Guest Editors Introduction

Distinguishing between apoptosis, necrosis, necroptosis and other cell death

modalities
This issue of Methods contains a series of articles that cover the
major techniques used to measure apoptosis, necrosis and varia-
tions thereof, at the population, single cell, organelle and molecular
level. Although we used to think of apoptosis as programmed or
regulated cell death and necrosis as strictly non-programmed or
unregulated cell death [1,2], data have emerged in recent years
which suggest that the boundaries between these two modes of
cell death can become blurred in certain situations [3,4].
To summarize, for those that are new to the eld, apoptosis is a
mode of cell death that is characterized by a series of morpholog-
ical and biochemical alterations to the cell architecture that pack-
age a cell up for removal by cells with phagocytic capacity [5].
Crucially, apoptotic cells are recognized by phagocytes and are en-
gulfed before they leak their contents. Thus, apoptosis ensures that
when a cell needs to be removed from a tissue, this occurs in an or-
derly fashion that minimizes disruption to neighboring cells. The
major consideration during apoptosis is that intracellular contents
do not leak into the extracellular space because this could (a) dam-
age surrounding cells, and (b) trigger inammation through release
of molecules with immune-activating activity, the so-called alar-
mins [5]. Thus, apoptosis is largely concerned with avoiding dis-
turbance to tissues in which there is ongoing homeostatic cell
death. Most of the biochemical and morphological changes that
typify apoptosis are the consequence of activation of a subset of
the caspase family of proteases (caspases 3, 6, 7, 8 and 9)
[1,5]. The latter caspases operate similar to a controlled demolition
squad, coordinating the packaging and disposal of cells in a manner
that minimizes damage to neighbors and the initiation of inam-
mation [5]. Methods for measuring apoptosis typically rely on
the detection of caspase-dependent events, such as exposure of
plasma membrane phosphatidylserine [6], that precede uptake of
vital dyes such as trypan blue or propidium iodide. Alternatively,
the striking morphological features of apoptotic cells (such as com-
paction and fragmentation of the cell nucleus), which are also ef-
fected through caspase activation, are still highly relevant for
methods for detecting this mode of cell death.
In contrast to apoptosis, necrosis is generally uncontrolled and
involves the sudden loss of membrane integrity, release of extra-
cellular contents, leading to activation of the immune system and
extensive inammation [2,4]. Necrosis is typically not associated
with caspase activation, although the exception to this is where
cell death follows aggressive activation of the inammatory subset
of caspases (caspases 1, 4 and 5), a mode of cell death termed
pyroptosis. Necrotic cell death bears none of the striking features
that characterize apoptotic cells, such as extensive membrane
blebbing and hypercondensation and fragmentation of the nucleus.
Instead, necrotic cells undergo extensive organelle and cell swell-
ing, leading to decondensation of nuclei which become markedly
less stained with uorescent dyes such as DAPI or propidium io-
dide. Thus, this mode of cell death is relatively easy to distinguish
from apoptosis on the basis of morphological criteria.
Necroptosis, which is a form of necrosis that appears to be dri-
ven as a consequence of the deregulated activity of specic mole-
cules, is under intensive investigation at present. This mode of cell
death is typically seen in response to engagement of certain mem-
bers of the death receptor subset of the TNF superfamily, particu-
larly of the TNF receptor itself, when caspase activity is inhibited
[7]. The caveat is that death receptor-induced necroptosis is only
observed in cells that express the kinase, RIPK3. In cells lacking
RIPK3, inhibition of caspase activity completely blocks TNF-in-
duced cell death, but in RIPK3-expressing cells results in a necro-
tic-like mode of cell death [4,8]. Necroptosis results from
excessive recruitment of RIPK3 onto RIPK1, thereby promoting
excessive RIPK1 kinase activity, which is cytotoxic. Thus, necropto-
sis represents somewhat of a kinase bomb that is set off within the
cell if the normal caspase wiring is interfered with. Under normal
circumstances, caspase-8 restrains recruitment of RIPK3 onto
RIPK1 in two ways, rst by cleaving RIPK1 and second by cleaving
the deubiquitinating enzyme CYLD. CYLD negatively regulates the
degree of RIPK1 ubiquitination that occurs in response to death
receptor engagement, an event that is involved in assembling sig-
naling complexes on RIPK1 to propagate death receptor-induced
NFjB activation. However, untrammeled CYLD activity can lead
to deubiquitination of RIPK1 and excessive recruitment of RIPK3
as a consequence. Thus, by cleaving RIPK1 and CYLD, caspase-8
ne-tunes the composition of the RIPK1/RIPK3 complex, which, if
perturbed, deregulates the RIPK1/RIPK3 kinase complex, resulting
in necroptosis. What is not clear at present is why. Moreover, the
physiological relevance of this mode of cell death is currently de-
bated but may occur upon infection with certain viruses that en-
code caspase inhibitors. Irrespective of the precise physiological
relevance of necroptosis, which is under investigation at present,
the major means of distinguishing conventional necrosis from nec-
roptosis is through probing for the involvement of RIPK1, or RIPK3,
where cell death occurs in the absence of caspase involvement.
This can be done either through knocking down expression of
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RIPK1 or RIPK3 using siRNA or shRNA or by means of the RIPK1
inhibitor necrostatin [7].
The issue begins with an overview of the morphological fea-
tures of apoptosis by Henry et al. [9]. Morphological features of cell
death still remain a key means of distinguishing apoptosis from
necrosis, with the former typically associated with extensive cell
shrinkage and plasma membrane blebbing and the latter typied
by cell swelling and gross membrane rupture. Henry et al., also de-
scribe ow cytometry-based methods for quantitating apoptosis
based upon DNA fragmentation, caspase activation and phosphati-
dylserine externalization, which are very well established methods
in the eld [9].
This is followed by a comprehensive discussion of the differ-
ences between apoptosis and necrosis by Vandenabeele and col-
leagues [10], who also describe a variety of assays that can be
used to discriminate between these different modes of cell death
with particular emphasis on the L929 cell line that is widely used
to study TNF-induced necroptosis. Bortoluci and colleagues de-
scribe a novel form of cell death, called pyroptosis, that has fea-
tures of apoptosis as well as necrosis and is induced through
delivery of bacterial agellin into the cytoplasm of macrophages
[11]. This form of cell death, which appears to be dependent on
activation of inammatory caspases, such as caspase-1, is rapid
and associated with the release of pro-inammatory cytokines
[11].
Mitochondria have a central role in the initiation of apoptosis
through releasing cytochrome c, which acts as a co-factor for the
assembly of a key caspase-activating complex, called the apopto-
some [1]. The Bcl-2 family of proteins regulate this cytochrome c
release step in a positive and negative way, with opposing factions
within this family ne-tuning the balance between life and death
[1]. In response to diverse cytotoxic insults, members of the BH3-
only subset of the Bcl-2 family can be mobilized to facilitate cyto-
chrome c release by triggering opening of the Bax/Bak channel
within the mitochondrial outer membrane. However, Bax/Bak
channel assembly can be countered by the pro-survival subset of
the Bcl-2 family, such as Bcl-2 and Bcl-xL. Mitochondrial outer
membrane permeabilization (MOMP), as a result of Bax/Bak activa-
tion represents a dening event where a cell commits to apoptosis
[1]. Waterhouse and colleagues detail high-throughput ow
cytometry-based methods to measure uptake of cationic dyes, as
well as cytochrome c translocation, to distinguish healthy cells
with intact mitochondria from dying cells where permeabilisation
of mitochondria occurs [12]. Biochemical analysis of apoptosis-
associated mitochondrial events, such as Bax/Bak activation, can
also be conveniently studied using puried populations of isolated
mitochondria, a topic which is covered by Chipuk and colleagues
[13]. The proximity of a tumor cell to undergo MOMP, due to the
interplay between members of the Bcl-2 family, may be one of
the reasons why chemotherapeutic drugs are effective on some
but not all cancers and is the topic of the article by Letai and col-
leagues [14]. They describe an assay developed by the Letai lab,
called BH3-proling, which utilizes peptides based on BH3 do-
mains of BH3-only proteins to measure the proximity of mitochon-
dria within specic tumor cell populations to MOMP and, by
extension, apoptosis. Proling of tumor cell mitochondria in this
way, prior to chemotherapy, may help to guide oncologists to-
wards drugs or drug combinations that elicit favorable cell death
responses, thereby sparing patients needless treatment cycles with
drugs that are ineffective for their particular tumor.
Along similar lines, it is also possible that responses to pertur-
bations in the cell death machinery may be subject to computa-
tional modeling in silico. Prehn and colleagues outline the use of
signal transduction models to quantify and temporally analyze
changes to particular proteins in response to apoptotic stimuli
[15]. In situations where the concentrations of all members of a cell
death pathway are known, useful predictions may be made con-
cerning how best to engage or manipulate such pathways for ther-
apeutic purposes.
The assembly of large multi-protein complexes mediate many
of the signaling events within a cell. Several of these complexes
have been identied as key mediators of apoptotic and necrotic cell
death including, the Death Inducing Signaling Complex (DISC) and
the apoptosome, with the necrosome and RIPoptosome identied
and extensively characterized in recent years. The precise compo-
sition of these complexes is currently an area of intense research
due to the increasing evidence that cell death outcomes are often
determined by subtle compositional and stoichiometric changes
within these complexes [4]. In this issue, MacFarlene and col-
leagues provide a detailed range of methodologies to characterize
the assembly of these complexes in vitro including afnity purica-
tion, sucrose fractionation, size exclusion chromatography and
reconstitution of the death-inducing complexes [16].
Moving onto methods used to evaluate cell death in vivo, Amar-
ante-Mendes and colleagues detail a method for the assessment of
cytotoxic T-lymphocyte (CTL) killing within the complex microen-
vironment in which immune responses take place [17]. This is par-
ticularly important given the conicting data published on CTL-
mediated cytotoxicity arising from studies performed in vitro ver-
sus in vivo.
This is followed by an article by Derry and colleagues on the
evaluation of cell death in the nematode worm C. elegans [18], a
model system that has been exploited very successfully for the
identication of many conserved components of cell death path-
ways. Derry and colleagues utilize uorescent markers in conjunc-
tion with RNAi screening to identify new genes and pathways that
regulate apoptosis in the worm germline [18].
To complete this issue of Methods, we turn our attention to the
use of genetically engineered mice in the study of cell death. There
is no doubt that gene-targeted mice have become an indispensible
resource in every area of biological research. The cell death eld is
no exception to this and such models have provided key insights
into the physiological roles and importance of many pro-apoptotic
and anti-apoptotic proteins in vivo. Gain-of-function as well as
loss-of-function gene mutations have helped to delineate cell
death pathways, as well as tease out redundant and non-redun-
dant functions of many apoptosis-related proteins. However, while
much has been learned from studies using knockout mice and de-
rived cell lines, Villunger and colleagues highlight the potential pit-
falls of interpreting data generated from different genetic
backgrounds [19]. While the use of knockouts clearly have their
place, it is often meaningless to simply compare cell death re-
sponses from cell lines derived from one genotype versus another.
Such crude comparisons, while supercially sophisticated, are of-
ten as crude as comparing one tumor cell line to another on the ba-
sis that they differ by only one expressed gene.
In conclusion, for those that wonder why it matters which way
a cell dies, it is useful to consider why multicellular organisms have
developed such complex strategies to regulate cell death. The ma-
jor driving forces appear to be the avoidance of collateral damage
to surrounding healthy neighbors, as well as the avoidance of
inammation. Or conversely, where it is desirable that the immune
system becomes activated, the exact opposite of this is true. Much
like in daily life, it is important to know whether a death has oc-
curred as a result of natural causes or foul play. Each scenario war-
rants a very different response by those close to the recently
deceased, as well as the authorities.
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Seamus J. Martin
Conor M. Henry
Molecular Cell Biology Laboratory,
Dept. of Genetics, Trinity College, Dublin 2, Ireland
Fax: +353 1 679 8558. (S.J. Martin)
E-mail address: martinsj@tcd.ie (S.J. Martin)
Guest Editors Introduction/ Methods 61 (2013) 8789 89