[Cancer Research 60, 3212-3217, June 15, 2000]
© 2000 American Association for Cancer Research
Experimental Therapeutics |
Mitochondrial Amplification of Death Signals Determines Thymidine Kinase/Ganciclovir-triggered Activation of Apoptosis1
Christian Beltinger2,
Simone Fulda2,
Thomas Kammertoens,
Wolfgang Uckert and
Klaus-Michael Debatin3
University Children’s Hospital, 89075 Ulm [C. B., S. F., K-M. D.], and Max-Delbrueck Center for Molecular Medicine, 13092 Berlin [T. K., W. U.], Germany
 |
ABSTRACT
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Previous clinical experience shows that the efficacy of suicide gene
transfer in tumor therapy is limited, resulting from inefficient gene
transfer or alternatively, from intrinsic resistance of the tumor
in vivo. Herpes simplex virus thymidine
kinase/ganciclovir (TK/GCV), a paradigmatic suicide gene therapy
system, has been described to exert its cytotoxic effect, at least in
part, by inducing apoptosis in target cells. Here, we report that
mitochondria amplify TK/GCV-induced apoptosis by regulating p53
accumulation and the effector phase of apoptosis. Treatment with TK/GCV
led to mitochondrial perturbations including loss of the mitochondrial
membrane potential and release of cytochrome c from
mitochondria into the cytosol, inducing caspase activation and nuclear
fragmentation. Inhibition of TK/GCV-induced mitochondrial perturbations
by Bcl-2 overexpression or by the mitochondrion-specific inhibitor
bongkrekic acid also strongly inhibited TK/GCV-induced activation of
caspases and apoptosis. TK/GCV-induced mitochondrial perturbations
depended on caspase activity possibly initiated by death receptor
signaling. Perturbation of mitochondrial function mediated accumulation
of wild-type p53 protein, since Bcl-2 overexpression, bongkrekic acid,
or inhibition of mitochondrial protein synthesis with chloramphenicol
strongly reduced TK/GCV-induced accumulation of wild-type p53 protein.
These findings suggest that TK/GCV therapy may be less efficient in
tumors in which the mitochondrial amplification of TK/GCV-induced
apoptosis is blocked, e.g., by Bcl-2 overexpression.
Given the low efficacy of currently used gene therapy systems, our data
on molecular mechanisms that regulate sensitivity or resistance toward
TK/GCV-induced cytotoxicity might have important implications to
improve the clinical application of suicide gene therapy.
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INTRODUCTION
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After an initial wave of enthusiasm, suicide gene therapy systems
such as the herpes simplex
TK/GCV4
system did not meet the expectations once they entered the clinical
stage. Low target specificity and low transfection efficacy of target
cells have been discussed to contribute to the limited success,
e.g., in the treatment of malignant brain tumors. However,
thus far the molecular basis for TK/GCV cytotoxicity is poorly
understood. Suicide gene therapy has been described to exert its
effect, at least in part, by inducing apoptosis in target cells
(1
, 2)
. Although DNA damage may be involved in the
initiation of the cell death response (3)
, the exact
mechanisms that regulate apoptosis signaling pathways in target cells
after suicide gene therapy have not yet been defined.
Apoptotic cell death is involved in the regulation of tissue
homeostasis in numerous physiological and pathological conditions.
Three apoptosis signaling systems play a major role in the regulation
of the initiation or the effector phase of apoptosis
(4, 5, 6, 7, 8, 9, 10, 11, 12)
: death receptor/ligand systems such as the CD95
system that are predominantly involved in the initiation phase of
apoptosis; caspases as effector molecules; and mitochondria that may
provide initiator as well as effector functions. Mitochondria have been
reported to play a key role in various apoptotic processes including
cell death induced by cytotoxic agents (5)
. Mitochondria
undergoing permeability transition release apoptogenic proteins such as
cytochrome c or apoptosis-inducing factor from the
mitochondrial intermembrane space into the cytosol, where they can
activate caspases and endonucleases. Mitochondrial function during
apoptosis seems to be controlled by the Bcl-2 family of proteins
(13)
. Bcl-2 and several of its homologues have been
localized to intracellular membranes including the mitochondrial
membrane. Overexpression of the antiapoptotic molecules Bcl-2 and
Bcl-XL has been found to confer resistance to
anticancer treatment. Bcl-2 and Bcl-XL may
inhibit apoptosis through the capacity to prevent permeability
transition and/or to stabilize the barrier function of the outer
mitochondrial membrane (14)
. These apoptosis pathways are
partially redundant, and because they are interconnected amplification
loops are possible.
In the present report, we investigated the role of mitochondria in
suicide gene therapy and found that mitochondria amplify TK/GCV-induced
apoptosis by regulating both the initiation and the effector phase of
apoptosis.
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MATERIALS AND METHODS
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Drugs.
GCV (Hoffmann La Roche, Grenzach, Germany) was provided as pure
substance and dissolved in water and stored at -80°C.
Cell Culture.
SHEP neuroblastoma cells transfected with control vector (pLXSN) or TK
(pHyTK) or double transfected with Bcl-2 and TK (pHyTK/Bcl-2) were
cultured in RPMI 1640 (Life Technologies, Inc., Eggenstein, Germany)
supplemented with 10% heat-inactivated FCS (Conco, Wiesbaden,
Germany), 10 mM HEPES (pH 7.3; Biochrom, Berlin, Germany),
100 U/ml penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin
(Life Technologies, Inc.), 2 mM L-glutamine
(Biochrom), 0.5 mg/ml G418 (Life Technologies, Inc.), or 0.5 mg/ml
hygromycin (Life Technologies, Inc.) as previously described (1
, 11
, 15) .
Determination of Apoptosis.
Quantification of DNA fragmentation was performed by FACS analysis of
propidium iodide-stained nuclei as previously described
(16)
. Cells were analyzed for DNA content by flow
cytometry (FACScan; Becton Dickinson, Heidelberg, Germany) with
CELLQuest software.
Inhibition of Drug-induced Apoptosis or Loss of
m by zVAD.fmk or BA.
The broad spectrum tripeptide inhibitor of caspases zVAD.fmk (Enzyme
Systems Products, Livermore, CA) was used at a concentration of
60 µM, and the mitochondrion-specific inhibitor BA was
used at a concentration of 50 µM (kindly provided by Dr.
Duine, University of Delft, Delft, the Netherlands).
Assessment of Mitochondrial Potential.
The cationic lipophilic fluorochrome DiOC6(3)
(460 ng/ml; Molecular Probes, Eugene, OR) was used to measure the
m. Cells were incubated for 12 min at
37°C in the presence of the fluorochrome, washed in PBS/1% FCS, and
immediately analyzed by flow cytometry (FACScan).
DiOC6(3)
was recorded in fluorescence 1. The
percentage of cells with low mitochondrial potential was calculated in
comparison with untreated control cells.
Western Blot Analysis.
Cells were lysed for 30 min at 4°C in PBS with 0.5% Triton X (Serva,
Heidelberg, Germany) and 1 mM PMSF (Sigma, Deisenhofen,
Germany) followed by high speed centrifugation. Membrane proteins were
eluted in buffer containing 0.1 M glycine, pH 3.0, and 1.5
M Tris, pH 8.8. Protein concentration was assayed with
bicinchoninic acid (Pierce, Rockford, IL). Protein, 40 µg/lane, was
separated by 12% SDS-PAGE and electroblotted onto nitrocellulose
membranes (Amersham Pharmacia, Freiburg, Germany). Equal protein
loading was controlled by Ponceau red staining of membranes. After
blocking for 1 h in PBS supplemented with 2% BSA (Sigma) and
0.1% Tween 20 (Sigma), immunodetection of caspase-8, caspase-3, PARP,
and cytochrome c protein was conducted with the use of mouse
anti-caspase-8 monoclonal antibody C15 [1:10 dilution of hybridoma
supernatant (17)
], mouse anti-caspase-3 monoclonal
antibody (1:1,000; Transduction Laboratories, Lexington, KY), rabbit
anti-PARP polyclonal antibody (1:10,000; Boehringer Mannheim, Mannheim,
Germany), or mouse anti-cytochrome c monoclonal antibody
(1:5,000; PharMingen, San Diego, CA) and goat anti-mouse IgG or goat
anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA).
ECL (Amersham) was used for detection.
Preparation of Mitochondria, Cytosolic Extracts, and Nuclei.
For isolation of mitochondria, cells (3 x
108/sample) were washed twice with ice-cold PBS
and resuspended with 5 volumes of buffer A [50 mM Tris, 1
mM EGTA, 5 mM 2-mercaptoethanol, 0.2% BSA, 10
mM KH2PO4 (pH
7.6), 0.4 M sucrose] and allowed to swell on ice for 20 min. Cells
were homogenized with 30 strokes of a Teflon homogenizer and
centrifuged at 10,000 x g for 10 min at
4°C. The resulting pellets were resuspended in buffer B [10
mM
KH2PO4 (pH 7.2), 0.3
mM mannitol, 0.1% BSA]. Mitochondria were
separated by sucrose gradient [lower layer, 1.6 M sucrose, 10
mM
KH2PO4 (pH 7.5), 0.1% BSA;
upper layer, 1.2 M sucrose, 10 mM
KH2PO4 (pH 7.5), 0.1%
BSA]. Interphases containing mitochondria were washed with buffer B at
18,000 x g for 10 min at 4°C, and the
resulting mitochondrial pellets were resuspended in buffer B. For
preparation of cytosolic extracts, cells (1 x
108/sample) were washed twice with ice-cold PBS,
resuspended with 1 volume of buffer A, and allowed to swell on ice for
20 min. Cells were homogenized with 30 strokes of a Dounce homogenizer
and centrifuged at 15,000 x g for 15 min at
4°C. The protein concentration of mitochondria or cytosolic extracts
was determined using bicinchoninic acid (Pierce, Rockford, IL). For
isolation of nuclei, cells were washed twice in ice-cold PBS,
resuspended in 10 volumes of buffer C (10 mM
PIPES, pH 7.4, 10 mM KCl, 2
mM MgCl2, 1
mM DTT [Merck, Darmstadt, Germany), 1
mM PMSF (Sigma), 10 µM
cytochalasin B (Sigma)], allowed to swell on ice for 20 min, and
homogenized in a Teflon homogenizer. Homogenates were layered over 30%
sucrose in buffer C and centrifuged at 800 x
g for 10 min. The resulting nuclear pellets were resuspended
in buffer C and washed three times. Nuclei were stored at -80°C in
aliquots of 108 nuclei/ml.
Cell-free System of Apoptosis.
For determination of nuclear fragmentation, nuclei
(103/µl) were incubated with mitochondria (1
µg/µl) in buffer D [10 mM HEPES (pH 7.4), 50
mM NaCl, 2 mM MgCl2, 5
mM EGTA, 1 mM DDT, 2 mM ATP
(Sigma), 10 mM phosphocreatine (Sigma), 50 µg/ml creatine
kinase (Sigma), 10 µM cytochalasin B] for 2 h at
37°C. Nuclei were stained with propidium iodide (10 µg/µl) and
analyzed by flow cytometry. For determination of mitochondrial
cytochrome c content, mitochondria were lysed in lysis
buffer, and proteins were separated by 15% SDS-PAGE. Western blot
analysis was performed as described above.
Inhibition of Mitochondrial Protein Synthesis.
Mitochondrial protein synthesis was inhibited by treating SHEP pHyTK11
cells with 50 µg/ml chloramphenicol (Sigma-Aldrich, Steinheim,
Germany) for 3 weeks in medium containing 110 µg/ml sodium pyruvate
(Biochrom), 50 µg/ml uridine (Sigma), and 5 mg/ml glucose (Sigma).
TK/GCV-induced Apoptosis during Inhibition of Mitochondrial
Protein Synthesis.
SHEP pHyTK11 cells (2 x 104)
in which mitochondrial protein synthesis had been
inhibited as described above were incubated with GCV in increasing
concentrations for 72 h in the presence of sodium pyruvate,
uridine, and glucose. Control SHEP pHyTK11 cells were also incubated in
medium containing the same concentrations of pyruvate, uridine, and
glucose starting 3 weeks prior and continued during the experiment.
Specific apoptosis was measured by enumerating propidium iodide stained
hypodiploid nuclei by flow cytometry.
p53 Induction by TK/GCV.
SHEP pHyTK11 cells (5 x 106) were
treated with 10 µM GCV for increasing times, with or
without 50 µg/ml chloramphenicol (Sigma-Aldrich). When the effect of
chloramphenicol was examined, both the chloramphenicol-treated cells
and the untreated controls were adapted to medium containing pyruvate,
uridine, and glucose starting 3 weeks prior and continued during the
experiment. Western blotting was performed as described above with
mouse anti-p53 monoclonal antibody (1:1000; Transduction Laboratories).
Mouse anti-
-tubulin monoclonal antibody (1:3000; Calbiochem, Bad
Soden, Germany) was used to control for equal gel loading.
Detection of Mitochondrial DNA Damage.
DNA was extracted using the QJAamp Blood Kit (Qiagen,
Hilden, Germany) according to the instruction of the manufacturer. To
detect mitochondrial DNA damage, the entire coding region of the
mitochondrial genome was amplified by long range PCR with the use of
primers 5'-CCCACAGTTTATGTAGCTTACCTCCTCA-3' (nucleotides 571–598 of the
Cambridge human mitochondrial DNA sequence) and 5'-TTGATTGCTGTACTT
GCTTGTAAGCATG-3' (nucleotides 16,220–16,193). Long range PCR was
performed with the Expand Long Template PCR System (Boehringer
Mannheim) according to the manufacturer’s instructions with the
following modifications. Denaturing temperature was 92°C, annealing
temperature was 65°C, extension time was 14 min during the first 10
cycles and 14 min plus 20 s for each consecutive cycle during the
subsequent 12 cycles. The final extension time was 14 min. The 15.6-kb
PCR products were resolved on a 0.7% agarose gel and visualized by
ethidium bromide. As a positive control, mitochondrial DNA damage was
induced in 1 x 106 SHEP pHyTK11
cells by treatment with
H2O2 (Mallinckrodt-Baker,
Deventer, the Netherlands) in concentrations ranging from 6.25 to 200
µM at 37°C for 1 h. To detect TK/GCV-induced DNA
damage, 1 x 106 SHEP pHyTK11
cells were treated with 10 µM GCV with increasing duration
before DNA extraction.
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RESULTS
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To investigate whether mitochondria are involved in
TK/GCV-triggered apoptosis, we studied apoptosis after GCV treatment in
TK- and Bcl-2-transfected cells or with BA, a specific inhibitor of
mitochondrial permeability transition pores. Induction of apoptosis in
response to treatment with GCV was strongly inhibited in SHEP
neuroblastoma cells transfected with both TK and Bcl-2 compared with
cells expressing TK, but not Bcl-2 (Fig. 1A
). Similarly, TK/GCV-triggered apoptosis was blocked in
TK-expressing cells in the presence of BA (Fig. 1A
),
indicating that mitochondria were involved in mediating TK/GCV gene
therapy-induced apoptosis.
To study the effect of mitochondrial activation on the cleavage of
caspases, we monitored cleavage of initiator (caspase-8) and effector
(caspase-3) caspases by Western blot analysis in TK- and
Bcl-2-transfected cells. After incubation with GCV, cleavage of the
effector caspase-3 and processing of the caspase substrate PARP were
inhibited in cells overexpressing Bcl-2 and TK compared with cells
expressing TK alone (Fig. 1B
). However, the initiator
caspase-8 was cleaved regardless of Bcl-2 overexpression (Fig. 1B
), suggesting that cleavage of caspase-8 occurred upstream
of mitochondria possibly as a result of death receptor aggregation.
To further define alterations in mitochondrial function after treatment
with TK/GCV, we assessed the mitochondrial transmembrane potential
m using the potential-sensitive
fluorochrome DiOC6(3)
. GCV treatment was
associated with a time-dependent loss of the
m in TK-transfected cells with a maximum at
96 h of GCV treatment, whereas no significant alteration of the
m was found in mock transfected control
cells (Fig. 2A
). In addition, loss of the m
upon TK/GCV gene therapy was strongly reduced in TK and Bcl-2
double-transfected cells or in the presence of BA (Fig. 2B
).
To dissect mitochondrial-driven effects of TK/GCV therapy from its
effect on other subcellular systems, we used a cell-free system.
Mitochondria were isolated from TK/GCV-treated cells and incubated with
cytosolic extracts prepared from untreated cells. In this experimental
setting, isolated mitochondria induced cleavage of caspase-8,
caspase-3, and PARP in cytosolic extracts (Fig. 3A
). In addition, mitochondria isolated from TK/GCV-treated
cells triggered nuclear fragmentation when incubated with nuclei
isolated from untreated cells (Fig. 3B
). However, cleavage
of caspases and nuclear fragmentation was blocked when mitochondria
isolated from TK/GCV-treated cells transfected with Bcl-2 were used
(Fig. 3, A and B
). These findings indicate that
mitochondria of TK/GCV-treated cells were able to trigger full
activation of apoptosis effector programs.
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Fig. 3. TK/GCV-induced mitochondrial alterations are involved in
activation of caspases and nuclear fragmentation. A,
TK/GCV-induced mitochondrial alterations mediate activation of
caspases. SHEP neuroblastoma cells transfected with pHyTK
(+TK), mock transfectants (-TK), or
pHyTK/Bcl-2 double transfectants (+TK/Bcl-2) were
treated with 10 µM GCV for 24 h. Mitochondria were
isolated and incubated with cytosolic extracts from untreated cells for
6 h. Proteins (40 µg/lane) were separated by 12% SDS-PAGE.
Immunodetection of caspases-3 and -8 and PARP protein was performed by
mouse anti-caspase-3 monoclonal antibody, mouse anti-caspase-8
monoclonal antibody, rabbit anti-PARP polyclonal antibody, and ECL.
B, TK/GCV-induced mitochondrial alterations mediate
nuclear fragmentation. SHEP neuroblastoma cells transfected with pHyTK
(+TK), mock transfectants (-TK), or
pHyTK/Bcl-2 double transfectants (+TK/Bcl-2) were
treated with 10 µM GCV for 24 h. Mitochondria were
isolated and incubated with nuclei from untreated cells. Nuclei
incubated with either mitochondria from untreated cells or treated with
GCV were used as control. Nuclear apoptosis was determined by FACS
analysis of propidium iodide-stained DNA content.
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On induction of apoptosis, mitochondria can release apoptogenic
factors such as cytochrome c into the cytosol which may
directly activate downstream effector caspases such as caspase-3
(18
, 19)
. Therefore, we next investigated cytochrome
c release after TK/GCV gene therapy. Treatment of
TK-transfected neuroblastoma cells resulted in a time-dependent release
of cytochrome c from mitochondria into the cytosol which was
inhibited in TK and Bcl-2 double-transfected cells (Fig. 4
). This suggests that mitochondria contribute to TK/GCV-triggered
apoptosis by releasing cytochrome c into the cytosol before
the breakdown of the mitochondrial membrane potential. Bcl-2 interfered
with TK/GCV-triggered apoptosis by inhibiting cytochrome c
release. TK/GCV-induced release of cytochrome c from
mitochondria into the cytosol was inhibited in cells incubated with
zVAD.fmk (Fig. 4
), indicating that mitochondrial dysfunctions in TK/GCV
gene therapy required activation of upstream caspases.
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Fig. 4. TK/GCV-induced apoptosis involves cytochrome
c release that is inhibited by Bcl-2 overexpression or
by inhibition of caspases. SHEP neuroblastoma cells transfected with
pHyTK (+TK), mock transfectants (-TK),
or pHyTK/Bcl-2 double transfectants (+TK/Bcl-2) were
treated with 10 µM GCV for indicated times in the
presence or absence of the broad range caspase inhibitor zVAD.fmk.
Mitochondria were isolated and lysed. Proteins (5 µg/lane) were
separated by 15% SDS-PAGE. Immunodetection of cytochrome
c was performed by mouse anti-cytochrome
c monoclonal antibody and ECL.
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Because GCV has been reported to cause chain termination and single
strand breaks on incorporation into nuclear DNA after conversion to
active metabolites by TK, we asked whether TK/GCV gene therapy would
have a direct damaging effect on mitochondrial DNA (3)
.
However, with a PCR-based method, no damage to mitochondrial DNA was
detected during treatment of cells with TK/GCV, whereas mitochondrial
DNA damage was detectable after incubation with
H2O2 known to induce DNA
damage (Fig. 5
). This suggests that TK/GCV therapy did not involve obvious damage to
mitochondrial DNA replication detectable by PCR.
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Fig. 5. Lack of damage to mitochondrial DNA replication
mediated by TK/GCV. SHEP pHyTK11 cells (1 x
106) were treated with H2O2
(A) or 10 µM GCV(B) in
concentrations indicated at 37°C for 1 h. Genomic DNA was
extracted, long range PCR of the mitochondrial genome was performed,
and the 15.6-kb PCR product was separated by 0.7% agarose gel
electrophoresis and visualized by ethidium bromide.
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To test whether mitochondrial DNA would play any role in TK/GCV-induced
apoptosis, we studied the effect of proteins encoded by the
mitochondrial genome on TK/GCV-induced apoptosis. To inhibit
mitochondrial protein synthesis, we used chloramphenicol which
specifically blocks synthesis of mitochondrial encoded proteins
(20)
. Treatment of TK-transfected neuroblastoma cells with
chloramphenicol resulted in inhibition of mitochondrial metabolic
function as measured by the dependence on uridine (data not shown).
Pyruvate was added to the medium because cells rendered respiratory
deficient by chloramphenicol depend on exogenous pyruvate.
Interestingly, pyruvate alone markedly decreased TK/GCV-induced
apoptosis, However, treatment of TK-transfected neuroblastoma cells
with chloramphenicol did not further inhibit TK/GCV-induced apoptosis,
indicating that proteins encoded by the mitochondrial genome and
involved in mitochondrial redox function did not contribute to
TK/GCV-induced apoptosis (Fig. 6
).
Because damage to nuclear DNA may involve accumulation of
wild-type p53 protein and because release of mitochondrial factors may
contribute to nuclear DNA damage, we then studied whether interfering
with mitochondrial functions would have any influence on p53
accumulation. Accumulation of wild-type p53 protein was reduced in TK-
and Bcl-2-transfected cells or in TK-transfected cells in the presence
of BA (Fig. 7
). In addition, accumulation of wild-type p53 protein after GCV
treatment was markedly diminished in cells in which mitochondrial
protein synthesis was blocked by chloramphenicol (Fig. 7
). This
suggests that mitochondria might be involved in mediating accumulation
of p53 protein after GCV treatment in the absence of detectable damage
to mitochondrial DNA replication.
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Fig. 7. Mitochondria are involved in TK/GCV-induced accumulation
of p53 protein. SHEP pHyTK11 cells (5 x
106) or pHyTK/Bcl-2 double transfectants (TK/Bcl-2) were
treated with 10 µM GCV for the time indicated, alone
(GCV), with 50 µg/ml chloramphenicol (GCV/CA), or with 50
µM BA (GCV/BA). Proteins were extracted, separated by
SDS-PAGE, and transferred to nitrocellulose membranes. p53 was detected
using p53 monoclonal antibody, avidin-horseradish peroxidase, and an
ECL kit. -Tubulin was detected to control for equal gel loading.
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DISCUSSION
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Thus far, clinical results with suicide gene therapy systems such
as the HSV-TK/GCV system did not fulfill the initial expectations for
an effective new therapeutic approach in the treatment of malignancies,
especially for patients with malignant brain tumors. Therefore, in
addition to improving target specificity and gene transfer efficacy, a
better understanding of the underlying mechanisms that regulate
sensitivity or resistance to TK/GCV-induced cytotoxicity may be
critical to improve its efficacy. The cytotoxic effect of TK/GCV
therapy is, at least in part, mediated by induction of apoptosis
(1
, 2)
. Here, we report that mitochondria are centrally
involved in the regulation of TK/GCV-induced apoptosis by amplifying
TK/GCV-induced activation of apoptosis pathways.
Treatment with TK/GCV triggered mitochondrial perturbations,
e.g., loss of the mitochondrial transmembrane potential and
cytochrome c release, leading to activation of apoptosis
effector programs. Inhibition of TK/GCV-induced mitochondrial
perturbations by Bcl-2 overexpression or by the mitochondrion-specific
inhibitor BA strongly reduced TK/GCV-induced apoptosis. Mitochondria
from TK/GCV-treated cells fully initiated activation of apoptosis
effector programs leading to DNA fragmentation and cell death by
releasing apoptogenic factors such as cytochrome c.
Cytochrome c has been described to form a complex together
with Apaf-1 and procaspase-9, resulting in activation of procaspase-9
and leading to cleavage of downstream caspases such as caspase-3 and
cleavage of CAD (caspase-activated DNase) or DNA fragmentation factor,
resulting in nuclear fragmentation (21
, 22)
. TK/Bcl-2
double-transfected cells that were blocked at the mitochondrial level
did not release cytochrome c from mitochondria and did not
activate caspases after treatment with GCV.
In addition to regulation of the effector phase of apoptosis,
mitochondria were also involved in mediating accumulation of wild-type
p53 protein, because accumulation of wild-type p53 after treatment with
TK/GCV protein was blocked by overexpression of Bcl-2, by blockade of
the mitochondrial megapore using BA, or by inhibition of synthesis of
mitochondrial encoded proteins. At present, the underlying mechanism(s)
regulating mitochondria-mediated p53 accumulation are not known and may
involve production of reactive oxygen species, release of soluble
factors from mitochondria or TK/GCV-induced damage to mitochondrial
DNA, although we could not detect obvious damage to mitochondrial DNA
replication. To investigate whether mitochondrial encoded respiratory
chain proteins contribute to TK/GCV-induced p53 accumulation and
apoptosis we blocked mitochondrial protein synthesis by the specific
inhibitor chloramphenicol. Pyruvate was added to the medium since cells
rendered respiratory deficient by chloramphenicol become auxotrophic
for pyruvate. Interestingly, pyruvate alone markedly decreased
TK/GCV-induced apoptosis, in line with a cytoprotective effect
described for pyruvate (23
, 24)
. Since pyruvate levels are
increased in hypoxic areas of solid tumors this may decrease
TK/GCV-induced apoptosis in vivo. Chloramphenicol treatment
ablated TK/GCV-induced p53 accumulation while not decreasing
TK/GCV-induced apoptosis. This may suggest that TK/GCV-mediated
apoptosis is independent of respiratory function and of p53. p53
independence during respiratory deficiency would contrast with our
previous finding that p53 is associated with TK/GCV-induced death
receptor aggregation during normal cellular respiration
(1)
. Alternatively, the cytoprotective effect of pyruvate
may have masked any decrease of apoptosis caused by chloramphenicol.
Chloramphenicol treatment markedly decreased tumor cell proliferation
(data not shown). This has been explained by decreased
G1-S transition linked to purine auxotrophy
caused by respiratory deficiency (25)
. Because the
efficacy of TK/GCV depends on replicating DNA, one would expect an
inhibition of TK/GCV-induced apoptosis in the presence of
chloramphenicol. As mentioned, this was not observed, suggesting that
purine auxotrophy induced by chloramphenicol led to sufficient
incorporation of the purine analogue GCV triphosphate despite decreased
DNA replication. Similar to cells experiencing chloramphenicol-induced
respiratory deficiency, cells in hypoxic tumor areas slow down at
G1 (26)
. Our finding that tumor
cells are not completely resistant to TK/GCV despite decreased
proliferation and increased pyruvate under conditions of respiratory
deficiency supports the use of TK/GCV for targeting hypoxic tumors
(27)
.
The molecular mechanisms leading to perturbations of mitochondria
in TK/GCV therapy are not understood. Although TK/GCV has been reported
to have a direct damaging effect on nuclear DNA by inhibiting DNA
polymerase resulting in termination of DNA synthesis, TK/GCV therapy
did not lead to damage of mitochondrial DNA replication detectable by
PCR. Moreover, TK/GCV therapy probably did not directly induce
perturbation of mitochondrial function. Caspases or caspase-dependent
signaling events operating upstream of mitochondria are required for
triggering mitochondrial permeability transition because
TK/GCV-triggered mitochondrial alterations such as loss of
mitochondrial membrane potential or release of cytochrome c
depended on caspase activity. In addition, activation of the initiator
caspase-8 was not blocked by Bcl-2 overexpression indicating that
cleavage of caspases occurred, at least to some extent, independent and
upstream of mitochondria in TK/GCV-treated cells. Bid, a recently
identified BH3 domain-containing molecule, or CAF (caspase-activated
factor) may link caspase-8 cleavage at the activated CD95 receptor to
mitochondrial pathways (28
, 29) .
Thus, mitochondria play a central role as amplifier of TK/GVC-induced
apoptosis by their effect on the effector phase of apoptosis and on p53
accumulation. Mitochondria trigger apoptosis effector systems such as
caspases through the release of cytochrome c into the
cytosol. Because p53 can mediate up-regulation of CD95 resulting in the
formation of the CD95 death-inducing signaling complex and activation
of the caspase cascade (1)
that may in turn lead to
mitochondrial perturbations, mitochondria might be part of a positive,
albeit redundant amplification loop in TK/GVC-induced apoptosis, at
least in some cell types.
The fact that TK/GCV therapy-induced cell death is strongly reduced in
cells overexpressing Bcl-2 might have important clinical implications
given the fact that a large proportion of brain tumors, a potential
target for TK/GCV therapy, express high levels of Bcl-2
(30)
. By demonstrating that mitochondria are amplifiers of
TK/GCV-induced apoptosis, our findings contribute to the molecular
understanding of TK/GCV gene therapy, a paradigmatic gene therapy
system. Furthermore, because TK/GCV gene therapy is a model of
drug-induced cytotoxicity apart from its clinical application, our
findings provide insights into molecular mechanisms of cytotoxicity by
showing that mitochondria are involved in the regulation of cell death
and accumulation of p53 protein.
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ACKNOWLEDGMENTS
|
|---|
We thank Carsten Scaffidi, Marcus E. Peter, and Peter H. Krammer
for anti-FLICE antibody. The technical assistance of H. Knauss and I.
Küttner is appreciated.
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FOOTNOTES
|
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The costs of publication of this article were defrayed in part 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.
1 Partially supported by grants (to K-M. D.) from
the Deutsche Forschungsgemeinschaft; the Bundesministerium für
Forschung and Technologie, Bonn; the Tumor Center Heidelberg/Mannheim;
and (to C. B. and K-M. D.) from the Deutsche Krebshilfe, Bonn. 
2 Both authors contributed equally to the work.
3 To whom requests for reprints should be
addressed, at University Children’s Hospital, Prittwitzstrasse 43,
89075 Ulm, Germany. Phone: 49 731 502 7700; Fax: 49 731 502 6681;
E-mail: klaus-michael. debatin{at}medizin.uni-ulm.de
4 The abbreviations used are: TK/GCV, thymidine
kinase/ganciclovir; BA, bongkrekic acid; DiOC6(3),
3,3'-dihexyloxacarbocyanide iodide; ECL, enhanced chemiluminescence;
FACS, fluorescence-activated cell sorting; m,
mitochondrial transmembrane potential; PARP, poly(ADP-ribose)
polymerase; zVAD.fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl
ketone.
Received 10/26/99.
Accepted 4/13/00.
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Top
ABSTRACT
INTRODUCTION
), mock
transfectants (
) or pHyTK/Bcl-2 double transfectants (
) were
treated with 10 µM GCV for 72 h in the absence or
presence (
) of 50 µM BA. Nuclear apoptosis was
determined by FACS analysis of propidium iodide-stained DNA content.
Percentage of specific apoptosis was calculated as follows:
100 x [experimental apoptosis (%) -
spontaneous apoptosis (%)/100% - spontaneous apoptosis
(%)]. Results are means of triplicates; similar results were obtained
in three separate experiments. B, inhibition of
TK/GCV-induced activation of caspases by Bcl-2 overexpression. SHEP
neuroblastoma cells transfected with pHyTK (+TK), mock
transfectants (-TK) or pHyTK/Bcl-2 double transfectants
(+TK/Bcl-2) were treated with 10 µM GCV
for indicated times. Proteins (40 µg/lane) isolated from cell lysates
were separated by 12% SDS-PAGE. Immunodetection of caspase-8,
caspase-3, and PARP protein was performed by mouse anti-caspase-8
monoclonal antibody, mouse anti-caspase-3 monoclonal, or rabbit
anti-PARP polyclonal antibody and ECL.
