[CANCER RESEARCH 56. 203.V20.18. May 1. 19%|
Apoptosis-associated Derangement of Mitochondrial Function in Cells Lacking
Mitochondrial DNA1
Philippe Marchetti, Santos A. Susin, Didier Decaudin, Susana Garnen, Maria Castedo, Tamara Hirsch,
Naoufal Zamzami, Javier Naval, Anna Senik, and Guido Kroemer2
CNRS-UPK420, IV rue Guy Miiquet, B.P. 8, F-94801 Villejuif, France [P. M., S. A. S.. D. D.. M. C.. T. H.. N. Z. A. S.. G. K.¡,and Department of Biochemistry and Molecular
Biology. University of Zaragoza, 50009 Zaragoza, Spain ¡S.G.. J. N. l
mtDNA1 dies from apoptosis in a Bcl-2-inhibitable
ABSTRACT
U937 cells lacking mitochondria!
DNA (p°cells) are auxotrophic
for
undine and pyruvate, hypersensitive
to hypoglycémie conditions, and
resistant to antimycin A-induced apoptosis. In spite of their obvious
metabolic defects, p°cells possess a normal mitochondria! transmembrane
potential, as well as a near-normal capacity to generate Superoxide anión
after menadione treatment. Similarly to p1 controls, p°cells undergo
apoptosis in response to tumor necrosis factor-a plus cycloheximide.
Detailed comparison of the apoptotic process in p+ and p" cells reveals
essentially the same sequence of events. In response to tumor necrosis
factor/cycloheximide,
cells first lose their mitochondria! transmembrane
potential (Ai//ml and then manifest late apoptotic alterations, such as
generation of reactive oxygen species and DNA fragmentation. Experi
ments involving isolated mitochondria from p* and p°cells confirm that
;< mitochondria can be induced to undergo permeability transition, a
process thought to account for the pre-apoptotic Ai//,,, disruption in cells.
Like p ' mitochondria, p°mitochondria contain a pre-formed soluble
factor that is capable of inducing eliminatili condensation in isolated
nuclei in vitro. This factor is released from mitochondria upon induction
of permeability transition by calcium or the specific ligand of the adenine
nucleotide translocator atractyloside. In conclusion, it appears that all
structures involved in the maintenance and pre-apoptotic disruption of
the Ai//,,,, as well as a mitochondria! apoptotic factor(s), are present in p°
fashion when
stimulated with the phosphotyrosine kinase inhibitor staurosporine or
cultured in the absence of PCS (14). Based on this observation, it has
been widely assumed that mtDNA. and by extension mitochondria,
would not be involved in the regulation of apoptosis.
Based on these premises, we decided to characterize cell lines
lacking mtDNA and to determine the putative contribution of defec
tive mitochondria to the apoptotic process. Our data indicate that
mitochondria from cells lacking mtDNA (p°cells) undergo the same
early apoptosis-associated changes as mitochondria from control (p+)
cells. In normal conditions, both p°and p+ cells possess an intact
Ai//m. This Ai//m is lost at an early pre-apoptotic stage when cells are
induced to die from apoptosis. In addition, both p°and p * mitochon
dria contain a pre-formed factor capable of inducing signs of nuclear
apoptosis (chromatin condensation and DNA fragmentation) in iso
lated nuclei in vitro. These data underline the participation of mito
chondria in the apoptotic process and indicate that all apoptotic
functions of mitochondria are products of the nuclear rather than
mitochondrial genes. Thus, our data confirm that mtDNA is not
important for the regulation of apoptosis yet underscore the probable
involvement of mitochondria in the apoptotic process.
cells and thus are controlled by the nuclear rather than by the mitochon
dria! genome. These findings underline the contribution of mitochondria
to the apoptotic process.
MATERIALS
INTRODUCTION
supplemented
AND METHODS
Generation of p°Cells and Culture Conditions. U937 cells were cultured
at 37°C in a humidified atmosphere containing 5* CO, in RPMI 1640
For many years, it has been believed that the nucleus would
constitute the prime target of the apoptotic process. Since 1994.
however, it has become clear that even anucleate cells can undergo
programmed cell death and that nonnuclear cytoplasmic structures
participate in the control of apoptosis (1. 2). Recently, it has been
found that cells undergo alterations in mitochondria! structure and
function early during the apoptotic process, i.e.. well before nuclear or
chromatin structures are affected (3-11). This applies to rather dif
ferent cell types: neurons (3). fibroblasts (4), thymocytes (6. 10),
peripheral T lymphocytes (5, 9. 10). B cells (5). and myelomonocytic
cells (7. 8). Before cells undergo nuclear apoptosis. they manifest a
reduction in the mitochondrial transmembrane potential (Ai//m). sug
gesting the involvement of mitochondria in the apoptotic process
(3-11). This idea has been corroborated by the observation that
cytoplasmic extracts enriched in mitochondria can induce apoptosis in
isolated nuclei in vitro (12, 13). Contrasting with this hypothesis,
however, it has been shown that a human fibroblast cell line lacking
Received 12/18/95: accepted 2/27/96.
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.
1This work was supported by grants from Association pour la Recherche sur le
Cancer. Agence Nationale de Recherche sur le SIDA. Centre National de la Recherche
Scientifique. Fondation pour la Recherche Médicale.Institut National de la Santéet de la
Recherche Médicale.NATO. LéoFoundation. Ministère de la Recherche (France).
Sidaction (to G. K.) and Dirección General de Ciencia y TecnologÃ-a(PB92-0355 lo i. N.).
S. A. S. received a fellowship from Ihe Spanish Ministry of Science.
2 To whom requests for reprints should be addressed.
with 10% FCS. 2 HIML-glutamine. 1(X)units/ml penicillin.
100
/j.g/ml streptomycin, 4.5 mg/ml glucose. 100 pig/ml sodium pyruvate. and 50
p,g/ml m ululo. To deplete mtDNA. cells were cultured during 6 months in the
presence of ethidium bromide (50 ng/ml). Four different suhlines were derived
and controlled for mlDNA depletion using a specific PCR reaction (15). Three
of four of these p°sublines conserved TNF receptor expression and demon
strated a comparable TNF sensitivity. The P subline morphologically resem
bles most closely the parental wild-type cell line, and results are shown for this
cell line. In all experiments, both p°and p+ control cells were maintained in
the same culture medium supplemented with glucose, pyruvate, and uridine.
Forty-eight h before experiments were performed, ethidium bromide was
routinely removed from the medium of p°cells.
Induction of Apoptosis. Cells were cultured during the indicated interval
in the presence of recombinant TNF-a (4 ng/ml: Research Diagnostics.
Flanders. NJ) plus CHX (0.5 fig/ml: Sigma Chemical Co.. St. Louis, MO),
antimycin A (100 /XM: Sigma), or glucose-free medium (Lite Technologies.
Inc.) plus 2-deoxy-D-glucose (2.5 min; Sigma; Ref. 16). followed by the
determination of apoptotic fealurcs. DNA fragmentation was assessed as
described by agarose gel electrophoresis of nuclear DNA (5 X Iflr cells/lane:
Ref. 17).
Cytofluorometric Analysis and Confocal Microscopy. To measure the
A<//m,cells were incubated at 37°Cduring 15 min in the presence of DiOC,,(3)
(40 nM; Molecular Probes: Ref. 4). To determine Superoxide anióngeneration,
cells were kept at 37°Cduring 15 min in the presence of HE (2 p.M: Molecular
' The abbreviations
used are: mtDNA. mitochondrial
factor; A(//,n, mitochondrial
3.3'-dihexyloxacarbocyanine
DNA: TNF. tumor necrosis
transmembrane potential: CHX. cycloheximide: DiOC,,(3).
iodide: HE. hydroethidine: mOCCP. carbonyl cyanide m-
chlorophenylhydra/one:
ROS. reactive oxygen species; Atr, atractyloside; BA. bongkrekic acid: RR. ruthenium red; p" cells, cells lacking mitcx-hondrial DNA; p' cells, cells
bearing an intact mitochondrial genome.
2033
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
APOPTOSIS
AND MITOCHONDRIA!.
FUNCTION
Probes; Ref. 7). After incubation with DiOC6(3) plus HE, cells were imme
diately analyzed in an Epics Profile II cytofluorometer (Coulter, Miami, FL).
In control experiments, cells were labeled after incubation in the presence of
the protonophore rnCICCP (50 /J.M; 30 min; Sigma) or the ROS-generating
RESULTS AND DISCUSSION
agent menadione (1 mM; 1 h; Sigma). DiOC6(3) fluorescence was recorded in
FL1, and HE was recorded in FL3. Confocal microscopy was performed on
cells labeled with either DiOC6(3)/HE or with ethidium bromide (50 ng/ml; 1
h) using a Meridian Acas 570 confocal microscope (Meridian Instrument Co.,
Okemos, MI). The frequency of cells having lost part of their chromosomal
DNA (hypoploid cells) was determined by the propidium iodine staining of
ethanol permeabilized cells, as described (18).
in Vitro Tests of Mitochondria! Permeability, Transition, and Genera
tion of Mitochondria! Supernatants. Mitochondria were purified on a Per
coli gradient (19) and were stored on ice in buffer B (400 mM mannitol, 10 mM
PO4H,K, 5 mg/ml BSA, and 50 mM Tris-HCl, pH 7.2) for up to 4 h at a
cationic lipophilic fluorochrome DiOC6(3) allows for the assessment
of the mitochondria! transmembrane potential (At//m;Refs. 5 and 24).
Normal control U937 cells incorporate 1 to 1.5 log more DiOC6(3)
than cells treated with the protonophore rnCICCP, indicating that most
of the dye incorporation is indeed driven by the inner mitochondria!
transmembrane proton gradient (Fig. 1A). A detailed comparison of
normal U937 cells (p+ cells) and U937 sublines lacking mtDNA (p°
cells) revealed no significant difference in the At|(m;p°U937 cells
Mitochondria! Inner Transmembrane Potential (Ai//,,,)and Mitochondrial Superoxide AniónGeneration in U937 p°Cells. The
concentration of 100 j¿gprotein/10 fjL\buffer. For determination of swelling,
mitochondria (10 /¿I)were resuspended in 90 /J.M CFS buffer [220 mM
mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM PO4H2K, 0.5 mM EGTA, 2 mM
MgCl2, 5 mM pyruvate, 0.1 mM phenylmethylsulfonyl
fluoride, 1 mM DTT,
and 10 mM HEPES-NaOH (pH 7.4); reagents from Sigma], and adsorption was
recorded at 540 nm in a Beckman DU 7400 spectrophotometer, as described
(20). Atr (final concentration, 5 mM; Sigma), BA (50 /IM; kindly provided by
Dr. J. A. Duine, Delft University. Delft, the Netherlands), CaCU (500 (¿M),
and/or RR ( 100 /XM;Sigma) were added to mitochondria. The loss of absorp
tion induced by 5 mM Atr within 5 min was considered as the 100% value of
large amplitude swelling. Supernatants (150,000 x g for 1 h at 4°C) of
mitochondria treated with Atr. BA, CaCU, and/or RR during 5 min at room
temperature were recovered and used in a cell-free system of apoptosis.
Cell-free System of Apoptosis. Nuclei from HeLa cells were purified on a
sucrose gradient, as described (21), and were resuspended in CFS buffer.
Nuclei were preserved at —¿
20°Cin 50% glycerol for up to 8 days, as described
(21, 22). Nuclei (1 X 10'//il final concentration) were mixed with the supernatants from mitochondria and were incubated for 90 min at 37°C,followed by
staining with 4'-6-diamidino-2-phenylindole
dihydrochloride (10 ¿¿M)
and
examination by fluorescence microscopy (23).
Statistical Analysis. Results were calculated as arithmetic means ±SEM,
and significance values were calculated by means of the Student t test.
P < 0.01 was regarded as significant.
possess a normal A(/*m(Fig. 1/4). In addition, the determination of
Superoxide aniónin p+ and p°cells does not reveal any major
differences. The superoxide-driven conversion of nonfluorescent li
pophilic HE into fluorescent hydrophilic ethidium is increased in the
presence of menadione, a substance that undergoes redox cycles in
mitochondria (Ref. 25; Fig. 15). Menadione enhances the rate of
HE—»ethidium
conversion both in p+ and p°cells. However, as
expected for partially respiration-deficient cells, the maximum
HE->ethidium conversion is lower in p°than in p+ cells (Fig. 15).
Thus, p°cells retain at least part of their capacity to generate superoxide aniónafter menadione treatment. In conclusion, it appears that
p°cells possess a normal Ai|;mas well as a near-normal capacity of
generating ROS species in response to the mitochondrion-targeted
drug menadione.
Early Ai//mLoss and ROS Generation in p°Cells Undergoing
Apoptosis. In contrastto some p°cell lines that become resistantto
the induction of necrosis in response to TNF (26, 27), U937 p°cells
stimulated with a combination of TNF and CHX exhibit hallmarks of
apoptosis such, as oligonucleosomal DNA fragmentation (Fig. 2A)
and loss of chromosomal DNA (hypoploidy; Fig. 2B). As described
(7, 8), p+ U937 cells treated with the apoptosis-inducing combination
of TNF plus CHX exhibit a reduction in the Ai|/m, as well as an
increase in Superoxide anióngeneration (Fig. 3-4, upper panels). As
described (28), TNF and CHX exhibit a clear synergistic effect on
B
Fig. I. Mitochondria! transmembrane potential (Ai//m) and
Superoxide anióngeneration in p°and p" U937 cells. Cells were
treated with the uncoupling agent mClCCP (A) or with the superoxide anion-generating agent menadione (B), followed by
labeling with the ii/im-sensitive dye DiOC6(3) or the Superoxide
anión oxidi/.able probe hydroethidine, respectively. Results are
representative for three different experiments. Similar results
were obtained with two additional U937 p°sublines (data not
shown).
o
cells
10°
Menadionetreated
cells
\
io-
io-4
log DiOC6(3)
10'
10°
IO1
10'
10-
log HE
2034
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
APOPTOSIS
AND MITOCHONDRIA!.
FUNCTION
(30, 31) accounts for the pre-apoptotic Ai/<lndisruption (7, 11). We,
therefore, tested whether purified p°and p+ mitochondria would
B
respond in a similar fashion to standard protocols for the induction of
permeability transition. As shown in Fig. 5A, no difference could be
detected in the permeability transition-dependent
colloidosmotic
swelling of isolated p°and p+ mitochondria induced by calcium or by
atractyloside, a specific ligand of the adenine nucleotide translocator
(32). As a control, calcium-induced permeability transition was in
hibited in the presence of RR, an inhibitor of the inner mitochondrial
membrane calcium uniport (33). Similarly, the Atr-induced perme
ability transition was inhibited in the presence of its antagonist BA,
both in p°and p+ mitochondria (Fig. 5/4).
TNF
CH
Further experiments involving a cell-free system of apoptosis (22)
revealed that mitochondria induced to undergo permeability transition
release a factor capable of inducing chromatin condensation in iso
lated nuclei. Mitochondria were treated with different inducers of
permeability transitions and centrifuged. After ultracentrifugation,
supernatants were recovered and added to isolated nuclei to determine
their effect on chromatin distribution. An apoptosis-inducing factor
was detectable in the supernatant of mitochondria treated with Atr or
Ca2"1"but not in control supernatants of mitochondria treated with
Atr/BA or Ca2+/RR (Fig. 5fl), indicating a strict correlation between
PI (DNA content)
Fig. 2. Apoptosis sensitivity of p°and p+ U937 cells. Cells were cultured in the
presence of TNF and/or CHX. followed by the determination of apoptotic parameters, i.e.,
DNA fragmentation (A; 12 h), or subdiploidy (B; 6 h). Oligonucleosomal DNA fragmen
tation was assessed by horizontal agarose gel electrophoresis (5 X IO5 cells/lane).
Subdiploidy was determined by ethidium bromide staining of ethanol-fixed cells. Control
cells maintained in standard culture conditions exhibited <5% hypoploidy. •¿
the addition
of the relevant substance.
permeability transition (Fig. 5A) and release of the pro-apoptotic
activity (Fig. 5ß).This apoptosis-inducing activity is destroyed by
proteinase K treatment, indicating that it is a protein.4 The addition of
antioxidants did not abolish the apoptosis-inducing activity of mito
chondrial supernatants, indicating that ROS are dispensable for nu
clear apoptosis induction (data not shown). Both the supernatant of
Atr- or calcium-treated p°and p+ mitochondria contain a similar
apoptosis induction, suggesting that they trigger different death path
amount of pro-apoptotic activity. No qualitative or quantitative dif
ways. This pattern was also obtained for p°cells (Fig. 3A, lower
ferences could be appreciated between supernatants from p°and p+
panels). Confocal microscopy confirmed the similarity between p°
mitochondria (Fig. 5, B and Q. In conclusion, p°mitochondria are not
and p+ cells. p°cells, the mitochondria of which fail to stain with
only fully capable of undergoing permeability transition but also
ethidium bromide, exhibit a normal distribution of intra-mitochondrial
contain normal quantities of a protein that induces apoptotic nuclear
DiOC,,(3) uptake (Fig. 35). After stimulation with TNF/CHX, a
changes in a cell-free system.
fraction of either p°or p+ cells lose DiOC6(3) incorporation and
Concluding Remarks. As mentioned in the Introduction to this
accelerate HE—»ethidiumconversion, giving rise to a diffuse
report, a human fibroblast cell line lacking mtDNA was shown to die
nonnuclear cytoplasmic staining (Fig. 3ß).In view of these results, we
from apoptosis in a Bcl-2-inhibitable fashion (14). Based on this
investigated whether alterations in mitochondria! function would pre
observation, it has been postulated that apoptosis itself and the anticede nuclear signs of apoptosis. Analysis of several time points
apoptotic activity of Bcl-2 would not be related to mitochondria. The
revealed that the loss of DiOC6(3) uptake precedes DNA fragmenta
data contained in this report disagree with this interpretation.
tion and hypoploidy, irrespective of the presence of mtDNA (Fig. 4/4).
As expected, U937 rapidly underwent apoptosis in response to a
p°cells tend to exhibit a slower kinetics of apoptosis induction than p°
combination of TNF and CHX, irrespective of their mtDNA status. p°
cells. The reasons for this difference are not understood. It is possible
U937 thus behave similar to p°L929 cells, which die in response to
that a reduced metabolic rate and/or generation of Superoxide anión TNF plus actinomycin D (26), exactly as control p+ cells do. How
(which mediates some of the TNF effects; Ref. 27) by p°cells could
account for this discrepancy. The sequence of events (first Ai|fm
disruption, then Superoxide anióngeneration and DNA fragmentation)
is essentially the same in p°and p+ cells (Fig. 4A). Control experi
ments confirmed that p°cells, which rely on enhanced glycolysis,
undergo apoptosis in the absence of glucose much more rapidly than
p+ controls (Fig. 4B). Moreover, p°cells are resistant to apoptosis
induction by antimycin A in conditions in which most p+ cells are
killed (Fig. 4ß).This is in accord with the fact that p°cells have a
reduced complex III activity and thus become resistant to antimycin
A, an inhibitor of respiratory chain complex III (29).
These data indicate that p°cells induced to undergo apoptosis
exhibit an early A<//mreduction that precedes DNA fragmentation,
exactly as this is the case in p+ control cells. Once the cells have lost
their Ai/»m,
they overproduce HE-detectable ROS.
Normal Permeability Transition and Induction of Nuclear
Apoptosis by p" Mitochondria. Our previous studies indicate that
ever, in contrast to L929 fibrosarcoma cells, which undergo necrosis
in response to TNF (27, 34), U937 pro-monocytes undergo apoptosis
(8, 16, 28), and this type of cell death can be induced in p°sublines,
even in the absence of inhibitors of macromolecule synthesis (15).
When induced to undergo apoptosis, p°U937 cells behave much as
wild-type p+ cells in the sense that they exhibit an early reduction in
At//m that precedes all other manifestations of apoptosis, such as
Superoxide anión hypergeneration, cell shrinkage, hypoploidy, DNA
fragmentation, and loss of plasma membrane integrity. It appears that
the molecular mechanisms accounting for the pre-apoptotic Ai//m
disruption, i.e., mitochondrial permeability transition (7, 11), are
operative in p°mitochondria and that p°mitochondria preserve their
capacity to release a factor causing isolated nuclei to undergo apop
totic chromatin condensation. Therefore, in line with the published
data (14), mtDNA does not encode any of the mitochondrial struc4 Unpublished data.
the opening of so-called mitochondria! permeability transition pores
2035
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
APOPTOSIS
A
ie°
AND MITOCHONDRIA!.
FUNCTION
Control, p+
TNF + CHX, p +
Control, p °
TNF + CHX, p°
10'
ie2
toJ
104 w° io1
i»2
DiOC6(3) (A(pm)
B
low,p+
Eth Br, p+
Eth Br, p°
O)11^ HEIow, p°
DiOC6(3)low
,p
+
°
DiOC6(3)low
10 urn
Fig. 3. Mitochondrial alterations occurring in p" and p1 U937 cells stimulated with TNF/CHX. Cells were cultured in the presence or absence
and p°cells, respectively), followed by simultaneous staining with DiOCft(3) and HE. A, cytofluorometric analysis. Results are representative for
plus CHX cause a significant (P < ().<XH) reduction of the DiOC6( 3) incorporation, as well as a significant increase in the HE—»ethidium
conversion,
microscopy of p°and p +. Untreated cells were labeled with ethidium bromide (which reveals mtDNA plus the nucleus) or DiOCft(3) plus HE.
bearing a DiOC(1(3)l"wHEtl":h phenotype are shown.
of TNF/CHX for 3 or 6 h (p^ cells
five independent experiments. TNF
both in p ' and p°cells. B, confocal
In addition. TNF/CHX-treated cells
2036
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
APOPTOSIS
KM)'
Fig. 4. Kinetics of mitochondria! and nuclear
alterations induced by TNF/CHX. A, chronology
of mitochondria! and nuclear alterations. p°and
p* U937 cells were cultured during the indicated
interval in the presence of TNF/CHX. followed by
assessment of the frequency of DiOCh(3)'"wHElow
and DiOC,,^)'""â„¢'"811(as in Fig. 3,4), and hypoploid cells (as in Fig. 2B). Results are the means
(bars, SEM) of three independent experiments.
For the loss of DiOC6(3) incorporation, the dif
ference between p° and p+ is significant
(P < 0.01) at 1.5 h of culture. For hypoploidy. the
difference is significant at 3 h. B. control experi
ments revealing the functional impact of the p°
genotype. p°and p^ U937 cells were cultured
during 12 h in the presence of 2-deoxy-i>glucose
or in the presence of antimycin A, followed by
assessment of the frequency of hypoploid cells.
Results are the means (harn. SEM) of three inde
pendent experiments. The apoptolic response to
both 2-deoxy-D-glucose and antimycin A is sig
nificantly different between p°and p* cells.
AND MITOCHONDRIAL
FUNCTION
DiOC6(3)1()WHEi E|DÌOC6<3)'°WHE+
80-
hypoploidy
I
60-1
«W
O
p status
+0+1.5+3+6+12O0O1.5O3O6O12
+ deoxygluc.
Antimycin A
time [hours]
tures/functions involved in the apoptotic cascade. Accordingly, the
proteins thought to participate in the formation of permeability tran
sition pore, i.e., the adenine nucleotide translocase, the peripheral
benzodiazepine receptor, and the voltage-dependent anión channel,
are all encoded by nuclear genes (30-32). The data presented in this
report also indicate that a pro-apoptotic factor is contained in mito
chondria and released upon induction of permeability transition. This
finding may resolve a controversy concerning cell-free systems of
apoptosis. Thus, in some systems, ultracentrifuged cytosolic prepara
tions are sufficient to induce apoptosis in isolated nuclei (22, 23).
whereas in other systems, mitochondria are required to induce nuclear
apoptosis (12, 13). It appears that mitochondria release a factor that
causes isolated nuclei to undergo apoptosis-like chromatin condensa
tion. This factor is a protein and operates even in the presence of
antioxidants (13), indicating that, as expected (35), ROS cannot ac
count for the apoptosis-inducing activity of mitochondria. If mito
chondria can be induced to release a soluble factor, which by itself is
sufficient to induce apoptosis in vitro, it is possible that the same
activity ultimately accounts for nuclear disintegration in all of these
systems. To address this hypothesis, it will be necessary to clone the
gene coding for this factor and to design specific reagents to inhibit its
expression and/or function. Our data indicate that the mitochondrionderived, apoptosis-inducing protein is a product of a nuclear rather
than a mitochondria! gene. Thus, although the mitochondrial genome
has no impact on the effector phase of apoptosis, it appears likely that
mitochondria play an active part in the death process. In this context,
it appears intriguing that proteins from the apoptosis-regulatory Bcl-2
family do localize to the outer mitochondrial membrane (36, 37),
B
DP
100-•S2
soo•*->
60aoOH
40"SSR"x,o-BAAtr.RRCa2+1lì•1•l•1
Ca2+
"•
•¿fr•HH•rtJ••
O 30 60 901201501800 30 60 90120150180
time (sec)
Fig. 5. Permeability transition and elaboration of an apoptotic factor by p°and p+ mitochondria. A, permeability transition of isolated mitochondria. Mitochondria derived from p°
and p* cells were monitored for changes in the absorbance (OD54„)
indicative of permeability transition-dependent swelling. As indicated by arrows, BA (50 /IM), RR ( 100 fil). Atr
(5 mM)or calcium (500 p.M)were added to isolated mitochondria. Results are plotted as the percentage of mitochondrial swelling, considering the Atr-induced drop in ODMnobtained
after 5 min as 100% maximum value. B, apoptosis-inducing activity released from p°and p* mitochondria. Mitochondria were treated with BA. RR. Atr. and/or calcium (same doses
as in A}for 5 min. Mitochondria were spun down, and ultracentrifuged supematants were tested for apoptosis-inducing activity on isolated HeLa nuclei. After a 60-min incubation period
at 37°C,nuclei were stained with the DNA intercalating dye 4'-6-diamidino-2-phenylindole dihydrochloride and examined in a fluorescence microscope. A minimum of 200 nuclei
was counted for each point by two independent investigators. Results are the means (hum, SEM) of three experiments. C. representative samples of nuclei treated with supematants
of isolated mitochondria. Examples of the dominant phenotype of nuclei (as in B, >70% of nuclei) are shown.
2037
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
APOPTOSIS
AND MITOCHONDRIAL
mainly to so-called contact sites with the inner membrane (36) in
which permeability transition pores are expected to form (30, 31, 38,
39). Indeed, at least in some experimental systems, bcl-2 is only
efficient as an apoptosis inhibitor when it is targeted to the mitochondrial compartment (40. 41).
In synthesis, our results clearly implicate mitochondria in the
apoptotic process. Because it is not possible to generate higher eukaryotic cells lacking mitochondria, indirect approaches, including
cell-free systems of (pre-)apoptosis, will be required to determine
whether mitochondria play a facultative or an obligatory role in the
apoptotic process.
ACKNOWLEDGMENTS
18.
19.
20.
21.
22.
23.
We are indebted to Dr. J. A. Duine (Delft University of Technology. Delft,
the Netherlands) for the gift of BA; to Zohar Mishal (CNRS, Villejuif, France)
for confocal microscopy; and to Maurice Geuskens (Université Libre de
Bruxelles, Brussels, Belgium) for electron microscopy.
REFERENCES
1. Jacobson, M. D.. Burne, J. F., and Raff, M. C. Programmed cell death and Bcl-2
proteclion in the absence of a nucleus. EMBO J.. 13: 1899-1910, 1994.
2. Schulze-Osthoff. K.. Walczak. H.. Droge. W., and Krammer. P. H. Cell nucleus and
DNA fragmentation are not required for apoptosis. J. Cell Biol., 127: 15-20, 1994.
3. Deckwerth, T. L., and Johnson, E. M. Temporal analysis of events associated with
programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth
factor. J. Cell Biol.. 123: 1207-1222. 1993.
4. Vayssière, J-L.. Petit. P. X.. Risler. Y.. and Mignotte. B. Commitment to apoptosis is
associated with changes in mitochondria! biogenesis and activity in cell lines condi
tionally immortalized with simian virus 40. Proc. Nati. Acad. Sci. USA, 91: 1175211756. 1994.
5. Zamzami, N., Marchetti. P.. Castedo, M.. Zanin, C., Vayssière, J-L., Petit. P. X.. and
Kroemer, G. Reduction in mitochondria! potential constitutes an early irreversible
step of programmed lymphocyte death in vivo. ¡.Exp. Med.. 181: 1661-1672. 1995.
6. Petit. P. X., LeCouer, H., Zorn, E., Dauguet. C.. Mignotte. B., and Gougeon. M. L.
Alterations of mitochondria! structure and function are early events of dexamethasone-induced Ihymocyte apoptosis. J. Cell Biol., 130: 157-167, 1995.
7. Zamzami. N.. Marchetti, P.. Castedo. M., Decaudin, D.. Macho. A., Hirsch, T.. Susin,
S. A.. Petit. P. X.. Mignotte, B., and Kroemer. G. Sequential reduction of mitochon
dria! transmembrane potential and generation of reactive oxygen species in early
programmed cell death. J. Exp. Med., 182: 367-377, 1995.
8. Cossarizza, A., Franceschi. C., Monti, D.. Salvioli, S., Bellesia, E., Rivabene. R.,
Biondo. L.. Rainaldi. G., Tinari. A., and Malorni. W. Protective effect of W-acetylcysteine in tumor necrosis factor-a-induced apoptosis in U937 cells: the role of
mitochondria. Exp. Cell Res.. 220: 232-240, 1995.
9. Macho. A., Castedo. M., Marchetti. P., Aguilar. J. J.. Decaudin, D., Zamzami. N..
Girard. P. M.. Uriel. J.. and Kroemer. G. Mitochondria! dysfunctions in circulating T
lymphocytes from human immunodeficiency virus-l carriers. Blood. 86: 2481-2487,
1995.
10. Castedo, M., Macho, A.. Zamzami, N., Hirsch, T., Marchetti. P.. Uriel, J., and
Kroemer. G. Mitochondria! perturbations define lymphocytes undergoing apoptotic
depletion in vivo. Eur. J. Immunol., 25: 3277-3284. 1995.
11. Kroemer, G., Petit, P. X., Zamzami, N.. Vayssière, J-L., and Mignotte, B. The
biochemistry of apoptosis. FASEB J.. 9: 1277-1287, 1995.
12. Newmeyer. D. D.. Farschon, D. M., and Reed, J. C. Cell-free apoptosis in Xenopus
egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched
in mitochondria. Cell, 79: 353-364, 1994.
13. Zamzami, Z., Susin. S. A., Marchetti, P., Hirsch, T., Gómez-Monterrey, 1., Castedo,
M., and Kroemer. G. Mitochondrial control of nuclear apoptosis. J. Exp. Med.. in
press. 1996.
14. Jacobson. M. D., Bume, J. F., King, M. P.. Miyashita, T., Reed. J. C.. and Raff. M. C.
Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature (Lond.), 361:
365-369, 1993.
15. Gamen, S., Anel, A.. Montoya, J.. Marzo, I., Pineiro, A., and Naval. J. mtDNAdepleted U937 cells are sensitive to TNF and Fas-mediated cytotoxicity. FEBS Lett..
376: 15-18, 1995.
16. Halicka, H. D.. Ardelt, B., Li. X.. Melamed. M. M., and Darzynkiewicz, Z. 2-Deoxyu-glucose enhances sensitivity of human histiocytic lymphoma U937 cells to apop
tosis induced by tumor necrosis factor. Cancer Res., 55: 444-449, 1995.
17. Gonzalo, J. A., Moreno de Alborán, I., Ales-Martinez, J. E.. Ales-Martinez, C.. and
24.
FUNCTION
Kroemer. G. Expansion and donai deletion of peripheral T cells induced by bacterial
superantigen is independent of the interleukin 2 pathway. Eur. J. Immunol., 22:
1007-1011, 1992.
Nicoletti, I., Migliorati. G.. Pagliacci. M. C., and Riccardi. C. A rapid simple method
for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
J. Immunol. Methods, 139: 271-280, 1991.
Boutry. M.. and Briquet. M. Mitochondrial modifications associated with the cytoplasmic male sterility in faba beans. Eur. J. Biochem., 727: 129-135, 1982.
Petronilli, V., Cola, C., Massari, S., Colonna, R., and Bernardi, P. Physiological
effectors modify voltage-sensing by the cyclosporin A-sensitive mitochondrial per
meability transition pore. J. Biol. Chem.. 268: 21939-21945, 1993.
Wood, E. R.. and Earnshaw, W. C. Mitotic chromatin condensation in vitro using
somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II.
J. Cell Biol.. ///: 2839-2850. 1990.
Lazebnik, Y. A.. Cole, S., Cooke, C. A., Nelson, W. G., and Eamshaw, W. C. Nuclear
events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis
of the active phase of apoptosis. J. Cell Biol., 1993: 7-22, 1993.
Lazebnik, Y. A.. Kaufmann. S. H.. Desnoyers, S., Poirier. G. G., and Earnshaw, W. C.
Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.
Nature (Lond). 371: 346-347, 1994.
Petit, P. X., O'Connor, J. E., Grunwald, D., and Brown, S. C. Analysis of the
membrane potential of rat- and mouse-liver mitochondria by flow cytometry and
possible applications. Eur. J. Biochem., 220: 389-397. 1990.
25. Richter, C. Pro-oxidanls and mitochondrial Ca~ ' : their relationship to apoptosis and
oncogenesis. FEBS Lett., 325: 104-107, 1993.
26. Schulze-Osthoff, K., Bakker, A. C.. Vanhaesbroeck, B.. Beyaert, R., Jacob, W. A.,
and Fiers, W. Cytotoxic activity of tumor necrosis factor is mediated by early damage
of mitochondrial functions. J. Biol. Chem., 267: 5317-5323. 1992.
27. Schulze-Osthoff, K.. Beyaert, R.. Vandevoorde, V.. Haegeman, G., and Fiers, W.
Depletion of the mitochondria! electron transport abrogates the cytotoxic and geneinductive effects of TNF. EMBO J., 12: 3095-3104, 1993.
28. Wright, S. C., Kumar, P., Tarn, A. W.. Shen, N.. Varma, M., and Larrick, J. W.
Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells. J.
Cell. Biochem., 48: 344-355, 1992.
29. Wolvetang, E. J., Johnson, K. L., Krauer. K.. Ralph. S. J., and Linnane, A. W.
Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS Lett.. 339: 40-44,
1994.
30. Bernardi, P.. Broekemeier, K. M., and Pfeiffer, D. R. Recent progress on regulation
of the mitochondrial permeability transition pore: a cyclosporin-sensitive pore in the
inner mitochondrial membrane. J. Bioenerg. Biomembr., 26: 509-517, 1994.
31. Zoratti, M., and Szabo, I. The mitochondrial permeability transition. Biochem.
Biophys. Acta Rev. Biomembr., 1241: 139-176, 1995.
32. Brandolin, G., Le-Saux, A., Trezeguet, V., Lauquinn, G. J.. and Vignais, P. V.
Chemical, immunological, enzymatic, and genetic approaches to studying the ar
rangement of the peptide chain of the ADP/ATP carrier in the mitochondrial mem
brane. J. Bioenerg. Biomembr., 25: 493-501. 1993.
33. Broekemeier. K. M., Krebsbach, R. J., and Pfeiffer. D. R. Inhibition of the mitochon
dria] Ca
uniporter by pure and impure ruthenium red. Mol. Cell. Biochem., 139:
33-40, 1994.
34. Schulze-Osthoff, K., Krammer, P. H., and Droge, W. Divergent signalling via
APO-I/Fas and the TNF receptor, two homologous molecules involved in physio
logical cell death. EMBO J.. 13: 4587-4596. 1994.
35. Jacobson, M. D.. and Raff, M. C. Programmed cell death and Bcl-2 protection in very
low oxygen. Nature (Lond.). 374: 814-816, 1995.
36. Krajewski. S., Tanaka, S.. Takayama. S., Schibler. M. J.. Fenton. W.. and Reed, J. C.
Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the
nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Can
cer Res., 53: 4701-4714, 1993.
37. Krajewski. S.. Krajewska, M., Shabaik, A., Wang, H. G.. Irie, S.. Fong, L.. and Reed.
J. C. Immunohistochemical analysis of in vivo patterns of bcl-x expression. Cancer
Res., 54: 5501-5507, 1994.
38. McEnery. M. W.. Snowman. A. M., Trifiletti. R. R.. and Snyder, S. H. Isolation of the
mitochondrial benzodiazepine receptor: association with the voltage-dependent anión
channel and the adenine nucleotide carrier. Proc. Nati. Acad. Sci. USA, 89: 31703174, 1992.
39. Kinnally. K. W.. Zorov, D. B., Antonenko. Y. N., Snyder. S. H.. McEnery, M. W..
and Tedeschi, H. Mitochondrial benzodiazepine receptor linked to inner membrane
ion channels by nanomolar actions of ligands. Proc. Nati. Acad. Sci. USA, 90:
1374-1378, 1993.
40. Tanaka, S., Saito, K., and Reed, J. C. Structure-function analysis of the Bcl-2
oncoprotein. Addition of a heterologous transmembrane domain to portions of the
Bcl-2ßprotein restores function as a regulator of cell survival. J. Biol. Chem.. 268:
10920-10926, 1993.
41. Nguyen. M., Branton, P. E.. Walton, P. A., Oltvai, Z. N.. Korsmeyer, S. J., and Shore,
G. C. Role of membrane anchor domain of Bcl-2 in suppression of apoptosis caused
by ElB-defective adenovirus. J. Biol. Chem., 269: 16521-16524. 1994.
2038
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
Apoptosis-associated Derangement of Mitochondrial Function
in Cells Lacking Mitochondrial DNA
Philippe Marchetti, Santos A. Susin, Didier Decaudin, et al.
Cancer Res 1996;56:2033-2038.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/56/9/2033
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.