A d v a n c e s in Molecular B i o l o g y (2): 57-66, 2008

istanbul Kultur University, Printed in Turkey \

www.advmolbiol.org

Review

A p o p t o s i s s i g n a l l i n g : A life or death d e c i s i o n

Gül Özcan Arıcan* Introduction

Istanbul University, Faculty of Science, Biology T o d a y w e k n o w t h a t m o r p h o l o g i c a l c h a n g e s are t h e
Department, Istanbul - Turkey result of the activation of an intracellular signal
transduction pathway, probably the only function of
Abstract w h i c h i s t o kill t h e cell a n d t o o r g a n i z e t h e d i s p o s a l o f
the body. The apoptotic pathway results in
Apoptosis occurs during the normal development of characteristic morphological features including cell
multicellular organisms and continues throughout shrinkage, chromatin condensation, formation of
adult life. C e l l s die in r e s p o n s e to a variety of s t i m u l i cytoplasmic blebs and apoptotic bodies a n d finally
and during apoptosis they do so in a controlled, phagocytosis of the apoptotic bodies by adjacent
regulated fashion. This makes apoptosis distinct from p a r e n c h y m a l c e l l s , n e o p l a s t i c cells a n d m a c r o p h a g e s
another f o r m of cell death called necrosis in w h i c h (Hacker, 2000). There are two major m e c h a n i s m s of
uncontrolled cell death leads to lysis of cells, cell death-necrosis and apoptosis. Cells that are
inflammatory responses and, potentially, to serious d a m a g e d b y e x t e r n a l injury u n d e r g o n e c r o s i s , w h i l e
health problems. M o r e recent evidence has indicated cells t h a t a r e i n d u c e d t o c o m m i t p r o g r a m m e d s u i c i d e
that apoptosis depends upon a tightly regulated because of internal and external stimuli undergo
cellular program for its successful initiation and apoptosis. Although understanding of the detailed
execution. Molecular participants in this p r o g r a m are signalling pathways that trigger apoptosis is
present in different subcellular compartments, i n c o m p l e t e , this p r o c e s s is c o n t r o l l e d by a n u m b e r of
including the plasma membrane, cytosol, complex proteins, which are activated by various
mitochondria and nucleus. The interplay a m o n g these triggers and arranged in sequential signalling modules.
compartments and the exchange of specific signaling A p o p t o s i s i s a n e v o l u t i o n a r i l y c o n s e r v e d f o r m o f cell
molecules are critical for the systematic progression d e a t h t h a t w a s first d e s c r i b e d b y Kerr a n d c o l l e a g u e s i n
o f a p o p t o s i s . T h e a b i l i t y t o m o d u l a t e t h e life o r d e a t h 1 9 7 2 (Kerr et al., 1972). P r o g r a m m e d cell d e a t h is
of a cell is recognized for its i m m e n s e therapeutic internal component of Caenorhabditis elegans
potential. Therefore, research continues to f o c u s on development. C. elegans, w a s shown to act d o w n ­
the elucidation and analysis of the cell cycle s t r e a m of Ced-3 a n d Ced-4 (Hortvitz, 1999). In this
m a c h i n e r y a n d signaling p a t h w a y s that control cell organism 1 0 9 0 s o m a t i c cells are g e n e r a t e d in the
c y c l e a r r e s t a n d a p o p t o s i s . For t h i s r e a s o n , t h e f i e l d o f f o r m a t i o n o f t h e a d u l t w o r m , o f w h i c h 131 o f t h e s e c e l l s
apoptosis research has been moving forward at undergo apoptosis or programmed cell death. By
i n c r e a s i n g l y rapid rate. The a i m of t h i s review a b o u t contrast, more complex animals can not survive
general overview of current knowledge on apoptosis w i t h o u t a p o p t o s i s : m u t a t i o n s t h a t inhibit a p o p t o s i s i n
signalling considers recent progress in understanding the fruitfly Drosophila melanogaster, for example, are
the nature of the suicide process and how it is lethal e a r l y in d e v e l o p m e n t , as a r e m u t a t i o n s in m i c e
controlled. t h a t inhibit a p o p t o s i s m a i n l y i n t h e d e v e l o p i n g b r a i n
(Raff, 1998). Apoptosis occurs normally during
development and aging and as a homeostatic
K e y w o r d s : A p o p t o s i s , Bcl-2, c a s p a s e , p r o g r a m m e d
m e c h a n i s m to maintain cell p o p u l a t i o n s in tissues.
cell d e a t h
Although there are a wide variety of stimuli and
conditions, both physiological and pathological, that
* Corresponding Author: are t r i g g e r a p o p t o s i s , not all cells will n e c e s s a r y d i e in
Istanbul Unversity, Faculty of Science, r e s p o n s e t o t h e s a m e s t i m u l u s . I n s o m e c a s e s it's t h e
Biology Department, Vezneciler Istanbul - Turkey
type of stimuli a n d /or t h e degree of stimuli that
Email: gozcan@istanbul.edu.tr
d e t e r m i n e s if cells d i e by a p o p t o s i s or n e c r o s i s . S i n c e

Received: November 1 1 , 2008; Accepted: November 18, 2008

 Apoptosis signalling
58 Arıcan

a p o p t o s i s t y p i c a l l y d o e s not i n d u c e n e i g h b o u r i n g cell linked to apoptosis. Apoptosis m e c h a n i s m s are highly

d e a t h , inflammation or tissue scarring, it is well suited complex and sophisticated, involving an energy-
for its role in normal cell turnover during dependent cascade of molecular events. Research
e m b r y o g e n e s i s and in adult tissue. Depending on the indicates that apoptosis occurs through two main
a p o p t o t i c s t i m u l i a n d t h e a f f e c t e d cell t y p e , this p r o c e s s p a t h w a y s . T h e first, referred to as the extrinsic or
c a n last f r o m a f e w h o u r s to a f e w d a y s ( Z a n g et al., cytoplasmic pathway, is triggered through the Fas
2 0 0 4 ; Elmore, 2007). Moreover, in s o m e cases, the death receptor, a m e m b e r of the tumor necrosis factor
e n g u l f m e n t o f a p o p t o t i c cells b y p h a g o c y t e s a p p e a r s t o ( T N F ) r e c e p t o r s u p e r f a m i l y (Ricci a n d El-Deiry, 2 0 0 7 ) .
b e n e c e s s a r y for D N A d e g r a d a t i o n , s u g g e s t i n g t h a t t h e T h e s e c o n d p a t h w a y i s t h e intrinsic o r m i t o c h o n d r i a l
Nuc-1 n u c l e a s e m a y b e e x p r e s s e d by, a n d fulfill its pathway that w h e n stimulated leads to the release of
f u n c t i o n w i t h i n , e n g u l f i n g cells ( R o b e r t s o n et al., 2 0 0 0 ) . cytochrome-c from the mitochondria a n d activation of
Apoptosis is a coordinated a n d often energy d e p e n d e n t t h e d e a t h s i g n a l (Pei et al., 2 0 0 3 ; W o u t e r s a n d C h i u ,
process that involves the activation of a g r o u p of 2007). B o t h p a t h w a y s c o n v e r g e t o a final c o m m o n
cysteine proteases called 'caspases' and a complex pathway involving the activation of a cascade of
c a s c a d e o f e v e n t s t h a t link t h e initiating s t i m u l i t o t h e proteases called caspases that cleave regulatory and
final d e m i s e o f t h e cell ( E l m o r e , 2 0 0 7 ) . s t r u c t u r a l m o l e c u l e s , c u l m i n a t i n g i n t h e d e a t h o f t h e cell
( F i g u r e 1).
Initiation of apoptosis
The extrinsic signaling pathways that initiate
Apoptosis is a multi-step, multi-pathway cell-death apoptosis involve transmembrane receptor-mediated
p r o g r a m m e t h a t i s i n h e r e n t i n e v e r y cell o f t h e b o d y . interactions. M e m b e r s of the T N F receptor family share
Many physiological growth-control mechanisms that similar cyteine-rich extracellular d o m a i n s . This death
g o v e r n cell p r o l i f e r a t i o n a n d t i s s u e h o m e o s t a s i s are d o m a i n p l a y s a critical role in t r a n s m i t t i n g t h e d e a t h

F i g u r e 1. Schematic representation of apoptotic events. The extrinsic pathways involved the delivery of granzyme B through perforin
to the cells as well as receptor ligation that triggers caspase-8 activation. Caspase-9 is activated following release of mitochondrial
components to form the Apaf complex in the instrinsic pathway. Some intracellular stres can also induce the activation of caspase-
1 2 . IAPs gene family can suppress these pathways either blocking the activation of caspase-9 or by directly inhibiting caspase-3
activity (Zang et al., 2004).

A d v a n c e s in M o l e c u l a r B i o l o g y
 Apoptosis signalling
Arıcan 59

s i g n a l f r o m t h e cell s u r f a c e t o t h e i n t r a c e l l u l a r s i g n a l i n g t h e latter role a r e not o n l y t h e B a x / B a k c h a n n e l , w h i c h

pathways. To date, the best-characterized ligands and is o p e n to d i r e c t r e g u l a t i o n by Bcl-2/Bcl-x L b u t a l s o a
corresponding death receptors include FasL/FasR, nonspecific pore in the inner mitochondrial m e m b r a n e ,
TNF-a/TNFR-1, Apo3L/DR3, Apo2L/DR4 and known as the mitochondrial permeability transition pore
A p o 2 L / D R 5 ( Z a n g et al., 2 0 0 4 ; E l m o r e , 2 0 0 7 ) . (MPTP) ( N i c h o l a s et al., 2 0 0 1 ; Igney and Krammer,
2002). Apoptosis through mitochondria can be inhibited
Chemotherapy, irradiation growth-factor depletion
o n d i f f e r e n t levels b y a n t i - a p o p t o t i c p r o t e i n s , i n c l u d i n g
and other stimuli can initiate a p o p t o s i s t h r o u g h t h e
t h e a n t i - a p o p t o t i c Bcl-2 f a m i l y m e m b e r s Bcl-2 a n d B c l -
m i t o c h o n d r i a l (intrinsic) p a t h w a y . T h e intrinsic s i g n a l i n g
x L a n d i n h i b i t o r s o f a p o p t o s i s p r o t e i n s ( l A P s ) , w h i c h are
p a t h w a y t h a t initiate a p o p t o s i s i n v o l v e a d i v e r s e a r r a y
regulated by Smac/DIABLO as shown in Figure 2
of non-receptor-mediated stimuli that produce
(Second mitochondria-derived activator of
i n t r a c e l l u l a r s i g n a l s t h a t act d i r e c t l y o n t a r g e t s w i t h i n
caspase/direct IAP binding protein) (Igney and
t h e cell are m i t o c h o n d r i a l - i n i t i a t e d e v e n t s ( W a n g , 2 0 0 1 ;
K r a m m e r , 2 0 0 2 ; v a n L o o et al., 2 0 0 2 ) .
v a n L o o e t al., 2 0 0 2 ; K a r b o w s k i a n d Y o u l e , 2 0 0 3 ) . P r o -
a p o p t o t i c Bcl-2 f a m i l y p r o t e i n s (for e x a m p l e , Bax, B i d ,
T h e r e i s a n a d d i t i o n a l p a t h w a y t h a t i n v o l v e s T-cell
B a d a n d B i m ) are i m p o r t a n t m e d i a t o r s o f t h e s e s i g n a l s
mediated cytotoxicity and perforin/granzyme
( H a n a d a et al., 1 9 9 5 ) . A c t i v a t i o n of m i t o c h o n d r i a l e a d s
d e p e n d e n t killing o f t h e c e l l . T h e p e r f o r i n / g r a n z y m e
to t h e r e l e a s e of c y t o c h r o m e c into c y t o s o l , w h e r e it
pathway can induce apoptosis via either g r a n z y m e B or
binds apoptotic protease factor 1 (APAF-1) to f o r m the
g r a n z y m e A ( P a r d o et al., 2 0 0 4 ) . T h e s e r i n e p r o t e a s e s
a p o p t o s o m e . A t t h e a p o p t o s o m e , t h e initiator c a s p a s e -
g r a n z y m e B and A are the most important c o m p o n e n t
9 is a c t i v a t e d . T h e r e f o r e , in a d d i t i o n to their role in
within the granules. Granzyme B can utilize the
cellular energy metabolism, mitochondria are now
m i t o c h o n d r i a l p a t h w a y for a m p l i f i c a t i o n o f t h e d e a t h
r e c o g n i z e d a s c e n t r a l p l a y e r s i n cell d e a t h . C r u c i a l for
signal by specific cleavage of Bid and induction of

F i g u r e 2. Many death signals converge onto mitochondria and release intermembrane space proteins. A variety of apoptotic stimuli
trigger mitochondria, which results in the release of apoptotic proteins including cytochrome c, AIF, endonuclease G, Smac/DIABLO
and Omi/HtrA2 (van Loo et al., 2002).

A d v a n c e s in M o l e c u l a r B i o l o g y
 Apoptosis signalling
60 Arıcan

c y t o c h r o m e c r e l e a s e ( B a r r y a n d B l e a c k l e y , 2 0 0 2 ) . In cysteine-aspartate proteases (caspases) and their

a d d i t i o n g r a n z y m e B c a n a l s o a c t i v a t e c a s p a s e - 3 . In involvement in signaling and executing apoptosis
this way, the upstream signaling pathways are i m p l i c a t e d t h e critical i m p o r t a n c e o f t h e s e e n z y m e s i n
b y p a s s e d and there is direct induction of the execution this form o f cell death (MarcFarlane and Williams,
phase of apoptosis. Reports are s u g g e s t e d that both 2004). The protein e n c o d e d by the Ced-3 g e n e w a s
the mitochondrial pathway and direct activation of f o u n d to be very similar to a h u m a n protein called
c a s p a s e - 3 are critical for g r a n z y m e B i n d u c e d killing interleukin-1 converting enzyme (ICE). ICE induces
( G o p i n g et al., 2 0 0 3 ) . G r a n z y m e A is a l s o i m p o r t a n t in a p o p t o s i s w h e n e x p r e s s e d i n cells a n d c r m A , a n ICE
cytotoxic T cell induced apoptosis and activates inhibitor s u p p r e s s e s a p o p t o s i s ; h o w e v e r , I C E is not a
caspase independent pathways (Elmore, 2007). The g r a n z y m e B s u b s t r a t e (Shi et al., 1 9 9 6 ) . T h e d i s c o v e r y
e x t r i n s i c , intrinsic a n d g r a n z y m e B p a t h w a y s c o n v e r s e a n d c h a r a c t e r i z a t i o n o f ICE p r o v i d e d n o c l u e s t o t h e
on the s a m e terminal, or execution pathway. Although role of ICE-like proteases in cell death. In most
the triggering of either the death receptor mediated situations, apoptosis is coordinated by caspases,
extrinsic pathway of apoptosis or the mitochondrial which dismantle the cell by targeting numerous
c y t o c h r o m e c - m e d i a t e d intrinsic p a t h w a y s o f a p o p t o s i s p r o t e i n s for l i m i t e d p r o t e o l y s i s , n o w w i d e l y k n o w n a s
i s d e p e n d e n t u p o n t h e d e a t h s t i m u l u s and/or t h e cell c a s p a s e s , p l a y k e y role i n a p o p t o s i s e x e c u t i o n ( K u m a r ,
type involved, there is crosstalk between the two 2007).
pathways. It has been shown that caspase-8 can
T o d a t e , a t least 1 4 m e m b e r s o f this f a m i l y h a v e
cleave Bid, a death-inducing m e m b e r of t h e Bcl-2
b e e n i d e n t i f i e d i n m a m m a l s a l t h o u g h not all o f t h e m
family. T h e truncated Bid in turn translocates to the
function during apoptosis. The caspase family
mitochondria and induces cytochrome c release which
members can be divided into three subgroups
subsequently results in caspase-9-dependent
d e p e n d i n g on inherent substrate specificity, d o m a i n
activation of executioner caspases (Chen and W a n g ,
c o m p o s i t i o n o r t h e p r e s u m e d role i n a p o p t o s i s : t h e y
2002; Wouters and Chiu, 2007). The fact that
i n c l u d e initiators in a p o p t o s i s ( c a s p a s e - 2 , 8, 9 a n d 10),
cytoplasm of activated cells depleted of Bid by
e x e c u t i o n e r s in a p o p t o s i s ( c a s p a s e - 3 , 6 a n d 7) a n d
immunoprecipitation was no longer able to induce
participants in cytokine activation (caspase-1, 4, 5, 1 1 ,
c y t o c h r o m e c release indicated that Bid w a s the only
12, 13 a n d 14). A h i e r a r c h i c a l r e l a t i o n s h i p is p o s t u l a t e d
effector molecule d o w n s t r e a m of caspase-8 that has t o e x i s t b e t w e e n t h e initiators a n d e x e c u t i o n e r s i n t h a t
such a capability. However, apoptosis triggered t h e initiators act u p s t r e a m o f t h e e x e c u t i o n e r s . T h e
t h r o u g h Fas w a s u n a f f e c t e d i n l y m p h o i d cells but w a s a c t i v a t e d e x e c u t i o n e r s c l e a v e k e y p r o t e i n s r e q u i r e d for
l a r g e l y r e d u c e d i n liver cells f r o m m i c e l a c k i n g B i d . the maintenance of homeostasis, leading to the
T h e s e findings s u g g e s t that the proteolysis of Bid m a y c o l l a p s e a n d d e m i s e o f t h e c e l l . All t h e initiator a n d
not be e s s e n t i a l for cell d e a t h in all cell t y p e s , but t h a t e x e c u t i o n e r c a s p a s e s h a v e e i t h e r a d i r e c t or i n d i r e c t
the crosstalk between the two caspase activation role i n t h e p r o c e s s i n g , p r o p a g a t i o n a n d a m p l i f i c a t i o n o f
pathways may help to amplify weaker apoptotic signals apoptotic signals that results in the destruction of
a n d a c c e l e r a t e s cell d e a t h e x e c u t i o n i n c e r t a i n cell c e l l u l a r s t r u c t u r e s ( Z a n g et al., 2 0 0 4 ) .
t y p e s s u c h as h e p a t o c y t e s ( Z a n g et al., 2 0 0 4 ) .
C a s p a s e s are s y n t h e s i z e d a s i n a c t i v e p r o e n z y m e s
Execution of apoptosis which are activated by cleavage at specific Asp
r e s i d u e s t o a c t i v e e n z y m e s c o n t a i n i n g b o t h large ( p 2 0 )
O n c e t h e a p o p t o t i c p r o g r a m i s a c t i v a t e d , i t initiates t h e
and small (p10) subunits. In some cases these
cell d i s a s s e m b l y p r o c e s s , w h i c h i n c l u d e s n u c l e a r D N A
s u b u n i t s are s e p a r a t e d by a linker r e g i o n of u n k n o w n
fragmentation, cytoplasm shrinkage, and exposure of
f u n c t i o n but w h i c h m a y b e i n v o l v e d i n r e g u l a t i o n o f t h e
'eat me' signal(s) on the cell surface to induce
activation of the caspase. The process a n d association
phacytosis by neighboring cells (Conradt and Xue,
model postulates that the p20-p10 heterodimer is
2005). During apoptosis, the controlled destruction of
derived from the s a m e p r o c a s p a s e molecule (Figure
t h e cell i s c o o r d i n a t e d , f r o m w i t h i n , b y t h e c a s p a s e
3 ) . S u b s e q u e n t r e m o v a l o f t h e linker a n d p r o d o m a i n s
family of cysteine proteases. This meeting focused on
allows the two heterodimers to form the tetrameric
t w o o f t h e m a j o r a p o p t o t i c p a t h w a y s : o n e initiated b y
the activation of death receptors and the other by s t r u c t u r e ( C h a n g a n d Y a n g , 2 0 0 0 ) . All c a s p a s e s are

s t r e s s - i n d u c i n g s t i m u l i . T h e d i s c o v e r y of a f a m i l y of c l e a v e d a t s p e c i f i c r e s i d u e s , raising t h e p o s s i b i l i t y t h a t
some caspases sequentially activate others, so

A d v a n c e s in M o l e c u l a r B i o l o g y
 Apoptosis signalling
Arican 61

F i g u r e 4. Activation of the caspase cascade. Apoptotic signal

F i g u r e 3. Caspase-3 tetramer structure in complex with Ac- trigger oligomerization of death adapter proteins. Active initia¬
DEV-CHO. The p17 subunits in red and pink, and bound tor caspases then process and activate effector procaspases.
inhibitors in yellow. The C termini of p17 and N termini of p12 Effector caspases cleave various death substrates to induce
are indicated (Chang and Yang, 2000). apoptosis (Chang and Yang, 2000).

establishing a h i e r a r c h y of c a s p a s e s . A model has safeguard m e c h a n i s m s to protect the organism against

been proposed in which caspase-8 has been termed u n w a n t e d a n d potential harmful cells w h e n c a s p a s e -

an 'initiator' protease, which activates an 'amplifier' mediated routes fail but can also be triggered in

p r o t e a s e s u c h a s c a s p a s e - 1 , w h i c h i n turn a c t i v a t e s a response to cytotoxic agents or other death stimuli.

'machinery' protease such as caspase-3 or caspase-7 Endoplasmic reticulum (ER) is an important sensor of

(Cohen, 1997). cellular stres that can withhold protein synthesis and
metabolism to restore cellular homeostasis. If the
Caspases are among the most specific d a m a g e t o t h e E R i s t o o e x t e n s i v e , this c a n initiate
endopeptidases. The sustrate specificity of human apoptosis via the unfolded protein response or via
caspases was determined using a systematic r e l e a s e of c a l c i u m into t h e c y t o p l a s m ( B r e c k e n r i d g e et
approach involving combinatory peptide fluorogenic al., 2003). This l e a d s to activation of c a s p a s e - 1 2 ,
substrates. Categorizing caspases based on their possibly via translocation of the Bcl-2 family m e m b e r
s u b s t r a t e s p e c i f i c i t y g i v e s a d i f f e r e n t result t h a n d o i n g Bim to the ER. In addition a n d independent of c a s p a s e -
so based on sequence homology among the caspases 12 activation, ER stres can induce permeabilization of
(Figure 4 ) . This difference is related to the fact that only the mitochondrial membrane and thus activate the
small n u m b e r s of amino acids determine the substrate classic apoptotic pathway as well as other
specificity. m i t o c h o n d r i a l d e a t h p a t h w a y s ( B r o k e r et al., 2 0 0 5 ) .
T h e a c t i v a t i o n o f c a s p a s e s m a y b e r e s p o n s i b l e for
the neurodegeneration associated with Alzheimer 's
Regulation of apoptosis
disease and several recent studies have suggested The apoptotic self-destruction machinery is tightly
that caspases may also play role in promoting controlled. Various proteins regulate the apoptotic
p a t h o g e n i c m e c h a n i s m s u n d e r l y i n g this d i s e a s e ( R o h n process at different levels. T h e m e m b e r s of the Bcl-2
and H e a d , 2009). However, there is now accumulating family, which regulate apoptosis at the mitochondrial
evidence indicating t h a t cell death can o c c u r in a level, are an important class of regulatory proteins.
programmed fashion but in complete absence and They can be divided into anti-apoptotic and pro-
independent of caspase activation. Caspase a p o p t o t i c p r o t e i n s a c c o r d i n g t o their f u n c t i o n ( H a n a d a
independent cell death pathways are important et al., 1995; Igney and Krammer, 2002). The

Advances in Molecular Biology

 Apoptosis signalling
62 Arican

complexity of the apoptotic program began to increase lack the N-terminal B H 4 d o m a i n but contain the other
with the discovery of Bcl-2 a gene whose product B H d o m a i n s . Finally, g r o u p III f a m i l y m e m b e r s , w h i c h
causes resistance to apoptosis in lymphocytes. Bcl-2 i n c l u d e B i d , B a d , Bik, B i m , Blk, Bmf, H r k , B n i p 3 , Nix,
w a s s h o w n to c o r r e c t p a r t i a l l y t h e p h e n o t y p e of a C. Noxa, P U M A , and Bcl-G, are a more heterogeneous
elegans m u t a t i o n in C e d - 9 , a cell s u r v i v a l g e n e t h a t collection of polypeptides that share limited s e q u e n c e
functions upstream of C e d - 4 and Ced-3. This finding h o m o l o g y only in their 15-amino acid B H 3 d o m a i n s ,
suggested an apparent one-for-one correlation w h i c h is s u m m a r i z e d in Figure 5. It has been o b s e r v e d ,
between the C. elegans and mammalian pro- and for example, that cytochrome c release during
antiapoptotic pathways (Nagata, 1997). Although the apoptosis in fibroblasts requires expression of either
precise intracellular localization of all proteins Bax or Bak, w h e r e a s the release of S M A C requires
c o n t a i n e d w i t h i n this f a m i l y is u n k n o w n , it is c l e a r t h a t expression of both. The mechanistic basis for this
Bcl-2 is located in mitochondrial, endoplasmic observation r e m a i n s to be d e t e r m i n e d ( O l i v e r et al.,
r e t i c u l u m a n d n u c l e a r m e m b r a n e o f d i f f e r e n t cell t y p e s 2005; Kaufmann, 2007).
and possesses an anti-apoptotic function. Subsequent
The anti-apoptotic members Bcl-2, Bcl-x , L and
analysis demonstrated that Bcl-2 overexpression
Mcl-1 prevent the release of mitochondrial proteins,
inhibits cell d e a t h ( V i e r a et al., 2 0 0 2 ) .
including cytochrome c, endonuclease G, and AIF, by
Since these early observations, approximately 20 inhibiting the pore-forming function of BH-3 d o m a i n -
related m a m m a l i a n polypeptides have been identified. containing Bcl-2 proteins. Data from human studies
On the basis of functional a n d structural criteria, these suggest that Bcl-2 family proteins might also participate
polypeptides can be divided into t h r e e g r o u p s , the in the regulation of the stress r e s p o n s e by interacting
antiapoptotic group I family members and the w i t h h e a t s h o c k p r o t e i n s ( L u c i a n o t e t al., 2 0 0 6 ; Z h a n g
p r o a p o p t o t i c g r o u p I I a n d g r o u p III f a m i l y m e m b e r s . et al., 2 0 0 5 ) .
G r o u p I family m e m b e r s , which include Bcl-2, Bcl-x , L

Apoptosis can also be regulated by intracellular

Bcl-w, Mcl-1, A1/Bfl1, Boo/Diva, Nrf3, and Bcl-B,
signal transduction pathways. Several c o m p o n e n t s of
generally contain four short, conserved BH (Bcl-2
the mitogen activated protein kinase ( M A P K ) pathway
homology) domains, B H 1 - B H 4 , and most contain a C-
extracellular regulated kinase-1/2, c-Jun N-terminal
terminal t r a n s m e m b r a n e d o m a i n that targets t h e m to
kinase, and p38 p a t h w a y s are differentially activated
the cytoplasmic surfaces of various intracellular
d e p e n d i n g o n t h e r e g i o n o f b r a i n a n d t i m i n g after injury.
membranes, including the outer mitochondrial
A c t i v a t i o n of t h e p r o t e i n k i n a s e C s i g n a l i n g p a t h w a y
m e m b r a n e and the endoplasmic reticulum. G r o u p II
has also been reported. Pro-survival intracellular signal
family m e m b e r s , which include Bax, Bak, and Bok/Mtd,
t r a n s d u c t i o n p a t h w a y s a r e a l s o a c t i v a t e d after b r a i n

F i g u r e 5. Schematic representation of Bcl-2 family members and related proteins in apoptosis. Group I polypeptides are antiapop-
totic and include Bcl-2, Bcl-xL, Bcl-w, M c l - 1 , A 1 , Boo/Diva, Nrf3, and Bcl-B. Among these, Mcl-1 is unique in lacking a BH4 domain
and containing a proline/glutamate/serine/threonine-rich (PEST) sequence, which is often seen in short-lived polypeptides. Group II
family members are proapoptotic and include Bax, Bak, and Bok/Mtd. Group III family members, which share limited sequence
homology only in their 15-amino acid BH3 domains, include Bid, Bad, Bik, Bim, Blk, Bmf, Hrk, Bnip3, Nix, Noxa, P U M A , and Bcl-G
(Kaufmann, 2007).

Advances in Molecular Biology

 Apoptosis signalling
Arican 63

injury ( Z a n g et al., 2 0 0 5 ) . In many tumors, genetic a downstream effect of c a s p a s e activation and is

d a m a g e a p p a r e n t l y fails t o i n d u c e a p o p t o s i s b e c a u s e d i s p e n s a b l e for cell killing. I n s t e a d , D N A f r a g m e n t a t i o n
the constituent cells have inactivated the g e n e that is a w a y of " h i d i n g t h e b o d y " a n d r e p r e s e n t s o n e of t h e
c o d e s for t h e p53 protein. This protein, i t will be multiple parallel execution pathways in apoptosis
recalled, c a n lead to activation of the cell's apoptotic (Chang and Yang, 2000).
m a c h i n e r y w h e n D N A is i n j u r e d ( D u k e et al., 1996).
T h e critical role t h a t p 5 3 p l a y s i s e v i d e n t b y t h e l a r g e Inhibitor of apoptosis family of
n u m b e r of t u m o r s t h a t b e a r a m u t a t i o n in this g e n e . proteins
Loss of p53 in many cancers leads to genomic
instability, i m p a i r e d cell c y c l e r e g u l a t i o n , a n d inhibition Many natural inhibitors of caspases are known,
o f a p o p t o s i s . A f t e r D N A d a m a g e , p 5 3 h o l d s t h e cell a t i n c l u d i n g s e v e r a l v i r a l a n d cellular p r o t e i n s t h a t e i t h e r
a checkpoint until the damage is repaired. If t h e act as direct inhibitors of caspases or block the
d a m a g e is irreversible, apoptosis is triggered. The activation of c a s p a s e s (Kumar, 2007).
m e c h a n i s m b y w h i c h p 5 3 p r o m o t e s a p o p t o s i s i s still
IAP g e n e f a m i l y is highly c o n s e r v e d in a w i d e r a n g e
not fully u n d e r s t o o d ( G h o b r i a l et al., 2 0 0 5 ) .
o f o r g a n i s m s r a n g i n g f r o m i n s e c t s t o h u m a n s . T h e first
IAP w a s d i s c o v e r e d i n b a c u l o v i r u s , w h e r e i t w a s s h o w n
Nuclear DNA fragmentation to be involved in suppressing the death of viral-infected
A p o p t o s i s , or p r o g r a m m e d cell d e a t h , is a m e c h a n i s m host cells. S . M a r t i n d i s c u s s e d t h e h i e r a r c h i c a l n a t u r e
b y w h i c h cells u n d e r g o d e a t h t o c o n t r o l cell p r o l i f e r a t i o n of the c a s p a s e activation c a s c a d e that is triggered by
or in response to DNA damage. The nuclear cellular s t r e s s . M a r t i n ' s t e a m h a v e s h o w n t h a t A p a f 1 ,
alterations, which are the pre-eminent ultrastructural caspase-9, caspase-3 and the X-linked inhibitor o f
changes of apoptosis, are often associated with apoptosis (XIAP) are the main constituents of the

i n t e r n u c l e o s o m a l c l e a v a g e of D N A , r e c o g n i z e d as a native 'apoptosome', a n d t h a t c y t o c h r o m e c is not

"DNA ladder" on conventional agarose gel stably associated with the active complex. Martin also

e l e c t r o p h o r e s i s a n d long c o n s i d e r e d as a b i o c h e m i c a l presented data obtained from global proteomic

hallmark of apoptosis. During apoptosis, nuclear D N A a n a l y s e s o f a p o p t o t i c cells a n d d i s c u s s e d t h e role o f

specific caspases within this cascade in targeting
i s c o n d e n s e d a n d d e g r a d e d into large (50 t o 3 0 0 kb)
cellular p r o t e i n s for d e g r a d a t i o n . T h e d a t a s u g g e s t t h a t
and subsequently into small oligonucleosomal
more than 400 proteins are targeted for limited
fragments of several hundred base pairs.
proteolysis during the terminal 'demolition' phase of
Internucleosomal cleavage of DNA now appears to be
apoptosis (MarcFarlane and Williams, 2004).
a relatively late e v e n t in t h e a p o p t o t i c p r o c e s s w h i c h in
s o m e m o d e l s m a y b e d i s s o c i a t e d f r o m e a r l y critical
T o d a t e , s e v e n m a m m a l i a n m e m b e r s o f t h e IAP
steps (Ghobrial et al., 2005; Elmore, 2007).
family have been identified; they are X I A P , c - I A P 1 , c-
B i o c h e m i c a l p u r i f i c a t i o n b y s e v e r a l g r o u p s led t o t h e
discovery of a caspase regulated DNase complex
termed DFF (DNA fragmentation factor) that is
QfOMfihtti
c o m p o s e d of a DNase termed C A D (caspase activated MA* II I I I •
D N a s e : a l s o n a m e d D F F 4 0 a n d C P A N ) a n d its inhibitor c
"* w
ll ll I I I Ml
I C A D (also n a m e d D F F 4 5 ) . I n h e a l t h y c e l l s , C A D i s
complexed with ICAD and functionally inactive. In
apoptotic cells, ICAD is cleaved by c a s p a s e - 3 a n d -7,
***n its.. I I II -I M
releasing C A D to degrade nuclear DNA. D F F induces
both chromatin condensation and D N A fragmentation
HUP II I I II 'I ]
in vitro, a n d cells f r o m m i c e d e f i c i e n t for D F F activity
exhibit neither of these classic apoptotic features w h e n
induced to die. Another caspase-3-activated factor, I I Blfl mold m R I N G flnjw

named acinus, induces chromatin condensation

without affecting D N A fragmentation. T h e task of D N A
F i g u r e 6. Schematic representation of IAP family members.
fragmentation appears to be separate from other
The proteins were originally identified in baculovirus and are
phenotypic aspects of apoptosis. D N A fragmentation is
structurally similar (Schulze-Osthoff et al., 1998).

A d v a n c e s in M o l e c u l a r B i o l o g y
 Apoptosis signalling
64 Arican

I A P 2 , N A I P , s u r v i v i n , livin a n d T s - I A P . All o f t h e s e mitochondria regulate apoptosis. From a therapeutic

proteins possess one or more 70- to 80-amino acid p o i n t o f v i e w , this k n o w l e d g e w o u l d a l l o w t h e r a t i o n a l
b a c u l o v i r a l IAP r e p e a t ( B I R ) d o m a i n s , t h e p r e s e n c e o f design a n d use of specific synthetic molecules that
w h i c h i s e s s e n t i a l for t h e a n t i - a p o p t o t i c f u n c t i o n o f t h e mimic Bcl-2-like proteins, IAP-binding factors or the
IAPs. Unlike the Bcl- 2 family of proteins, w h i c h exert action m o d e o f S m a c b . T h e s e m o l e c u l e s m a y not
their regulatory effects through the mitochondria, the necessarily provoke apoptosis, but they might sensitize
I A P s d i r e c t l y b i n d a n d inhibit c a s p a s e - 3 , 7 , a n d 9 but cells to apoptotic stimuli, allowing more efficient cancer
not o t h e r c a s p a s e s . I n a d d i t i o n t o t h e BIR d o m a i n , m o s t therapies. Are the BH3- only proteins the major signal
o f t h e I A P s c o n t a i n a R I N G z i n c - f i n g e r ( R Z F ) n e a r their transducers? Or are they only part of a more
carboxyl terminus (Figure 6). T h e balance b e t w e e n complicated network of proteins? To answer these
IAPs and their negative regulators is postulated to q u e s t i o n s , w e will n e e d t o d e v e l o p m o r e s o p h i s t i c a t e d
constitute another apoptotic checkpoint, possibly strategies to identify other players, either through

downstream to that comprised by pro- and anti- b i o c h e m i c a l a s s a y s o r g e n e t i c s c r e e n s . O n l y t h e n will

apoptotic Bcl-2 family proteins (Elmore, 2007; w e b e g i n t o s e e t h e big p i c t u r e o f w h a t i s h a p p e n i n g

W a u t h e r s and Chiu, 2007). Overall IAPs have a central w h e n c e l l s d e c i d e w h e t h e r t o live o r d i e .

role i n r e g u l a t i n g a p o p t o s i s . O v e r e x p r e s s i o n o f o n e o r
m o r e o f t h e I A P s o r d o w n r e g u l a t i o n o f their n e g a t i v e References
regulator proteins may help suppress apoptosis by
Barry M and Bleackley RC. Cytotoxic T lymphocytes:
a l l o w i n g u n r e s t r i c t e d I A P activity ( Z a n g et al., 2 0 0 4 )
all roads l e a d to d e a t h . Nat Rev Immunol. 2 : 4 0 1 ¬
409, 2002.
Concluding remarks and future
directions Breckenridge D G , Germain M, Mathai JP, Nguyen M
and Shore GC. Regulation of apoptosis by
Apoptosis is an essential component of most endoplasmic reticulum pathways. Oncogene. 22:
developmental abnormalities and human diseases and, 8608-8618, 2003.
in m a n y cases, the underlying c a u s e of the resulting
Bröker LE, Kruyt F A E and Giaccone G. Cell death
pathology. An increased understanding of signalling in
i n d e p e n d e n t of caspases: A review. Clin Cancer
apoptosis continues to be a prominent research focus
Res. 11:9: 3155-3162, 2005.
w i t h i n this f i e l d . A p o p t o s i s is a u b i q u i t o u s w a y of cell
death in both physiological and pathological conditions. C h a n g H Y a n d Y a n g X . P r o t e a s e s for C e l l S u i c i d e :
During the past decade there have been major Functions and Regulation of Caspases.
advances in our understanding of the fundamental Microbiology and Molecular Biology Reviews. 64:4:
mechanisms of the apoptotic death program, especially 821-846, 2000.
the function of three families of proteins including
C h e n M a n d W a n g J . Initiator c a s p a s e s i n a p o p t o s i s
caspases, Bcl-2 and IAPs. Although significant
s i g n a l i n g p a t h w a y s . Apoptosis. 7 : 3 1 3 - 3 1 9 , 2 0 0 2 .
progress has been m a d e to specifically define and
characterize the participation of nuclear c h a n g e s in Cohen G M : Caspases. The executioners of apoptosis.
a p o p t o t i c cell d e a t h , this a r e a i s still relatively y o u n g i n Biochem J. 3 2 6 : 1-16, 1 9 9 7 .
s o far a s a k n o w l e d g e o f e s s e n t i a l , c o m p u l s o r y e v e n t s
C o n r a d t B a n d X u e D . P r o g r a m m e d cell d e a t h . E D :
t h a t m u s t o c c u r i n o r d e r t o elicit n u c l e a r c o n d e n s a t i o n
S e y d o u x G a n d P r i e s s J R . WormBook. 1-13, 2 0 0 5 ,
and fragmentation and D N A breakdown is lacking. An
The C. Elegans community, USA.
understanding of the mechanisms controlling and
i m p l e m e n t i n g a p o p t o s i s is m o r e t h a n a m a t t e r of m e r e D u k e R C , O j c i u s D M a n d Y o u n g J D E . Cell S u i c i d e i n
scientific interest. In comparison to the execution Health and Disease. Scientific American. 80-87,
phase of the mitochondrial apoptotic pathway, we k n o w 1996.
a little a b o u t h o w t h e u p s t r e a m s i g n a l i n g p a t h w a y s t o
E l m o r e S. A p o p t o s i s : A r e v i e w of p r o g r a m m e d cell
the mitochondria are regulated. H o w are the signals
death. Toxicologic Pathology. 35: 495-516, 2007.
from either developmental cues or damage signals
transduced to and integrated in the mitochondria? Ghobrial IM, Witzig TE, and Adjei AA. Targeting
These questions will be solved in a near future, Apoptosis Pathways in Cancer Therapy. CA,
permitting a general v i e w on the m e c h a n i s m by which Cancer J Clin. 55:178-194, 2005.

Advances in Molecular Biology

 Apoptosis signalling
Arican 65

Goping IS, Barry M, Liston P, Sawchuk T, SK, Lik C Y J , Sasaki T, Elia A J , Cheng HYM,
constantinescu G, Michalak K M , Shostak I, Roberts R a v a g n a n L , Feri K F , Z a m z a m i N , W a k e h a m A ,
DL, Hunter A M , Korneluk R and Bleackley RC. Hakem R, Yoshida H, Kong YY, Mak TW, ZuÀnÀ
G r a n z y m e B-induced apoptosis requires both direct J C , K r o e m e r G a n d P e n n i n g e r J M . E s s e n t i a l role o f
c a s p a s e a c t i v a t i o n a n d relief o f c a s p a s e i n h i b i t i o n . the mitochondrial apoptosis-inducing factor in
Immunity. 18:355-365, 2003. programmed cell death. Nature. 410:540-554,
2001.
H a c k e r G. The m o r p h o l o g y of a p o p t o s i s . Cell Tissue
Res. 3 0 1 : 5 - 1 7 , 2 0 0 0 . Oliver CL, Miranda M B , Shangary S, Land S, W a n g S
a n d J o h n s o n D E . (-)-Gossypol acts directly on the
H a n a d a M, A i m e - S e m p e C, Sato T and Reed JC.
mitochondria to overcome Bcl-2 and Bcl-X - L
Structure-function analysis of Bcl-2 protein.
mediated apoptosis resistance. Molecular Cancer
I d e n t i f i c a t i o n o f c o n s e r v e d d o m a i n s i m p o r t a n t for
Therapeutics 4:23-31, 2005.
homodimerization with Bcl-2 and
heterodimerization with Bax. J Biol Chem. 270\ Pardo J, B o s q u e A, B r e h m R, Wallich R, Naval J,
11962-11969, 1995. Müllbacher A, Anel A and Simon M M . Apoptotic
p a t h w a y s are selectively activated by g r a n z y m e A
H o r t v i t z H R . G e n e t i c c o n t r o l o f p r o g r a m m e d cell d e a t h
and/or granzyme B in CTL-mediated t a r g e t cell
in t h e nematode Caenorhabditis elegans. Cancer
lysis. The Journal of Cell Biology. 167(3): 4 5 7 - 4 6 8 ,
Res. 5 9 : 1 7 0 1 - 1 7 0 6 , 1 9 9 9 .
2004.
Igney FH and K r a m m e r PT. Death and Anti-death:
Pei W , L i o u A K F , a n d C h e n J . T w o c a s p a s e - m e d i a t e d
Tumour resistance to apoptosis. Nature Reviews.
a p o p t o t i c p a t h w a y s i n d u c e d b y r o t e n o n e toxicity i n
2:277-288, 2002.
c o r t i c a l n e u r o n a l c e l l s . FASEB. 1 7 : 5 2 0 - 5 2 2 , 2 0 0 3 .
Karbowski M and Youle RJ. Dynamics of mitochondrial
Raff M. Cell s u i c i d e for b e g i n n e r s . Nature. 3 9 6 : 1 1 9 ¬
m o r p h o l o g y in healthy cells a n d during apoptosis.
122, 1998.
Cell Death and Differentiation. 10:870-880, 2003.
Ricci MS a n d El-Deiry W S . T h e Extrinsic P a t h w a y of
K a u f m a n n S H . T h e Intrinsic P a t h w a y o f A p o p t o s i s . Part
Apoptosis. Part I: Apoptosis and Alternative M o d e s
I: Apoptosis and Alternative M o d e s of Cell Death.
o f C e l l D e a t h . E D : G e w i r t z D A , Holt S E a n d G r a n t
E D : G e w i r t z D A , H o l t S E a n d G r a n t S . Apoptosis,
S. Apoptosis, Senescence, and Cancer. 31-54,
Senescence, and Cancer. 3-30, 2007, Humana
2 0 0 7 , H u m a n a P r e s s Inc, T o t o w a , N e w J e r s e y .
P r e s s Inc, T o t o w a , N e w J e r s e y .
Robertson J D , Orrenius S a n d Zhivotovsky B. Nuclear
Kerr JF, Wylie AH and Currie A R . Apoptosis: a basis
Events in Apoptosis. Journal of Structural Biology.
biological phenomenon with wide-ranging
129: 3 4 6 - 3 5 8 , 2000.
i m p l i c a t i o n s in t i s s u e k i n e t i c s . Br J Cancer. 2 6 : 2 3 9 ¬
257, 1972. Rohn TT and H e a d E. C a s p a s e s as therapeutic targets
in A l z h e i m e r ' s D i s e a s e : Is it T i m e to "cut" to t h e
Kumar S. Caspases and their many biological
c h a s e ? Int J Clin Exp Pathol. 2 : 1 0 8 - 1 1 8 , 2 0 0 9 .
functions. Cell and Differentiation. 14:1-2, 2007.
S c h u l z e - O s t h o f f K, F e r r a r i D, L o s M, W e s s e l b o r g S a n d
L u c i a n o t F, Z h a i D, Z h u X, B a i l l y - M a i t r e B, R i c c i J - E ,
Peter M E . Apoptosis signaling by death receptors.
Satterthwait AC and Reed JC. Cytoprotective
Eur J Biochem. 2 5 4 : 4 3 9 - 4 5 9 , 1998.
peptide humanin binds and inhibits p r o a p o p t o t i c
Bcl-2/Bax family protein BimEl. The Journal of S h i L, C h e n G, M a c D o n a l d G, B e r g e r o n L, Li H, M i u r a
Biological Chemistry 2 8 0 : 1 5 8 2 5 - 1 5 8 3 6 , 2006. M, R o t e l l o R J , Miller D K , Li P, S e s h a d r i T, Y u a n J
a n d G r e e n b e r g A H . Activation of an interleukin 1
MacFarlane M and Williams AC. Apoptosis and
converting enzyme-dependent apoptosis pathway
disease: a life or death decision. EMBO Rep.
by granzyme B. Immunology. 93: 11002-11007,
5(7):674-678, 2004.
1996.
N a g a t a S. Apoptosis by Death Factor Review. Cell.
van Loo G, Saelens X, van Gurp M and MacFarlane M,
8 8 : 3 5 5 - 3 6 5 , 1997.
Martin SJ and Vandenabeele P. The role of
Nicholas J N , Susin SA, Daugas E, Stanfordk W L , Cho mitochondrial factors in apoptosis: a Russian

Advances in Molecular Biology

 Apoptosis signalling
66 Arican

roulette with m o r e t h a n o n e bullet. Cell Death and

Differentiation. 9:1031-1042, 2002.

V i e r a H L A , B o y a P, C o h e n I, El H a m e l C, H a o u z i D,
Druillenec S, Belzacq A - S , Brenner C, R o q u e s B
and Kroemer G. Cell permeable BH3-peptides
o v e r c o m e the cytoprotective effect of Bcl-2 a n d Bcl-
X . L Oncogene. 2 1 : 1 9 6 3 - 1 9 7 7 , 2002.

Wang X. The expanding role of mitochondria in

apoptosis. Genes & Development. 15:2922-2933,
2001.

W o u t e r s BG and Chiu RK. Evaluating the Importance

o f A p o p t o s i s a n d O t h e r D e t e r m i n a n t s o f Cell D e a t h
and Survival. Part I: Apoptosis and Alternative
M o d e s o f C e l l D e a t h . E D : G e w i r t z D A , Holt S E a n d
Grant S. Apoptosis, Senescence, and Cancer. 54¬
7 2 , 2 0 0 7 , H u m a n a P r e s s Inc, T o t o w a , N e w J e r s e y .

Zhang X, Chen Y, Jenkins LW, Kochanek PM and

Clark RSB. Bench-to-bedside review:
Apoptosis/programmed cell death triggered by
t r a u m a t i c b r a i n injury. Critical Care 9 : 6 6 - 7 5 , 2 0 0 5 .

Z h a n g A , W u Y , Lai H W L a n d Y e w D T . A p o p t o s i s -
A Brief Review. Neuroembryology. 3:47-59, 2004.

A d v a n c e s in M o l e c u l a r B i o l o g y