Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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aggregates to be active. After being activated, generally by initiator

caspases, the effector caspases are responsible for the death signal ampliﬁ-

cation, for example, caspase 9 activates others caspases, like caspase 3,

which in turn cleaves and activates more caspase 9, thus amplifying the

apoptotic signal (Slee et al., 1999).

Effector caspases are activated by a transactivation mechanism, which is

characterized by the catalytic action of a mature caspase on a procaspase

(Thornberry et al., 1997; Earnshaw et al., 1999; Slee et al., 1999). Never-

theless, their activation can also occur by the action of other proteases.

Granzyme B, a serine-protease, also has proteolytic speciﬁcity for aspartic

acid residues. It is able to cleave and directly activate caspase 3 (Darmon et

al., 1995). Cathepsin B, a lysosomal protease, cleaves and activates procas-

pase 11 (Schotte et al., 1998).

Effector caspases also interact with other molecules besides caspases.

These caspases interact and cleave key regulatory and structural proteins

(Earnshaw et al., 1999) that can be directly inactivated, directly activated,

or can modulate the function of other proteins as a result of cleavage. The

main substrates directly inactivated are structural proteins, which lose

their function, like cytoskeleton proteins (actin, gelsolin, Æ-foldrin); com-

ponents of gap junctions (-cathenin, plakoglobin), and nuclear proteins

(lamin A and B); proteins involved in metabolism and DNA repair (DNA

topoisomerase II, PARP); signaling proteins, like transcriptional activators

(NFkB) and kinases (Akt, FAK); and antiapoptotic proteins (Bcl-2, Bcl-

). The cleavage of cytoskeleton and gap junction proteins results in cells

becoming spherical and detaching from the surface and from neighbor

cells. The cleavage of lamin A and B contributes to the break up of the

nucleus into vesicles. Examples of proteins activated after cleavage are the

caspases themselves, proapoptotic proteins, like Bid and Bax, and kinases

(PAK2, MEKK1). These two kinases, when activated, are capable of

activating the SAPK/JNK pathway, which increases the transcription of

proapoptotic genes under the control of the transcription factor c-Jun.

Caspases can also modulate the activity of speciﬁc proteins by inactivating

inhibitors of these proteins, such as DNAse CAD/DFF40, which is

constantly inactivated by its inhibitor ICAD. This inhibitor is a caspase 3

substrate, and CAD/DFF40 release results in chromosome cleavage at

internucleosomal spaces. These irreversible proteolytic events are respon-

sible for the morphological changes displayed by apoptotic cells.

Caspase activation occurs as a late and common step in all cells under-

going apoptosis. Nevertheless, there are many initial pathways that can

result in caspase activation. Probably, each distinct pathway is triggered

by different apoptotic stimuli. In mammalian cells, the apoptotic response

is usually mediated by the intrinsic and extrinsic pathways, depending on

the origin of the death signal. The intrinsic pathway can further be divided

into mitochondrial and ER stress pathways.

The Bcl-2 family and the intrinsic mitochondrial pathway

Besides its role as the energy-generating organelle, the mitochondrion has

recently emerged as the center of conversion of cellular life and death

signals. This organelle contains, in its intermembrane space, apoptogenic

162 Animal Cell Technology

factors, like cytochrome c, AIF (apoptosis-inducing factor), procaspases 2,

3, and 9, Smac/DIABLO (second mitochondrial activator of caspases/

direct inhibition of apoptosis protein IAP binding protein with low pI),

Omi/HtrA2, and endonuclease G (Gross et al., 1999). In the presence of

apoptotic signals, these factors are released into the cytoplasm and a few

of them participate in caspase activation. This apoptotic pathway centered

on the mitochondria is known as the intrinsic mitochondrial or mitochon-

dria-dependent caspase activation pathway.

Proteins of the Bcl-2 family are responsible for the maintenance or

release of these factors from the mitochondria into the cytoplasm. For this

reason, this family and the caspase family are considered as the main

regulators of the apoptosis process (Gross et al., 1999; Cory and Adams,

2002; Kuwana and Newmeyer, 2003). To date, at least 20 members of the

Bcl-2 family have been identiﬁed, which can be divided into two main

groups, depending on their function. The proapoptotic group contains

members (e.g. Bax, Bcl-X

, Bak, Bad, Bid, Bik) that induce the release of

apoptogenic factors from the mitochondria into the cytoplasm, resulting

in apoptosis. On the other hand, the antiapoptotic group contains proteins

(e.g. Bcl-2, Bcl-X

, Mcl-1, Bcl-w, Boo) that are responsible for the

maintenance of these factors inside the mitochondria, inhibiting the

apoptotic process (Adams and Cory, 1998).

Members of the Bcl-2 family share one or more Bcl-2 homology (BH)

domains, named BH1, BH2, BH3, and BH4 (Adams and Cory, 1998). It is

not yet clear which structural features determine if these proteins possess

pro- or anti-apoptotic activities. However, some studies revealed that the

BH3 domain is a critical domain for the proapoptotic members (Chitten-

den et al., 1995). Besides BH domains, some contain a hydrophobic

domain in the C-terminal region, which is essential for the attachment to

intracellular membranes, like the outer mitochondrial, nuclear, and endo-

plasmic reticulum membranes (Krajewski et al., 1993; Nguyen et al., 1993).

In the absence of a death signal, most of the pro- and anti-apoptotic

members are located in separate subcellular compartments. Anti-apoptotic

proteins are inserted in intracellular membranes, mainly the mitochondrial

membrane, while some proapoptotic members are located in the cyto-

plasm or cytoskeleton in an inactive form. They are activated and translo-

cated by apoptotic stimuli to their place of action to perform their

functions (Gross et al., 1999).

A remarkable feature of the Bcl-2 proteins is their ability to interact

with one another to form either homo- or hetero-dimers (Oltvai et al.,

1993). The formation of hetero-dimers between pro- and anti-apoptotic

members suggests a competitive neutralization of their activities. A

healthy cell maintains a balance between these groups of proteins and a

destabilization in their relative concentration determines, at least in part,

the decision for cell survival or cell death (Gross et al., 1999).

Proapoptotic proteins can be further classiﬁed according to their BH

domains (Figure 7.5). Some members, such as Bax and Bak, contain multi-

ple domains and others, like Bid, Bad, Bim, and Bmf, contain only the

BH3 domain (Gross et al., 1999; Kuwana and Newmeyer, 2003). These

structural differences also reﬂect differences in their function. Multi-

domain proteins directly induce outer mitochondrial membrane permeabi-

Mechanisms of cell proliferation and cell death in animal cell culture

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163

lization with consequent release of apoptogenic factors into the cytoplasm

(Desagher et al., 1999; Zong et al., 2001). On the other hand, the proteins

with only the BH3 domain are considered the essential initiators of the cell

death program (Huang and Strasser, 2000; Bouillet and Strasser, 2002).

When activated by apoptotic stimuli, the BH3-only proteins have two

fates: (1) some are targeted to the outer mitochondrial membrane and

heterodimerize with Bcl-2 and Bcl-X

, neutralizing the action of these

BH3BH4 BH1 BH2

BH3 BH1 BH2

BH3

BH3 BH3

Bcl-2

Bcl-x

Bcl-w

Mcl-1

Boo/Diva

Bax

Bak

Bok/Mtd

Bid

Bad

Bim

Bmf

Bik

Hrk/DP5

Blk

Nip3

BNip3/Ni

Puma

Noxa

Antiapoptotic

Proapoptotic

Multidomain

BH3-only

Figure 7.5

Classiﬁcation scheme of the Bcl-2 family members. TM refers to the hydrophobic

C-terminal region, which is probably a transmembrane domain (adapted from

Kuwana and Newmeyer, 2003).

164 Animal Cell Technology

antiapoptotic proteins and allowing, indirectly, multidomain proapoptotic

Bax and Bak proteins to release apoptogenic factors from the mitochon-

dria; (2) others, besides the previously described function, are also respon-

sible for the direct activation of the multidomain proteins, as is the case for

Bid (Desagher et al., 1999; Eskes et al., 2000) and probably Bim (Letai

et al., 2002). At least two events seem to be critical for Bax and Bak to

release apoptogenic factors into the cytoplasm homodimerization and

insertion into the outer mitochondrial membrane (Bouillet and Strasser,

2002).

It is not clear whether each member of this subgroup is activated by a

particular stimulus and through a speciﬁc mechanism, or whether their

roles are redundant. Nevertheless, it is possible that different BH3-only

domain proteins, or their combinations, are critical for apoptosis in differ-

ent cell types.

Tridimensional structure analysis of some Bcl-2 family members, such

as Bcl-X

, Bcl-w, Bax, and Bid, surprisingly revealed that pro- and anti-

apoptotic proteins share common structures (Kuwana and Newmeyer,

2003). The BH3 domain is buried inside the molecule, and it has been

suggested that it is essential for activity of the proapoptotic members and

has to be exposed to render the protein active. Therefore, in a healthy cell,

proapoptotic members are inactive, with the BH3 domain hidden inside

the molecule. However, by receiving apoptotic signals, they undergo a

conformational change, exposing this domain and, thus, acquiring pro-

apoptotic activity.

In some molecules, such as Bid, Bax, Bak, Bmf, and Bim, the N-terminal

region acts as an inhibitory domain, hiding the BH3 domain (Gross et al.,

1999). Bid must be cleaved by caspase 8, and its truncated form (without

the N-terminal) translocates to the mitochondria to interact with Bax and/

or Bak, activating them (Li et al., 1998; Desagher et al., 1999). Prior to the

apoptotic signal, Bmf and Bim are found associated with cytoskeleton

complexes by the N-terminal region. In the presence of these signals, they

dissociate from these complexes and translocate to the mitochondria to

bind Bcl-2 and Bcl-X

, antagonizing their antiapoptotic activity (Puthala-

kath et al., 1999, 2001). Bax and Bak require the interaction with some

BH3-only proteins to derepress their N-terminal domain, exposing not

only their BH3-only domain, but also a C-terminal hydrophobic domain,

which allows them to become integral proteins in the outer mitochondrial

membrane and induce the release of apoptogenic factors (Goping et al.,

1998; Desagher et al., 1999). Bad is phosphorylated at two serine residues

(Ser-112 and Ser-136), which allows it to be sequestered by the cytosolic

protein 14-3-3, keeping it inactivated (Zha et al., 1996). In the presence of

apoptotic signals, Bad is dephosphorylated, resulting in its dissociation

from the 14-3-3 protein and its translocation to the outer mitochondrial

membrane to bind to Bcl-2 and Bcl-X

(Kelekar et al., 1997; Ottilie et al.,

1997). It is suggested that Bad phosphorylation regulates the BH3 domain

exposure (Zha et al., 1997). Antiapoptotic proteins can also be converted

to proapoptotic if they expose their BH3 domains (Cheng et al., 1997).

However, not all proapoptotic members are regulated post-translation-

ally. Some, such as Noxa, Puma, and HRK, are regulated transcriptionally.

Noxa and Puma are regulated by the p53 protein and, therefore, are critical

Mechanisms of cell proliferation and cell death in animal cell culture

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165

for an apoptosis process induced by DNA damage (Oda et al., 2000;

Nakano and Vousden, 2001), while HRK is regulated by the JNK-

dependent mechanism (Harris and Johnson, 2001). Some post-translation-

ally regulated proteins can also be regulated transcriptionally, such as Bax,

which can also be regulated by p53 (Miyashita and Reed, 1995). Antiapop-

totic members, like Bcl-X

, Mcl-1, A-1 and, less frequently, Bcl-2, can

also be regulated transcriptionally (Gross et al., 1999).

The exact manner of how proapoptotic proteins induce the release of

apoptogenic factors from the mitochondrial intermembrane space into the

cytoplasm and how antiapoptotic proteins prevent it remains obscure.

The protection conferred by antiapoptotic members may occur by

their direct binding to proapoptotic members, sequestering the BH3-

only proteins and therefore preventing the activation of Bax and Bak or

directly neutralizing the activity of the multidomain proteins (Oltvai

and Korsmeyer, 1994). The heterodimerization between members of

Bcl-2 family occurs by the insertion of the BH3 domain of the

proapoptotic protein into a hydrophobic pocket formed by the BH1,

BH2, and BH3 domains on the surface of the antiapoptotic protein

(Sattler et al., 1997). Bcl-2 and Bcl-X

do not prevent Bid-induced

conformational change of Bax and Bak (Desagher et al., 1999). How-

ever, they block the release of apoptogenic factors from the mitochon-

dria. Some mutants of Bcl-X

that have lost the ability to form

heterodimers with Bax and Bak can still suppress cell death by apopto-

sis, suggesting the existence of a protection mechanism independent of

heterodimer formation (Cheng et al., 1996).

In the literature, two main mechanisms have been proposed that could

explain how Bax and Bak induce the release of apoptogenic factors,

especially cytochrome c. The ﬁrst model of outer mitochondrial mem-

brane permeabilization predicts the occurrence of homo-oligomerization

of Bax (probably four molecules) and Bak, resulting in the formation of

channels just wide enough for the passage of cytochrome c. The passage

of other apoptogenic factors is still contested (Antonsson et al., 1997;

Saito et al., 2000; Kuwana et al., 2002; Kuwana and Newmeyer, 2003).

The second model is based on the activity regulation of pre-existent

channels, such as the permeability transition pore (PTP) (Marzo et al.,

1998; Narita et al., 1998; Kuwana and Newmeyer, 2003). PTP is a

multiprotein channel, formed by components of both the outer and inner

mitochondrial membranes and matrix proteins, including VDAC (vol-

tage-dependent anion channel, also known as mitochondrial porin), ANT

(adenine nucleotide translocator), and cyclophilin D, respectively

(Crompton et al., 1999). Bax, Bak, and Bcl-X

have been found to

interact with VDAC (Narita et al., 1998; Shimizu et al., 1999), and Bax

to interact with ANT (Marzo et al., 1998). The PTP opening would be

followed by the swelling of the mitochondrial matrix and the rupture of

the outer membrane. In this context, the release of apoptogenic factors

would not be speciﬁc and would occur indirectly, as a result of the

rupture of the outer membrane. When PTP is induced, the inner

mitochondrial membrane potential (˜łm) becomes dissipated, leading to

the loss of mitochondrial functions, such as energy production and

protein import.

166 Animal Cell Technology

Some authors suggest that the mitochondria, especially the inner mem-

brane, remain intact during apoptosis. Thus, it is suggested that PTP

activation is not involved or that cells starting to die by apoptosis could

switch to necrosis, therefore activating the PTP (Antonsson, 2001). Never-

theless, it is important to remember that these contradictory results can

occur since apoptosis can be activated by distinct mechanisms in different

cell types by different apoptotic signals. It is also possible that both

mechanisms are correct. Bax and Bak oligomers can form channels for the

initial release of cytochrome c, followed by a larger ﬂux through PTP. In

both cases, antiapoptotic proteins Bcl-2 and Bcl-X

seem to inhibit the

formation of both kinds of channels.

Following the outer mitochondrial membrane permeabilization, the

apoptogenic factors are released into the cytoplasm. Among them, cyto-

chrome c has an important role in caspase activation, because it is the

cofactor for assembling a large caspase 9 activating complex in the

cytoplasm, called apoptosome. Along with cytochrome c, the Apaf-1

protein and dATP or ATP are required to form this complex in the

cytoplasm (Hill et al., 2003).

Apaf-1 consists of three functional domains: an N-terminal CARD, a

central NBD (nucleotide-binding domain), and WD-40 repeats at the C-

terminal region (Figure 7.6). In the absence of cytochrome c, Apaf-1 exists

as a monomer in a compact, auto-inhibited form (Hu et al., 1998; Acehan

et al., 2002). When cytochrome c and dATP (or ATP) are present, Apaf-1

is forced into a more open conformation, facilitating the oligomerization

with adjacent Apaf-1 molecules (Jiang and Wang, 2000) and the cyto-

chrome c association with the WD-40 repeats (Acehan et al., 2002). The

apoptosome assembly is illustrated in Figure 7.6.

It has been suggested that the apoptosome is formed by the oligomer-

ization of seven Apaf-1 molecules, resulting in a wheel-like structure. This

structure comprises a central hub connected to seven radial spokes, as

shown in Figure 7.6. The model suggests that the ring is composed of

seven Apaf-1 CARD domains held together in close proximity. Apaf-1

central and C-terminal regions form spokes projecting outward from the

hub (Qin et al., 1999; Acehan et al., 2002). The procaspase 9 is recruited

into the apoptosome through interaction with Apaf-1 with the procaspase

9 CARD domains, at a 1:1 proportion (Budihardjo et al., 1999; Jiang and

Wang, 2004). The procaspase 9 aggregation leads to autoproteolysis (Saleh

et al., 1999). Caspase 9 and the apoptosome form an active holoenzyme,

responsible for the activation of downstream effector caspases, such as

caspases 3 and 7 (Bratton et al., 2001).

After caspase 9 activation, the death signal is propagated by downstream

caspase activation. Caspase 9 directly activates caspases 3 and 7. Caspase 3,

in turn, processes and activates caspases 2 and 6 and also caspase 9,

therefore amplifying the death signal. Caspase 6 cleaves and activates

caspases 8 and 10 (Slee et al., 1999).

Besides cytochrome c and procaspase 9, other important apoptogenic

factors are also released from the mitochondria, such as SMAC/DIABLO,

Omi/HrtA2, AIF, and endonuclease G. The function of SMAC/DIABLO

and Omi/HrtA2 is to activate caspase by suppressing the caspase inhibi-

tory activity of IAP (Du et al., 2000; Verhagen et al., 2000). The protein

Mechanisms of cell proliferation and cell death in animal cell culture

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167

family IAP, which contains members like survivin, xIAP, cIAP1, cIAP2,

inhibits caspase activity by directly binding to the active enzymes. These

proteins contain one or more BIR (baculovirus IAP repeat) domains,

which are responsible for the caspase inhibitory activity. In healthy cells,

it is likely that IAP proteins serve to inhibit residual or unwanted caspase

activity. SMAC/DIABLO directly binds to BIR domains of IAPs, inhibit-

ing their functions. Since Omi/HtrA2 is a serine-protease, it proteolyti-

cally cleaves and inactivates IAP proteins.

When AIF and endonuclease G are released into the cytoplasm, they

directly translocate to the nucleus and induce DNA fragmentation and

subsequent chromosomal condensation, a remarkable morphological fea-

ture of the apoptotic process. AIF induces chromatin digestion into large

fragments of approximately 50 kb, probably by activating a nuclear

DNAse. Therefore, these proteins are important for the caspase-indepen-

dent apoptosis pathway.

CARD

NBD

WD-40 repeat region

Apoptotic stimulus

Mithocondrion

Cytochrome c

Apaf-1

dATP/ATP

Apoptosome

Procaspase-9

Active caspase-3/7 Procaspase-3/7

Figure 7.6

Scheme of the mechanism of apoptosome formation. (A) The Apaf-1 molecule

possesses three domains: CARD, NBC, and WD-40 repeats. (B) In the absence of

cytochrome c, Apaf-1 may exist in a compact, autoinhibited form, with the CARD

region buried between the lobes of WD-40 repeats. The binding of cytochrome c

displaces the CARD region from the WD-40 repeats, forcing the molecule into a

more open conformation. The interaction with dATP/ATP prevents the

reassociation of CARD to the lobes of WD-40 repeats, facilitating the interaction

with other Apaf-1 molecules for the apoptosome assembly. Procaspases 9 are

recruited by a CARD:CARD interaction, leading to their autoprocessing (adapted

from Hill et al., 200 3, and Jiang and Wang, 2004).

168 Animal Cell Technology

The intrinsic ER stress-induced pathway

The ER is an organelle in which most proteins acquire their tertiary and

quaternary structures. It is also the Ca

2þ

storage site (Rao et al., 2004).

Under certain conditions, such as disruption of Ca

2þ

homeostasis, hypoxia

or ischemia, or when there is an overload of proteins, an accumulation of

unfolded proteins occurs in the ER. This accumulation activates a com-

pensatory mechanism called ER stress response or unfolded protein

response (UPR). This response consists of four distinct steps: a cell cycle

arrest in G

/S phase; attenuation of protein synthesis to prevent further

protein aggregation and accumulation in this organelle; induction of ER-

localized chaperone proteins to assist protein folding; and activation of a

protein degradation mechanism to eliminate unwanted protein aggregates.

However, if the ER stress cannot be bypassed, it culminates in apoptosis

(Szegezdi et al., 2003).

Since this ER pathway was discovered recently, not much is known

about its signaling mechanism. However, it is clear that this pathway can

unleash the apoptotic process through three distinct mechanisms. The ﬁrst

mechanism is dependent on a transcription factor, the GADD153/CHOP.

Although no target genes have been identiﬁed to date, it has been

speculated that GADD153/CHOP can decrease Bcl-2 expression

(McCullough et al., 2001).

The second mechanism is dependent on caspase 12 activation. Never-

theless, it is still not clear how this activation occurs. Preliminary

studies showed a caspase activating complex containing VCP and ALG-

2 proteins, which also possess apoptotic activities. This has suggested

the existence of a caspase 12 activating complex, similar to the apopto-

some, which was designated eraptosome (Hoppe et al., 2002; Kilic et

al., 2002). The adaptor protein TRAF2 may be involved in procaspase

12 aggregation, resulting in its cleavage and activation (Yoneda et al.,

2001). The procaspase 12 may also be processed by caspase 7, through a

cleavage in the middle of its prodomain, which leads to autoprocessing

between the prodomain and the large subunit, and between the large

and small subunits of caspase 12 (Szegezdi et al., 2003). Furthermore, it

was also suggested that caspase 12 can be processed by calpain. The

disruption of Ca

2þ

homeostasis leads to calpain activation, which in

turn translocates from the cytoplasm to the ER and cleaves off the

caspase 12 CARD prodomain. After this processing, this caspase is

autoprocessed into large and small subunits (Szegezdi et al., 2003).

Once activated, caspase 12 cleaves and activates the procaspase 9,

triggering the remaining caspase proteolytic cascade. Calpain can also

cleave Bid.

The third mechanism is Ca

2þ

-dependent and also involves the intrinsic

mitochondrial pathway. Some apoptotic stimuli induce Ca

2þ

release from

the ER, and this process may be regulated by the Bcl-2 family members

that reside in this organelle (Szegezdi et al., 2003). Bax and Bak seem to

induce Ca

2þ

release, while antiapoptotic members, such as Bcl-2, seem to

reduce this process. Ca

2þ

ions released from the ER eventually accumulate

in mitochondria, which induces permeabilization of the outer mitochon-

drial membrane through PTP formation. Release of apoptogenic factors

Mechanisms of cell proliferation and cell death in animal cell culture

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169

into the cytoplasm causes activation of caspase 9 and mitochondria-

mediated apoptosis. Apoptogenic Bap31 protein, which resides in the ER,

can be cleaved by caspase 8 and also seems to induce Ca

2þ

release from

this organelle (Breckenridge et al., 2003).

The extrinsic or death receptor-induced pathway

The extrinsic pathway consists of a series of events initially induced by

death receptors located on the cell surface. It is initiated by interaction of

extracellular death ligands with their respective receptors, located on the

surface of the plasma membrane. The death ligands are members of the

tumor necrosis factor (TNF)/nerve growth factor (NGF) superfamily.

TNF-R1, Fas (Apo-1/CD95), TRAIL-R1, TRAIL-R2, and NGF-R are

examples of death receptors. They are transmembrane proteins consisting

of an external domain, where the ligand associates, and a cytoplasmic

domain, which contains the DD (death domain).

The death ligands, such as FasL, are typically homotrimeric molecules.

When they bind to their receptors, which are monomers, they induce

aggregation and trimerization of the receptors, resulting in their cytoplas-

mic domains becoming physically closer together. The close proximity of

the DDs results in the recruitment of adaptor proteins located in the

cytoplasm, such as FADD and TRADD. These adaptor proteins also

possess a DD and bind to the DD of the death receptors. FADD binds

mainly to Fas, while TRADD binds preferentially to TNF-R1. After

TRADD binds to TNF-R1, FADD can bind to TRADD. FADD also

possesses another domain, besides DD, which is called DED. Procaspases

8 and 10 (initiator caspases) also possess the DED and are able to bind to

the adaptor protein through their DED, therefore assembling the DISC

(death-inducing signaling complex) (Figure 7.7).

The DISC-induced procaspase assembly results in the autoactivation of

caspases 8 and 10. The DISC process of caspase activation seems to be

analogous to the apoptosome process of caspase 9 activation. Caspase 8, in

turn, cleaves and activates caspase 3, which is responsible for the apoptotic

signal ampliﬁcation with subsequent cell collapse.

Some cell types maintain a low level of caspase 8 in the cytoplasm.

Therefore, in the presence of apoptotic stimuli, this small amount of

caspase 8 is activated in the DISC complex, and the subsequent activation

of effector caspases is not possible. These cell types are called type II and

also require the mitochondrial pathway activation. These two pathways

can communicate through the cleavage of a Bcl-2 member, Bid, by caspase

8 (Li et al., 1998). Cell lines that have a higher level of caspase 8 in the

cytoplasm are called type I. In these cells, Bcl-2 family members do not

regulate the death receptor-mediated pathway.

The proteolytic cleavage of Bid removes its N-terminal portion, exposing

the BH3 domain (Li et al., 1998). The truncated Bid protein, called tBid or

p15, translocates to the mitochondria, where it interacts with Bax or Bak,

inducing a conformational change in these proteins (Desagher et al., 1999).

This conformational change is necessary for the permeabilization of the

outer mitochondrial membrane and the subsequent release of the apopto-

genic factors into the cytoplasm, resulting in caspase 9 activation.

170 Animal Cell Technology

7.6.2 Molecular strategies for apoptosis control

Since apoptosis occurs under the control of several genes, the molecular

manipulation of the signaling cascade could block apoptosis progression

and prolong cell viability. Maintaining a high cell viability in bioreactors is

of great interest for biotechnological processes. Therefore, many research-

ers in this area are devoted to the development of genetically modiﬁed cell

lines with increased resistance to apoptosis.

The most common strategies consist of developing recombinant cell

lines expressing antiapoptotic genes that regulate the two main families of

proteins involved in the apoptotic cascade: the Bcl-2 and caspase families.

The Bcl-2 gene has been the most widely studied and its overexpression

in cells has been described. It has the ability to protect several industrially

important cell lines, such as hybridomas, myelomas and CHO, BHK, and

COS cells, against apoptotic stimuli that are typical of a cell culture

Death receptors

TNFa

APO3L

TNFR1

APO3L/DR3

FAS

FasL FasL

DR4,5

TRADD

FADD

FADD ?

Caspase 8

Caspase 9

Caspase 8

Caspase 10

Death receptors

pathway

Apoptosis

Bid cleavage

Mitochondrion Bax

Bax/BAK

Bak

Bcl-2/Bcl-x

Cytochrome c

Mitochondrial

pathway

Apoptosome

Figure 7.7

Caspase activation through the death receptor-induced pathway. The activation of

initiator caspases 8 and 10 by the death receptors results in t he activation of

effector caspases 3, 6, and 7. In type II cell lines, the activation of these initiator

caspases also results in Bid cleavage and, therefore, in the activation of the

mitochondrial pat hway.

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