Environmental and Molecular Mutagenesis 00:000^000 (2010)
Mitochondria as Decision-Makers in Cell Death
Institute for Biomedical Research, Kaunas University of Medicine,
Mitochondria play an essential role in both cell outer membrane permit the translocation of proapop-
health and death. Increasing experimental evidence totic proteins into cytosol, how mitochondria ‘‘make
suggests that mitochondria are involved in active decisions’’ on the mode of cell death, and how they
control of cell death processes at several levels regulate caspase activation by changing the redox
including (1) mitochondrial membrane permeabiliza- state of cytosolic cytochrome c. The interventions into
tion and release of proapoptotic proteins, (2) post- these processes may constitute an important strategy
cytochrome c regulation of caspase activation, and for the pharmacological prevention of unwanted cell
(3) supply of energy for execution of death program. death in various pathologies or, conversely, for facili-
The purpose of this review is to discuss the main tation of anticancer therapy. Environ. Mol. Mutagen.
mechanisms by which alterations in mitochondrial 00:000–000, 2010. V 2010 Wiley-Liss, Inc.
Key words: apoptosis; necrosis; cytochrome c; mitochondrial membrane; permeability transition
INTRODUCTION mitochondrial generation of ROS and reactive nitrogen
species [Brown, 2007; Erusalimsky and Moncada, 2007;
Mitochondria are membranous organelles that play an Murphy, 2009], the roles of Bcl-2 family proteins in
essential role in cellular bioenergetics, thermogenesis, induction and regulation of apoptosis [Kroemer et al.,
heme biosynthesis, lipid catabolism, calcium homeostasis, 2007; Ott et al., 2009]. In this review, we will restrict
and in other metabolic activities. Therefore, structurally ourselves to three aspects of mitochondria-cell death inter-
and functionally intact mitochondria are crucial for cell actions, and we will present a brief glimpse at the main
health. Moreover, mitochondria are increasingly recog- mechanisms explaining how mitochondria signal to initi-
nized as mediators of cell death and both forms of ate cell death by changing the intactness of their mem-
death––apoptosis and necrosis––require active participa- branes, how they ‘‘make decisions’’ on the mode of cell
tion of this organelle. death, and how they regulate caspase activation at post-
In recent years, a signiﬁcant understanding of the mul- cytochrome c level.
tiple roles of mitochondria has been reached, and now it
is well established that mitochondria may be involved
in regulation of cell death processes at several levels PERMEABILIZATION OF MITOCHONDRIAL MEMBRANES
including: AS THE MAIN CHECKPOINT OF APOPTOSIS
– cytosolic calcium homeostasis (disturbances of which The main event in mitochondrial signaling and control
may initiate cell death program); of apoptosis is permeabilization of mitochondrial outer
– generation of reactive oxygen (ROS) and nitrogen membrane and the release of proapoptotic proteins into
– mitochondrial membrane permeabilization and release Grant sponsors: British Heart Foundation, Lithuanian State Science and
of proapoptotic proteins; Studies Foundation.
– supply of energy; *Correspondence to: Vilmante Borutaite, Institute for Biomedical
– regulation of apoptosome formation and caspase acti- Research, Kaunas University of Medicine, Eiveniu str. 4, Kaunas LT-
vation by the redox state of cytosolic cytochrome c. 50009, Lithuania. E-mail: email@example.com
Received 28 October 2009; provisionally accepted 22 January 2010; and
Recently, excellent reviews were published summariz- in ﬁnal form 27 January 2010
ing current knowledge on mitochondrial roles in cellular DOI 10.1002/em.20564
calcium homeostasis and cell death [Pinton et al., 2008], Published online in Wiley InterScience (www.interscience.wiley.com).
V 2010 Wiley-Liss, Inc.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
the cytosol. This step is considered by many investigators undergoes a conformational change and oligomerization
as ‘‘a point of no return.’’ There are a few reasons for resulting in the release of proteins from mitochondria
such a statement. First, the release of cytochrome c from [Valentijn et al., 2008; Ivashyna et al., 2009].
mitochondria leads to rapid activation of caspases in cyto- Bak also undergoes conformational changes in response
sol which then execute the degradation of cellular struc- to proapoptotic signals and when activated can form
tures. Second, permeabilization of mitochondrial inner homo or heterooligomeric complexes involving Bax or
and outer membranes may lead to cell death even when BH3-only proteins [Grifﬁths et al., 2001]. In healthy cells,
caspases are not activated due to mitochondrial dysfunc- Bak is thought to be kept inactive by forming complexes
tion and decline in ATP or due to release of proapoptotic with antiapoptotic proteins, particularly Bcl-XL or Mcl-1
factors (AIF, endonuclease G, etc.) that cause caspase-in- [Willis et al., 2005]. In addition, it has been shown that
dependent cell death [Susin et al., 1999]. Bak interacts with the mitochondrial outer membrane pro-
Mitochondrial membrane permeabilization is a complex tein voltage dependent anion channel (VDAC2), and this
process and several mechanisms have been described that inhibits the membrane-permeabilizing function of Bak
include the following: [Cheng et al., 2003].
BH3-only proteins on their own usually do not cause
– Bcl-2-family protein-dependent, membrane permeabilization, rather, they act in concert
– Mitochondrial permeability transition pore (MPTP)-de- with Bax/Bak and serve as activators or facilitators
pendent, (reviewed in [Kroemer et al., 2007]). Bad, Bim, and Bid
– Lipid-dependent permeabilization. are suggested to act in this way. Recently, it has been
revealed that Bim binds directly to a speciﬁc region in
Permeabilization by Bcl-2 Family Proteins Bax molecule causing its activation [Gavathiotis et al.,
2008]. Bid has a function linking the extrinsic (plasma
Bcl-2 family includes both pro and antiapoptotic pro- membrane receptor mediated) and intrinsic (mitochondria
teins. The common feature of the family is that the pro- mediated) pathways of apoptosis so that caspase-8 is acti-
teins share one or more Bcl-2 homology (BH) domains. vated through the cell membrane death receptor complex
The antiapoptotic proteins in the family are Bcl-2, Bcl- cleaves Bid. The truncated form of Bid (tBid) then trans-
XL, Mcl-1, and others containing four BH domains. The locates to mitochondria where it interacts with Bak/Bax
main proapoptotic proteins include multidomain Bak and causing disruption of intactness of the mitochondrial outer
Bax, containing three BH domains, and so called BH3- membrane. Most of the other BH3-only proteins are
only proteins Bad, Bid containing one BH domain [Cory thought to promote apoptosis by binding and thus sup-
and Adams, 2002]. pressing activities of antiapoptotic Bcl-2 proteins.
Bak and Bax are considered to be the main players in How Bax/Bak proteins permeabilize the mitochondrial
mitochondrial outer membrane permeabilization as the outer membrane is a question under debate. Most investi-
expression of at least one of them is required for apopto- gators agree that at least two mechanisms can be
sis to proceed. If either Bax or Bak is eliminated alone, involved. First, Bax/Bak may form channels in the mito-
such cells are sensitive to apoptosis and undergo normal chondrial outer membrane. This is supported by ﬁndings
mitochondrial membrane permeabilization. In contrast, that these proteins can form ion-conducting channels in
cells lacking both Bax and Bak are resistant to apoptosis artiﬁcial membranes [Scorrano and Korsmeyer, 2003;
induced by variety of mitochondria-related stimuli [Wei Breckenridge and Xue, 2004]; however, there is question
et al., 2001]. Bak is an integral mitochondrial outer mem- whether such channels can permit translocation of pro-
brane protein, whereas Bax resides in the cytosol of teins out of the intermembrane space. Another hypothesis
healthy cells under physiological conditions. Various apo- suggests that Bax/Bak oligomers, instead of forming
ptotic stimuli, including growth factor withdrawal, DNA channels/pores, upon insertion into membranes cause
damage, staurosporine, etc., result in Bax translocation to destabilization of lipid bilayers thus enabling translocation
mitochondria where it assembles into high-molecular of intermembrane proteins [Basanez et al., 2002].
weight complexes [Hsu and Youle, 1998; Desagher et al., The mechanism(s) by which the antiapoptotic proteins
1999; Nechushtan et al., 2001]. To induce mitochondrial of Bcl-2 family perform their prosurvival roles are even
membrane permeabilization, Bax has to be inserted into less elucidated. The main mechanism seems to be the
the outer membrane and undergo a conformational sequestration of proapoptotic BH3-only proteins or neu-
change. Most of the literature recognize exposure of the tralization of activated Bax/Bak in mitochondrial mem-
alpha-1 N-terminal and alpha-9 membrane-targeting branes [Lucken-Ardjomande and Martinou, 2005; Kroemer
sequence in Bax molecule as a type of conformational et al., 2007]. In addition, Bcl-2 proteins reside in the endo-
activation prior to membrane insertion [Kim et al., 2009], plasmic reticulum where they can regulate Ca21 homeo-
though there is evidence suggesting that Bax inserts into stasis [Distelhorst and Shore, 2004], and thus can regulate
the mitochondrial membrane as a monomer and then induction of mitochondrial permeability transition.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Mitochondria in Cell Death 3
Permeabilization by Lipids to contribute to beta-amyloid toxicity in Alzheimer’s dis-
ease [Du et al., 2008; Resende et al., 2008], skeletal mus-
Majority of the literature concerning mitochondrial cle cell death in Ullrich congenital muscular dystrophy
membrane permeabilization during apoptosis is focused and Bethlem myopathy [Palma et al., 2009] and other dis-
on proteins. Other possible molecules that may mediate eases.
membrane permeabilization are less investigated though Mitochondrial permeability transition (MPT) ﬁrst was
in recent years there is an increasing interest in the role discovered as a phenomenon when energized mitochon-
of lipids. Indeed, lipid composition of mitochondrial dria undergo large amplitude but reversible swelling upon
membranes changes during apoptosis, as well as in many exposure to high calcium concentration [Hunter and
pathological situations and aging [Garcia Fernandez et al., Haworth, 1979a,b]. Later studies [Bernardi and Petronilli,
2002; Lucken-Ardjomande and Martinou, 2005; Chicco 1996; Halestrap et al., 1998; Crompton, 1999] have
and Sparagna, 2007; Lesnefsky and Hoppel, 2008]. One revealed that MPT is caused by opening of nonspeciﬁc
of the most common alterations in membrane lipids is channels/pores in the mitochondrial inner membrane
cardiolipin peroxidation that can be mediated by mito- allowing ions and small (up to 1.5 kDa) molecules to
chondrially produced ROS. Cardiolipin is particularly sen- freely move and equilibrate across the mitochondrial inner
sitive to oxidation by ROS or RNS because of high con- membrane. The consequences of MPT pore opening are
tent of unsaturated fatty acids. Cardiolipin is mainly loss of mitochondrial membrane potential and uncoupling
located in the mitochondrial inner membrane, but it was of oxidative phosphorylation, increased generation of
also found to be present in the outer membrane [Hovius ROS, and depletion of ATP. Perturbations in mitochon-
et al., 1993] where it appears to interact with Bcl-2 pro- drial inner membrane permeability to small electrolytes
teins. Another mechanism for cardiolipin peroxidation while maintaining high concentration of proteins inside
was recently proposed by Kagan et al.  and was the matrix lead to osmotic swelling of mitochondria and
suggested to involve cytochrome c which forms com- eventually to the rupture of the mitochondrial outer mem-
plexes with cardiolipin that catalyze H2O2-dependent car- brane thus permitting the release of large molecules from
diolipin peroxidation during apoptosis. Cardiolipin peroxi- the intermembrane space.
dation may affect interactions of Bax and tBid with mito- An elevated concentration of Ca21 in the mitochondrial
chondrial membranes and in this way may be involved in matrix is an indispensable inducer of MPTP and addi-
mitochondrial outer membrane permeabilization [Lucken- tional factors such as ROS, RNS, and high phosphate con-
Ardjomande and Martinou, 2005]. However, the role of centrations or depletion of the adenine nucleotide pool are
cardiolipin in functions of Bcl-2 proteins remains conten- considered activators of MPTP. They sensitize mitochon-
tious [Polcic et al., 2005]. dria to Ca21 so that MPTP opening can occur at lower
In some pathological situations (e.g., heart ischemia), Ca21 concentrations. Cyclosporine A and sanglifehrin A
the intracellular and intramitochondrial accumulation of are known to block MPTP opening. In experimental set-
free fatty acids is observed. They may induce mitochon- tings, the sensitivity to cyclosporine A (particularly to its
drial membrane permeabilization through activation of non-immunosuppressive derivatives that do not inhibit
mitochondrial permeability transition pore. Alternatively, calcineurin) is usually considered an indication for MPTP
saturated free fatty acids, such as palmitic and stearic, in involvement in cell death.
complexes with elevated Ca21 concentrations have been The molecular composition of the MPTP remains a
shown to induce mitochondrial outer membrane perme- puzzle despite wide interest and intensive studies carried
abilization due to formation of speciﬁc, cyclosporine- out over the last decades. It has been proposed that the
insensitive pores [Belosludtsev et al., 2006]. Another core structural components of MPTP include a matrix
lipid, ceramide, has also been shown to form lipid chan- protein cyclophilin D (CypD), an inner membrane protein
nels in mitochondrial membranes capable of releasing ap- adenine nucleotide translocator (ANT), and a VDAC in
optotic factors from mitochondria [Siskind, 2005]. the outer mitochondrial membrane. This model implies
Whether these lipids have a role during apoptosis in vivo that MPTP spans across the inner and outer membranes
awaits experimental clariﬁcation. of mitochondria. Several other proteins such as peripheral
benzodiazepine receptors, creatine kinase, and hexokinase
Mitochondrial Permeability Transition were proposed to be involved in regulation of the activity
of MPTP. Such models explained the main features of
This type of permeabilization of mitochondrial mem- MPTP but were largely speculative, lacking complete mo-
branes is most relevant and best described in ischemic lecular evidence. Recent studies using transgenic mice de-
pathologies (heart ischemia-reperfusion, stroke, etc., ﬁcient in particular putative components of MPTP did not
[Duchen et al., 1993; Grifﬁths and Halestrap, 1993; Ma- shed as much light on the structure of MPTP as one
tsumoto et al., 1999; Borutaite et al., 2003; Hausenloy would have expected. It was found that mitochondria
et al., 2003; Halestrap et al., 2004]), but it is also known lacking all three isoforms of VDAC (VDAC1, VDAC2,
Environmental and Molecular Mutagenesis. DOI 10.1002/em
and VDAC3) undergo normal MPT in response to Ca21 chondrial membrane potential. This eventually should
or ROS that was indistinguishable from wild-type mito- lead to depletion of cellular ATP resulting in necrosis. It
chondria, and in accordance, cell death was found to be is generally accepted that MPTP is involved in necrotic
unaltered in ﬁbroblasts lacking all VDACs [Baines et al., rather than apoptotic cell death. On the other hand, there
2007]. Therefore, it was concluded that VDAC is not a is ample evidence that cells can be rescued from apopto-
constituent of MPTP. Likewise, mitochondria from sis by MPTP inhibitors indicating that MPTP is also
ANT1/ANT2 double knockout mice still exhibit Ca21- involved in apoptosis [Hortelano et al., 1997; Borutaite
induced cyclosporin A-sensitive MPTP, though pore open- et al., 2000, 2003; Zamzami et al., 2005]. It has been pro-
ing is less sensitive to Ca21 and insensitive to ADP, car- posed that MPTP may exist in several distinct permeabil-
boxyatractyloside, bongkrekic acid, known modulators of ity states ranging from a low conductance (allowing per-
MPTP [Kokoszka et al., 2004]. This would suggest that meability to ions only) to a high conductance that permits
ANT is not a necessary structural component of MPTP. translocation of a bigger molecules (<1,500 Da) across
However, the shortcoming of these experiments was that mitochondrial membranes [Zoratti and Szabo, 1995; Ichas
it was not possible to completely knockdown all isoforms and Mazat, 1998]. Then it would be possible that persis-
of ANT (such animals are not viable), and even if double tent, wide MPTP opening leading to mitochondrial outer
knockouts were used there still remained the possibility membrane rupture would cause necrotic cell death when
that another isoform was present and could execute a glycolytic ATP supply is not adequate, whereas transient,
pore forming function. small amplitude MPTP openings without cellular ATP
In contrast to VDAC and ANT, genetic ablation of depletion could lead to apoptosis. A key question here is
CypD resulted in desensitization of liver, heart, and brain how the mitochondrial outer membrane is permeabilized
mitochondria to Ca21-induced MPTP in vitro [Baines to release intermembrane proteins leading to apoptosis as
et al., 2005; Nakagawa et al., 2005]. In accordance, MPTP relates primarily to the inner mitochondrial mem-
hearts, hepatocytes, or neuronal cells from CypD-knock- brane? One of the explanations may be that MPTP com-
out mice were found to be resistant to ischemia-reperfu- ponents interact with activated proapoptotic Bcl-2 family
sion injury- or oxidative stress-induced necrosis known to proteins to form a speciﬁc pore rather than nonspeciﬁc
be mediated by MPTP [Baines et al., 2005; Nakagawa rupture of the outer membrane. Several studies have
et al., 2005; Schinzel et al., 2005]. However, CypD lack- reported cooperation between Bax and various compo-
ing cells died normally in response to apoptotic stimuli nents of MPTP in regulation of apoptosis [Marzo et al.,
activating the extrinsic or Bax-mediated cell death path- 1998; Adachi et al., 2004]. In contrast, other studies have
way [Baines et al., 2005; Nakagawa et al., 2005]. There- shown that Bcl-2 family proteins and MPTP are regulated
fore, it seems almost certain that CypD is an essential and function independently of each other in mitochondrial
component of MPTP. However, what still remains uncer- permeabilization [Polster et al., 2001; Kuwana et al.,
tain is how CypD, a soluble matrix enzyme with a pep- 2002; De Marchi et al., 2004]. There is also a possibility
tidyl-prolyl cis-trans isomerase activity, may cause per- that MPTP may lead to a mixed form of cell death that
meabilization of the mitochondrial inner membrane. There has both apoptotic and necrotic features. This may happen
still must be some membrane component to MPTP. in situations when MPTP-induced cytochrome c release
Recent studies by Halestrap’s and Bernardi’s groups sug- causes rapid activation of caspases and some time later
gest that such component may be a phosphate carrier of MPTP-induced mitochondrial dysfunction and depletion
the mitochondrial inner membrane [Basso et al., 2008; of glycolytic ATP results in disruption of plasma mem-
Halestrap, 2009]; however, the exact role of this carrier in brane integrity while caspases remain activated. The
MPTP formation remains unproven until it is demon- mixed form of cell death is often observed in primary
strated that knockdown of this protein affects MPTP func- cells and tissues in vivo and has been suggested as to be
tion. Another hypothesis explaining permeabilization of called ‘‘necraptosis’’ [Lemasters, 1999].
the mitochondrial inner membrane during MPT suggests
that the pore in the MPT may be formed by any oxidized/ CONSEQUENCES OF PERMEABILIZATION OF
misfolded carrier protein of the inner membrane including MITOCHONDRIAL MEMBRANES
ANT, phosphate carrier or even some new unidentiﬁed
mitochondrial protein [He and Lemasters, 2002; Hale- Whatever the mechanism, permeabilization of the mito-
strap, 2009]. chondrial outer membrane results in the release of inter-
Even if we do not know the exact structure of MPTP, membrane proteins into the cytosol: cytochrome c, apo-
there is no doubt that MPTP plays a signiﬁcant role in ptosis-inducing factor (AIF), Smac/DIABLO, HtrA2/Omi,
both apoptotic and necrotic cell death. MPTP dissipates etc. When in the cytosol, cytochrome c binds to the apo-
the protonmotive force, causing uncoupling of oxidative ptosis activating factor 1 (Apaf-1), which then recruits
phosphorylation and reversal of the ATP synthase reaction and activates procaspase-9 within the multiprotein com-
so that glycolytic ATP is hydrolyzed to maintain mito- plex called ‘‘apoptosome’’ [Yang et al., 1997]. The func-
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Mitochondria in Cell Death 5
tion of Smac/DIABLO (direct inhibitor of apoptosis-bind- ptosis is not well understood. The proposed mechanism
ing protein with a low isoelectric point) and HtrA2 (high for hypoxia-induced apoptosis suggests that an increase in
temperature requirement protein 2) is to neutralize the en- AMP/ATP ratio is sensed by AMP-dependent protein ki-
dogenous inhibitors of caspases (IAP, inhibitor of apopto- nase (AMPK). Thus, a decline in ATP, caused by hy-
sis proteins) and by doing so to promote caspase activa- poxia, ischemia or other insults may bring about the acti-
tion. AIF and endonuclease G translocate from the inter- vation of AMPK leading to phosphorylation and activa-
membrane space to the nucleus and cause caspase- tion of mitogen-activated protein kinase p38. The latter
independent cell death with some features of apoptosis has been shown to cause translocation of cytosolic Bax to
[Susin et al., 1999]. mitochondria in cultured cardiac myocytes exposed to
There is some evidence suggesting that not all proteins simulated ischemia [Capano and Crompton, 2006]. At the
translocate from mitochondria at once but that there is se- same time, AMPK upregulates glucose uptake and acti-
lectivity in the release of cytochrome c and Smac/DIA- vates glycolytic enzymes [Kahn et al., 2005] stimulating
BLO, AIF, or endonuclease G though the mechanism is glycolytic production of ATP, and thus maintaining a suf-
not clear [Huang et al., 2001; Mattson et al., 2008]. In ﬁcient ATP level for the execution of apoptosis.
neuronal cells, it has been observed that AIF and endonu- When cells receive signals for apoptosis and the degra-
clease G are released from mitochondria much later than dative processes are started the depletion of ATP usually
cytochrome c [Beart et al., 2007; Diwakarla et al., 2009] diverts the mode of cell death from apoptosis to necrosis.
and the release may be mediated by caspases or calpains This has been shown in various cell types (Jurkat cells,
[Polster et al., 2005] as it was sensitive to inhibitors of cortical neurons, human bronchial epithelial cells, etc.)
these proteases whereas the release of cytochrome c was using typical apoptotic stimuli such as staurosporine, acti-
insensitive. These data suggest that the loss of cyto- nomycin D, antibodies against CD95, Fas ligands, etc.
chrome c from mitochondria is not mediated by caspases; [Eguchi et al., 1997; Leist et al., 1999a,b], inhibitors of
however, activated caspases may have a feed-back effect the mitochondrial respiratory chain (cyanide, menadione,
on mitochondria promoting the release of additional fac- cigarette smoke, etc. [Li et al., 2005; van der Toorn et al.,
tors into the cytosol ensuring completion of apoptotic cell 2007]). In all these models, cell death that was started as
death. Moreover, activated caspase-3 has been shown to apoptosis ended up as necrosis when cellular ATP was
cleave the 75 kDa subunit (NDUF1) of complex I of the depleted by inhibiting mitochondrial respiration by NO or
mitochondrial respiratory chain resulting in inhibition of cigarette smoke, by overexpressing mitochondrial uncou-
its activity [Ricci et al., 2004]. Cleavage and inactivation pling protein 2, or by incubating cells in glucose-free
of complex I causes ROS production that seem to be media with inhibitors of mitochondrial ATPases
essential for immune tolerance induction by apoptotic (depleting glycolytically produced ATP). These studies
cells [Kazama et al., 2008]. Therefore, mitochondria not also point to the fact that the source of ATP––mitochond-
only trigger a cell death program by releasing cytochrome rially produced or glycolytic––is not important for apopto-
c and other proapoptotic proteins, but they also determine sis to proceed.
the impact of apoptotic cells on the immune system. Related to this, one might speculate that endogenous
inhibitors of mitochondrial respiration such as NO may
MITOCHONDRIA AND THE SWITCH OF THE also be potent inhibitors of apoptosis reversing it into
MODE OF CELL DEATH more damaging necrosis. This may be particularly rele-
vant to cells that are primarily dependent on mitochon-
Apoptosis is a highly organized and coordinated form drial energy production (i.e., cardiomyocytes, neurons)
of cell death, and as such, requires energy. Mitochondria and may happen in inﬂammatory pathologies which are
can facilitate apoptosis by supplying ATP for execution associated with production of proapoptotic cytokines and
of the program. On the other hand, a moderate fall in cel- simultaneous stimulation of NO production. In these
lular ATP level by itself may trigger apoptosis. For cases, cytokine-induced apoptosis may be switched to
instance, it has been shown that when cells were exposed ATP-depletion-caused necrosis due to NO-induced inhibi-
to oligomycin (an inhibitor of ATP synthase) and deoxy- tion of mitochondrial respiration when glycolytic capacity
glucose (which caused partial inhibition of glycolysis) the of the cells is low or inhibited. Necrotic cell death is usu-
intracellular level of ATP markedly decreased, and such ally more deleterious to the tissues and organs because it
cells died by apoptosis several hours later when ATP lev- causes rupture of the affected cells further propagating
els were recovered by removal of deoxyglucose [Izyumov inﬂammatory damage. In a similar manner, an environ-
et al., 2004]. If ATP was not restored, necrotic cell death mental pollutant cigarette smoke, the components of
proceeded due to lack of energy. This indicates that a which cause inhibition of mitochondrial respiratory func-
decrease in ATP can be recognized by cells as a signal tion, has been proposed to exacerbate development of
for apoptosis but the execution of it required ATP. How chronic obstructive pulmonary disease [van der Toorn
the decrease in cellular ATP results in initiation of apo- et al., 2007]. This pathology involves increased lung cell
Environmental and Molecular Mutagenesis. DOI 10.1002/em
apoptosis that may be converted to cell-destructive and 2009]. The main sites of ROS production in mitochondria
more injurious necrosis in smoking patients. Aging-related are complex I and complex III of the respiratory chain.
diseases may as well be related to an ATP-dependent Besides, ROS can be generated in mitochondria by two
switch in the cell death mode. Low levels of ATP other enzymes––monoamine oxidase and p66Shc [Cadenas
observed in aging cells due to suppressed activity of the and Davies, 2000; Di Lisa and Bernardi, 2005; Giorgio
mitochondrial oxidative phosphorylation system may per- et al., 2005]. Normally, p66Shc resides in the cytoplasm,
mit apoptotic stimuli to cause necrosis and this may be however, under certain conditions upon phosphorylation
the reason why aged organisms are more vulnerable to by protein kinase Cb it translocates to mitochondria where
oxidative stress [Miyoshi et al., 2006]. it directly oxidizes cytochrome c producing H2O2
As discussed earlier, numerous studies have provided [Giorgio et al., 2005]. Therefore, it is feasible that mito-
sufﬁcient evidence that apoptosis requires ATP; however, chondrial ROS can be involved in regulation of cell death
it is less obvious which particular steps in the apoptotic and determination of cell death mode. However, one
program are critically dependent on cellular ATP levels. might question whether mitochondria are able to produce
One of the ATP requiring steps is formation of caspase-9 enough ROS for such function in vivo. To this point, we
activating complex, apoptosome, consisting of Apaf-1, have to admit that today there is little knowledge about
cytochrome c, and procaspase-9. ATP acts as a cofactor the actual levels of mitochondrial ROS in vivo or about
in this process binding to Apaf-1; however, dATP is bet- their quantitative importance relative to other sources of
ter suited for this function. Other potential ATP-requiring ROS in induction or regulation of cell death processes
apoptotic events include protein kinase-mediated phospho- [Murphy, 2009].
rylation of proteins involved in apoptosis, enzymatic hy- Experimental evidence indicates that mitochondria can
drolysis of cellular components, chromatin condensation, mediate the shift from apoptosis to necrosis. From the
and formation of apoptotic bodies (for review see [Skula- clinical point of view, it would be desirable to reverse
chev, 2006]). this phenomenon: from un-controlled necrosis to apoptosis
In principle, mitochondria may perform the switch in which may be better controlled and which may proceed
the mode of cell death by another mechanism involving without signiﬁcant damage to the tissues. This would be
ROS. There is extensive literature indicating that an especially beneﬁcial in ischemic pathologies in the heart
increase in endogenous production or exposure of cells to and brain. During heart or brain ischemia, cells in the
exogenous ROS induces apoptosis through the mitochon- center of the injury usually die by necrosis resulting in
drial pathway involving translocation of cytochrome c to local inﬂammation and scar formation. Apoptosis nor-
the cytosol and activation of caspases. However, continu- mally occurs in surrounding noninfarcted areas and by
ous presence of low concentrations of H2O2 or bolus this form of cell death only single damaged cells are
additions of higher amounts of H2O2 have been shown to removed without expansion of tissue damage. If necrosis
inhibit caspases particularly when they actively cleave could be switched to apoptosis, that would be an impor-
their substrates [Borutaite and Brown, 2001; Hampton tant protective mechanism extending the time-window for
et al., 2002; Barbouti et al., 2007]. Inactivation of cas- therapeutic treatment and preventing deep tissue damage
pases is most likely to be due to reversible oxidation of [Ueda, 2009]. There are some indications from in vitro
thiol groups present in the active sites of caspases. Sup- experiments that if the glycolytic capacity of the cells
pression of caspase activity diverts apoptosis to necrosis was supported by supplying glucose or intermediate
at high concentrations of H2O2 [Borutaite and Brown, metabolites such as phosphoglycerate to the inﬂamed and
2001; Medan et al., 2005] or, in situations of continuous hypoxic aorta, ischemic retina or brain, this resulted in
exposure to low levels of H2O2, cell death proceeds as a prevention of necrosis [Ueda and Fujita, 2004; Borutaite
caspase-independent form though retaining typical fea- et al., 2005]. Similarly, in macrophages exposed to nitric
tures of apoptosis––cell shrinkage and chromatin conden- oxide, the necrotic cell death (which resulted from the si-
sation [Barbouti et al., 2007]. This may happen when the multaneous inhibition of mitochondrial respiration and
apoptotic stimulus causes the release of AIF and other glycolysis) can be reverted into apoptosis by supplying
proapoptotic proteins from mitochondria. glycolytic intermediates [Borutaite and Brown, 2003].
Although there is evidence that ROS may perform the Recent studies by Fujita and Ueda  revealed that
switch between modes of cell death, the source of ROS pro-thymosyn a (a multifunctional protein of oncogenic
for this function––mitochondrial or from other cellular origin) can prevent necrosis induced by simulated ische-
sites––is not yet determined. Mitochondria are one of the mia in neuronal cultures or by cerebral ischemia in exper-
major sites for ROS production in cells, and inhibition of imental animals by preventing depletion of cellular ATP
mitochondrial respiratory chain by various means (inhibi- while stimulating apoptosis. It was then possible to rescue
tors of complex I, III or cytochrome oxidase, loss of cyto- the cells from death in the presence of several neurotro-
chrome c from mitochondria, etc.) usually leads to an phins, including neuronal growth factor (NGF) [Ueda,
increase in ROS production [Droge, 2002; Murphy, 2009]. Whether such approaches can be applied in clinical
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Mitochondria in Cell Death 7
situations and what is the actual mechanism of the protec- cytochrome c affected caspase activation it would be irrele-
tion needs to be clariﬁed as this may have important ther- vant in the cell because cytochrome c in the cytosol would
apeutic implications in stroke and other ischemic patholo- always be fully reduced. During the years, this was the
gies. main concept concerning the role of cytochrome c redox
state in caspase activation. However, this somehow neglects
MITOCHONDRIA AND REGULATION OF APOPTOSIS the fact that the cytoplasm of cells contains mitochondria
AFTER CYTOCHROME C RELEASE with strong capacity to oxidize cytochrome c and in certain
conditions when the mitochondrial outer membrane is per-
In apoptosis, cytochrome c is a key signaling molecule meabilized (as happens during apoptosis) they may oxidize
sent by mitochondria to the cytosol where it participates external cytochrome c. In support of this, we have found
in the formation of the caspase-activating complex–– that cytochrome c added to the cytosol of apoptotic cells,
apoptosome. Cytochrome c is a redox active enzyme con- induced by staurosporine, is rapidly and fully oxidized, and
taining heme as a cofactor and for the apoptotic function this was in contrast to the nonapoptotic cytosol that reduced
a complete holoenzyme is essential. The enzyme can exist added cytochrome c [Borutaite and Brown, 2007]. The oxi-
in interconvertable reduced or oxidized forms depending dation of external cytochrome c was apparently mediated
on the redox state of environment. In mitochondria, cyto- by mitochondrial cytochrome oxidase as this activity was
chrome c is reduced by complex III and rapidly oxidized completely suppressed by an inhibitor of cytochrome oxi-
by cytochrome oxidase so that when the respiratory chain dase––azide [Borutaite and Brown, 2007]. This suggested
is fully active there is no increase in steady-state reduc- that the permeabilization of the mitochondrial outer mem-
tion of cytochrome c [Hancock et al., 2001]. It is likely brane to cytochrome c during apoptosis enables cytochrome
that cytochrome c which is released from mitochondria in oxidase to oxidize cytosolic cytochrome c, thus promoting
the early stages of apoptosis will be in an oxidized form caspase activation.
and in this form it would participate in assembly of the Interestingly, during apoptosis mitochondria may ac-
apoptosome. In support of this, there were several studies quire another mechanism to directly oxidize cytochrome
showing that oxidized cytochrome c rapidly activated cas- c. As it was mentioned earlier, this may happen after
pases-9 and -3 in a cytosolic fraction, whereas cyto- translocation of p66Shc to mitochondria where it acts as
chrome c reduced by dithiothreitol, cysteine, or glutathi- an oxidoreductase oxidizing cytochrome c and producing
one was completely ineffective at activating the caspases H2O2 [Giorgio et al., 2005]. Thus, there may be several
[Hancock et al., 2001; Suto et al., 2005]. In agreement, mitochondrial activities maximizing oxidation of cyto-
we found that oxidation of cytochrome c by supplemented chrome c, which is released from mitochondria when cells
cytochrome oxidase stimulates caspase activation in cyto- are committed to apoptosis.
sols prepared from macrophage J774 or HeLa cells, The redox state of cytosolic cytochrome c may also
whereas reduction of cytochrome c by added TMPD (tet- depend on the redox status of the environment outside of
ramethyl-phenylenediamine) or yeast lactate dehydrogen- the mitochondria. Different cell types have been shown to
ase/cytochrome c reductase (which selectively reduces have different cytosolic cytochrome c reducing activity
cytochrome c in the presence of lactate) blocks caspase and this may also change during apoptosis. Recently,
activation [Borutaite and Brown, 2007]. Vaughn and Deshmukh  have shown that injection
On the other hand, there were studies apparently showing of tBid into mouse embryonic cells or sympathetic neu-
that the redox state of cytochrome c had no effect on cas- rons deprived of NGF induced rapid apoptosis whereas it
pase activation and apoptosis [Kluck et al., 1997; Hampton did not cause any neuronal death in the presence of NGF
et al., 1998]. It has been shown that reduced and oxidized though there was similar cytochrome c release from mito-
cytochrome c added to cytosolic extracts were equally chondria in all three cases. This was shown to be poten-
effective in activation of caspases and that the Fe of cyto- tially caused by the higher cytosolic capacity of healthy,
chrome c could be replaced by a redox inactive metal with NGF-maintained neurons to reduce cytochrome c (and
no effect on proapoptotic function of the enzyme [Kluck thus prevent caspase activation) than that of NGF-
et al., 1997; Hampton et al., 1998]. However, the cytosol deprived cells. Cytosolic cytochrome c reducing capacity
has strong capacities to both reduce and oxidize cytochrome of neurons and cancer cells was found to be high (com-
c, so that reduced or oxidized cytochrome c can be brought pared to mouse embryonic ﬁbroblasts used in that study)
to the same redox state within a few minutes of addition, and dependent on the redox state of intracellular glutathi-
and the efﬁcacy of redox inactive cytochrome c shows only one which, in turn, was dependent on glucose metabolism
that cytochrome c does not have to change redox state to be by the pentose phosphate pathway maintaining cellular
effective [Borutaite and Brown, 2007]. A study by Hampton NADPH levels [Vaughn and Deshmukh, 2008]. Studies in
et al.  showed that cytochrome c added to cytosol of our laboratory have demonstrated that inhibition of cyto-
normal, nonapoptotic cells was rapidly reduced and this led chrome oxidase was able to increase cytochrome c-reduc-
investigators to conclude that even if the redox state of ing activity only in homogenates from cells at early stages
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Fig. 1. Cytoplasmic cytochrome c-reducing activity decreases during ap-
optosis. HeLa cells were incubated with/without 2 lM staurosporine for
4–7 hrs. Whole cell homogenates were prepared and incubated with
added 10 lM cytochrome c. Cytochrome c reduction rate was assayed
following spectral changes in the interval of wavelengths 500–600 nm
for 5 min and measuring the height of characteristic peak at 550 nm as
described in Borutaite and Brown .
of apoptosis (e.g., after 4 hr incubation with staurospor-
ine, Fig. 1). In later phases of apoptosis (7 hr with stauro-
sporine, Fig. 1), cytochrome c reducing activity was lost
and could not be recovered by inhibiting cytochrome oxi-
dase with azide. The loss of cytochrome c activity in the
cytosol correlated with a decrease in cellular NADPH
content which was found to be normal in early apoptotic
cells but was almost completely exhausted after 6 hr of
incubation of the cells with staurosporine [Baniene and
Borutaite, unpublished data] suggesting that NADPH may
be one of the factors involved in the regulation of the re-
dox status of cytochrome c after its release from mito-
There are several possible mechanisms by which the re-
dox state of cytochrome c may regulate apoptosis and
they were recently reviewed in Brown and Borutaite
; however, they are mainly speculative and need
thorough experimental investigations.
The cytochrome c-mediated pathway of apoptosis is im-
portant in many pathophysiological conditions including is-
Fig. 2. Mitochondria in the regulation of cell death and survival. (A)
chemic pathologies, atherosclerosis, endothelial damage in Mitochondrial outer membrane permeabilization by Bcl-2 proteins, lipids
diabetes, and neurodegenerative disorders. In contrast, or MPTP causes the release of cytochrome c. Cytochrome c can be oxi-
many cancer cells developed various mechanisms of sup- dized by cytochrome oxidase (COX) or other mitochondrial activities
pression of this pathway. It is important to understand how and in this oxidized form it binds to Apaf-1 forming apoptosome which
activates caspases leading to apoptosis (when there is enough ATP, i.e.,
this pathway of apoptosis can be regulated at the late, post-
produced by glycolysis). When activated, caspases cleave and inactivate
cytochrome c release level, so that interventions can be complex I of the mitochondrial respiratory chain increasing production
designed to prevent or to facilitate cell death. Factors that of ROS that, in turn, leads to induction of immunotolerance of apoptotic
are capable of oxidizing cytosolic cytochrome c may cells. (B) When released into the cytosol, cytochrome c may be reduced
render cancer cells more sensitive to apoptosis induced by by various chemicals or enzymes. Reduced cytochrome c is inactive in
initiation of apoptosis and therefore leads to cell survival. (C) MPTP
chemotherapy. On the other hand, there are various bioac- and related loss of cytochrome c from mitochondria may result in mito-
tive plant components that may directly reduce cytochrome chondrial dysfunction and ATP depletion (when glycolysis is insufﬁcient
c and thus may be potent means to prevent or at least delay or inhibited) causing necrosis.
unwanted apoptosis in other pathologies.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Mitochondria in Cell Death 9
CONCLUDING REMARKS Beart PM, Lim ML, Chen B, Diwakarla S, Mercer LD, Cheung NS,
Nagley P. 2007. Hierarchical recruitment by AMPA but not by
There is a rapidly growing body of evidence indicating staurosporine of pro-apoptotic mitochondrial signaling in cultured
that mitochondria are the active players not only in cell cortical neurons: Evidence for caspase-dependent/independent
cross-talk. J Neurochem 103:2408–2427.
health but also in various forms of cell death (Fig. 2). De- Belosludtsev K, Saris NE, Andersson LC, Belosludtseva N, Agafonov A,
spite fascinating new discoveries of the multiple roles of Sharma A, Moshkov DA, Mironova GD. 2006. On the mecha-
mitochondria in cell death, there are still considerable nism of palmitic acid-induced apoptosis: The role of a pore
challenges to be solved. We still need better understand- induced by palmitic acid and Ca21 in mitochondria. J Bioenerg
ing of the molecular mechanisms of permeabilization of Biomembr 38:113–120.
Bernardi P, Petronilli V. 1996. The permeability transition pore as a mi-
the mitochondrial outer membrane, particularly whether
tochondrial calcium release channel: A critical appraisal. J Bioen-
there is one general mechanism of permeabilization erg Biomembr 28:131–138.
involving interactions of Bcl-2 proteins, MPTP compo- Borutaite V, Brown GC. 2001. Caspases are reversibly inactivated by
nents and lipid molecules, or there are multiple ways of hydrogen peroxide. FEBS Lett 500:114–118.
disruption of the integrity of mitochondrial membranes Borutaite V, Brown GC. 2003. Nitric oxide induces apoptosis via hydro-
gen peroxide, but necrosis via energy and thiol depletion. Free
that may be activated by different apoptotic signals in
Radic Biol Med 35:1457–1468.
various types of cells and conditions. It is unclear whether Borutaite V, Brown GC. 2007. Mitochondrial regulation of caspase acti-
mitochondrially produced ROS are important in regulation vation by cytochrome oxidase and tetramethylphenylenediamine
of cell death, and if so, by which mechanisms? It would via cytosolic cytochrome c redox state. J Biol Chem 282:31124–
be desirable to have a full understanding of how the 31130.
Borutaite V, Morkuniene R, Brown GC. 2000. Nitric oxide donors,
switch between the modes of cell death can be precisely
nitrosothiols and mitochondrial respiration inhibitors induce cas-
controlled in various pathologic conditions. The question pase activation by different mechanisms. FEBS Lett 467:155–
of the function of cytochrome c in the apoptosome is not 159.
completely resolved either. New innovative methods are Borutaite V, Jekabsone A, Morkuniene R, Brown GC. 2003. Inhibition
required to measure changes in the redox state of cyto- of mitochondrial permeability transition prevents mitochondrial
dysfunction, cytochrome c release and apoptosis induced by heart
solic cytochrome c. This would be helpful in solving con-
ischemia. J Mol Cel Cardiol 35:357–366.
troversy concerning the redox regulation of apoptosome- Borutaite V, Moncada S, Brown GC. 2005. Nitric oxide from inducible
dependent caspase activation in the cells and in determin- nitric oxide synthase sensitizes the inﬂamed aorta to hypoxic
ing factors that may control the redox state of cytosolic damage via respiratory inhibition. Shock 23:319–323.
cytochrome c. The possible pharmacological interventions Breckenridge DG, Xue D. 2004. Regulation of mitochondrial membrane
permeabilization by BCL-2 family proteins and caspases. Curr
into the late, post-cytochrome c phase of apoptosis would
Opin Cell Biol 16:647–652.
promise the development of novel strategies in treatment Brown GC. 2007. Nitric oxide and mitochondria. Front Biosci 12:1024–
of various diseases. 1033.
Brown GC, Borutaite V. 2008. Regulation of apoptosis by the redox
state of cytochrome c. Biochim Biophys Acta 1777:877–881.
REFERENCES Cadenas E, Davies KJ. 2000. Mitochondrial free radical generation, oxi-
dative stress and aging. Free Radic Biol Med 29:222–230.
Adachi M, Higuchi H, Miura S, Azuma T, Inokuchi S, Saito H, Kato S, Capano M, Crompton M. 2006. Bax translocates to mitochondria of heart
Ishii H, et al. 2004. Bax interacts with the voltage dependent cells during simulated ischemia: Involvement of AMP-activated
anion channel and mediates ethanol-induced apoptosis in rat he- and p38 mitogen-activated protein kinases. Biochem J 395:57–64.
patocytes. Am J Physiol 287:G695–G705. Cheng EH, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ. 2003.
Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton VDAC2 inhibits BAK activation and mitochondrial apoptosis.
MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, Robbins Science 301:513–517.
J, Molkentin JD. 2005. Loss of cyclophilin D reveals a critical Chicco AJ, Sparagna GC. 2007. Role of cardiolipin alterations in mito-
role for mitochondrial permeability transition in cell death. Na- chondrial dysfunction and disease. Am J Physiol Cell Physiol
ture 434:658–662. 292:C33–C44.
Baines CP, Kaiser RA, Sheiko T, craigen WJ, Molkentin JD. 2007. Volt- Cory S, Adams JM. 2002. The Bcl2 family: Regulators of the cellular
age-dependent anion channels are dispensable for mitochondrial- life-or-death switch. Nat Rev Cancer 2:647–656.
dependent cell death. Nature Cell Biol 9:550–555. Crompton M. 1999. The mitochondrial permeability transition pore and
Barbouti A, Amorgianiotis C, Kolettas E, Kanavaros P, Galaris D. 2007. its role in cell death. Biochem J 341:233–249.
Hydrogen peroxide inhibits caspase-dependent apoptosis by inac- `
De Marchi U, Campello S, Szabo I, Tombola F, Martinou JC, Zoratti M.
tivating procaspase-9 in an iron-dependent manner. Free Radic 2004. Bax does not directly participate in the Ca21-induced per-
Biol Med 43:1377–1387. meability transition of isolated mitochondria. J Biol Chem 279:
Basanez G, Sharpe JC, Brandt TB, Hardwick JM, Zimmerberg J. 2002. 37415–37422.
Bax-type apoptotic proteins porate pure lipid bilayers through a Desagher S, Osen Sand A, Nichols A, Eskes R, Montessuit S, Lauper S,
mechanism sensitive to intrinsic monolayer curvature. J Biol Maundrell K, Antonsson B, Martinou JC. 1999. Bid-induced con-
Chem 277:49360–49365. formational change of Bax is responsible for mitochondrial cyto-
Basso E, Petronilli V, Forte MA, Bernardi P. 2008. Phosphate is essen- chrome c release during apoptosis. J Cell Biol 144:891–901.
tial for inhibition of the mitochondrial permeability transition Di Lisa F, Bernardi P. 2005. Mitochondrial function and myocardial
pore by cyclosporin A, by cyclophilin D ablation. J Biol Chem aging. A critical analysis of the role of permeability transition.
283:26307–26311. Cardiovasc Res 66:222–232.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Distelhorst CW, Shore GC. 2004. Bcl-2 and calcium: Controversy He L, Lemasters JJ. 2002. Regulated and unregulated mitochondrial per-
beneath the surface. Oncogene 23:2875–2880. meability transition pores: A new paradigm of pore structure and
Diwakarla S, Nagley P, Hughes ML, Chen B, Beart PM. 2009. Differen- function? FEBS Lett 512:1–7.
tial insult-dependent recruitment of the intrinsic mitochondrial Hortelano S, Dallaporta B, Zamzami N, Hirsch T, Susin SA, Marzo I, Bosca
pathway during neuronal programmed cell death. Cell Mol Life L, Kroemer G. 1997. Nitric oxide induces apoptosis via triggering
Sci 66:156–172. mitochondrial permeability transition. FEBS Lett 410: 373–377.
Droge W. 2002. Free radicals in the physiological control of cell func- Hovius R, Thijssen J, van der Linden P, Nicolay K, de Kruijff B. 1993.
tion. Physiol Rev 82:47–95. Phospholipid asymmetry of the outer membrane of rat liver mito-
Du H, Guo L, Fang F, Chen D, Sosunov AA, McKhann GM, Yan Y, chondria. Evidence for the presence of cardiolipin on the outside
Wang C, Zhang H, Molkentin JD, Gunn-Moore FJ, Vonsattel JP, of the outer membrane. FEBS Lett 330:71–76.
Arancio O, Chen JX, Yan SD. 2008. Cyclophilin D deﬁciency Hsu YT, Youle RJ. 1998. Bax in murine thymus is a soluble monomeric
attenuates mitochondrial and neuronal perturbation and amelio- protein that displays differential detergent-induced conformations.
rates learning and memory in Alzheimer’s disease. Nat Med J Biol Chem 273:10777–10783.
14:1097–1105. Huang X, Zhai D, Huang Y. 2001. Dependence of permeability transi-
Duchen MR, McGuinness O, Brown LA, Crompton M. 1993. On the tion pore opening and cytochrome c release from mitochondria
involvement of a cyclosporin A sensitive mitochondrial pore in on mitochondria energetic status. Mol Cell Biochem 224:1–7.
myocardial reperfusion injury. Cardiovasc Res 27:1790–1794. Hunter DR, Haworth RA. 1979a. The Ca21-induced membrane transi-
Eguchi Y, Shimizu S, Tsujimoto Y. 1997. Intracellular ATP levels deter- tion in mitochondria. I. The protective mechanisms. Arch Bio-
mine cell death fate by apoptosis or necrosis. Canc Res 57:1835– chem Biophys 195:453–459.
1840. Hunter DR, Haworth RA. 1979b. The Ca21-induced membrane transi-
Erusalimsky JD, Moncada S. 2007. Nitric oxide and mitochondrial sig- tion in mitochondria. III. Transitional Ca21 release. Arch Bio-
naling: From physiology to pathophysiology. Arterioscler Thromb chem Biophys 195:468–477.
Vasc Biol 27:2524–2531. Ichas F, Mazat JP. 1998. From calcium signaling to cell death: Two con-
Fujita R, Ueda H. 2007. Prothymosin-alpha1 prevents necrosis and apo- formations for the mitochondrial permeability transition pore.
ptosis following stroke. Cell Death Differ 14:1839–1842. Switching from low- to high-conductance state. Biochim Biophys
Garcia Fernandez M, Troiano L, Moretti L, Nasi M, Pinti M, Salvioli S, Acta 1366:33–50.
Dobrucki J, Cossarizza A. 2002. Early changes in intramitochon- ´ ´
Ivashyna O, Garcıa-Saez AJ, Ries J, Christenson ET, Schwille P, Schle-
drial cardiolipin distribution during apoptosis. Cell Growth Differ singer PH. 2009. Detergent-activated BAX protein is a monomer.
13:449–455. J Biol Chem 284:23935–23946.
Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG, Tu Izyumov DS, Avetisyan AV, Pletjushkina OY, Sakharov DV, Wirtz KW,
HC, Kim H, Cheng EHY, Tjandra N, Walensky LD. 2008. BAX Chernyak BV, Skulachev VP. 2004. ‘‘Wages of fear’’: Transient
activation is initiated at a novel interaction site. Nature threefold decrease in intracellular ATP level imposes apoptosis.
455:1076–1081. Biochim Biophys Acta 1658:141–147.
Giorgio M, Migliaccio E, Orsini F, Paolucci D, Moroni M, Contursi C, Pel- Kagan VE, Tyurin VA, Jiang J, Tyurina YY, Ritov VB, Amoscato AA, Osi-
licci G, Luzi L, Minucci S, Marcaccio M, Pinton P, Rizzuto R, Ber- pov AN, Belikova NA, Kapralov AA, Kini V, Vlasova II, Zhao Q,
nardi P, Paolucci F, Pelicci PG. 2005. Electron transfer between Zou M, Di P, Svistunenko DA, Kurnikov IV, Borisenko GG. 2005.
cytochrome c and p66Shc generates reactive oxygen species that Cytochrome c acts as a cardiolipin oxygenase required for release of
trigger mitochondrial apoptosis. Cell 122:221–233. proapoptotic factors. Nat Chem Biol 1:223–232.
Grifﬁths EJ, Halestrap AP. 1993. Protection by cyclosporin A of ische- Kahn BB, Alquier T, Carling D, Hardie DG. 2005. AMP-activated pro-
mia/reperfusion-induced damage in isolated rat hearts. J Mol Cell tein kionase: Ancient energy gauge provides clues to modern
Cardiol 25:1461–1469. understanding of metabolism. Cell Metab 1:15–25.
Grifﬁths GJ, Corfe BM, Savory P, Leech S, Esposti MD, Hickman JA, Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA.
Dive C. 2001. Cellular damage signals promote sequential 2008. Induction of immunological tolerance by apoptotic cells
changes at the N-terminus and BH-1 domain of the pro-apoptotic requires caspase-dependent oxidation of high-mobility group box-
protein Bak. Oncogene 20:7668–7676. 1 protein. Immunity 29:21–32.
Halestrap AP, Clarke SJ, Javadov SA. 2004. Mitochondrial permeability Kim H, Tu HC, Ren D, Takeuchi O, Jeffers JR, Zambetti GP, Hsieh JJ,
transition pore opening during myocardial reperfusion—A target Cheng EH. 2009. Stepwise activation of Bax and Bak by tBid,
for cardioprotection. Cardiovasc Res 61:372–385. Bim, and PUMA initiates mitochondrial apoptosis. Mol Cell
Halestrap AP. 2009. What is the mitochondrial permeability transition 36:487–499.
pore? J Mol Cell Cardiol 46:821–831. Kluck RM, Martin SJ, Hoffman BM, Zhou JS, Green DR, Newmeyer
Halestrap AP, Kerr PM, Javadov S, Woodﬁeld KY. 1998. Elucidating DD. 1997. Cytochrome c activation of CPP32-like proteolysis
the molecular mechanism of the permeability transition pore and plays a critical role in a Xenopus cell-free apoptosis system.
its role in reperfusion injury of the heart. Biochim Biophys Acta EMBO J 16:4639–4649.
1366:79–94. Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J, Jones DP, Mac-
Hampton MB, Zhivotovsky B, Slater AFG, Burgess DH, Orrenius S. Gregor GR, Wallace DC. 2004. The ADP/ATP translocator is
1998. Importance of the redox state of cytochrome c during cas- not essential for the mitochondrial permeability transition pore.
pase activation in cytosolic extracts. Biochem J 329:95–99. Nature 427:461–465.
Hampton MB, Stamenkovic I, Winterbourn CC. 2002. Interaction with Kroemer G, Galluzzi L, Brenner C. 2007. Mitochondrial membrane per-
substrate sensitises caspase-3 to inactivation by hydrogen perox- meabilization in cell death. Physiol Rev 87:99–163.
ide. FEBS Lett 517:229–232. Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M,
Hancock JT, Desikan R, Neill SJ. 2001. Does the redox status of cyto- Schneiter R, Green DR, Newmeyer DD. 2002. Bid, Bax, lipids
chrome C act as a fail-safe mechanism in the regulation of pro- cooperate to form supramolecular openings in the outer mito-
grammed cell death? Free Radic Biol Med 31:697–703. chondrial membrane. Cell 111:331–342.
Hausenloy DJ, Duchen MR, Yellon DM. 2003. Inhibiting mitochondrial Leist M, Single B, Naumann H, Fava E, Simon B, Kuhnle S, Nicotera P.
permeability transition pore opening at reperfusion protects 1999a. Inhibition of mitochondrial ATP generation by nitric oxide
against ischaemia-reperfusion injury. Cardiovasc Res 60:617–625. switches apoptosis to necrosis. Exp Cell Res 249:396–403.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Mitochondria in Cell Death 11
Leist M, Single B, Naumann H, Fava E, Simon B, Kuhnle S, Nicotera P. ˜
Ricci JE, Munoz-Pinedo C, Fitzgerald P, Bailly-Maitre B, Perkins GA,
1999b. Nitric oxide inhibits execution of apoptosis at two distinct Yadava N, Schefﬂer IE, Ellisman MII, Green DR. 2004. Disrup-
ATP-dependent steps upstream and downstream of mitochondrial tion of mitochondrial function during apoptosis is mediated by
cytochrome c release. Biochem Biophys Res Commun 258:215–221. caspase cleavage of p75 subunit of complex I of the electron
Lemasters JJ. 1999. Necraptosis and the mitochondrial permeability tran- transport chain. Cell 117:773–786.
sition: Shared pathways to necrosis and necraptosis. Am J Physiol Schinzel AC, Takeuchi O, Huang Z, Fisher JK, Zhou Z, Rubens J, Hetz
276:G1–G6. C, Danial NN, Moskowitz MA, Korsmeyer SJ. 2005. Cyclophilin
Lesnefsky EJ, Hoppel CL. 2008. Cardiolipin as an oxidative target in D is a component of mitochondrial permeability transition and
cardiac mitochondria in the aged rat. Biochim Biophys Acta mediates neuronal cell death after focal cerebral ischemia. Proc
1777:1020–1027. Natl Acad Sci USA 102:12005–12010.
Li L, Prabhakaran K, Mills EM, Borowitz JL, Isom GE. 2005. Enhance- Scorrano L, Korsmeyer SJ. 2003. Mechanisms of cytochrome c release
ment of cyanide-induced mitochondrial dysfunction and cortical by proapoptotic BCL-2 family members. Biochem Biophys Res
cell necrosis by uncoupling protein-2. Toxicol Sci 86:116–124. Commun 304:437–444.
Lucken-Ardjomande S, Martinou JC. 2005. Newcomers in the process of Siskind LJ. 2005. Mitochondrial ceramide and the induction of apoptosis.
mitochondrial permeabilization. J Cell Sci 118:473–483. J Bioenerg Biomembr 37:143–153.
Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira Skulachev VP. 2006. Bioenergetic aspects of apoptosis, necrosis and
HL, Prevost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G. mitoptosis. Apoptosis 11:473–485.
1998. Bax and adenine nucleotide translocator cooperate in the Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM,
mitochondrial control of apoptosis. Science 281:2027–2031. Mangion J, Jacotot E, Costantini P, Loefﬂer M, Larochette N,
Matsumoto S, Friberg H, Ferrand-Drake M, Wieloch T. 1999. Blockade Goodlett DR, Aebersold R, Siderovski DP, Penninger JM,
of the mitochondrial permeability transition pore diminishes Kroemer G. 1999. Molecular characterisation of mitochondrial
infarct size in the rat after transient middle cerebral artery occlu- apoptosis-inducing factor. Nature 397:441–446.
sion. J Cereb Blood Flow Metab 19:736–741. Suto D, Sato K, Ohba Y, Yoshimura T, Fuji J. 2005. Suppression of the pro-
Mattson MP, Gleichmann M, Cheng A. 2008. Mitochondria in neuro- apoptotic unction of cytochrome c by singlet oxygen via a heme re-
plasticity and neurological disorders. Neuron 60:748–766. dox state-independent mechanism. Biochem J 392:399–406.
Medan D, Wang L, Toledo D, Lu B, Stehlik C, Jiang BH, Shi X, Roja- Ueda H. 2009. Prothymosin a and cell death mode switch, a novel target
nasakul Y. 2005. Regulation of Fas (CD95)-induced apoptotic for the prevention of cerebral ischemia-induced damage. Pharma-
and necrotic cell death by reactive oxygen species in macro- col Therapeut 123:323–333.
phages. J Cell Physiol 203:78–84. Ueda H, Fujita R. 2004. Cell death mode switch from necrosis to apo-
Miyoshi N, Oubrahim H, Chock PB, Stadtman ER. 2006. Age-dependent ptosis in brain. Biol Pharm Bull 27:950–955.
cell death and the role of ATP in hydrogen peroxide-induced apo- Valentijn AJ, Upton JP, Gilmore AP. 2008. Analysis of endogenous Bax
ptosis and necrosis. Proc Natl Acad Sci USA 103:1727–1731. complexes during apoptosis using blue native PAGE: Implica-
Murphy MP. 2009. How mitochondria produce reactive oxygen species? tions for Bax activation and oligomerization. Biochem J
Biochem J 417:105–111. 412:347–357.
Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata van der Toorn M, Slebos DJ, de Bruin HG, Leuvenink HG, Leuvenink
H, Inohara H, Kubo T, Tsujimoto Y. 2005. Cyclophilin D-de- HG, Bakker SJ, Gans RO, Koeter GH, van Oosterhout AJ, Kauf-
pendent mitochondrial permeability transition regulates some ne- man HF. 2007. Cigarette smoke-induced blockade of the mito-
crotic but not apoptotic cell death. Nature 434:652–658. chondrial respiratory chain switches lung epithelial cell apoptosis
Nechushtan A, Smith CL, Lamensdorf I, Yoon SH, Youle RJ. 2001. Bax into necrosis. Am J Physiol Lung Cell Mol Physiol 292:L1211–
and Bak coalesce into novel mitochondria-associated clusters dur- L1218.
ing apoptosis. J Cell Biol 153:1265–1276. Vaughn AE, Deshmukh M. 2008. Glucose metabolism inhibits apoptosis
Ott M, Norberg E, Zhivotovsky B, Orrenius S. 2009. Mitochondrial tar- in neurons and cancer cells by redox inactivation of cytochrome
geting tBid/Bax: A role for the TOM complex. Cell Death Differ c. Nat Cell Biol 10:1477–1483.
16:1075–1082. Wei MC, Zong WX, Cheng EH, Panoutsakopoulou V, Ross AJ, Roth
Palma E, Tiepolo T, Angelin A, Sabatelli P, Maraldi NM, Basso E, Forte KA, MacGregor GR, Thompson CB, Korsmeyer SJ. 2001. Proa-
MA, Bernardi P, Bonaldo P. 2009. Genetic ablation of cyclophilin poptotic BAX, BAK: A requisite gateway to mitochondrial dys-
D rescues mitochondrial defects and prevents muscle apoptosis in function and death. Science 292:727–730.
collagen VI myopathic mice. Hum Mol Genet 18:2024–2031. Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM,
Pinton P, Giorgi C, Siviero R, Zecchini E, Rizzuto R. 2009. Calcium Huang DCS. 2005. Proapoptotic Bak is sequestered by Mcl-1 and
and apoptosis: ER-mitochondrial Ca21 transfer in the control of Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins.
apoptosis. Oncogene 27:6407–6418. Genes Dev 19:1294–1305.
Polcic P, Su X, Fowlkes J, Blachly-Dyson E, Dowhan W, Forte M. 2005. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones
Cardiolipin and phosphatidylglycerol are not required for the in DP, Wang X. 1997. Prevention of apoptosis by Bcl-2: Release of
vivo action of Bcl-2 family proteins. Cell Death Differ 12:310–312. cytochrome c from mitochondria blocked. Science 275:1129–
Polster BM, Kinnally KW, Fiskum G. 2001. BH3 death domain peptide 1132.
induces cell type-selective mitochondrial outer membrane perme- Zamzami N, Larochette N, Kroemer G. 2005. Mitochondrial permeabil-
ability. J Biol Chem 276:37887–37894. ity transition in apoptosis and necrosis. Cell Death Differ
Polster BM, Basanez G, Etxebarria A, Hardwick JM, Nichols DG. 2005. 12:1478–1480.
Calpain I inducescleavage and release of apoptosis-inducing fac- Zoratti M, Szabo I. 1995. The mitochondrial permeability transition. Bio-
tor from isolated mitochondria. J Biol Chem 280:6447–6454. chim Biophys Acta 1241:139–176.
Resende R, Ferreiro E, Pereira C, Resende De Oliveira C. 2008. Neuro-
toxic effect of oligomeric and ﬁbrilar species of amyloid-beta pep-
tide 1–42: Involvement of endoplasmic reticulum calcium release Accepted by—
in oligomer-induced cell death. Neuroscience 155:725–737. D. Wilson