text] [PDF]
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M. Christmann, M. T. Tomicic, and B. Kaina Phosphorylation of mismatch repair proteins MSH2 and MSH6 affecting MutS{alpha} mismatch-binding activity Nucleic Acids Res., May 1, 2002; 30(9): 1959 - 1966. [Abstract] [Full Text] [PDF]
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 S. Mohr, X. Xi, J. Tang, and T. S. Kern Caspase Activation in Retinas of Diabetic and Galactosemic Mice and Diabetic Patients Diabetes, April 1, 2002; 51(4): 1172 - 1179. [Abstract] [Full Text] [PDF]
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K. Ochs, J. Lips, S. Profittlich, and B. Kaina Deficiency in DNA Polymerase {beta} Provokes Replication-dependent Apoptosis via DNA Breakage, Bcl-2 Decline and Caspase-3/9 Activation Cancer Res., March 1, 2002; 62(5): 1524 - 1530. [Abstract] [Full Text] [PDF]
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J. Lips and B. Kaina DNA double-strand breaks trigger apoptosis in p53-deficient fibroblasts Carcinogenesis, April 1, 2001; 22(4): 579 - 585. [Abstract] [Full Text] [PDF]
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 T. R. Dunkern and B. Kaina Cell Proliferation and DNA Breaks Are Involved in Ultraviolet Light-induced Apoptosis in Nucleotide Excision Repair-deficient Chinese Hamster Cells Mol. Biol. Cell, January 1, 2002; 13(1): 348 - 361. [Abstract] [Full Text] [PDF]
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