Apoptosis Me

8/4/2019 Apoptosis Me

http://slidepdf.com/reader/full/apoptosis-me 1/9

Metabolic Engineering 5 (2003) 124–132

Metabolic engineering of apoptosis in cultured animal cells:

implications for the biotechnology industry

Joaquim Vives,1 Sandra Juanola, Jordi Joan Cairo ´ , and Francesc Go ` diaÃ

Deptartament d’Enginyeria Quı mica (UAB), Escola Tecnica Superior d’Enginyeria, E.T.S.E., Universitat Autonoma de Barcelona,

U.A.B., 08193 Bellaterra, Barcelona, Spain

Received 13 November 2002; accepted 15 April 2003

Abstract

Animal cells have been widely used to obtain a wide range of products for human and animal healthcare applications. However,

the extreme sensitivity of these cells in respect to changes experienced in their environment is evidenced by the activation of a gene-

encoded program known as apoptosis, resulting in their death and destruction. From the bioprocess angle, losses in cell viability

bring lower productivities and higher risks of product degradation. Consequently, many research efforts have been devoted to the

development of apoptosis protective mechanisms, including the metabolic engineering of apoptosis pathways, that has proven

effective in diminishing programmed cell death in a variety of biotechnological relevant cell lines. This review is focused especially in

the encouraging initial results obtained with the over-expression of cloned anti-apoptosis genes, from both endogenous and viral

origin interfering at mitochondrial and initiator caspases levels.

r 2003 Elsevier Science (USA). All rights reserved.

Keywords: Apoptosis; Metabolic engineering; Animal cell culture; Caspases; Bcl-2 homologues; Caspase-inhibitors; Bcl-2; Bcl-XL; KSBcl-2;

BHRF-1; X-IAP; p35; Caspase 9-dominant negative

1. Introduction

Animal cells are widely used in industrial processes as

sophisticated cell factories for over-production of

proteins with many applications in diagnosis, therapeu-

tics, downstream processing, and other fields of bio-

technological interest, since they are able to perform

post-translational modifications as well as protein

folding in an authentic manner (Reiter and Blu ¨ ml,

1994).

However, in spite of its potential, cells cultivated in

bioreactors are typically subjected to a number of

stimuli that lead to their demise, therefore limiting the

productivity of these bioprocesses (Al-Rubeai and

Singh, 1998). Mammalian cells, in particular, are

extremely sensitive to changes in their environment,

responding to insults such as nutrient deprivation,

growth factor withdrawal, oxygen limitation, and

excessive shear stress levels by activating an intrinsic

death cascade process known as programmed cell

death (PCD) or apoptosis (Mastrangelo, 1999). This

mechanism of cell death occurs as a sequence of events

under the control of a number of cellular genes,

transcription factors, enzymes, and signaling molecules

that culminate in a series of well known and easily

recognizable morphological, biochemical and molecular

changes which may be broadly and chronologically

defined. For a detailed discussion of such features the

reader is directed to recent reviews on the subject

(Hengartner, 2000; Cory and Adams, 2002). Briefly,

apoptosis is morphologically characterized by a reduc-

tion in cellular volume, membrane blebbing, nuclear

fragmentation, and finally disintegration of the cell

into a number of membrane enclosed apoptotic bodies

(Fig. 1). In fact, apoptosis is the principal form of cell

death in large-scale cultures (Franek and Dolnikova ´ ,

1991; Mercille and Massie, 1994; Singh et al., 1994)

and is very likely responsible for the significant losses

in revenue to the biopharmaceutical industries

(Mastrangelo and Betenbaugh, 1998).

Since this death program is gene-encoded, metabolic

engineering of commercial relevant cell lines blocking its

ARTICLE IN PRESS

ÃCorresponding author.

E-mail address: [email protected] (F. Godia).1Current address: Institute for Stem Cell Research, Roger Land

Building, King’s Buildings, University of Edinburgh, EH9 3JQ

Edinburgh, Scotland, UK.

1096-7176/03/$- see front matterr 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S1096-7176(03)00024-7



progress could prolong cell viability in bioreactors

during production of diagnostic and therapeutic mole-

cules (Fussenegger and Bailey, 1998; Go ` dia and Cairo ´ ,

2002). The object of this paper is to briefly review recent

advances for limiting apoptosis in mammalian cell

cultures by a genetic modification approach that could

lead to more robust cell lines, to be more efficiently used

in bioprocesses.

ARTICLE IN PRESS

Fig. 1. Electron micrographs of murine hybridoma cells undergoing apoptosis. (A) Viable cell showing spherical nucleus (n) in which chromatin is

not condensed; some of the main cell organelles and cellular structures are easily recognizable, such as mitochondria (m) and endoplasmic reticulum

(er), (B ), Cell undergoing an early stage of apoptosis; nuclear chromatin compacts in uniformly dense masses along the nuclear envelope; the

cytoplasm begins to shrink following the cleavage of lamins and actin filaments; mitochondria and endoplasmatic reticulum remain intact, ( C ) Cell

on an advanced phase of apoptosis: nuclear condensation can also be observed following the breakdown of chromatin and nuclear structural

proteins; organelles are damaged, (D), Cell on a final phase of apoptosis: nucleus is fragmented into various pieces including, in many cases,

condensed chromatin; cell adopts an irregular shape that leads eventually to the formation of membrane-bound apoptotic bodies. KB26.5 hybridoma

(Sanfeliu et al., 1996) cell samples were fixed, dehydrated and embedded following the araldite protocol (Glauert and Glauert, 1958). Sections on

grids were observed in Hitachi H-7000 electron microscope.

J. Vives et al. / Metabolic Engineering 5 (2003) 124–132 125



2. Triggering events and apoptosis pathways

Limitations in nutrients such as glucose and amino

acids have been shown to trigger apoptosis in batch and

fed-batch cultures of hybridomas, Chinese hamsterovary (CHO), NS0, and Sf-9 cells (Vomastek and

Franek, 1993; Mercille and Massie, 1994; Perreault

and Lemieux, 1994; Singh et al., 1994; Simpson et al.,

1998; Tinto et al., 2002). In addition to this, other insults

leading to apoptosis are the absence of serum, which

deprives cells of essential growth factors and cytokines

and is typically imposed on cell lines during their use at

large-scale operations for biosafety reasons, as well as

the lack of protein and lipids, or high osmolarity and

concentrations of toxic by-products from the cellular

metabolism (Al-Rubeai and Singh, 1998).

Although inhibiting apoptosis during the signalingphase can be quite effective in certain instances as the

intervention is in the earliest stage, the main drawback

of the strategies employed to do so is that they are cell

type- and stimulus-specific. Thus a more practical

method of blocking PCD may be to change the action

of the final execution pathway. Such an approach could

have wide range of applications in the cell culture

industry where cells are exposed to a variety of insults,

each utilizing a unique signal transduction pathway but

mostly channeling into a reduced number of pathways

in the execution phase. Interfering with an event

common to most cases of PCD might therefore provide

a stronger defense against apoptotic death, allowing for

increased cell viability and product yields. Despite the

fact that much of the apoptotic cascade remains unclear,

intense investigation has provided a preliminary descrip-

tion of the effector phase. In this sense, researchers in

many different fields have dissected the critical steps in

the apoptotic pathways. Currently it is known that

apoptosis occurs as a series of events that can be

subdivided into four distinct phases: initiation, commit-

ment, amplification and demolition (Slee et al., 1999).

Efforts to block PCD should be focused on the

interference of the final common pathway of apoptosis

immediately upstream the point after which death

signals became irreversible. Strategies employed to

accomplish this consist of engineering cells to express

anti-apoptosis genes at two different levels: protective

Bcl-2 family members and caspase-inhibitors, as indi-

cated in Fig. 2.

3. Use of protective members of Bcl-2 family

The most common manipulation reported in the

literature to date is the over-expression of the Bcl-2

protein. It has been the first and best studied of the anti-

apoptosis genes (Tsujimoto et al., 1985; Vaux et al.,

1988). Moreover, Bcl-2 is the founding member of a

family of structurally related proteins that play a crucial

role in the modulation of the cell death process (Reed,

1998), which includes both death inducers (such as Bakand Bax, among others) and death suppressors (such as

Bcl-2 and Bcl-XL, among others). There are consider-

able interactions between different members of this

family and with other cellular proteins, and it is

suggested that these interactions play an important role

in the regulation of apoptosis. Specifically, interactions

between pro- and anti-apoptotic proteins, and the

relative ratio of the two groups of proteins determine

whether a cell remains viable or enters into apoptosis

(Korsmeyer, 1995; Cory and Adams, 2002). Although

the specific mechanism of action of these anti-apoptotic

gene products remains unclear, certain functions of Bcl-

2 and Bcl-XL are well described. For instance, it is

accepted that both can heterodimerize with Bax, a

related death promoter, in an effort to render Bax

ineffective (Oltvai et al., 1993; Chao et al., 1995).

Furthermore, Bcl-2 can prevent the release of cyto-

chrome c from the mitochondria (Kluck et al., 1997;

Yang et al., 1997), an important step in the apoptotic

cascade which is thought to lead to the activation of the

caspases (proteases which are largely responsible for

the destruction of the cell) through the cytochrome c/

Apaf-1/caspase 9 apoptosome. However, recent studies

suggest that Bcl-2 prevents caspase activation program

independently of the apoptosome. This finding would

ARTICLE IN PRESS

DEATHSTIMULI

Pro-apoptotic Bcl-2family members

Bcl-2CED-4-likeadaptors ?

Initiatorcaspases

Mitochondrialdamage

Cyt c/Apaf-1/ Caspase 9

Caspase 3

CELLDESTRUCTION

Caspase 7

Protective Bcl-2 familymembers

Inhibitors of caspases

block

activation

Fig. 2. Model for the control of apoptosis (as recently proposed by Marsden et al., 2002) and location of the anti-apoptosis activity of the protective

members of the Bcl-2 family and caspase-inhibitors.

J. Vives et al. / Metabolic Engineering 5 (2003) 124–132126



indicate that the function of the apoptosome would

amplify rather than initiate the caspase cascade (Mars-

den et al., 2002).

In any case, over-expression of bcl-2 and its protective

homologues in industrial relevant cell lines (such as

hybridomas, myelomas, baby hamster kidney (BHK),

CHO, and COS cells) has been proven to protect againstnumerous apoptosis-inducing elements during typical

culture limiting conditions, including nutrient depriva-

tion (Itoh et al., 1995; Singh et al., 1996; Mercille et al.,

1999; Mastrangelo et al., 2000b; Vives et al., 2003),

serum withdrawal (Fassnacht et al., 1998; Mastrangelo

et al., 2000b), oxygen limitation (Simpson et al., 1997;

Singh et al., 1997), elevated shear levels (Perani et al.,

1998), accumulation of toxic metabolites, such as

ammonia (Mastrangelo et al., 2000b), and virus infec-

tion (Mastrangelo et al., 2000a). In contrast, exogenous

expression of bcl-2 was found to be of little consequence

in other cell lines such as Sf-9 insect cells and HeLa cells,where viability was not improved by over-expressing this

gene (Fujita et al., 1997; Mitchell-Logean and Mur-

hammer, 1997).

Table 1 summarizes a subset of recent data where

Bcl-2 family members have been over-expressed in a

variety of cell types used in large-scale cell culture. As

mentioned above, in nearly all cases these genetic

modifications protected cells from apoptosis, but the

cultures exhibited varying phenotypes. In some cases,

cell density increases while in others growth arrest is

observed. More importantly, recombinant protein pro-

duction increases in some cases, but not in others. Thereasons for these differences in the actual cell lines and

the specific culture conditions employed will have a

significant effect on the outcome of such cell line

engineering strategies. Moreover, Table 1 clearly

shows that most of the studies published to date

have been limited to the over-expression of the bcl-2

and bcl-xL genes. Interestingly, their gene products

are subject to regulatory mechanisms leading to

the loss of their apoptosis protection effectiveness.

Such mechanisms may include: phosphorylation,

ubiquitin-mediated proteolysis, caspase-mediated

cleavage, protein-protein interactions and changes in

subcellular localization (Fadeel et al., 1999). As an

example, Bcl-2 is degradated by caspase 3 cleavage into

a 23 kDa fragment that enhances cell death instead of

protecting cells from apoptosis when the apoptosis

inducing conditions are maintained (Cheng et al., 1997).

The processing of Bcl-2 by its large unstructured

loop was found to correlate with reduced cell viabilities

following external factors leading to apoptosis

(Figueroa et al., 2001). These studies indicate that

the cells regulate anti-apoptosis protein levels and

these processing events can limit the effectiveness of

cell death inhibition strategies in mammalian cell culture

systems.

Thus, genetic modifications of cell lines of biotechno-

logical interest with mutants, such as loop-deleted

Bcl-XL and Bcl-2 (Charbonneau and Gauthier, 2001;

Figueroa et al., 2001), and viral homologues of Bcl-2,

such as BHRF-1, KSBcl-2, and E1B 19 K (Mercille et al.,

1999; Vives et al., 2003), opens new possibilities to

increase the robustness of these cells under cultureconditions. The rationale of this strategy is based on

maintaining the original function of such viral proteins,

that is, to protect the infected host from cell death in

order to favor the replication of the virus, therefore

ensuring the completion of the virus life cycle and the

subsequent infection of other cells (Hardwick, 2001).

4. Inhibition of caspases

Another highly conserved family of apoptosis reg-

ulators is the caspase proteases (Fig. 2). These aspartate-specific proteases are critically involved in the apoptotic

process in mammalian cells and have an important role

in the commitment, amplification and demolition phases

of the death program (Slee et al., 1999). These enzymes

exist as zymogens composed of a prodomain, and large

and small catalytic subunits. Generation of active

caspases requires accurate processing of internal aspar-

tic residues to liberate the prodomain and produce the

two chain active enzyme (reviewed by Nicholson, 1999).

Various authors have shown protection against nutrient

deprivation in hybridomas and NS0 cells (McKenna and

Cotter, 2000; Tinto ´ et al, 2002), and virus infection of rat carcinomal cell line (Mastrangelo et al., 1999) by the

addition to the medium of caspase-specific peptidic

inhibitors that bind to the catalytic site of these

proteases (Ekert et al., 1999). Again, cell line depen-

dency was observed, while no improvement in produc-

tivity could be demonstrated in NS0 cells (McKenna

and Cotter, 2000), 3.9-fold higher productivities were

observed in a rat carcinomal cell line maintained in a

growth medium with z-VAD-fmk (Mastrangelo et al.,

1999).

In addition to these results, the use of specific caspase-

inhibitors in hybridomas demonstrated that the action

of caspases in the apoptosis cascade is located upstream

of the point after which irreversible commitment to

death occurs in nutrient deprived cultures and that the

treatment of these cultures with a combination of

peptide compounds (namely, acetyl-Asp-Glu-Val-Asp-

aldehyde (Ac-DEVD-cho) and N-benzyloxycarbonyl-

Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk)) enabled

not only a transitory suppression of the progress

of apoptosis under nutrient deprivation conditions but

also made possible the recovery of cell culture even after

a period of 36 h under glutamine depletion (Tinto ´ et al.,

2002). However, the continuous addition of chemicals to

the culture medium can become expensive and may

ARTICLE IN PRESS




ARTICLE IN PRESS

T a

b l e 1

E f f e c t o f t h e o v e r - e x p r e s s i o n o f B c l - 2 a n d c a s p a s e - i n h i b i t o r s o n a v a r i e t y o f c e l l l i n e s o f b i o t e c h n o l o g i c a l i n t e r e s t

G e n e ( s )

e x p r e s s e d

H o s t c e l l

I m p r o v e m e n t i n

O b s e r v a t i o n s

R e f e r e n c e s

V i a b

i l i t y

P r o d u c t i v i t y

B c l - 2

M y e l o m a

N

o

–

B c l - 2 f a i l e d t o a f f e c t t h e d e c l i n e p h a s e c h a r a c t e r i s t i c s a n d s e r u m

d e p e n d e n c e o f N S 0 c e l l s .

M u r r a y e t a l . ( 1 9 9 6 )

Y

e s

–

H i g h l e v e l s

o f B c l - 2 e x p r e s s i o n b u t o n l y m o d e r a t e e n h

a n c e m e n t o f

s u r v i v a l w i t

h r e s p e c t t o t h e c o n t r o l c u l t u r e i n l o w s e r u m c o n d i t i o n s

F u j i t a e t a l . ( 1 9 9 7 )

Y

e s

Y e s

I n c r e a s e d m a x i m u m c e l l n u m b e r a n d c u l t u r e d u

r a t i o n

T e y e t a l .

( 2 0 0 0 b )

H y b r i d o m a

Y

e s

Y e s

E x t e n d e d v

i a b l e c u l t u r e p e r i o d a n d e n h a n c e d s p e c i fi c

p r o d u c t i v i t y

I t o h e t a l .

( 1 9 9 5 )

Y

e s

N o

H i g h e r

m e m b r a n e i n t a c t i n d e x a t i n c r e a s i n g d i l u t i o n s t e p s

F a s s n a c h t e t

a l . ( 1 9 9 8 )

Y

e s

Y e s

E n g i n e e r e d

c e l l s w e r e a d a p t e d t o g r o w i n h i g h o s m o l a

r i t y m e d i u m

s h o w i n

g a 1 0 0 %

h i g h e r p r o d u c t i v i t y t h a n c o n t r o l c u l t u r e

P e r a n i e t a

l . ( 1 9 9 8 )

Y

e s

Y e s

I m p r o v e d c e l l u l a r r o b u s t n e s s i n i n t e n s i v e c u l t u r e p r o c e s s e s

F a s s n a c h t e t

a l . ( 1 9 9 9 )

Y

e s

N o

A l t e r e d c e l l - c y c l e d i s t r i b u t i o n

S i m p s o n e t

a l . ( 1 9 9 9 )

Y

e s

Y e s

E f f e c t e n h a n c e d b y c o - e x p r e s s i o n o f B a g

T e r a d a e t a l . ( 1 9 9 9 a )

C H O

Y

e s

–

E x t e n d e d c u l t u r e d u r a t i o n

G o s w a m i e t

a l . ( 1 9 9 9 )

N

o

Y e s

H i g h e r c e l l

d e n s i t i e s , b u t n o b e n e fi c i a l e f f e c t o n t h e d e c l i n e p h a s e ;

D N A l a d d e r i n g w a s s t i l l o b s e r v e d

F u s s e n e g g e r e

t a l . ( 2 0 0 0 )

Y

e s

N o

I n c r e a s e d c u l t u r e d u r a t i o n

T e y e t a l .

( 2 0 0 0 a )

S f 9 / H i g h F i v e

N o / N o

N o / Y e s

B c l - 2 c o u

l d i n h i b i t t r a n s c r i p t i o n o f g e n e s u n d e r t h e

c o n t r o l o f

p o l y h e d r i n p r o m o t e r

M i t c h e l l - L o g e a n a n d M u r h a m m e r ( 1 9 9 7 )

B c l - 2 ,

B c l - X L

C H O

Y

e s

Y e s

P r o t e c t i o n f r o m a l p h a v i r u s v e c t o r s i n d u c e d a p o

p t o s i s

M a s t r a n g e l o e t

a l . ( 2 0 0 0 a , b

)

B c l - 2 ,

K S B c l -

2 , B H R F - 1

H y b r i d o m a

Y

e s

–

E x t e n d e d c u l t u r e d u r a t i o n u n d e r G l n d e p l e t i o n i n d u c e d a p o p t o s i s ;

d i d n o t a s s e s s p r o d u c t i v i t y

V i v e s e t a l . ( 2 0 0 3 )

B c l - X L

H y b r i d o m a

Y

e s

–

S u b s t a n

t i a l i n c r e a s e i n v i a b i l i t y i n s t a t i o n a r y b a t c h c u l t u r e

C h a r b o n n e a u a n d

G a u t h i e r ( 2 0 0 0 )

Y

e s

–

U s e o f a z i n c - i n d u c i b l e e x p r e s s i o n s y s t e m

J u n g e t a l .

( 2 0 0 2 )

E 1

B 1 9 K

C H O

Y

e s

–

V a c c i n a v i r u s - i n d u c e d a p o p t o s i s w a s p r e v e n t e d

I n k e t a l .

( 1 9 9 5 )

N S 0 m y e l o m a

Y

e s

N o

E x t e n t o f p r o t e c t i o n a g a i n s t G l n d e p l e t i o n i n d u c e d a p o

p t o s i s a n d i n

t h e l a t e p h a s e o f b a t c h c u l t u r e s

M e r c i l l e e t a l . ( 1 9 9 9 )

X - I A P

2 9 3 ,

C H O

Y

e s

–

S i g n i fi c a n t p r o t e c t i o n a g a i n s t v a r i o u s i n s u l t s

S a u e r w a l d e t a l .

( 2 0 0 2 ,

2 0 0 3 )

C r m A

C H O

Y

e s

–

O n l y s l i g h t

d e l a y o f c e l l d e a t h i s a c h i e v e d .

E n g i n e e r e d

C r m A o f f e r s

b e t t e r p r o t e c t i o n a g a i n s t a v a r i e t y o f a p o p t o t i c s t i m u l i

T e r a d a e t a l . ( 1 9 9 9 b ) , S a u e r w a l d e t a l . ( 2 0 0 3 )

p 3 5

C H O

Y

e s

–

D e l a y e d c e l l d e a t h i n c u l t u r e

T e r a d a e t a l . ( 1 9 9 9 b )

S f 9

Y

e s

–

P r o f o u n d r e s i s t a n c e t o n u t r i e n t d e p r i v a t i o n

L i n e t a l .

( 2 0 0 1 )

C a

s p a s e 9 -

D N

C H O

Y

e s

Y e s

D r a m a t i c r e s i s t a n c e o f e n g i n e e r e d c e l l s t o a v a r i e t y o

f a p o p t o t i c

s t i m u l i a s w e l l a s i n c r e a s i n g p r o d u c t i v i t y i n b i o r

e a c t o r s

v a n d e G o o r e t a l . ( 2 0 0 1 )

– I n d i c a t e s t h a t t h e s t u d y d i d n o t a s s e s s t h e e f f e c t o f a n t i - a p o p t o t i c g e n e e x p r e s s i o n o n p r o d u c t i v i t y .




affect cell growth. Fortunately, a number of natural

caspase-inhibitor genes have been identified so far in

both eukaryotes and viruses (Deveraux et al., 1999; Ekert

et al., 1999). For this reason, genetic strategies directed

against regulatory points located upstream of effector

caspases to avoid their activation are of interest in order

to generate cell lines more robust in front of apoptoticstimuli (Go ` dia and Cairo ´ , 2002). To achieve this goal,

several proteins encoded by anti-apoptotic genes, such as

baculovirus p35, crma, x-iap and caspase 9-dominant

negative, have recently been described and shown to

inhibit apoptosis in some cell types (Beidler et al., 1995;

Pan et al., 1998; Deveraux et al., 1999; Table 1).

Expression of X-linked inhibitor of apoptosis protein

(X-IAP) in CHO and 293 human embryonic kidney

(HEK) cells inhibited apoptosis significantly and de-

monstrated that cells engineered to express this protein

protected against various insults, including virus infec-

tion and serum deprivation (Sauerwald et al., 2002).Moreover, mutants of X-IAP and cytokine response

modifier (CrmA) (namely, X-IAP-BIR123NC and

CrmA-DQMD) provide even better protection against

apoptosis from multiple insults than their wild-type

counterparts (Sauerwald et al., 2003).

Expression of caspase 9-dominant negative in CHO

cells resulted in a dramatic resistance of these cells to a

variety of apoptotic stimuli as well as prolonged

viability and increased productivity in bioreactors (van

de Goor et al., 2001). Similarly, expression of p35

delayed cell death in CHO cells as well (Terada et al.,

1999b). Moreover its expression in stable Sf-9 cell linesshowed a profound resistance to nutrient deprivation

(Lin et al., 2001). On the other hand, expression

of CrmA, from cowpox virus (Salvesen and Dixit,

1997), only slightly delayed cell death in CHO cells

(Terada et al., 1999b).

5. Stability of the expression of anti-apoptotic genes

The constitutive high-level expression of anti-apopto-

tic genes has been shown to have detrimental effects on

genomic stability of some types of cultured cells by

preventing p53-induced apoptotic death of cells bearing

genetic abnormalities such as mutation or mitotic

damage (Cherbonnel-Lasserre et al., 1996; Minn et al.,

1996). The non-deletion of these abnormal cells could

give rise to the accumulation of variant cells during the

long-term culture period necessary to prepare the cell

stocks used for production, with potential adverse

consequences on the monoclonality of the cell line and

of the antibody product or recombinant protein.

Inducible gene expression may be used to avoid this

problem (Jung et al., 2002). Thus anti-apoptotic gene

expression could be restricted to the late phase of batch

and fed-batch cultures by exogenous control using

regulatable promoters. A comprehensive list of such

promoters has been recently reviewed in Fussenegger,

(2001).

It should be also pointed out the necessity to improve

this type of approach in order to ensure long-term

expression of anti-apoptosis genes. Many research

groups are currently designing and evaluating a varietyof viral and non-viral vectors as well as expression

concepts for their safety, high-level transduction, trop-

ism and sustained expression in a variety of therapeu-

tically relevant cell lines (Crystal, 1995; Nishikawa and

Huang, 2001; Mitta et al., 2002).

6. Downstream processing considerations

In addition to providing the opportunity for pro-

longed protein production in those cases in which a real

enhancement is achieved, the over-expression of anti-

apoptosis genes may present certain advantages even in

the absence of productivity enhancements. For example,

extensions in cell lifetimes may limit the amount of cell

debris associated with a particular process, thereby

simplifying purification schemes. Since product purifica-

tion often represents a significant fraction of total

production cost, limiting downstream processing steps

could lead to more cost-effective bioprocesses.

The final use of the products obtained from apopto-

sis-resistant cell lines in biotechnological processes could

be diagnostic or therapeutic. Particularly in the latter

case, downstream processing should ensure the absence

of both oncogenes and viral contents on the final

product, as in fact is done in many other cases in

Biotechnology Industry (Sofer, 1995). Therefore the use

of oncogenes and viral gene products should not

represent any problem for any potential final product

contamination.

7. Use of genetic engineered cells

Results summarized in this paper demonstrate that

the incorporation of a genetic protection enables the

delay of apoptosis in those cells that otherwise would dieand accumulate in the bioreactor. Moreover, such

engineered cells can be rescued after prolonged times

being exposed to apoptosis inducing conditions such as

nutrient deprivation, growth factors withdrawal and

oxygen limitations, among other insults which can be

present in any standard bioprocess (Fig. 3).

8. Conclusions

There are many causes of apoptosis in eukaryotic cell

culture and more knowledge in this area is likely to lead

to additional pathway-based strategies to prevent cell

ARTICLE IN PRESS




death. Recent studies have focused on the over-expression of Bcl-2 family members, either endogenous

or viral. Since caspase-inhibitors act at a different step in

the apoptotic pathway than Bcl-2 and its homologues,

further work should be done to examine whether co-

expression of these genes could synergistically enhance

cell viability and therefore prolong the culture perfor-

mance (as shown by Terada et al., 1999a). A single

action is unlikely to eliminate apoptosis and enhance

productivity in all cell culture systems. Clearly, many

proteins are involved in this process and it is likely that a

combination of modifications in the expression of a

number of these proteins will be required to achieve the

desired outcomes of increased density, viability and

productivity. Such genetic modifications will enable the

construction of recombinant cell lines with advanta-

geous properties. The understanding of the molecular

processes of apoptosis in cultured cells is therefore a

prerequisite for the successful development of high

producer bioprocesses with cultured cells.

Acknowledgments

The present work has been developed in the frame-

work of the ‘‘Centre de Refere ` ncia en Biotecnologia’’

(Generalitat de Catalunya) and supported by the ‘‘Plan

Nacional de Biotecnologı ´a’’ (BIO97-0542). J.V. is a

recipient of a fellowship from CIRIT (1999FI00779).

Further reading

Al-Rubeai, M., Singh, R.P., 1998. Apoptosis in cell culture. Curr.Opin. Biotechnol. 9, 152–156.

Beidler, D.R., Tewari, M., Friesen, P.D., Poirier, G., Dixit, V.M.,

1995. The baculovirus p35 protein inhibits Fas- and tumor necrosis

factor-induced apoptosis. J. Biol. Chem. 270, 16526–16528.

Chao, D.T., Linette, G.P., Boise, L.H., White, L.S., Thompson, C.B.,

Korsmeyer, S.J., 1995. Bcl-XL and Bcl-2 repress a common

pathway of cell death. J. Exp. Med. 182, 821–828.

Charbonneau, J.R., Gauthier, E.R., 2000. Prolongation of murine

hybridoma cell survival in stationary batch culture by Bcl-xL

expression. Cytotechnology 34, 131–139.

Charbonneau, J.R., Gauthier, E.R. 2001. Protection of hybridoma

cells against apoptosis by a loop domain-deficient Bcl-xL protein.

Cytotechnology 37, 41–47.

Cheng, E.H.-Y., Kirsch, D.G., Clem, R.J., Ravi, R., Kastan, M.B.,

Bedi, A., Ueno, K., Hardwick, J.M., 1997. Conversion of Bcl-2 to a

Bax-like death effector by caspases. Science 278, 1966–1968.

Cherbonnel-Lasserre, C., Gauny, S., Kronenberg, A., 1996. Suppres-

sion of apoptosis by Bcl-2 or Bcl-xL promotes susceptibility to

mutagenesis. Oncogene 13, 1489–1497.

Cory, S., Adams, J.M., 2002. The Bcl2 family: regulators of the cellular

life-or-death switch. Nat. Rev. Cancer 2, 647–656.

Crystal, R.G., 1995. Transfer of genes to humans: early lessons and

obstacles to success. Science 270, 404–410.

Deveraux, Q.L., Stennicke, H.R., Salvesen, G.S., Reed, J.C., 1999.

Endogenous inhibitors of caspases. J. Clin. Immunol. 19, 388–398.

Ekert, P.G., Silke, J., Vaux, D.L., 1999. Caspase inhibitors. Cell Death

Differ. 6, 1081–1086.

Fadeel, B., Zhivotovsky, B., Orrenius, S., 1999. All along the

watchtower: on the regulation of apoptosis regulators. FASEB J.13, 1647–1657.

Fassnacht, D., Ro ¨ ssing, S., Franek, F., Al-Rubeai, M., Po ¨ rtner, R.,

1998: Effect of Bcl-2 expression on hybridoma cell growth in

serum-supplemented, protein-free and diluted media. Cytotechnol-

ogy 26, 219–225.

Fassnacht, D., Ro ¨ ssing, S., Singh, R.P., Al-Rubeai, M., Po ¨ rtner, R.

1999. Influence of bcl-2 on antibody productivity in high cell

density perfusion cultures of hybridoma. Cytotechnology 30,

95–105.

Figueroa, B. Jr., Sauerwald, T.M., Mastrangelo, A.J., Hardwick, J.M.,

Betenbaugh, M.J. 2001. Comparison of Bcl-2 to a Bcl-2 deletion

mutant for mammalian cells exposed to culture insults. Biotechnol.

Bioeng. 73, 211–222.

Franek, F., Dolnikova ´ , J. 1991. Nucleosomes occurring in protein-free

hybridoma cell culture. Evidence for programmed cell death. FEBSLett. 284, 285–287.

Fujita, T., Terada, S., Fukuoka, K., Kitayama, A., Ueda, H., Suzuki,

E. 1997. Reinforcing apoptosis-resistance of COS and myeloma

cells by transfecting with Bcl-2 gene. Cytotechnology 25, 25–33.

Fussenegger, M. 2001. The impact of mammalian gene regulation

concepts on functional genomic research, metabolic engineering,

and advanced gene therapies. Biotechnol. Prog. 17, 1–51.

Fussenegger, M., Bailey, J.E., 1998. Molecular regulation of cell-cycle

progression and apoptosis in mammalian cells: implications for

biotechnology. Biotechnol. Prog. 14, 807–833.

Fussenegger, M., Fassnacht, D., Schwartz, R., Zanghi, J.A., Graf, M.,

Bailey, J.E., Po ¨ rtner, R., 2000. Regulated overexpression of the

survival factor bcl-2 in CHO cells increases viable cell density in

batch culture and decreases DNA release in extended fixed-bed

cultivation. Cytotechnology 32, 45–61.

ARTICLE IN PRESS

insult

S u b - l e t h a l i n j u r y

v i a b i l i t y

time

recovery

P r o t e c t i v e

t i m e - w i n d o w

Apoptosis

commitment

Fig. 3. Viability profiles of cell cultures depending on the robustness of

the cells after exposition to apoptosis-inducing insults (adapted from

Vaux and Strasser, 1996). Although cells can respond to several insults

by activating the apoptosis program (—), cells receiving a transient, or

low-level exposure to the agent (sub-lethal injury), as well as being

protected by over-expressing anti-apoptosis genes (protective time-

window), may be able to recover when re-exposed to non-inducing

apoptosis conditions ( Á Á Á ). Cells not protected against apoptosis

irremediably die once entered into the commitment step of the death

program.




Glauert, A.M., Glauert, R.H., 1958. Araldite as an embedding

medium for electron microscopy. J. Biophys. Biochem. Cytol.

4,191–194.

Godia, F., Cairo, J.J., 2002. Metabolic engineering of animal cells.

Bioprocess Biosys. Eng. 24, 289–298.

van de Goor, J., Hamilton, R., Dixit, V., 2001. Methods for Making

Recombinant Proteins Using Apoptosis Inhibitors. International

Patent Application WO 01/23592, April 5, 2001.Goswami, J., Sinskey, A.J., Steller, H., Stephanopoulos, G.N., Wang,

D.I., 1999. Apoptosis in batch cultures of Chinese hamster ovary

cells. Biotechnol. Bioeng. 62, 632–640.

Hardwick, J.M., 2001. Apoptosis in viral pathogenesis. Cell Death

Differ. 8, 109–110.

Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407,

770–776.

Ink, B.S., Gilbert, C.S., Evan, G.I., 1995. Delay of vaccinia virus-

induced apoptosis in nonpermissive Chinese hamster ovary cells by

the cowpox virus CHOhr and adenovirus E1B 19K genes. J. Virol.

69, 661–668.

Itoh, Y., Veda, H., Suzuki, E., 1995. Overexpression of bcl-2,

apoptosis supressing gene: prolonged viable culture period of

hybridoma and enhanced antibody production. Biotechnol.Bioeng. 48, 118–122.

Jung, D., Cote, S., Drouin, M., Simard, C., Lemieux, R. 2002.

Inducible expression of Bcl-XL restricts apoptosis resistance to the

antibody secretion phase in hybridoma cultures. Biotechnol.

Bioeng. 79, 180–187.

Kluck, R.M., Bossy-Wetzel, E., Green, D.R., Newmeyer, D.D., 1997.

The release of cytochrome c from mitochondria: a primary site for

Bcl-2 regulation of apoptosis. Science 275, 1132–1136.

Korsmeyer, S.J., 1995. Regulators of cell death. Trends Genet. 11,

101–105.

Lin, G., Li, G., Granados, R.R., Blissard, G.W., 2001. Stable cell lines

expressing baculovirus P35: resistance to apoptosis and nutrient

stress, and increased glycoprotein secretion. In vitro Cell. Dev.

Biol. Anim. 37, 293–302.

Marsden, V.S., O’Connor, L., O’Reilly, L.A., Silke, J., Metcalf, D.,Ekert, P.G., Huang, D.C., Cecconi, F., Kuida, K., Tomaselli, K.J.,

Roy, S., Nicholson, D.W., Vaux, D.L., Bouillet, P., Adams, J.M.,

Strasser, A., 2002. Apoptosis initiated by Bcl-2-regulated caspase

activation independently of the cytochrome c/Apaf-1/caspase-9

apoptosome. Nature 419, 634–637.

Mastrangelo, A.J., 1999. Inhibition of apoptosis in mammalian cell

culture: the biotechnological relevance of limiting cell death. In: Al-

Rubeai, M., (Ed.), Cell Engineering, Vol. 1. Kluwer Academic

Publishers, Dordrecht, The Netherlands, pp.162–185.

Mastrangelo, A.J., Betenbaugh, M.J., 1998. Overcoming apoptosis:

new methods for improving protein-expression systems. TIBTECH

16, 88–95.

Mastrangelo, A.J., Zou, B., Hardwick, J.M., Betenbaugh, M.J., 1999.

Antiapoptosis chemicals prolong productive lifetimes of mamma-lian cells upon Sindbis virus infection. Biotechnol. Bioeng. 65,

298–305.

Mastrangelo, A.J., Hardwick, J.M., Bex, F., Betenbaugh, M.J., 2000a.

Part I. Bcl-2 and Bcl-xL limit apoptosis upon infection with

alphavirus vectors. Biotechnol. Bioeng. 67, 544–554.

Mastrangelo, A.J., Hardwick, J.M., Zou, B., Betenbaugh, M.J., 2000b.

Part II. Overexpression of bcl-2 family members enhances survival

of mammalian cells in response to various culture insults.

Biotechnol. Bioeng. 67, 555–565.

McKenna, S.L., Cotter T.G., 2000. Inhibition of caspase activity

delays apoptosis in a transfected NS/0 myeloma cell line.


Mercille, S., Massie, B., 1994. Induction of apoptosis in nutrient-

deprived cultures of hybridoma and myeloma cells. Biotechnol.

Bioeng. 44, 1140–1154.

Mercille, S., Jolicoeur, P., Gervais, C., Paquette, D., Mosser, D.D.,

Massie, B., 1999. Dose dependent reduction of apoptosis in

nutrient limited cultures of NS/0 myeloma cells transfected

with the E1B-19K adenoviral gene. Biotechnol. Bioeng. 63,

516–528.

Minn, A.J., Boise, L.H., Thompson, C.B., 1996. Expression of Bcl-xL

and loss of p53 can cooperate to overcome a cell cycle checkpoint

induced by mitotic spindle damage. Genes Dev. 10, 2621–2631.Mitchell-Logean, C., Murhammer, D.W., 1997. bcl-2 expression in

Spodoptera Frugiperda Sf-9 and Trichoplusia Ni BTI-Tn-5B1-4

insect cells: Effect on recombinant protein expression and cell

viability. Biotechnol. Bioeng. 56, 380–390.

Mitta, B., Rimann, M., Ehrengruber, M.U., Ehrbar, M., Djonov, V.,

Kelm, J., Fussenegger, M., 2002. Advanced modular self-inactivat-

ing lentiviral expression vectors for multigene interventions

in mammalian cells and in vivo transduction. Nucleic Acids Res.

30, e113.

Murray, K., Ang, C.-E., Gull, K., Hickman, J.A., Dickson, A.J., 1996.

NS0 myeloma cell death: Influence of bcl-2 overexpression.


Nicholson, D.W., 1999. Caspase structure, proteolytic substrates, and

function during apoptotic cell death. Cell Death Differ. 6,

1028–1042.

Nishikawa, M., Huang, L., 2001. Nonviral vectors in the new

millennium: delivery barriers in gene transfer. Hum. Gene Ther.

12, 861–870.

Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 hetero-

dimerizes in vivo with a conserved homolog, Bax, that accelerates

programmed cell death. Cell 74, 609–619.

Pan, G., O’Rourke, K., Dixit, V.M., 1998. Caspase-9, Bcl-XL, and

Apaf-1 form a ternary complex. J. Biol. Chem. 273, 5841–5845.

Perani, A., Singh, R.P., Chauhan, R., Al-Rubeai, M., 1998. Variable

functions of bcl-2 in mediating bioeator stress-induced apoptosis in

hybridoma cells. Cytotechnology 28, 177–188.

Perreault, J., Lemieux, R., 1994. Essential role of optimal protein-

synthesis in preventing the apoptotic death of cultured B-cell

hybridomas. Cytotechnology 13, 99–105.Reed, J.C., 1998. Bcl-2 family proteins. Oncogene 17, 3225–3236.

Reiter, M., Blu ¨ ml, G., 1994. Large-scale mammalian cell culture. Curr.

Opin. Biotechnol. 5, 175–179.

Salvesen, G.S., Dixit, V.M., 1997. Caspases: intracellular signaling by

proteolysis. Cell 91, 443–446.

Sanfeliu, A., Cairo ´ , J.J., Casas, C., Sola ` , C., Go ` dia, F., 1996. Analysis

of nutritional factors and physical conditions affecting growth and

monoclonal antibody production of the hybridoma KB-26.5 cell

line. Biotechnol. Prog. 12, 209–216.

Sauerwald, T.M., Betenbaugh, M.J., Oyler, G.A., 2002. Inhibiting

apoptosis in mammalian cell culture using the caspase inhibitor

XIAP and deletion mutants. Biotechnol. Bioeng. 77, 704–716.

Sauerwald, T.M., Oyler, G.A., Betenbaugh, M.J., 2003. Studying of

caspase inhibitors for limiting death in mammalian cell culture.Biotechnol. Bioeng. 81, 329–340.

Simpson, N.H., Milner, A.N., Al-Rubeai, M., 1997. Prevention of

hybridoma cell death by bcl-2 during sub-optimal culture condi-

tions. Biotechnol. Bioeng. 54, 1–16.

Simpson, N.H., Singh, R.P., Perani, A., Goldenzon, C., Al-Rubeai,

M., 1998. In hybridoma cultures, deprivation of any single amino

acid leads to apoptotic death, which is suppressed by the expression

of the bcl-2 gene. Biotechnol. Bioeng. 59, 90–98.

Simpson, N.H., Singh, R.P., Emery, A.N., Al-Rubeai, M., 1999. Bcl-2

over-expression reduces growth rate and prolongs G1 phase in

continuous chemostat cultures of hybridoma cells. Biotechnol.

Bioeng. 64, 174–186.

Singh, R.P., Al-Rubeai, M., Gregory, C.D., Emery, A.N., 1994. Cell

death in bioreactors: a role for apoptosis. Biotechnol. Bioeng. 44,

720–726.

ARTICLE IN PRESS




Singh, R.P., Emery, A.N., Al-Rubeai, M., 1996. Enhancement

of survivability of mammalian cells by over expression of

the apoptosis suppressor gene bcl-2. Biotechnol. Bioeng. 52,

166–175.

Singh, R.P., Finka, G., Emery, A.N., Al-Rubeai, M., 1997.

Apoptosis and its control in cell culture systems. Cytotechnology

23, 87–93.

Slee, E.A., Adrain, C., Martin, S.J., 1999. Serial killers: orderingcaspase activation events in apoptosis. Cell Death Differ. 6,

1067–1074.

Sofer, G., 1995. Validation of biotechnology products and processes.

Curr. Opin. Biotechnol. 6, 230–234.

Terada, S., Komatsu, T., Fujita, T., Terakawa, A., Nagamune, T.,

Takayama, S., Reed, J.C., Suzuki, E., 1999a. Co-expression of

bcl-2 and bag-1, apoptosis suppressing genes, prolonged viable

culture period of hybridoma and enhanced antibody production.

Cytotechnology 31, 143–151.

Terada, S., Yata, T., Fukuoka, K., Aisawa, H., Fujita, T., Nagamune,

T., Suzuki, E, 1999b. Improvement of mammalian cell survival by

apoptosis-inhibiting genes and caspase inhibitors for effective use

of mammalian cells. Seibutsu-kogaku kaishi 77, 2–11.

Tey, B.T., Singh, R.P., Piredda, L., Piacentini, M., Al-Rubeai, M.,

2000a. Influence of bcl-2 on cell death during the cultivation of a

Chinese hamster ovary cell line expressing a chimeric antibody.


Tey, B.T., Singh, R.P., Piredda, L., Piacentini, M., Al-Rubeai, M.,

2000b. Bcl-2 mediated suppression of apoptosis in myeloma NS0

cultures. J. Biotechnol. 79, 147–159.

Tinto ´ , A., Gabernet, C., Vives, J., Prats, E., Cairo ´ , J.J., Cornudella, L.,

Go ` dia, F., 2002. The protection of hybridoma cells from apoptosis

by caspase inhibition allows culture recovery when exposed to non-

inducing conditions. J. Biotechnol. 95, 205–214.

Tsujimoto, Y., Gorham, J., Cossman, J., Jaffe, E., Croce, C.M., 1985.The t(14;18) chromosome translocations involved in B-cell neo-

plasms result from mistakes in VDJ joining. Science 229, 1390–1393.

Vaux, D.L., Strasser, A., 1996. The molecular biology of apoptosis.

Proc. Natl. Acad. Sci. USA 93, 2239–2244.

Vaux, D.L., Cory, S., Adams, J.M., 1988. Bcl-2 gene promotes

haemapoietic cell survival and cooperates with c-myc to immorta-

lize pre-B-cells. Nature 335, 440–442.

Vives, J., Juanola, S., Cairo, J.J., Prats, E., Cornudella, L., Godia, F.,

2003. Protective effect of viral homologues of bcl-2 on hybridoma cells

under apoptosis-inducing conditions. Biotechnol. Prog. 19, 84–89.

Vomastek, T., Franek, F., 1993. Kinetics of development of

spontaneous apoptosis in B cell hybridoma cultures. Immunol.

Lett. 35, 19–24.

Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng,

T., Jones, D.P., Wang, X., 1997. Prevention of apoptosis by Bcl-2:

release of cytochrome c from mitochondria blocked. Science 275,

1129–1136.

ARTICLE IN PRESS


Apoptosis Me

Documents

Transcript of Apoptosis Me