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Department of Radiation Oncology, University Hospital Zurich, CH-8091 Zurich, Switzerland [S. R., C. G., S. B., M. P.]; Laboratory for Biochemistry, Federal Institute of Technology, 8091 Zurich, Switzerland [S. R., K. W.]; Novartis Pharma Inc., 4002 Basel, Switzerland [D. F.]; and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 [M. S. S., S. W. L.]
Abstract
Caspases are a family of cysteine proteases that constitute the apoptotic cell death machinery. We report the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by the protein kinase C (PKC) inhibitors staurosporine (STP) and PKC-412. In our genetically defined tumor cells, treatment with low doses of STP or the conventional PKC-specific inhibitor PKC-412 in combination with irradiation (5 Gy) potently reduced viability, enhanced mitochondrial cytochrome c release into the cytosol, and specifically stimulated the initiator caspase-9. Whereas treatment with each agent alone had a minimal effect, combined treatment resulted in enhanced caspase-3 activation. This was prevented by broad-range and specific caspase-9 inhibitors and absent in caspase-9-deficient cells. The tumor suppressor p53 was required for apoptosis induction by combined treatment but was dispensable for dose-dependent STP-induced caspase activation. These results demonstrate the requirement for an intact caspase-9 pathway for apoptosis-based radiosensitization by PKC inhibitors and show that STP induces apoptosis independent of p53.
Introduction
Depending on the stimulus that initiates a death program, different caspase cascades, the core of the apoptotic program, are activated (1) . Whereas death ligand-mediated receptor activation facilitates the clustering and autoprocessing of caspases (initiator caspase-8) at the plasma membrane, other stress stimuli activate caspases at intracellular sites (2) . Mitochondrial cytochrome c is released into the cytosol on cellular stress, where, in the presence of ATP/dATP, it associates with Apaf-1, which processes procaspase-9 to its active form (3 , 4) . This, in turn, activates the effector caspase-3, leading to cellular apoptotic morphology.
Induction of DNA double-strand breaks was considered the major mechanism of IR3 -induced cell death. However, more recent studies focus on multiple signal transduction cascades generated at the site of DNA damage and at the plasma membrane, which trigger cells to undergo apoptosis (5) . Apoptosis by IR has been demonstrated in many cell types. Cellular radiation resistance may be linked to the overexpression of antiapoptotic proteins or the loss of apoptosis-inducing proteins like the tumor suppressor protein p53. Radioresistance of tumor cells lacking p53 may be a consequence of a diminished ability to undergo apoptosis in vitro and in vivo (6) . Cellular stresses such as growth factor deprivation, DNA-damaging agents, and irradiation rapidly activate p53 as a transcriptional activator. Various p53-inducible genes are known, but the specific apoptotic signaling network induced by p53 is only now emerging (7, 8, 9) .
To overcome treatment resistance of tumor cells is a formidable task for radiobiology. The use of chemical modifiers as radiosensitizers in combination with low-dose irradiation may increase the therapeutic effect by overcoming a high apoptotic threshold. Here, growth-promoting PKC represents an interesting target. The multigene family of PKC codes for serine/threonine kinases that act as transducers for various lipid second messengers in the regulation, transduction, and propagation of cell-proliferative stimuli. Previous studies have indicated the PKC family to be an interesting target not only for a single treatment modality but also in combination with additional chemotherapeutic agents and IR. The cellular response to stress comprises both proapoptotic and antiapoptotic pathways, and concomitant inhibition of growth-promoting or antiapoptotic pathways promotes cell death. PKC inhibitors such as STP and UCN-01 are potent inducers of apoptosis but also sensitize tumor cells to antimetabolites or cytotoxic agents. On the other hand, PKC stimulation by phorbol esters can rescue different cell types from cell death induced by glucocorticoid and growth factor withdrawal (10 , 11) . Different PKC inhibitors sensitize tumor cells for irradiation (12 , 13) , but mechanistic studies with STP or other PKC inhibitors as inducers of apoptosis in combination with irradiation are scarce.
Here we analyze the execution of apoptosis with the PKC inhibitor STP in combination with IR in genetically defined tumor cells that mimic the genetic conditions of developing tumors. Biochemical analysis of these processes complement the cell biological approach to demonstrate the importance of an intact cytochrome c-dependent caspase-9/-3 pathway for STP-induced radiosensitization and provide a model of treatment resistance.
Results
STP and IR Cooperatively Induce Apoptosis.
We used E1A/ras-transformed MEFs to determine the cytotoxic effect of
agents STP and IR. Quantitative analysis of dead cells was performed
using the trypan blue exclusion assay. Treatment with a low dose of STP
(20 nM) or 5 Gy of irradiation induced minimal cell death
at an early time point (6 h), and even 24 h after treatment, only
15% of the cells were trypan blue positive. On the other hand,
combined treatment with STP and IR induced cell death in 30% of the
cell population at the 6 h time point, and 24 h after
treatment, up to 90% of the total cell population was no longer viable
(Fig. 1A)
. This massive induction of cell death correlated with a
drastic reduction in metabolic activity of the treated cell population
after combined treatment (more than 90% loss of metabolic
activity; Fig. 1B
). Metabolic activity was assessed with the
alamar blue assay, a proliferation assay comparable to the
tetrazolium-based
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay]
quantification of cell metabolism. The comparative results of cell
death and reduced metabolic activity on combined treatment indicate
that combined treatment not only inhibited the proliferative activity
but induced massive cell killing. STP or IR as a single agent alone
only inhibited metabolic activity to 59% and 51%, respectively, in
comparison with untreated cells. The more pronounced reduction of
proliferative activity by STP or IR as a single treatment in
comparison with the minimal effect on cell viability suggests a
possible contribution of cell cycle arrest induced by each of the two
agents (Fig. 1B)
.
|
Characteristic morphological changes indicative of apoptosis
appeared after combined treatment. Cells started to round up 6 h
after treatment and detached as single cells from the surface (data not
shown). To assess the mode of cell death, the effector protease
activity (caspase-3-like/DEVDase activity) induced as part of an
apoptotic process was measured in the cytosolic S-100 fraction 6 h
after the different treatments. Using Ac-DEVD-pNA as a colorimetric
caspase-3 substrate, only minimal DEVDase activity was observed after
treatment with STP (20 nM) or IR (5 Gy) alone, and the
DEVDase activity was not further enhanced at later time points (data
not shown). On the other hand, combined treatment increased
caspase-3-like activity at least 10-fold (Fig. 2A)
. Because an appropriate 
-murine caspase-3 antibody that
recognizes both the zymogen and activated form of caspase-3 was not
available, immunoblotting was carried out with an antibody that only
recognizes active caspase-3. Combined treatment resulted in an increase
of the active large p17 fragment and p19 fragment, corresponding to
enhanced caspase activation. (Fig. 2B)
. In parallel, caspase
activation was detected by cleavage of endogenous PARP. Partial
cleavage of PARP could be detected only after combined treatment with
STP and irradiation (Fig. 2C)
.
|
, compare with Fig. 1A
). Members of the Bcl-2
family prevent activation of the cytochrome c-dependent
caspase cascade (reviewed in Ref. 14
), thereby increasing
the endogenous apoptotic threshold. Using a retroviral construct,
Bcl-2-overexpressing cells were generated, and the cytotoxic effect of
combined treatment and DEVDase activation were tested with the
proliferation assay. Bcl-2-overexpressing cells were up to 4 times more
resistant than control cells (cells infected with an empty vector), and
caspase-3-like activity was drastically reduced compared with control
cells (Fig. 3, B and C)
.
|
–C). These results suggested that the cytochrome
c-activated caspase-9 pathway may be a key element for the
cooperative induction of apoptosis by combined treatment.
|
. Similarly, immunoblotting demonstrated that processing of
caspase-9 into the cleaved form (Mr
35,000–37,000) occurred on combined exposure (Fig. 5B)
,
whereas only the proform (Mr 46,000)
was detectable on single treatment.
|
. Furthermore, overexpression of Bcl-2 also abrogated
caspase-9 activation on treatment with STP and IR (data not shown),
similar to the effect of Bcl-2 on DEVDase activity (see above).
To further explore the relevance of the cytochrome
c-dependent caspase-9 pathway for the cooperative induction
of apoptosis, E1A/ras-transformed MEFs derived from caspase-9 knockout
were treated with STP and irradiation. Again, experiments were
performed with transformed cells obtained from uncloned mass culture to
avoid clonal variations. In the caspase-9-deficient cells, activation
of the effector caspase-3 was assessed by immunoblotting. The
processed large fragment of activated caspase-3
(Mr 17,000–19,000) was not detectable
after treatment with STP and IR alone or in combination, indicating the
requirement for an intact caspase-9 pathway (Fig. 6A
, compare with Fig. 2B). Furthermore,
in comparison to E1A/ras-transformed caspase-9 wild-type cells, the
cytotoxic effect of combined treatment with STP and IR was abolished up
to 75% in these caspase-9-deficient cells (Fig. 6B)
.
|
, ß, and 
PKC subtypes but
has an IC50 that is up to 10x higher than that
for STP (15)
. This clinically relevant STP
derivative is presently being tested as an antitumor agent in clinical
Phase I/II studies. When used as a single agent, PKC-412 (200
nM) only slightly decreased the proliferative
activity of this tumor cell population but also sensitized the cells
for combined treatment with IR. Although the antiproliferative effect
of PKC-412 was present at a later time point than that seen with STP
(48 h versus 24 h), the same initial mechanism of
apoptosis induction was observed but was shifted to a later time
point (18 h versus 6 h). The level of cytochrome
c released into the cytosol was drastically increased on
combined treatment with IR and PKC-412, and caspase-9 was cooperatively
activated (Fig. 7A
–C). These results support the observation that
inhibition of PKC is responsible for STP-based radiosensitization and
is mediated via the mitochondrial death pathway.
|
. Likewise, only a small increase in DEVDase activity was
observed after treatment with STP (20 nM) alone,
and no cooperative effect on the level of DEVDase activity was observed
after combined treatment in the p53-deficient cells (data not shown).
Due to the different response to STP and IR in the p53-deficient cells,
the amount of proliferation and DEVDase activity was assessed after
treatment with increasing concentrations of IR and STP alone. High
doses of irradiation did not alter proliferation 24 h after
treatment or DEVDase activity (data not shown). However, a
dose-dependent reduction in proliferative activity was observed in the
p53-deficient cell population concomitant with increasing DEVDase
activity and processing of the executioner caspase-3 upon treatment
with increasing STP concentrations (Fig. 8
,
B–D). These results suggest that the tumor
suppressor p53 is dispensable for STP-induced apoptosis but
is required for IR-induced apoptosis and the enhanced effect
of STP and IR.
|
We describe the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by PKC inhibitors. Several lines of evidence demonstrate that specific activation of the cytochrome c-regulated caspase-9 cascade is required for the cooperative cytotoxic effect of combined treatment with IR and a low dose of STP or the cPKC-specific derivative N-benzoyl-STP. Combined treatment resulted in mitochondrial release of cytochrome c into the cytosol, enhanced caspase-9 and caspase-3 activation, and cleavage of the endogenous substrate PARP. Treatment with each single agent alone only minimally activated this apoptotic pathway and resulted in only a partial reduction of cell proliferation. The relevance of apoptosis as a mode of cell death induced by combined treatment is corroborated by the specific requirement for an intact caspase-9 pathway and by the fact that pretreatment with broad-range and caspase-9-specific caspase inhibitors reversed the combined cytotoxic effect.
STP is a well known inducer of apoptosis, but at doses 25–100 times higher when applied as a single agent (16) . Irradiation alone can also induce apoptosis in cell lines like thymocytes at this dose level (17) , although we observed only minimal biochemical and morphological signs of apoptosis in the tumor cells used here when irradiation was used as a single agent. However, we cannot exclude that the partial reduction of proliferative activity observed is secondary to low activation of the apoptotic machinery. Thus, combined treatment with a low dose of STP (or PKC-412) and irradiation can cooperatively activate the apoptotic machinery, leading to enhanced cell death. These experiments were performed in E1/ras-transformed MEFs, exploiting genetically defined conditions. Similar results were obtained with rat embryo fibroblasts transformed with the human-related myc/ras oncogenes (data not shown).
Currently, two death pathways are considered to transduce a stress response to the apoptotic machinery. Whereas the clustering of plasma membrane death receptors activates initiator caspase-8, death receptor-independent apoptotic stimuli induce the release of mitochondrial cytochrome c to facilitate activation of initiator caspase-9 in the presence of dATP and Apaf-1 (1 , 2) . Combined treatment specifically activates the cytochrome c-mediated caspase cascade involving initiator caspase-9 and effector caspase-3. Combined treatment with IR and STP, and even more prominent with the cPKC-specific inhibitor N-benzoyl-STP induced already at the level of mitochondrial cytochrome c-release, a supra-additive response, leads to the enhanced activation of this pathway. This cytotoxic effect on combined treatment was reversed by pretreatment with a caspase-9-specific inhibitor or was even absent in caspase-9-deficient cells. Activated caspase-8 can directly stimulate the effector caspases, or it can induce the caspase 9-pathway via cleavage of Bid to induce the release of cytochrome c (18, 19, 20) . Neither initiator caspase-8 nor caspase-1 was activated after combined treatment. Thus, the lack of any caspase-8 stimulation indicates that the death receptor pathway is not involved in this stress response and suggests that an intact caspase-9 pathway is both necessary and sufficient for the apoptotic response to STP in combination with IR. The strict requirement for an intact caspase-9 pathway was confirmed by the lack of response in caspase-9-deficient cells.
Gene knockout studies directed against Apaf-1 and caspase-9
demonstrated the importance of these proteins for cell type- and
tissue-specific apoptosis induced by serum depletion, -irradiation,
etoposide, or hypoxia. Furthermore, these studies put Apaf-1 and
caspase-9 downstream of p53 and cytochrome c release in this
apoptotic signal transduction cascade (9
, 21
, 22)
,
suggesting that cytochrome c release is not always a lethal
event (9)
. p53 induces apoptosis through mitochondrial
cytochrome c release, and a lack of p53 is known to result
in resistance to IR-induced apoptosis (8
, 12)
. In our
otherwise isogenic but p53-deficient E1A/ras-transformed tumor cell
line, no cytochrome c was released into the cytosol, and
neither caspase-9 nor caspase-3 was activated on combined treatment
with PKC inhibitors and IR. Likewise cytochrome c was only
released after treatment with high doses of IR in wild-type p53
transformed cells (data not shown). Thus, these treatment modalities
require intact p53 to initiate the apoptotic machinery induced at the
site of the mitochondria. On the other hand, treatment with a high dose
of STP induced cytochrome c release and activated downstream
caspases even in p53-deficient cells, clearly demonstrating a
p53-independent and dose-dependent mechanism for PKC inhibitor-induced
cytochrome c release. These results suggest that STP might
act upstream of cytochrome c release but downstream of p53.
Thus, mechanistically, a specific combined treatment modality does not
simply correspond to a dose increase of either single agent alone. PKC
inhibitors sensitize tumor cells for IR-induced apoptosis by overcoming
an apoptotic threshold that is too high for a low dose of IR
alone. However, apoptosis induction by combined treatment
requires both an intact apoptotic pathway and wild-type p53,
whereas high doses of PKC inhibitors do not depend on the latter.
PKC-regulated signal transduction cascades cooperate with IR-induced pathways to overcome the apoptotic threshold, leading to cytochrome c-mediated caspase activation. Thus far, we do not understand at which level upstream or at the site of the mitochondria PKC-regulated processes interact with IR-induced signal transduction, leading to synergistic cytochrome c release and caspase-9 activation. Antiapoptotic members of the Bcl-2-family might prevent the release of cytochrome c from mitochondria, whereas some proapoptotic family members such as Bax and Bid induce cytochrome c release (reviewed in Ref. 14 ). Both Bax-dependent and -independent induction of apoptosis by p53 have been demonstrated in vitro (14 , 23) . We could not find any difference in the protein levels of Bax, Bcl-2, or Bcl-xL on treatment in our cells (data not shown), but we cannot exclude that conformational changes of these proteins or of other family members not investigated here modulate the treatment sensitivity of our cells.
Activation of PKC is known to promote cell survival and to protect against cell death. However, the exact role of the multiple PKC isoforms responsible for the growth-promoting effect is far from clear. For example, overexpression of PKC can abrogate stress-induced apoptosis by enhanced activation of the phosphatidylinositol 3'-kinase/Akt pathway (24) and concomitant phosphorylation of the proapoptotic Bcl-2 family member Bad (25 , 26) . Thus, PKC inhibitors might result in decreased Akt activity, thereby affecting the equilibrium of proapoptotic and antiapoptotic members of the Bcl-2-family and down-regulating an apoptotic threshold in tumor cells (12 , 26 , 27) . PKC inhibitor-mediated radiosensitization might also be mechanistically linked to the hydrolysis of sphingomyelin and the generation of the apoptotic second messenger ceramide. Ceramide is increased on irradiation but prevented by PKC activators such as 12-O-tetradecanoylphorbol-13-acetate (5 , 28, 29, 30) . Ceramide can also directly induce the release of cytochrome c from mitochondria (31) . Due to the numerous downstream targets of PKC, it will be important to elucidate which of the major growth-promoting and/or antiapoptotic cellular signal transduction cascades contribute to the STP-induced and the more specific PKC-412-induced radiosensitizing effect. We are currently investigating the level on which IR-induced signal transduction cascades merge with PKC-mediated pathways. This merge can be either upstream or at the level of the mitochondria to cooperatively induce the release of cytochrome c.
Treatment response often depends on the individual oncogenic background, but a disrupted apoptotic pathway could also interfere with treatment success. Combination of different antitumoral treatment modalities is often advantageous to limit unspecific toxicities frequently observed by an exceedingly high single treatment regimen. Here we demonstrate that PKC-mediated radiosensitization may overcome an apoptotic threshold but requires an intact caspase-9 pathway. A detailed molecular understanding of how single and combined treatment modalities induce and execute apoptosis will advance our knowledge of the apoptotic signaling network and will be important for successful treatment.
Materials and Methods
Cell Cultures, Irradiation, and STP/PKC-412 Treatment.
Clonal selected MEFs transformed with the two oncogenes E1A and T24
H-ras (32)
were used at low passage numbers and cultured
in a 5% CO2 atmosphere in DMEM containing 10%
FCS and 10% bovine calf serum (Hyclone Laboratories) supplemented with
penicillin and streptomycin. Uncloned mass cultures of
E1A/ras-transformed wild-type, p53-/-, and caspase-9-/- MEFs were
prepared as described previously (33)
and cultured in DMEM
containing 10% FCS supplemented with penicillin and streptomycin.
Irradiation was carried out at room temperature using a Pantak
Therapax 300 kV X-ray unit at 0.7 Gy/min. STP (Sigma) and PKC-412
(Novartis Pharma AG) were dissolved in DMSO and diluted in DMEM
containing 10% or 20% FCS-412. For combined treatments, cells were
preincubated with STP or PKC for 1 h before irradiation.
Bcl-2-overexpressing Cell Lines.
BOSC23 cell were transfected with a total of 30 µg of pBabe(puro)
plasmid DNA or its derivative (containing mouse bcl-2 cDNA;
Ref.34
) by using the calcium phosphate coprecipitation
method as described previously (35)
. Medium containing the
retrovirus was harvested 24–30 h after removal of the precipitate and
used to infect the E1A/ras-transformed MEFs at 40% confluence.
Puromycin selection (2 µg/ml medium) to obtain stably transfected
cells was initiated 30 h after infection, and pools of
puromycin-resistant cells (uncloned mass cultures) were analyzed for
bcl-2 overexpression with -bcl-2 antibodies (Upstate Biotechnology).
Trypan Blue Viability Assay.
Floating and adherent cells were collected at the indicated times, spun
down, and resuspended in PBS. The cells were then diluted 1:5 with
0.4% trypan blue solution (Sigma) and scored under a light microscope.
The results presented represent the mean plus SD of two independent
experiments, with a minimum of 500 cells scored per treatment.
Proliferation Assay.
Tumor cell proliferation was assessed 24 or 48 h after treatment
with the colorimetric alamar blue assay based on the detection of
metabolic activity (Biosource International, Camarillo, CA). Absorption
was measured at 570 and 600 nm using a Dynatech MR5000
spectrophotometer. Results are expressed as the mean plus SD of at
least two experiments in triplicate.
Caspase Inhibitor Assay.
The broad-range caspase inhibitor Z-VAD-FMK (10–100 µM;
Calbiochem) and the caspase-9-specific inhibitor Z-LEHD-FMK (100–200
µM; Calbiochem) dissolved in culture medium were added to
cells 1 h before treatment. Results are expressed as the mean plus
SD of at least two experiments in triplicate.
Cell Fractionation.
Cells were harvested by centrifugation at 1,800 x g
for 10 min at 4°C and washed with ice-cold PBS. The cell pellet was
suspended in 5 volumes of ice-cold buffer A [20
mM HEPES-KOH (pH 7.5), 10
mM KCl, 1.5 mM
MgCl2, 1 mM sodium EDTA, 1
mM sodium EGTA, 1 mM DTT,
250 mM sucrose, and 0.1 mM
phenylmethylsulfonyl fluoride] supplemented with protease inhibitors
(5 µg/ml pepstatin A, 10 µg/ml leupeptin, and 2 µg/ml aprotinin).
After sitting on ice for 15 min, the cells were disrupted by douncing
15 times in a Dounce homogenizer. Cell lysates were centrifuged at
1,000 x g for 10 min at 4°C (crude nuclear pellet),
and the supernatant was further centrifuged at 100,000 x
g for 1 h. The resulting supernatant (S-100 fraction)
and pellet (mitochondrial fraction) were stored at -80°C.
In Vitro Caspase Assay.
To determine caspase-3-like activity, 50–80 µg of protein from the
S-100 fraction were incubated at 37°C with the colorimetric caspase-3
substrate Ac-DEVD-pNA (100 µM; Calbiochem) and 1
mM dATP in a final volume of 120 µl. Measurements in the
presence of the caspase-3 inhibitor Ac-DEVD-CHO (20 µM;
Calbiochem) served to correct for unspecific background activity. To
determine caspase-1 and -8 activity, the S-100 fraction was incubated
at 37°C with the colorimetric caspase-1 substrate Ac-YVAD-pNA (100
µM; Calbiochem) or the colorimetric caspase-8 substrate
Ac-IETD-pNA (200 µM; Calbiochem), in a final volume of
120 µl. Caspase-9 activity was measured with the R&D Systems
colorimetric caspase-9 assay kit using Ac-LEHD-pNA as a substrate.
Cleavage of the caspase substrates was monitored at 405 nm using a
Dynatech MR5000 spectrophotometer. Results are expressed as the mean
plus SD of at least three experiments performed in duplicate.
Antibodies and Western Blots.
Mouse monoclonal antibody clone 7H8.2C12 recognizing murine cytochrome
c was purchased from Research Diagnostics, Inc. or
PharMingen. Rabbit polyclonal anti-PARP antibody was obtained from
Upstate Biotechnology, rabbit polyclonal anti-caspase-9 antibody was
obtained from PharMingen, and rabbit polyclonal anti-cleaved caspase-3
antibody was obtained from New England Biolabs. Cellular
proteins (100–200 µg) from the different fractions were
precipitated, resolved by SDS-PAGE, and blotted onto
polyvinylidene difluoride membranes. Antibody detection was achieved
using enhanced chemiluminescence (Amersham) and a horseradish
peroxidase-conjugated secondary antibody
according to the manufacturer’s protocol.
Acknowledgments
We thank E. Peterhans for providing us with the Bcl-2 cDNA, W. Krek for preparation of the retroviral constructs, and A. Marti for stimulating discussions.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 S. R. was supported by a predoctoral fellowship
by the Baugarten Stiftung, and S. B. and M. P. were supported by a
grant from the Swiss and Zurich Cancer Leagues. 
2 To whom requests for reprints should be
addressed, at Department of Radiation Oncology, University Hospital
Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. Phone:
41-12558549; Fax: 41-12554435; E-mail: pum{at}dmr.usz.ch
3 The abbreviations used are: IR, ionizing
radiation; PKC, protein kinase C; STP, staurosporine; MEF, mouse embryo
fibroblast; PARP, poly(ADP-ribose) polymerase; pNA,
p-nitroanilide; FMK, fluoromethyl ketone; cPKC,
conventional PKC; DEVDase activity, protease activity for
caspase-3 substrate Ac-DEVD-pNA.
Received for publication 6/15/00. Revision received 7/25/00. Accepted for publication 8/ 3/00.
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) and 24 h (
)
after treatment with STP (20 nM) and IR (5 Gy) alone or in
combination. B, proliferation was assessed with the
alamar blue assay 24 h after treatment with STP (20
nM) and IR (5 Gy) alone or in combination.
) was measured 24 h
after combined treatment with IR (5 Gy) in the presence of increasing
doses of STP (5–20 nM). C, caspase-3-like
activities were measured in Bcl-2-overexpressing cells (
