Contraryto the view that caspase-mediated apoptosis represents the standard programmed cell death, recent studies indicate that an apoptotic morphologycan be produced independent of casp
Trang 1R E V I E W A R T I C L E
Programmed cell death
Apoptosis and alternative deathstyles
Cinthya Assunc¸a˜o Guimara˜es* and Rafael Linden
Instituto de Biofı´sica da UFRJ, Rio de Janeiro, Brazil
Programmed cell death is a major component of both
normal development and disease The roles of cell death
during either embryogenesis or pathogenesis, the signals
that modulate this event, and the mechanisms of cell
demise are the major subjects that drive research in this
field Increasing evidence obtained both in vitro and
in vivosupports the hypothesis that a variety of cell death
programs maybe triggered in distinct circumstances
Contraryto the view that caspase-mediated apoptosis
represents the standard programmed cell death, recent
studies indicate that an apoptotic morphologycan be
produced independent of caspases, that autophagic
exe-cution pathways of cell death may be engaged without either the involvement of caspases or morphological signs
of apoptosis, and that even the necrotic morphologyof cell death maybe consistentlyproduced in some cases, including certain plants Alternative cell death programs mayimplynovel therapeutic targets, with important consequences for attempts to treat diseases associated with disregulated programmed cell death
Keywords: programmed cell death; apoptosis; autophagy; necrosis; neurodegenerative diseases
Introduction
Programmed cell death is a major component of both
normal development and disease [1–11] The roles of cell
death during either embryogenesis or pathogenesis, the
signals that induce or regulate this event, and the
mecha-nisms of cell demise are common subjects that drive research
in this field [12–17] The purpose of this article is to review
major morphological, biochemical and molecular hallmarks
of distinct forms of programmed cell death, and to examine
the limits of some prevailing views of cell death modes and
mechanisms
The classical ultrastructural studies of Kerr and coworkers
[18] provided evidence that cells mayundergo at least two
distinct types of cell death: The first type is known as necrosis,
a violent and quick form of degeneration affecting extensive cell populations, characterized bycytoplasm swelling, destruction of organelles and disruption of the plasma membrane, leading to the release of intracellular contents and inflammation A remarkablydistinct type of cell death was called apoptosis, identified in single cells usually surrounded byhealthy-looking neighbors, and characterized bycell shrinkage, blebbing of the plasma membrane, maintenance of organelle integrity, and condensation and fragmentation of DNA, followed byordered removal through phagocytosis [18,19] During the last 30 years, cell death has usuallybeen classified within this dichotomy The work of Kerr and collaborators stirred interest in programmed cell death, both because it provided a visible object (the apoptotic profile) to be consistentlyapproached
in experimental studies prior to the disappearance of the dead cells, as well as due to the evidence provided for controlled events that justifythe operational definition of ÔprogrammedÕ [20] At a first approximation, necrosis was attributed to accidental, uncontrolled degeneration, whereas apoptosis presented the defining characteristics of a cell death program Indeed, manyresearch groups began to consider apoptosis and programmed cell death as a single entity, despite the knowledgeable criticism of pioneers in the field [21] Nonetheless, this simplified and generalized scheme neglects the exceptions; for example, morphologies
of cell death that do not fit in the original classification (reviewed in [22]) On the other hand, evidence is now available for multiple alternative cell death pathways, as well as for cross-talk of intracellular mechanisms involved in distinct aspects of cell degeneration This review will focus
on the growing evidence that, besides apoptosis, autophagic and necrotic forms of cell degeneration maybe pro-grammed, and underlie cell death either in isolation or combined with mechanisms of apoptosis
Correspondence to R Linden, Instituto de Biofı´sica da UFRJ,
Centro de Cieˆncias da Sau´de, bloco G, Cidade Universita´ria,
21949–900, Rio de Janeiro, Brazil Tel.: + 55 21 25626553,
Fax: + 55 21 22808193, E-mail: rlinden@biof.ufrj.br
Abbreviations: AIF, apoptosis inducing factor; DAPk,
death-associ-ated protein kinase; DRP, DAPk-reldeath-associ-ated protein kinase; FADD,
Fas-associated protein with death domain; IAP, inhibitor of
apoptosis; LEI, leucocyte elastase inhibitor; L-DNase II,
LEI-DNase II; MPT, mitochondrial permeabilitytransition;
PCD, programmed cell death; PI3K, phosphatidylinositol-3-kinase;
TNFa, tumor necrosis factor-alpha; TUNEL, TdT-mediated
biotin-dUDP nick-end labeling.
Note: a website is available at http://www.biof.ufrj.br/neurogen
*Present address: The Hebrew Universityof Jerusalem, Department of
Biological Chemistry, Institute of Life Sciences, The Edmond J Safra
Campus, Givat Ram, Jerusalem 91904 Israel.
(Received 12 January2004, revised 17 February2004,
accepted 10 March 2004)
Trang 2Defining features of programmed cell death
Despite the tremendous impact of research in apoptosis
upon the understanding both of cellular and molecular
mechanisms of cell demise, as well as of mechanisms of
degenerative diseases, the confusion between apoptosis and
programmed cell death has somewhat obscured the field
Regardless of whether this paradox is attributable to either
disconnection of modern science from its philosophical
foundations [23] or to a more trivial neglect of classical
papers (reviewed in [20]), it is likelythat progress in the
identification and understanding of nonapoptotic forms
of programmed cell death mayhave been unnecessarily
delayed
Indeed, well before the upsurge in the understanding of
mechanisms of apoptosis, a clear warning had been issued
to avoid confusion between the form of cell death called
apoptosis, and the concept of programmed cell death as a
sequence of events, but not necessarilythose that led to the
morphologyof apoptosis [21]
Although the original work that led to the concept of
programmed cell death was carried out in developing
organisms [24–29], there is nothing
intrinsicallydevelop-mental in the concept The apoptotic form has been long
identified in adult tissues [18], and there is no evidence that
anyparticular form of cell death, much less the operational
concept of programmed cell death, can be attributed
exclusivelyto either developing or mature cells Conversely,
it has been argued that apoptotic forms of cell death induced
bycytotoxic drugs or physical stimuli could not be taken as
programmed cell death because the latter represents normal
degeneration that is part of the life of an organism [30]
However, those instances of induced degeneration reflected
no less an orderlysequence of cellular events than naturally
occurring cell death in the form of apoptosis found in
developing organisms
Thus, a simple and noncommittal definition of
pro-grammed cell death as Ôa sequence of events based on
cellular metabolism that lead to cell destructionÕ is likely
both to preserve the concept as originallydefined, as well as
to discard decorative qualifications based on particular
experimental findings A disturbing example of the latter is
the requirement for protein synthesis [31,32], that had a
great impact in the acceptance of cell death as controlled by
gene expression (and thus ÔgeneticallyprogrammedÕ) Not
onlydo manycells die under inhibition of either
transcrip-tion or translatranscrip-tion in a controlled wayindistinguishable
from that underlying cell death dependent on protein
synthesis [33–35], but the rapid progress in the
understand-ing of post-translational mechanisms of cell death has
largelyovershadowed transcriptional control and even the
classical requirement for protein synthesis [36,37] The
caveat that the sequence of events in programmed cell death
must be based on cell metabolism allows for the ironythat
even the time between hitting a cell with a hammer and the
death of the former is finite, notwithstanding that the
intervening events are not resolvable with current
tech-niques
Acceptance of the minimalist concept should help
attribute appropriate weight to alternative forms of
pro-grammed cell death, despite the overwhelming dominance
of apoptosis in the literature
Multiple mechanisms of apoptosis
Cell death with apoptotic morphologycan be triggered byseveral stimuli, including intracellular stress and receptor-mediated signaling These signals feed into an evolutionarilyconserved intracellular machineryof execu-tion [36,38], the mechanisms of which have mainlybeen traced to the activityof the caspase familyof cy steine-proteases [39–41]
Caspase-mediated apoptotic cell death has been extensivelyreviewed, e.g [16,36,38,42–44] Briefly, the caspases are synthesized as zymogens and upstream signals convert these precursors into mature proteases Initiator caspases (caspase-1, -2, -4, -5, -8, -9, -10 and -14] are activated via oligomerization-induced autoprocessing [45–50], while effector caspases [caspase-3, -6 and -7] are activated byother proteases, including initiator caspases and granzyme B Proteolytic cleavage of cellular substrates byeffector caspases largelydetermines the features of apoptotic cell death ([51–53]; reviewed in [54–56])
Three major pathways have been identified according to their initiator caspase: the death receptor pathwayinvolving caspase-8 [57], the endoplasmic reticulum stress pathway attributed to activation of caspase-12 [58], and the mito-chondrial pathway, in which various signals can trigger the release of harmful proteins bymitochondria into the cytoplasm, leading to activation of caspase-9 and down-stream cleavage of caspase-3, -7 or -6 [46,59–62]
Although caspase-3 is widelyinvolved in the execution of apoptosis [63], its effector functions maybe dispensable for apoptotic-like cell death [64,65] The use of either pharma-cological inhibitors or knockout animals further showed that cells can trigger alternative mechanisms of cell demise For example, sympathetic and dorsal root ganglion neurons deprived of nerve growth factor (NGF) die in a caspase-2-dependent manner, but the same neurons derived from caspase-2 knockout mice still die following nerve growth factor deprivation, this time depending on activation of caspase-9, which does not occur in wild-type mice [66] Thus, rather than a single linear mechanism, alternative caspase-mediated pathways may be activated for apoptotic cell death, depending on whether a preferential caspase is blocked It is likelythat the network of intrinsic regulatory pathways that impinge upon the activity of caspases, such as the inhibitors of apoptosis (IAPs) and IAP-binding proteins [67], mayregulate the choice between alternative pathways
in normal cells, depending on metabolic state, stage of differentiation and other conditions
In addition, caspase inhibition fails to block programmed cell death with apoptotic morphologyin several experimental models [68–72] For example, the ultrastructural features of apoptosis inducing factor (AIF)-induced cell death represent
an example of a slight variation from the standard pattern of apoptotic morphology, which appears to be independent of caspase activation ([73]; see also [74]) Cell death pathways independent of caspase activation have been described, for example, even in some forms of cell death induced either bythe Bcl-familyprotein Bax [75], as well as in cell death involving the activation of other proteases, such as calpain [76], proteasome [77] and serine proteases
The latter enzymes have an important role in early chromatin cleavage [78], and are activated in the classical
Trang 3model of apoptosis of thymocytes induced by
glucocortic-oids [79] Serine proteases participate in a cell death pathway
that involves the activation of the endonuclease leucocyte
elastase inhibitor (LEI)-DNase II (L-DNase II), and is not
inhibited in HeLa cells bypancaspase inhibitors [80]
Activation of L-DNase II was first described in lens cell
differentiation, which is related to apoptosis [81] The
activation of this enzyme also occurs under other
physio-logical conditions, such as the death of retinal cells during
development [82]
The keymolecule of this pathwayis LEI, which is a
member of the superfamilyof protease inhibitors called
serpins (serine protease inhibitors) In its active form, LEI
inhibits elastase, cathepsin G and probablyother proteases
[83] LEI can undergo post-translational modifications
either under acidic pH or bythe action of proteases,
including elastase Once LEI is exposed to these conditions,
a decrease in the molecular mass is observed simultaneously
with the appearance of endonuclease activity[84] The
DNase generated bythe action of the serine protease
elastase was named L-DNase II, as it shows dependence on
the same ions and pH required byDNase II
Recent reports shows that the serine protease Omi/HtrA2
is a mitochondrial direct X-chromosome-linked inhibitor of
apoptosis protein (XIAP)-binding protein, which is released
from mitochondria upon induction of apoptosis together
with cytochrome c and Smac/Diablo ([85,86]; reviewed in
[87]), and its release can be inhibited byBcl-2 [88] These
data suggest that in some cases there maybe a cooperative
action between serine proteases and caspases in the
execu-tion of cell death
The data show that the classicallydefined apoptotic
morphologycan be achieved either byactivation of
caspases, or through the mediation of other families of
proteases [Fig 1], although the exact cytological features
of cell demise mayvaryslightlyamong these various forms
of apoptosis
Autophagy and autophagic cell death
As part of normal development, cells depend on a strictly regulated balance of protein synthesis and degradation, as well as organelle biogenesis and dismantlement While proteasome-mediated degradation is responsible for most of the protein recycling, the turnover of organelles is mainly attributed to autophagy[89]
Autophagyoccurs in manyeukaryotic cell types, where organelles and other cell components are sequestered into lysosomes and degraded The lysosome is a cellular compartment enriched in hydrolases able to cleave proteins, lipids, nucleic acids and carbohydrates that may lead
to organelle degradation through macroautophagy[90] Autophagyhas been described both as a means to resist starvation, and as part of cellular remodeling during differentiation, metamorphosis, aging, cell transformation, physiological whole-organ changes such as growth of the uterus during pregnancyand its atrophyafter childbirth, as well as in the removal of anomalous cellular components that accumulate following toxic insults or during cell death [91] In the nervous system, for example, morphological signs of autophagyare observed in physiological processes, such as the removal of outer segments of retinal photo-receptors bythe pigment epithelium [92], which is not associated with cell death
Notwithstanding, autophagic profiles identified byultra-structural features have been associated with cell death in certain circumstances [22] Cells in the earlystages of autophagycontain several autophagic vacuoli, and both the nucleoplasm and the cytoplasm appear slightly darkened, although nuclear structure still appears normal Mitochon-dria and the endoplasmic reticulum are sometimes dilated, and the Golgi apparatus is often enlarged The plasma membrane loses specializations such as microvilli and junctional complexes, and blebbing can occur In several cases, an intense endocytosis is observed, and this probably
Fig 1 Multiple pathways to apoptosis, both dependent and independent of the activation of caspases The diagram summarizes the major components
of the pathways reported to underlie cell death in various types of cells and tissues.
Trang 4leads to a reduction in the area of the plasma membrane.
During late stages, both the number and size of vacuoli
increase, and manyof them contain myelin figures or are
filled with lipids, which appear as pale grayinclusions in the
cytoplasm [22]
The nucleus of a cell undergoing autophagic cell death
can become pyknotic and identifiable as such by light
microscopy, either in early or in late stages of the
degenerative process Nevertheless, this nuclear
condensa-tion is neither as common nor as remarkable as that of
apoptosis The late autophagic cell debris is frequently
removed byheterophagy, but this tends to occur in verylate
stages, and seems to be less conspicuous than the clearance
of apoptotic bodies [22]
Autophagic cell death is not an exclusive feature of
multicellular organisms In the protozoan pathogen
Leish-mania donovani, treatment with antimicrobial peptides
induced cytoplasmic vacuolization and dismantling of the
cellular organization without disruption of the plasma
membrane, with no nuclear fragmentation or DNA
laddering, and independent of caspase-like activity Instead,
monodansylcadaverine, a biochemical marker of
auto-phagy, specificallylabeled the vacuoles induced
byanti-microbial peptides [93]
Endostatin, an inhibitor of angiogenesis, was shown to
induce the formation of autophagic vacuoles in endothelial
cells Cell death was not prevented byantioxidants or
caspase inhibitors, but was reduced by3-methyladenine, a
specific inhibitor of autophagy, and serine and cysteine
lysosomal protease inhibitors [94] Neuregulin (NRG; a
ligand of ErbB), also activates ErbB-2/ErbB-3 heterodimers
and induces cell death of prostate cancer LNCaP cells
Neuregulin-induced cell death was not inhibited
bybroad-spectrum caspases inhibitors, but was blocked
by3-methyl-adenine [95]
Ionizing radiation induced a dose-dependent suppression
of cell proliferation and autophagic cell changes in several
glioblastoma multiform cell lines [96] Arsenic trioxide, an
agent that causes remission in patients with acute
promyelo-cytic leukemia and multiple myeloma without severe
side-effects, was shown to inhibit proliferation of glioma cell
lines The G2/M arrest was accompanied byultrastructural
features of autophagy, and was inhibited by the autophagy
inhibitor bafilomycin A1, whereas general caspase inhibitors
did not block As2O3-induced cell death [97] In
neurobla-stoma cells, dopamine leads to autophagic changes
charac-terized bythe presence of numerous cytoplasmic vacuoles
with inclusions, and accompanied bymitochondrial
aggre-gation, activation of the stress-response kinases SAPK/JNK
and p38, and increased a-synuclein expression Both cell
viabilityand the increase in a-synuclein expression were
prevented byantioxidants, bythe specific inhibitors of
p38 and SAPK/JNK, and by3-methyladenine [98] Thus,
various agents can lead to autophagic cell death in tumor
cell lines
Indeed, Bursch and collaborators [99] had long since
shown that the MCF-7 breast carcinoma cell line, which
does not express caspase-3 [100], undergoes autophagic cell
death upon treatment with tamoxifen More recent work
showed that in apoptotic cell death induced bytyrphostin
A25 in the human colon cancer cell line HT29/HI1, early
stages of the death process are associated with
depolymer-ization of actin and degradation of intermediate filaments
In contrast, during tamoxifen-induced autophagic cell death
of MCF-7 cells, intermediate and microfilaments are redistributed, but largelypreserved even beyond the stage
of nuclear collapse [101] These data support the concept that autophagic cell death is a separate form of programmed cell death that is distinctlydifferent from apoptosis
In keeping with this interpretation, intensive irradiation led to up to 30% cell death in MCF-7 cells without anysigns
of apoptosis In this case, cell death was accompanied bythe formation of acidic vesicular organelles and lamellar structures, which was prevented by3-methyladenine How-ever, following low-dose irradiation, the presence of acidic vesicular organelles correlated with an increased chance of survival, suggesting that moderate signs of autophagymay
be associated with a defensive reaction of nonlethally damaged cells [102] The data are consistent with the view that nonlethal injurycan trigger an autophagic defensive reaction, whereas harsh treatment of certain cells can lead to cell death largelydependent on autophagyitself
The first step of autophagyis the formation of an autophagosome, which occurs when a portion of the cytoplasm is engulfed by a double membrane vacuole that does not contain either acid phosphatases or aryl-sulphatase activity The double membrane is derived from ribosome-free areas of rough endoplasmic reticulum [91] After a maturation period that includes the acidification of the vacuole, hydrolases are inserted into the autophagosome by fusion with pre-existing lysosomes or elements deriving from the Golgi complex This process appears to involve mannose-6-phosphate receptors located in the autophago-some membrane, resulting in the formation of a degradative vacuole limited bya single membrane named an autolyso-some [103] Vacuole formation can be regulated byamino acids and hormones and bystress [104,105]
Similarlyto yeast [90], autophagyin mammalian cells is highlydependent on phosphorylation events In mammalian hepatocytes, the phosphorylation of the ribosomal protein S6 correlates stronglywith inhibition of macroautophagy [106] The activityof the p70S6-kinase is regulated bythe mTor kinase, and inhibition of S6 phosphorylation caused byinactivation of mTor with rapamycin induces autophagy even under nutrient-rich conditions In yeast, inhibition of the Tor2 kinase results in activation of protein phosphatase 2A and induction of autophagy[107] Also, in hepatocytes, the effect of the phosphatase inhibitor okadaic acid upon protein phosphatase 2A inhibits the autophagic process [108] Furthermore, various classes of phosphatidylinositol-3-kinase (PI3K) control the autophagic pathwayin distinct ways: class IA PI3K inhibits cytoplasm sequestration and degradation, while class III stimulates the sequestration of cytoplasm, implicating the PI3K familyas keyregulators of the autophagic pathway[109]
In a recent study, Inbal and coworkers [110] showed that the expression of death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1, members of a familyof Ca2+/calmodulin-regulated Ser/Thr death kinases, triggered two major caspase-independent cytoplasmic events These were membrane blebbing, a feature common
to various forms of cell death, and extensive autophagy, which is typical of autophagic cell death Furthermore, either the expression of the dominant negative mutant of
Trang 5DRP-1 or DAPk antisense mRNA reduced autophagy
induced byantiestrogens, amino acid starvation, or
admin-istration of interferon-c The finding of DRP-1 inside the
autophagic vesicles suggests a direct involvement of this
kinase in the process of autophagy
Liang and collaborators [111] showed that beclin-1, a
protein that interacts with bcl-2, promotes autophagyin
both an autophagy-defective yeast cell line and in the
MCF-7 cell line, which normallydoes not express detectable
levels of beclin-1 It was also shown that beclin-1 expression
is frequentlylow in epithelial breast carcinomas, but is
widelyexpressed in normal tissue The authors suggested
that beclin-1 maybe a mammalian gene for autophagy, and
that it inhibits tumorigenesis through activation of this cell
death pathway[111] Kihara and coworkers [112] suggested
that beclin-1 is a component of the PI3K complex, which is
also required for autophagy, and that beclin-1 and PI3K
control autophagyas a complex at the Trans Golgi network
[112] However, in contrast with the poor autophagic
response of MCF-7 cells to amino acid deprivation [111],
strong autophagic responses were elicited in this cell line
both by tamoxifen [101] and by irradiation [102] Thus,
either beclin-1 can be replaced byother autophagy-inducing
proteins, or alternative pathways of autophagymayoperate
under various stimuli
The death of cerebellar Purkinje cells in lurcher animals is
due to a mutation in GluRd2 that results in its constitutive
activation (GluRd2-Lc) Yue and collaborators [113]
showed that GluRd2, nPIST and beclin-1 interact, and that
autophagycan be induced bynPIST and beclin-1 synergy
and bythe mutated GluRd2-Lc, but not bythe wildtype
GluRd2 Dying lurcher Purkinje cells displayed
morpholo-gical features of autophagy in vivo, providing evidence both for a direct link between GluRd2-Lc receptor and the autophagic pathwayin these cells [113], and that beclin-1 can exert its autophagic functions through interaction with multiple proteins in the cells [111–113]
Current evidence therefore indicates that that in at least some instances, autophagymaylead to a caspase-inde-pendent program of cell demise that fits the concept of programmed cell death, subject to complex, multivariate control [Fig 2] The next section will examine its relation-ship with apoptosis
Apoptosis vs autophagy
Notwithstanding the abundant evidence for a role for both autophagyand apoptosis in various diseases, their interplay
in those pathologies is not yet fully understood In Parkinson’s disease, the ultrastructural studyof neurons
of the substantia nigra of affected patients showed signs of autophagy, as well as apoptosis [114] However, it has been shown that expression of a-synuclein mutants, a condition frequentlyfound among certain families with Parkinson’s disease, induces autophagic cell death with no caspase activation, due to alterations of the ubiquitin-dependent protein degradation system [115] This suggests that there maybe no obligatoryinterdependence between apoptosis and autophagyin the pathologyof this disease
In Alzheimer’s disease, the endosomal–lysosomal system was found to be enlarged and highlyactivated, showing that endocytosis and/or autophagy are accelerated in the neu-rons of affected brains, even at earlystages The early activation of this system could be related to major
Fig 2 A summary diagram of the major components identified in pathways leading to autophagy These maylead to either cell death
or recoveryfrom an insult such as aminoacid deprivation.
Trang 6etiological factors of the disease, such as defective
mem-brane proteins, apolipoprotein E function, or altered
processing of the amyloid precursor protein [116] In
Huntington’s disease, it was suggested that the
accumula-tion of a mutant isoform of the protein huntingtin in
lysosomal compartments can activate cell death by
auto-phagy[117,118] Finally, in experimental prion disease,
bovine or mice brains inoculated with scrapie-infected brain
tissue showed signs of autophagy, suggesting that the
accumulation of the protease-resistant prion protein
iso-form can lead to sequestration and autophagyof portions of
the cytoplasm and eventually to neuron loss [119]
Further-more, giant vacuoli were observed in experimental prion
disease induced in hamsters The incidence of these vacuoli
correlates with the inoculation pathway, the intensity of the
process and the incubation period, and theyare most
numerous following intracerebral inoculation The
emer-gence of vacuoli was chronologicallyrelated to the
appear-ance of fibrils associated with prion disease, suggesting that
this process maybe related to disturbed protein turnover
and processing of the prion protein [120] Despite the
evidence for autophagy, there are several reports of
apoptotic cells detected by in situ nick-end DNA labeling
in human cases of all Alzheimer’s, Huntington’s and
Creutzfeldt–Jakob diseases [121–123]
The mode of cell death in neurodegenerative disorders
remains a matter of controversy[124], and it is possible that
both apoptotic and nonapoptotic cell death coexist in the
brains of affected patients
This problem is further complicated byclaims that the
TdT-mediated biotin-dUDP nick-end labeling (TUNEL)
technique maystain not onlyapoptotic cells [125,126] For
example, cell death of keratinocytes induced by 5-fluoruracil
exhibits autophagic ultrastructural features, such as
cyto-plasm vacuolation and chromatin detachment from the
nuclear membrane, alongside apoptotic features such as
TUNEL staining and both a decrease in size and increase
in granularityobserved bycytometric analysis [127] The
authors suggested that in this case the TUNEL-positive cells
were dying by autophagy, and cast doubt on the
identifi-cation of apototic cell death solelyusing nick-end labeling
techniques, such as in some of the neuropathological
surveys cited above The data show that the incidence of
mixed cell death features maybe more common among
various cell types than predicted by previous studies
The same stimulus can sometimes lead to the activation
of distinct and independent cell signaling pathways
Tumor necrosis factor-alpha (TNFa)-induced cell death
in an acute T-lymphoblastic leukaemic cell line developed
with an apoptotic pattern that was preceded
byauto-phagy The compound 3-methyladenine, which inhibits the
formation of autophagosomes, also inhibited the cytolysis
and DNA fragmentation induced byTNFa However,
inhibition of the fusion of lysosomes with
autophago-somes byasparagine did not block TNFa-induced
apop-tosis, and amino acid and protein deprivation enhanced
TNFa-induced autophagy, but not apoptosis These data
suggest that, at least in this case, earlystages of autophagy
are required for, but do not necessarilyresult in, TNF-a
induced apoptosis [128], and also that a cell can switch
between apoptosis and autophagyas the dominant form of
cell death
However, there are clear examples of interdependence between apoptosis and autophagy Following damage to peripheral nerves in adult rats, oligodendrocytes undergo ultrastructural features of apoptotic cell death, but mem-brane-bound cytoplasmic organelles typical of autophagy were also noticed in the cytoplasm of these cells [129] In sympathetic neurons from the superior cervical ganglia, substantial autophagic activitywas activated bypro-apop-totic factors, and treatment of these cells with 3-methyl-adenine decreased their rate of apoptosis [130] This was also observed in serum-deprived PC12 cells, and was accompanied bychanges in the activityof lysosomal proteases, particularlycathepsins B and D [131]
Alternative pathways of execution of cell death induced byblockade of protein synthesis were shown among a relativelyhomogenous population of postmitotic undiffer-entiated cells in the developing retinal tissue Inhibition of protein synthesis induced in the immature retina various post-translational, mitochondria-dependent pathways of cell death In one pathwayautophagyprecedes the sequen-tial activation of caspases-9 and -3, and DNA fragmenta-tion, while, in parallel, caspase-6-dependent mechanisms led
to a TUNEL-negative form of cell death Evidence was also provided for additional cell death mechanisms dependent
on caspase-9 activity, which may be engaged upon selective inhibition of execution caspases [132] These results support the hypothesis of interdependence of autophagy and caspase-dependent cell death pathways
Camougrand and coworkers [133] showed in yeast that the expression of Bax induces cell death with characteristics
of both apoptosis and autophagy, providing a newly identified function of Bax in autophagic cell death [133] Developmental cell death of motoneurons in the hawkmoth Manduca sextadepends on caspase activation and the loss
of mitochondrial function, showing an accumulation of autophagic bodies and vacuoles Motoneurons displayed a normal nuclear ultrastructure, without chromatin conden-sation, although theywere found to be TUNEL-positive, which is diagnostic of fragmented DNA These results indicate that the steroid-induced, caspase-dependent moto-neuron cell death exhibits intermingled features of both autophagyand apoptosis [134] In the fruitflyDrosophila, autophagic cell death depends on steroid-regulated genes encoding transcription regulators, which appear to activate other genes involved in cell degeneration The latter include genes that function in apoptosis, such as caspases, showing that caspase function is required for autophagic cell death during Drosophila development (for a review of apoptosis and autophagyinterplayduring Drosophila development see [135,136])
The autophagyinhibitor 3-methyladenine also increased the sensitivityof HT-29 colon cancer cells to apoptosis induced bysulindac sulfide, an inhibitor of cyclo-oxygenase Mutants that have a low rate of autophagywere more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells, and the rate of cytochrome c release was higher
in mutant cells than in HT-29 cells, suggesting that autophagycould delayapoptosis bysequestering mito-chondrial death-promoting factors such as cytochrome c [137] In this context, autophagymayrepresent an attempt
of the cell to recover from a noxious stimulus, rather than a necessarystage of execution of cell death
Trang 7The available data thus indicate that cells maydie bya
caspase-independent autophagic process either with or
without showing signs of apoptosis In some cases, cells
maychoose between the autophagic and the apoptotic
execution pathways Nevertheless, several experiments show
that the limits between apoptosis and autophagymaybe
tenuous, and suggest either considerable overlap or
inter-dependence of both programs of cell demise
Mitochondria and cell deaths
Mitochondria have a central role both in cellular
homeo-stasis and pathological conditions, as not onlydo they
serve as the major energyfactoryof living cells, but theycan
also either trigger or amplifythe signals that lead to cell
death [46,59–62] Permeabilization of the mitochondrial
membrane has been associated with cell death byapoptosis
[59,138], bymechanisms that are not yet completely
elucidated (reviewed in [139,140]) Induction of permeability
transition in the inner mitochondrial membrane maybe
accompanied byrelease of cytochrome c, Smac/Diablo,
AIF and endonuclease G, all of which lead to
mitochon-dria-dependent apoptotic forms of cell death [141–146]
Notwithstanding, the collapse of the mitochondrial
trans-membrane potential as a consequence of permeability
transition is not a universal earlyfeature of apoptosis
[147–149]
Although mitochondrial involvement in apoptosis is
broadlydocumented, permeabilization of mitochondria
seems to be an event also shared byautophagyand
necrosis Mitochondria from hepatocytes spontaneously
depolarize after nutrient deprivation before their capture
byan acid lysosomal compartment, suggesting that a
permeabilitytransition occurs before the normal
autopha-gic process [150] The authors proposed that cells respond
to mitochondrial permeabilitytransition (MPT) in a
graded manner When MPT occurs onlyin a few
mitochondria, autophagyis activated, leading to lysosomal
degradation of the affected organelles and cessation of the
signals that stimulate autophagy When a larger number of
mitochondria are permeabilized apoptosis occurs, probably
due to the higher concentration of molecules such as
cytochrome c and AIF in the cytoplasm And finally, when
virtuallyall mitochondria in the cell are affected, MPT
promotes necrosis, attributed to the uncoupling of
oxida-tive phosphorylation and accelerated ATP hydrolysis by
mitochondrial ATPase [151] This interpretation is
consis-tent with the hypothesis that autophagy, apoptosis and
necrosis are part of a continuum of degenerative events
[152,153]
Programmed necrosis
The death and elimination of cells byapoptosis remains
unnoticed by the body’s immune system, while the release of
the intracellular content of necrotic cells into the
extracel-lular space induces an inflammatoryresponse, constituted
bythe activation of resident phagocytes and attraction of
leukocytes into the necrotic area [18] Until recently,
necrosis has often been viewed as an accidental and
uncontrolled cell death process Nevertheless, there is
growing evidence that necrotic and apoptotic forms of cell
death mayshare more similarities than originallythought (reviewed in [16,154–156])
Necrosis has indeed been found to be a potential substitute for apoptosis during development Develop-ment-related loss of spinal cord and brainstem neurons was not impaired upon genetic deletion of both caspase-3 and caspase-9, although the morphologyof the forebrain suffered a marked perturbation [157] Furthermore, the loss
of interdigital cells in the mouse embryo, a prototype of programmed cell death, still occurs bynecrosis either upon caspase inhibition bydrugs, or in mice bearing a mutation in the apoptosis protease-activating factor 1 (APAF-1) gene Such necrotic cell death was also observed in normal wild-type mice [158]
Several apoptosis-related molecules have been implicated
in necrosis-like forms of cell death The antiapoptotic proteins Bcl-2 and Bcl-xL as well as caspase inhibitors delaynecrotic cell death induced bycyanide, rotenone or antimycin A [159] Bcl-2 can also protect neural cells from necrotic cell death induced bythe depletion of glutathione [160] These results indicate that Bcl-2/Bcl-xL and caspases modulate not onlyapoptotic but also some forms of necrotic cell death
In a Jurkat-derived cell line (JB-6) that is deficient in caspase-8, the forced multimerization of Fas-associated protein with death domain (FADD) induced caspase-independent cell death No DNA fragmentation was observed and dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis [161] In other studies, caspases-3 and -7 were activated in cell death
of murine L929 fibrosarcoma cells transfected with human Fas receptor, but peptide inhibitors of caspase-1 and -3 failed to block cell degeneration, and microscopical analysis showed features of necrosis after a delayof 3 h [162] These results suggest the existence of two distinct pathways of cell death triggered bythe engagement of Fas receptors, which maylead either to classical caspase-dependent apoptosis or
to necrosis
Chi and coworkers [163] showed that the expression of oncogenicallymutated ras gene in human glioma and gastric cancer cell lines causes a necrotic-like cell death characterized bycytoplasmic vacuoles derived from lyso-somes Dying cells had relatively well-preserved nuclei that were negative for TUNEL staining This oncogenic Ras-induced cell death occurred in the absence of caspase activation and was not inhibited bythe overexpression of Bcl-2 [163]
These morphological descriptions of cell death recall the recentlysuggested concept of necrosis-like programmed cell death (PCD), where this term was used to Ô… define PCD in the absence of chromatin condensation, or at best with chromatin clustering to specklesÕ [16] It seems that the cell death often classified as aborted apoptosis, where PCD
is initiated in the presence of caspase inhibitors, also meets this requirement because cells end up dying by alternative routes that are independent of caspase activation (reviewed
in [164])
Biochemical mechanisms of the execution of necrosis are beginning to be identified in plants Recessive genetic mutations in the Rn locus in soybean lead to progressive browning of the root, accompanied byincidence of necrotic
Trang 8as well as apoptotic cell death within the same tissue, but in
distinct cells [165] The appearance of necrotic cells in the
root preceded visible browning and lesion formation at the
macroscopic level, exhibiting a flocculent nucleoplasm,
increased vacuolation, condensed cytoplasm and presence
of swollen malformed organelles [165]
Rcr3 is a plant disease-resistance protein that recognizes
pathogens and activates plant defenses This protein is a
secreted papain-like cysteine endopeptidase and is
specific-allyrequired for Cf-2 function, e.g resistance to pathogens
in tomato, a hypersensitive response that results in cell
death Genetic analysis showed that Rcr3 is allelic to the Ne
(Necrosis) gene, which suppresses the Cf-2-dependent
auto-necrosis induced byplant pathogens [166] These data may
represent, at least in plants, the first examples of genes
related to the induction of necrotic-like PCD
It is not known whether anyof the necrosis-related genes
of plants have homologs in animal cells However,
compo-nents of a necrotic program are beginning to emerge
Integration of the BH3 protein BNIP3 to mitochondria
triggers a necrotic-like form of PCD [167] The necrotic-like
cell death triggered byengagement of the Fas/FADD signal
transduction system in mammalian cells was reported to
depend on the kinase RIP as an effector molecule [168]
These data support the hypothesis that, besides
uncontrol-lable necrosis that mayoccur following either massive
mechanical aggression or harsh chemical treatment of
tissues, a program of cell demise with morphological
characteristics of necrosis maybe found with components
largelydistinct from either apoptosis or autophagy
Conclusion and perspectives
Increasing evidence obtained in manymodel systems both
in vitroand in vivo supports the hypothesis that a variety of
cell death programs maybe triggered in distinct
circum-stances [Fig 3] Contraryto the widelyheld view that
caspase-mediated apoptosis represents the standard PCD, recent studies indicate that an apoptotic morphologycan be produced without the involvement of caspases, that auto-phagic execution pathways of cell death may be engaged without either the involvement of caspases or morphological signs of apoptosis, and that even the necrotic morphologyof cell death maybe consistentlyproduced in some cases, including the natural historyof at least some plants Particularlyin the case of autophagic cell death, but to a lesser extent also in the case of controlled necrosis, components have been identified at the molecular level that justifythe assumption of an intracellular program mediating either form of cell death under upstream command Distinct from the idea that a requirement for protein synthesis defines geneticallyprogrammed cell death, the ÔprogramsÕ appear in general to be readyfor action Together with abundant evidence that the apoptosis execution pathways are essentiallyindependent of protein synthesis, both autophagyand programmed necrosis have so far been traced to post-translational signal transducers, such as protein kinases and phosphatases In all of these cases, it would appear that onlythe simplest definition of PCD, much like the original concept of Ôa sequence of events… that lead to death of the cellÕ [20] would resist close scrutiny There is much to be said in favor of undecorated concepts, which more often than not represent the essential features upon which myriad variations can be identified, quite typical of biological systems
Despite the long-standing evidence for alternative cell death strategies, it is still often assumed that caspase-mediated apoptosis is either the major, or the most frequent, form of PCD Although statistics mayeventuallyprove this point, this is byno means warranted Indeed, the dominance
of apoptosis among the published examples of PCD maybe due to the fact that apoptosis is the onlyform of cell death that has long been categorized not onlyon the basis of its defining ultrastructural changes, but also on the basis of
Fig 3 A summary diagram of the various
forms of degeneration that may follow cell stress
or damage Distinct forms of programmed cell
death are indicated in italics within the boldly
outlined boxes Cell demise is the usual
out-come of either the somewhat variable forms of
apoptosis mediated bymultiple, alternative
pathways, or of programmed necrosis.
Autophagymaylead to cell death either
directlyor through apoptosis, as well as
mediate cellular recovery.
Trang 9cytological features recognizable by either conventional
light microscopyor byrelativelysimple histochemical or
immunohistochemical techniques Reviewers frequently
demand TUNEL or caspase assays as tests of the nature
of programmed cell death, and are usuallysatisfied with a
simple positive, unqualified response, even though this
positive identification is often achieved for onlysome of the
dying cells in either naturally occurring or experimentally
induced cell death
More thorough examination of the forms of cell death in
each circumstance is needed to assess, for example, the view
that morphologicallydistinct pathways of cell death maybe
part of a continuum of degenerative events Onlyrecently,
techniques such as the monodansylcadaverine assay have
become available to test for autophagic cell death in cell
populations [169] and no simple assayis as yet available for
the positive identification of necrosis Thus, it remains to be
evaluated how frequentlythe alternative cell death forms
also occur where apoptosis has been positivelyidentified,
whether it is caspase-mediated or not
A further and pressing issue is how the autophagic and
apoptotic pathways interact Evidence is available both for
independent as well as for tandem programs for each of
these forms of cell dismantlement The issue is complicated
bythe fact that the presence of autophagic vacuoles per se is
not enough evidence that the cell is committed to
degener-ation, because autophagymaybe part of a cell defense
mechanism
Admittedly, the rather unpopular view that apoptosis,
or at least caspase-mediated apoptosis, maywell have
been grosslyoverrated has a significant chance to
underlie a minorityof cases, after all But it has already
been worthwhile in the form of increasing awareness
concerning alternative cell death styles These must be
criticallyevaluated, in lieu of the prevailing assumption
that the overwhelming and excellent science behind
apoptosis mayhave definitivelyset the scene for all the
hot stories related to programmed cell death Alternative
cell death programs most probablyimplynovel
thera-peutic targets, and have important consequences for
attempts to treat diseases associated with disregulated
programmed cell death
Acknowledgements
Research in the authors’ lab was supported byCNPq, FAPERJ,
PRONEX-MCT, and a fellowship from the John Simon Guggenheim
Foundation to R.L We are indebted to Richard Lockshin for critical
reading of this manuscript.
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