(Received for publication, July 13, 1995; and in revised form, December 14, 1995)
From the
In many cell types, position in the cell cycle appears to play a
role in determining susceptibility to apoptosis (programmed cell
death), and expression of various cyclins and activation of
cyclin-dependent kinases (CDKs) have been shown to correlate with the
onset of apoptosis in a number of experimental systems. To assess the
role of CDK-mediated cell cycle events in apoptosis, we have expressed CDK dominant negative mutants in human HeLa cells. Dominant
negative mutants of CDC2, CDK2, and CDK3 each suppressed apoptosis induced by both staurosporine and tumor
necrosis factor , whereas a dominant negative mutant of CDK5 was without effect. Like CDC2 and CDK2, CDK3 was shown to form a
complex with cyclin A in vivo. CDK5 did not bind cyclin A to
any detectable extent. Overexpression of wild type CDC2, CDK2, CDK3, or cyclin A (but not cyclin B) markedly
elevated the incidence of apoptosis in BCL-2
cells, which otherwise fail to respond to these agents. These
results help identify cell cycle events that are also important for
efficient apoptosis.
A number of studies suggest that apoptosis is linked with cell
cycle events(1) . Within the organism, apoptosis is found
primarily in proliferating tissues (2, 3) and is
associated with induction of proliferation-associated
genes(4, 5, 6) . Quiescent fibroblasts are
resistant to cytotoxic lymphocyte-induced apoptosis(7) , and
resting peripheral blood T cells are resistant to activation-induced
death by ligation of the T cell receptor(8) . Blocking
proliferation with c-myc antisense oligonucleotides blocks
activation-induced death of T cells(9) , and E1A mutants that are defective in induction of DNA synthesis also fail
to induce apoptosis in susceptible cells(10) . Cells undergoing
apoptosis frequently seem to do so from late in G or early
in S phase whether the stimulus is ligation of antigen
receptors(11, 12) , growth factor
deprivation(5, 13, 14) , or restoration of
wild type p53 function(15, 16) . Arrest in G
or early G
suppresses apoptosis in response to a wide
range of
agents(9, 14, 15, 16, 17, 18, 19, 20) ,
whereas arrest late in G
or in S phase can accelerate or
potentiate apoptosis (15, 16, 18, 19) . These data imply
the existence of molecules present in late G
and S phase
whose activities facilitate the execution of apoptosis.
Cell
proliferation is regulated by cell cycle-specific synthesis of cyclins
and activation of cyclin-dependent kinases (CDKs). ()Response to growth factors and passage through G
is mediated by D-type cyclins, whereas control of entry into S
phase (commencement of DNA replication) is regulated by protein kinases
associated with cyclin E and cyclin A (see reviews in (21) and (22) ). Cyclin D- and cyclin E-associated kinases phosphorylate
pRB, the protein product of the retinoblastoma susceptibility gene,
allowing E2F-dependent transcription of genes whose products are
required for DNA replication (e.g. DNA polymerase
),
whereas cyclin A-associated kinases participate in the phosphorylation
of substrates associated with formation of the replication fork (e.g. replication factor A(21, 22) ). We have
shown previously that arrest of HeLa cells in late G
or
early S phase greatly potentiates the apoptosis-inducing ability of a
variety of agents(19) . Induction of apoptosis was uniformly
associated with activation of cyclin A-dependent kinases but not
activity associated with cyclins E or B(19) . These results
suggested that cyclin A might act as a cell cycle-dependent facilitator
of apoptosis. To define at a molecular level cell cycle events that
modify the ability to undergo apoptosis, wild type and dominant
negative mutants of CDKs (23) were transiently expressed in
HeLa cells prior to exposure to apoptosis-inducing agents. We report
that dominant negative mutants of CDC2, CDK2, and CDK3 suppressed apoptosis in response to both staurosporine
and TNF-
. In addition, overexpression of the wild type form of
these kinases or cyclin A circumvented the anti-apoptosis activity of
the oncogene BCL-2.
Figure 4:
Expression of wild type CDKs and cyclin A
overcomes the inhibitory effect of CDK-dns on TNF--induced
apoptosis. Cells were transfected with the combination of plasmids
indicated and treated and analyzed as described in the legend to Fig. 1. Transfections contained wild type and dominant negative
plasmids at a 2:1 ratio, respectively (see ``Experimental
Procedures''). Transfected cells were identified by
-galactosidase activity, and apoptotic cells were identified by
phase microscopy. The percentage of inhibition of apoptosis was
calculated as [1 - (% of apoptosis in the presence of
CDK-dn/% of apoptosis with lacZ alone)]
100. The
data are the the means ± S.E. for six (cdc2-dn, cdk2-dn, and
cyclin A) and four (cdk3-dn) independent
determinations.
Figure 1:
Inhibition of TNF-- and
staurosporine-induced apoptosis by dominant negative CDK mutants. a and b, HeLa cells co-transfected with lacZ and
an excess of either pCMVcdk2-dn (a) or pCMVcdk5-dn (b). 48 h after transfection, cells were induced to undergo
apoptosis by addition of 100 ng/ml TNF-
and 30 µg/ml
cycloheximide for 3 h, at which time they were fixed and stained with
X-gal for
-galactosidase activity. Note the characteristic
rounded, shriveled appearance of apoptotic cells. Arrowheads identify cells displaying
-galactosidase activity. c and d, HeLa cells were co-transfected with lacZ
and pCMVcdk2-dn and induced to undergo apoptosis 48 h later by
treatment with 500 ng/ml staurosporine and 30 µg/ml cycloheximide
for 5 h and then fixed and stained for indirect immunofluorescence
using an anti-
-galactosidase antibody. c, fluorescent DNA
staining with Hoechst 33258; d, anti-
-galactosidase
immunofluorescence staining of the same cells. Note that the
characteristic apoptotic nuclei are present only in cells not
expressing the transfection marker (arrowheads). e and f, HeLa cells were transfected with pCMVcdk3-dn-HA.
Cells were treated with staurosporine and cycloheximide and stained
with an anti-HA antibody. e, DNA staining with Hoechst 33258; f, anti-HA immunofluorescence staining of the same cells. Note
that the sole apoptotic cell (arrowhead) is the only cell not
expressing cdk3-dn-HA.
Figure 2:
Quantitation of apoptosis in transfected
cells. 300-600 cells prepared as described in Fig. 1were
counted by phase contrast or fluorescence microscopy and scored as
normal or apoptotic on the basis of morphology. The data for
TNF--induced apoptosis are the means ± S.E. for seven
independent determinations; those for staurosporine-induced apoptosis
are for three independent determinations. sham, cells taken
through the transfection procedure in the absence of added
DNA.
Figure 3:
Association of cdk3 with cyclin
A. HeLa cells were transfected with pCMVcdk2, pCMVcdk3, HA
epitope-tagged pCMVcdk2-dn-HA, pCMVcdk3-dn-HA, pCMVcdk5-dn-HA, or
pCMVcdk3-dn-HA in the presence of a 10-fold excess of pCMVcdk3. 48 h
following transfection, cell extracts were immunoprecipitated with
cyclin A antibody(19) . Western blots of SDS-polyacrylamide gel
electrophoresis-resolved immunoprecipitates were probed with anti-HA
antibody and detected with [I]protein A.
HA-immunoreactive species are indicated by the arrowhead.
Figure 5:
Overexpression of wild type CDKs and
cyclin A increases apoptosis in the presence of BCL-2. A HeLa
line (clone HB14)(19) , which stably overexpresses human BCL-2 under control of the SV40 enhancer and
promoter(24) , was transfected with the plasmids indicated and
treated with cycloheximide and TNF- as described in the legend to Fig. 1. Transfected cells were identified by
-galactosidase
activity, and apoptotic cells were identified by phase microscopy (lacZ, cdc2, cdk2, cdk3, and cyclin A). Cyclin B-transfected
cells were visualized by indirect immunofluorescence staining using an
antibody to the myc-epitope tag, with apoptotic cells
identified by Hoechst staining. NT, not transfected (cells
processed in parallel but without LipofectACE treatment). The data are
the means ± S.E. for two (cdk3, cyclin A, and cyclin B) or three (NT, lacZ, cdc2, and cdk2) independent
determinations.
We have shown that three different dominant negative CDK mutants are capable of blocking apoptosis in response to two
general inducers of apoptosis, TNF- and staurosporine. The
inhibitory effect could be overcome by co-expression of cyclin A or by
the wild type form of any of the other three wild type CDKs.
This functional redundancy suggests that the dominant negative mutants
exert their effects via competition for a common activating factor or
factors, a conclusion further supported by the finding that
co-transfection of two or more CDK-dns was no more effective than
single CDK-dns for suppressing apoptosis. It is possible that this
shared activating factor is cyclin A, because all three mutants can
bind this cyclin.
These data are consistent with other published
results suggesting that events downstream of cyclin A-dependent kinase
activation are important for apoptosis. For example, cyclin A is
transcribed in response to two inducers of apoptosis, c-myc and E1A(47, 48, 49, 50) .
Elevated cyclin A expression from an inducible promoter can induce
apoptosis in serum-starved fibroblasts(48) , and cyclin
A-dependent kinases are specifically activated when apoptosis is
induced in T cell lines by human immunodeficiency virus tat(51) and in HeLa cells by a variety of physiological and
pharmacological agents(19) . In target cells killed by granzyme
B, CDC2 activity was increased(52) , including activity
associated with cyclin A. ()Use of a temperature-sensitive CDC2 mutant significantly reduced apoptosis in this
system(52) , and transfection of target cells with wee1, a tyrosine kinase that phosphorylates and inactivates
CDKs, also inhibited apoptosis(53) .
Results from
experiments performed with dominant negative CDK mutants must be
interpreted cautiously. Such mutants may be expected to exert
pleiotropic effects due to their interference with the cell cycle.
Although we have shown that overexpression of cyclin A or its wild type
catalytic partners can drive apoptosis to high levels in BCL-2 cells in the absence of gross cell
cycle perturbations, these data do not demonstrate a direct involvement
of cyclins or CDKs in apoptosis. The biochemical pathways that emanate
from activation of any cyclin-CDK complex are most certainly complex,
wide-ranging, and intricately involved in many different facets of cell
proliferation. However, these findings do identify cell cycle
components whose activity, when modulated, can promote or impede
apoptosis. The existence of such components supports the notion that
specific cell cycle events must be completed before apoptosis can occur
in an efficient manner and suggests that cell cycle regulatory proteins
might be useful therapeutic targets for manipulating apoptosis.