Apoptosis and Cell Culture Technology
241
proteases play important roles in apoptosis. For example, the overexpression of
the murine Ice gene induces apoptosis in several cell lines [69]. Moreover, the
cowpox virus crmA gene product, a potent inhibitor of ICE, has been shown to
protect cells against apoptosis induced by growth factor removal [70]. Several
other protease inhibitors have been reported to block apoptosis in many cells
types [37]. However, using TPCK (serine protease inhibitor) and BTEE (trypsin
and chymotrypsin-like enzymes inhibitor) we have been unable to obtain any
positive inhibition of either spontaneous or induced apoptosis in hybridoma
cultures. Many members of the ICE family have been recently identified and it
seems that more than one gene is activated to ensure effective cell elimination
(for review see [72]). To what extend the activation of ICE family proteases is
involved in apoptosis is still unknown. Evan et al. [25] asked two questions
which need to be answered to understand the possible mechanism by which ICE
acts: a) How do the genes that suppress or predispose cells to apoptosis and
survival factors regulate the activities of the ICE proteases? b) What are the
consequences of ICE protease activity and what substrates are cleaved and to
what biological end?
Some of the genes involved in the regulation of cell proliferation have been
also implicated in the control of apoptosis. For examples, the p53 tumor
suppressor gene which plays a central role in the regulation of cell proliferation,
has been shown to be required for mediating apoptosis induced in DNAdamaged mice [72]. p53 functions by arresting the cell in G1 phase after DNA
damage, which allows time for repair to proceed. If damage remains unrepaired
the cell will then undergo apoptosis. This action of p53 has been ascribed to its
ability to induce expression of WAF1/CIP1, which encodes an inhibitor of G1
cyclin-dependent kinases [73]. However, p-53 is not involved in the control of
apoptosis during the normal development of mice.
Another gene that stimulate cell proliferation and is involved in apoptosis is
c-myc (see above). Research by Gerard Evan and co-workers indicated that the
c-myc gene product gives the cell two option: proliferate or die but the outcome
depends on external survival signals such as the presence of growth factors (for
review see [25]). In addition, inhibition of c-myc expression by antisense
oligonucleotide in T-cell hybridoma cells inhibits activation-induced apoptosis
[74]. The requirement of c-myc, like that of p-53, is not universal for the
induction of apoptosis. Moreover, the effect of both genes in inducing apoptosis
is cell type and stimuli specific.
4.3 Inhibition of Apoptosis
The other class of genes invovled in the regulation of apoptosis represents those
genes which confer resistance to apoptosis. The best known example of these
'survival genes' is the mammalian protooncogene bcl-2, a functional equivalent
to the ted-9 gene in C. elegans. The action of bcl-2 was demonstrated by cloning
breakpoint in the chromosome translocation, t(14; 18); characteristic of the
241
proteases play important roles in apoptosis. For example, the overexpression of
the murine Ice gene induces apoptosis in several cell lines [69]. Moreover, the
cowpox virus crmA gene product, a potent inhibitor of ICE, has been shown to
protect cells against apoptosis induced by growth factor removal [70]. Several
other protease inhibitors have been reported to block apoptosis in many cells
types [37]. However, using TPCK (serine protease inhibitor) and BTEE (trypsin
and chymotrypsin-like enzymes inhibitor) we have been unable to obtain any
positive inhibition of either spontaneous or induced apoptosis in hybridoma
cultures. Many members of the ICE family have been recently identified and it
seems that more than one gene is activated to ensure effective cell elimination
(for review see [72]). To what extend the activation of ICE family proteases is
involved in apoptosis is still unknown. Evan et al. [25] asked two questions
which need to be answered to understand the possible mechanism by which ICE
acts: a) How do the genes that suppress or predispose cells to apoptosis and
survival factors regulate the activities of the ICE proteases? b) What are the
consequences of ICE protease activity and what substrates are cleaved and to
what biological end?
Some of the genes involved in the regulation of cell proliferation have been
also implicated in the control of apoptosis. For examples, the p53 tumor
suppressor gene which plays a central role in the regulation of cell proliferation,
has been shown to be required for mediating apoptosis induced in DNAdamaged mice [72]. p53 functions by arresting the cell in G1 phase after DNA
damage, which allows time for repair to proceed. If damage remains unrepaired
the cell will then undergo apoptosis. This action of p53 has been ascribed to its
ability to induce expression of WAF1/CIP1, which encodes an inhibitor of G1
cyclin-dependent kinases [73]. However, p-53 is not involved in the control of
apoptosis during the normal development of mice.
Another gene that stimulate cell proliferation and is involved in apoptosis is
c-myc (see above). Research by Gerard Evan and co-workers indicated that the
c-myc gene product gives the cell two option: proliferate or die but the outcome
depends on external survival signals such as the presence of growth factors (for
review see [25]). In addition, inhibition of c-myc expression by antisense
oligonucleotide in T-cell hybridoma cells inhibits activation-induced apoptosis
[74]. The requirement of c-myc, like that of p-53, is not universal for the
induction of apoptosis. Moreover, the effect of both genes in inducing apoptosis
is cell type and stimuli specific.
4.3 Inhibition of Apoptosis
The other class of genes invovled in the regulation of apoptosis represents those
genes which confer resistance to apoptosis. The best known example of these
'survival genes' is the mammalian protooncogene bcl-2, a functional equivalent
to the ted-9 gene in C. elegans. The action of bcl-2 was demonstrated by cloning
breakpoint in the chromosome translocation, t(14; 18); characteristic of the