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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 416-424
Granulocyte-Macrophage Colony-Stimulating Factor Rescues TF-1
Leukemia Cells From Ionizing Radiation-Induced Apoptosis Through a
Pathway Mediated by Protein Kinase C
By
Mary L. Kelly,
Yan Tang,
Nitsa Rosensweig,
Sanda Clejan, and
Barbara S. Beckman
From the Interdisciplinary Program in Molecular and Cellular Biology,
Tulane University, New Orleans, LA; the Department of Pharmacology,
Tulane University School of Medicine, New Orleans, LA; and the
Department of Pathology and Laboratory Medicine, Tulane University
Medical Center, New Orleans, LA.
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ABSTRACT |
Protein kinase C (PKC) activity has a recognized role in mediating
apoptosis. However, the role of individual PKC isoforms in apoptosis is
poorly defined. Therefore, we investigated the translocation of
individual PKC isoforms during radiation-induced apoptosis with and
without rescue from apoptosis by granulocyte-macrophage colony-stimulating factor (GM-CSF) in the human erythroleukemia cell
line TF-1. PKC was translocated from the particulate to cytosolic
fraction of TF-1 cells within 5 minutes of treatment with
apoptosis-inducing levels of ionizing radiation. However, this
postirradiation translocation did not occur when cells were rescued
from apoptosis by GM-CSF. Furthermore, treatment of cells with Gö
6976, an inhibitor of classical PKC isoforms, abrogated the rescue
effect of GM-CSF. The calcium-independent novel PKC isoform, PKC
appeared to be degraded in both the particulate and cytosolic fractions
of TF-1 cells after treatment with apoptosis-inducing levels of
ionizing radiation in either the presence or absence of GM-CSF rescue.
Levels of ceramide, a lipid mediator of apoptosis, were measured at 2, 4, 8, 10, and 60 minutes after treatment with ionizing radiation and
were substantially reduced in TF-1 cells rescued from apoptosis by
GM-CSF compared with apoptotic TF-1 cells. The largest decrease in
ceramide production seen was at 4 minutes postirradiation, with a 46%
reduction in ceramide levels in TF-1 cells rescued from apoptosis by
GM-CSF compared with those in apoptotic TF-1 cells. Because ceramide
has been shown to affect PKC subcellular distribution, these data
implicate a role for ceramide in mediating the rapid postirradiation
translocation and inhibition of PKC in TF-1 cells not rescued from
apoptosis by GM-CSF. Expression of the antiapoptotic protein Bcl-2
doubled in TF-1 cells rescued from apoptosis by GM-CSF, but did not
increase in unrescued cells. Our findings suggest that activated PKC
and increased expression of Bcl-2 after  irradiation determine
survival in TF-1 cells rescued from apoptosis with GM-CSF and that
PKC plays a role in mediating signals involved in sensing cellular damage and/or regulation of cell damage repair.
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INTRODUCTION |
PROTEIN KINASE C (PKC) is a family of
serine/threonine kinases with closely related structure and function.
The 12 known members of the PKC family are divided into three
categories on the basis of their activation requirements: the classical
PKCs (cPKC), which are Ca2+-dependent and phorbol
ester-requiring, (the ,  1, 2, and isoforms); the novel
PKCs (nPKC), which are Ca2+-independent, but still require
phorbol ester for optimal activity, (the ,  ,  , and  isoforms); and the atypical PKCs (aPKCs), which are
Ca2+-independent and phorbol ester-independent, but are
designated as PKC based on their kinase activity and sequence homology.
PKC acts as an intracellular mediator in a wide variety of cellular processes including growth factor activation,1 hormonal
response,2 neurotransmission,3 cell cycle
regulation,4-5 proliferation,6-8 differentiation,9-11 and tumor promotion.12
Additionally, expression of PKC isoforms is highly dependent on cell
type and differentiation state.13-16 For these reasons, it
is hypothesized that individual PKC isoforms mediate a variety of
biological effects.
PKC is known to activate c-Ras, c-Raf, and c-Mos, all of which are
associated with radioresistance, thus implicating PKC signaling pathways in radiation-induced cellular responses and tumor
promotion.17,18 Furthermore, PKC-mediated signaling
pathways have been implicated during apoptosis induced by a wide
variety of environmental stimuli including ionizing
radiation.19-26 Additionally, the effect of activating or
inhibiting PKC on cellular sensitivity to apoptosis is cell type and
differentiation state-dependent.27-29 Because PKC isoform
expression is also cell type and differentiation
state-dependent,14-16 it is likely that the cell-specific
nature of the apoptotic response to PKC activation and/or
inhibition is the result of alterations in the expression
and/or activity of individual PKC isoforms.
Recent studies suggest a role for individual PKC isoforms in mediating
apoptosis-associated phenomena: growth factor rescue from
apoptosis,26 ceramide-induced apoptosis,30 and
alterations in Bcl-2 expression.31 To identify a role for
individual PKC isoforms in mediating apoptosis, we investigated the
subcellular distribution and protein levels of PKC isoforms during
ionizing radiation-induced apoptosis and rescue from apoptosis by
growth factor, granulocyte-macrophage colony-stimulating factor
(GM-CSF). We demonstrated that PKC was translocated from the
particulate to cytosolic fraction during apoptosis in the human
leukemia cell line TF-1, and that this translocation did not occur when
cells were rescued from apoptosis by the addition of growth factor. These data identify a role for PKC in control of TF-1 cell survival and/or entry into apoptosis. Our data also identify differences in ceramide levels between TF-1 cells dependent on whether cells were
incubated in the absence or presence of GM-CSF after treatment with
ionizing radiation. Because ceramide has been shown to affect PKC
activity and/or translocation and because the changes in ceramide were concomitant with changes in PKC, these data support an
interaction between ceramide and PKC during the early stages of
apoptosis. Furthermore, our data suggest that PKC might regulate apoptosis by altering Bcl-2 levels, because increased Bcl-2 expression was seen with rescue from apoptosis by GM-CSF. Additionally, PKC appears to be degraded in both the particulate and cytosolic fractions of TF-1 cells after exposure to ionizing radiation in either the presence or absence of GM-CSF rescue at 1 and 6 hours postirradiation. Because PKC was degraded in both apoptotic and GM-CSF rescued TF-1
cells, this suggests a role for PKC in the cellular response to
radiation-induced damage, rather than in direct control of the
apoptotic process.
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MATERIALS AND METHODS |
Cell Culture
TF-1 cells were cultured in RPMI 1640 with 20% fetal bovine serum and
50 U/mL GM-CSF (Sandoz, Houston, TX). Cells were grown at 37°C in a
humidified 5% CO2 incubator.
Protein Determination
Protein of all samples was determined using the Bicinchoninic protein
assay kit from Pierce (Rockford, IL) with bovine serum albumin as the
protein standard.
Preparation of Whole Cell Lysates
A total of 10 × 106 cells were washed once in cold
phosphate-buffered saline (PBS). Cell pellets were suspended in sample
loading buffer (62.5 mol/L Tris-HCl, pH 6.8, 2% [wt/vol] sodium
dodecyl sulfate [SDS], 10% [vol/vol] glycerol, 5% [vol/vol]
-mercaptoethanol, 0.01% [wt/vol] bromophenol blue), and sonicated
for 1 minute on ice.
Preparation of Cellular Extracts
Preparation of cytosolic and particulate fractions for immunoblotting.
Cytosolic and membrane fractions were prepared as
described32-33 with modifications. A total of 15 × 106 cells were washed in cold PBS to remove growth factor
and serum. Cells were resuspended in a hypotonic lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 2 mmol/L EGTA, 1 mmol/L EDTA, 10 mg/mL
leupeptin, 10 mg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride
[PMSF]) and passed 10 times through a 25-gauge needle on ice. The
resultant cell lysate underwent two rounds of low speed centrifugation, first at 1,000g and then at 2,000g, at 4°C for 10 minutes each to remove the nuclear fraction. The supernatant was
centrifuged at 60,000g for 1 hour at 4°C, the pellet of
which comprises the particulate fraction and the supernatant of which
contains the cytosolic fraction. Proteins were extracted from the
particulate fraction by the addition of hypotonic lysis buffer with 1%
Triton X-100 and gentle agitation.
Preparation of nuclear proteins for immunoblotting.
Nuclei were isolated by an adapted method of Neumann et
al.34 A total of 10 × 106 cells were
washed once in cold sucrose-TKM buffer (0.25 mol/L sucrose, 0.05 mol/L
Tris-HCl, pH 7.5, 0.025 mol/L KCl, 0.005 mol/L MgCl2) and
resuspended in sucrose-TKM buffer containing 0.1% (wt/vol) Triton
X-100 and 1.0 mmol/L PMSF. Cells were disrupted by Dounce homogenization on ice and nuclei harvested at 1,000g at
4°C. Nuclei were washed in cold sucrose-TKM buffer and stored at
 70°C. To extract nuclear proteins, nuclei were resuspended
at 2 × 107 nuclei/mL high salt buffer (0.5 mol/L
NaCl, 0.05 mol/L MgCl2, 0.1 mol/L Tris-HCl, pH 7.5, 1.0 mmol/L PMSF, 25 mg/mL DNase, 12.5 mg/mL RNase, 1 mg/mL aprotinin,
2 mg/mL leupeptin), incubated at 37°C for 20 minutes, and
chilled on ice. Extracts were centrifuged at 12,000g at 4°C
to remove nuclear debris and supernatants were stored at
70°C.
Immunoblotting
The samples (50 µg protein) were denatured by boiling in Laemmli
sample buffer for 3 minutes and separated by electrophoresis on a 7.5%
polyacrylamide gel and transferred electrophoretically to a
nitrocellulose membrane. The membrane was blocked with a PBS-Tween
(0.05%) solution with 5% (wt/vol) low fat dry milk at 4°C
overnight. The membrane was incubated with a solution of recombinant rabbit anti-PKC isoform-specific antibody (1:100 dilution; Oxford, Oxford, MI) or goat antihuman Bcl-2 antibody (1:2,000 dilution; generous gift of Dr John C. Reed, Burnham Institute, La Jolla, CA) and
incubated for 2 hours at room temperature (RT). Blots were washed in
PBS-Tween solution and incubated with goat antirabbit antibodies
conjugated to horseradish peroxidase (1:30,000 dilution; Oxford) for 30 minutes at RT. Following four washes with PBS-Tween solution,
immunoreactive proteins were detected using the ECL chemiluminescence
system (Amersham, Arlington Heights, IL) and recorded by fluorography
on Hyperfilm (Amersham), according to the manufacturer's instructions.
In some cases, the fluorograms were quantitated using a BioRAD
(Hercules, CA) GS-670 laser densitometer and the Molecular Analyst
software program (BioRAD).
Cell Irradiation
Cells were irradiated at a rate of 6.4 Gy/minute in a GammaCell
127Ce irradiator. Just before irradiation, TF-1 cells in
mid- to late-logarithmic growth were washed once in PBS and resuspended in normal growth media lacking GM-CSF or containing GM-CSF (100 U/mL)
as indicated. Following irradiation, cells were incubated at 37°C
at a cell density of 2.5 × 105 cells/mL for 6 hours
(for DNA fragmentation analysis) or 48 hours (for cytochemical
detection of apoptosis) unless otherwise indicated.
Electrophoretic Examination of DNA Fragmentation
DNA fragmentation analysis was performed as described35
with modifications. A total of 4 × 106 TF-1 cells
were resuspended in Nicolletti lysis buffer (10 mmol/L Tris-HCl, 100 mmol/L NaCl, 1 mmol/L EDTA, 1% [wt/vol] SDS) to which fresh
proteinase K (1 mg/mL) had been added and incubated overnight at
45°C. The digest was heated to 55°C for 1 hour and nucleic
acids phenol/chloroform extracted. Nucleic acids were subjected to
RNase A (0.15 mg/mL) digestion for 1 to 2 hours at 37°C. DNA was
phenol/chloroform extracted, concentrated with ethanol and separated on
a 2% agarose-TBE gel at 80 V for 2 hours. DNA was stained with
ethidium bromide (1 mg/mL) and visualized on a UV transilluminator.
Cytochemical Detection of Apoptotic Cells
Cells undergoing apoptosis were detected based on the morphologic
changes seen in the nuclear chromatin associated with the apoptotic
process. Nuclei were visualized using the DNA-binding fluorochrome
bisbenzimide (Hoechst 33258; Sigma, St Louis, MO). Cells were stained
as described36 with modifications. A total of 3.0 × 106 cells were harvested at 300g for 10 minutes and
washed one time in PBS. The cell pellet was resuspended in 50 mL 10%
formalin in PBS and incubated for 10 minutes at RT. Fixed cells were
then washed one time in PBS, resuspended in 15 mL of the fluorescent dye bisbenzimide (50 mg/mL in PBS), and incubated for 15 minutes at RT.
Cells were stored at 4°C in the dark until use. Cells were examined
for apoptotic morphology using an Olympus BH-2 (Melville, NY)
fluorescent microscope fitted with appropriate filters using Oncor
(Gaithersburg, MD) visual systems software. Cells displaying nuclear
fragmentation and chromatin condensation with an intact plasma membrane
were considered apoptotic. Significance of differences in the numbers
of apoptotic versus nonapoptotic cells between groups were tested by
 2 analysis.
Measurement of Ceramide 1-Phosphate Levels
Extraction and measurement of ceramide were performed as
described37 by assay of 32P incorporated upon
phosphorylation of ceramide to ceramide 1-P by diacylglycerol (DAG)
kinase. Lipids were extracted by the method of Bligh and
Dyer.38 Lipids were dissolved in chloroform, and samples
corresponding to 200 mg were stored under N2 until the time
of assay. Ceramide stored within the organic phase of the extract was
resuspended in 20 mL of 7.5% -octyl--D-glucopyranoside; 5 mmol/L
cardiolipin; 1 mmol/L diethylenetriamine pentacetic acid (Sigma).
Thereafter, 40 µL of purified DAG kinase in enzyme buffer (dithiothreitol; 1.5 mol/L NaCl, 250 mmol/L sucrose, and 15% glycerol, pH 7.4) was added to the organic phase extract.
[32P]adenosine triphosphate (ATP) (20 µL 10 mmol/L;
1,000 dpm/pmol), in buffer, was added to start the reaction. After 30 minutes at 22°C, the reaction was stopped by extraction of lipids
with 1 mL of chloroform:methanol:hydrochloric acid (100:100:1;
vol/vol). Buffered saline solution (170 mL; 135 mmol/L NaCl; 1.5 mmol/L CaCl2, 0.5 mmol/L MgCl2, 5.6 mmol/L glucose,
and 10 mmol/L HEPES, pH 7.2) and 30 mL of 100 mmol/L EDTA were added.
The lower organic phase was dried under N2. Ceramide 1-P
was resolved by TLC using CHCl3:CH3OH:acetic
acid (65:15:5, vol/vol) as solvent, detected by autoradiography, and
the incorporated 32P was quantified by phosphoimaging (Fugi
BAS1000; Fugi Medical Systems, New Castle, DE). The level
of ceramide 1-phosphate was determined by comparison to a concomitantly
run standard composed of known amounts of ceramide.
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RESULTS |
GM-CSF Suppresses Ionizing Radiation-Induced Apoptosis in TF-1
Cells
Because TF-1 cells can be rescued from growth factor
deprivation-induced apoptosis by the addition of interleukin-3
(IL-3),39 we initially investigated whether TF-1 cells
could be rescued from ionizing radiation-induced apoptosis by the
addition of GM-CSF, another growth factor on which TF-1 cells are
dependent for proliferation. Ionizing radiation induced apoptosis in
TF-1 cells in a dose-dependent manner, as demonstrated by DNA
fragmentation and cytochemical analysis
(Fig 1 and
Table 1, respectively). The addition of
GM-CSF (100 U/mL) to culture medium at the time of irradiation led to significant rescue of TF-1 cells from radiation-induced apoptosis, as
demonstrated by 2 analysis of apoptosis induction
between cells treated with the same dose of radiation in either the
presence or absence of GM-CSF (Table 1). GM-CSF produced significant
rescue of cells from apoptosis even at doses of radiation up to 25 Gy
(P < .0001; Table 1). The concentration of GM-CSF necessary
to rescue cells from apoptosis was twice that used for routine cell
culture. GM-CSF deprivation alone did not produce significant increases
in apoptosis between nonirradiated cells cultured in either the
presence or absence of GM-CSF (P = .1269; Table 1).
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| Fig 1.
Rescue of TF-1 cells from apoptosis with GM-CSF. TF-1
cells were treated with radiation (0, 7.5, 10, 15, or 25 Gy) at a rate of 6.41 Gy/minute in either the presence or absence of GM-CSF (100 U/mL) in complete RPMI supplemented with 20% FBS. Cells were subsequently incubated for 6 hours in complete RPMI supplemented with
20% FBS in either the presence or absence of growth factor. Postincubation, DNA was harvested for DNA fragmentation analysis. DNA
was separated at 80 V for 2 hours on a 2% agarose-TBE gel, stained
with EtBr, and visualized using UV transmission. This is a
representative gel of three experiments.
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Effects of Ionizing Radiation-Induced Apoptosis and Rescue From
Apoptosis on PKC Isoform Expression and Subcellular Distribution
To assess which PKC isoforms might be associated with ionizing
radiation-induced apoptosis and rescue from apoptosis by GM-CSF in TF-1
cells, PKC isoform-specific activity was measured indirectly by
monitoring PKC isoform translocation between cytosolic and particulate
fractions. TF-1 cells predominantly express the , 2, , ,
and  isoforms of PKC, but do not express the 1 or isoforms of
PKC, as determined by immunoblot (Fig 2).
Therefore, subcellular distribution and levels of PKC, 2, ,
, and were determined by immunoblot at 5 minutes, and 1 and 6 hours postirradiation in either the presence or absence of rescuing
levels of GM-CSF. Cells were irradiated with 5 Gy radiation, which
was the lowest dose of radiation tested that resulted in apoptotic
induction and from which TF-1 cells could be rescued from apoptosis by
GM-CSF (Fig 1 and Table 1). While there were no consistent observable changes in the subcellular distribution or levels of PKC2, , or
with irradiation or rescue from radiation-induced apoptosis by
GM-CSF (data not shown), there were reproducible changes in the
subcellular distribution of PKC and in cytosolic and particulate PKC levels.
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| Fig 2.
PKC isoform expression in TF-1 cells. TF-1 whole cell
lysates were analyzed for expression of PKC, 1, 2, , ,
 , and isoforms by immunoblotting. This is a representative blot
of two experiments.
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Under normal culture conditions, ie, in the presence of GM-CSF, PKC
resided predominantly in the particulate fraction
(Fig 3). After treatment of TF-1 cells with
radiation in the absence of GM-CSF rescue, PKC was translocated
from the particulate to cytosolic cell fraction at 5 minutes and was
gradually translocated back to the particulate fraction by 6 hours. The
level of PKC in the cytosolic fraction increased at 5 minutes by
2.05- ± 0.88-fold when compared with unirradiated control cells
incubated in the absence of GM-CSF, as determined by densitometric
analysis. This change was concomitant with a decrease in PKC levels
in the particulate fraction to 78.0% ± 6.1% of control levels at
5 minutes postirradiation. By 6 hours, PKC levels in the cytosolic
and particulate fractions of cells irradiated in the absence of growth
factor returned to levels comparable to those of unirradiated control
cells incubated in the absence of growth factor and harvested at the
same time. The immediate translocation of PKC to the cytosolic
fraction seen in irradiated TF-1 cells incubated in the absence of
GM-CSF was not seen in cells that were irradiated in the presence of GM-CSF (Fig 3). PKC levels remained relatively unchanged in the particulate fractions of TF-1 cells irradiated in the presence of
GM-CSF when compared with control cells, which had not been irradiated,
but were harvested at the same times. These results were consistent for
three immunoblots.
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| Fig 3.
The effect of apoptosis on PKC and distribution in
cytosolic and particulate fractions. TF-1 cells were treated with radiation (5 Gy), incubated in either the presence or absence of GM-CSF
(100 U/mL) in complete RPMI supplemented with 20% FBS, and harvested
at 5 minutes and 1 and 6 hours postirradiation. Nonnuclear cytosolic
and particulate fractions were analyzed for PKC isoform expression
by immunoblotting. This is an autoradiograph of a representative blot
of three showing PKC expression.
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PKC levels decreased in both the cytosolic and particulate fractions
of TF-1 cells irradiated in either the presence or absence of GM-CSF
rescue (Fig 3). By 1 hour postirradiation, PKC levels in the
cytosolic and particulate fractions of TF-1 cells irradiated in the
absence of GM-CSF decreased to 61% ± 2.7% and 71% ± 9.7%, respectively, of those in the same fractions of unirradiated control cells treated otherwise in the same manner, as determined by
densitometric analysis of immunoblots (n = 3). Similar decreases were
seen in the cytosolic and particulate fractions of TF-1 cells
irradiated in the presence of GM-CSF. At 1 hour postirradiation,
cytosolic levels of PKC of TF-1 cells irradiated in the presence of
GM-CSF decreased to 69.0% ± 3.2% of those of control cells and
those in the particulate fraction dropped to 49.0% ± 6.5% of
PKC levels in control cells. PKC levels continued to decrease up
to 6 hours posttreatment in both cellular fractions of cells irradiated
either in the presence or absence of GM-CSF rescue. These changes were consistent for three immunoblots. Similar decreases in PKC levels were seen in nuclear fractions and whole cell lysates of PKC at 1 and 6 hours postirradiation (data not shown).
Effect of Ionizing Radiation Induced-Apoptosis on Ceramide
Production
Because ceramide has been shown to inactivate cellular
PKC,40 ceramide production was examined during ionizing
radiation-induced apoptosis and rescue from apoptosis by GM-CSF.
Ceramide levels in apoptotic TF-1 cells and TF-1 cells rescued from
apoptosis by GM-CSF were expressed as a ratio of control unirradiated
cells incubated in the presence or absence of GM-CSF, respectively
(Fig 4). For all times observed, ceramide
production was reduced in TF-1 cells rescued from apoptosis by GM-CSF
compared with that in apoptotic cells. The largest differences were
seen at 4 minutes postirradiation, with a 46% reduction in ceramide
levels of rescued cells compared with apoptotic cells. At all other
time points observed, the levels of ceramide produced in TF-1 cells
rescued from apoptosis by GM-CSF were 20.8% ± 6.0% lower than
those in apoptotic TF-1 cells. The differences in ceramide production
between cells irradiated in the presence or absence of GM-CSF are
noteworthy because it is thought that ceramide is produced as a direct
product of ionizing radiation-induced membrane lipid peroxidation.
These data imply that ceramide production after irradiation is not a sole consequence of membrane damage and that ceramide production is to
some extent regulated by cellular mechanisms, which may be influenced
by other external factors.
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| Fig 4.
The effect of GM-CSF rescue from apoptosis on ceramide
levels in TF-1 cells. TF-1 cells were treated with radiation (5 Gy) in either the presence or absence of GM-CSF (100 U/mL) in complete RPMI
supplemented with 20% FBS, and harvested at 2, 4, 8, 10, and 60 minutes postirradiation. Extraction and measurement of ceramide was
performed as described37 by assay of 32P
incorporated upon phosphorylation of ceramide to ceramide 1-P by DAG
kinase. Ceramide 1-P was resolved by TLC using
CHCl3:CH3OH:acetic acid (65:15:5, vol/vol) as
solvent, detected by autoradiography and the incorporated
32P was quantified by phosphoimaging (Fugi BAS1000, Fugi
Medical Systems). The level of ceramide was determined by comparison to a concomitantly run standard composed of known amounts of ceramide. Ratios were derived by comparison to ceramide levels of unirradiated TF-1 cells grown in either the presence or absence of GM-CSF. The
values were derived from duplicate experiments and error bars represent
standard deviations into which mean values were incorporated.
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Effect of the Calcium-Dependent PKC Isoform Inhibitor, Gö
6976, on Apoptosis
Because different effects were seen in PKC translocation in both the
presence and absence of GM-CSF rescue from apoptosis (Fig 3), this
suggested that PKC is involved in mediating early apoptotic or cell
survival signaling pathways in TF-1 cells. Because attempts to
genetically alter PKC levels in TF-1 cells were unsuccessful, the
cPKC inhibitor, Gö 6976, was used to further investigate the role
of PKC in mediating ionizing radiation-induced apoptosis and GM-CSF
rescue from apoptosis in TF-1 cells. Gö 6976 is a bisindolemaleimide compound, which has been shown to inhibit PKC and
at nanomolar concentrations.41 In TF-1 cells, Gö
6976 (5 mmol/L) reduced total 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced PKC activity in TF-1 cells by 55.1% as
examined by the method of Mirallia et al42 (data not
shown). The remaining PKC activity seen was most probably due to the
activity of other PKC isoforms expressed by TF-1 cells that were
unaffected by Gö 6976.
TF-1 cells were treated with 5 mmol/L Gö 6976 for 20 minutes
before irradiation and treated subsequently as described in Table 1. In
the absence of Gö 6976 pretreatment, TF-1 cells irradiated at a
dose of 10 Gy were rescued from apoptosis by the addition of GM-CSF to
culture medium at the time of irradiation as demonstrated by
significant differences (P < .0001) in the number of TF-1
cells undergoing apoptosis between cells receiving the same dose of
radiation, but cultured postirradiation in the presence and absence of
GM-CSF (Table 2). Pretreatment of TF-1 cells with Gö 6976 abrogated the GM-CSF rescue phenomenon as indicated by the lack of significant differences in the ratio of
nonapoptotic to apoptotic cells between cells receiving the same dose
of radiation, but cultured postirradiation in the presence and absence
of GM-CSF (P = .6144). Additionally Gö 6976 pretreatment did not significantly alter apoptotic induction in unirradiated TF-1
cells incubated in either the presence or absence of GM-CSF (P = .4543).
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Table 2.
Cytochemical Analysis of the Abrogation of Rescue of
TF-1 Cells From Apoptosis by Treatment With Gö 6976
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Rescue of TF-1 Cells From Apoptosis by GM-CSF Correlates With
Increased Levels of the Antiapoptotic Protein Bcl-2
Because it has been suggested that IL-3 suppression of apoptosis in the
myeloid cell line NFS/N1. H-7 is a result of cPKC-mediated alterations
in Bcl-2 levels31 and because IL-3 suppression of TF-1 cell
apoptosis is mediated through alterations in Bcl-2
levels,39 it is likely that GM-CSF rescue from apoptosis in
TF-1 cells is also modulated by Bcl-2 levels. For that reason, TF-1
cells were examined to see if Bcl-2 levels were altered by GM-CSF
rescue from ionizing radiation-induced apoptosis. GM-CSF-mediated
rescue from apoptosis produced a 1.97 ± 0.34-fold increase in Bcl-2
levels by 24 hours when compared with Bcl-2 levels of unirradiated
control cells, while radiation-induced apoptosis in the absence of
growth factor rescue did not significantly alter Bcl-2 levels (95% ± 6.0% of control levels), as determined by densitometric analysis (Fig 5).
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| Fig 5.
The effect of GM-CSF rescue from apoptosis on Bcl-2
levels in TF-1 cells. TF-1 cells were treated with radiation (5 Gy), incubated in either the presence or absence of GM-CSF (100 U/mL) in complete RPMI supplemented with 20% FBS, and harvested at 24 hours
postirradiation. Whole cell lysates were analyzed for Bcl-2 expression
by immunoblotting. The autoradiograph shows one of two blots assaying
Bcl-2 expression.
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| |
DISCUSSION |
PKC is an important mediator in the control of apoptotic signaling
pathways. These studies show that individual PKC isoforms play an
integral role in apoptosis and rescue from apoptosis by GM-CSF in the
human leukemia cell line, TF-1. Treatment of these cells with
apoptosis-inducing levels of ionizing radiation resulted in alterations
in the subcellular distribution of PKC that were not seen when cells
were rescued from apoptosis by GM-CSF. In particular, PKC resided
predominantly in the particulate fraction of TF-1 cells in both normal
culture conditions and in irradiated TF-1 cells rescued from apoptosis
by the addition of GM-CSF. However, PKC was translocated from the
particulate to the cytosolic fraction of irradiated TF-1 cells in the
absence of GM-CSF rescue (Fig 3). Additionally, PKC levels decreased
in both the particulate and cytosolic fractions of TF-1 cells
irradiated in either the presence or absence of rescuing levels of
GM-CSF (Fig 3). These results are similar to observations in the
IL-6-dependent plasmacytoma cell line, PCT, in which suppression of
IL-6 deprivation-induced apoptosis is mediated through PKC and
.43 PKC was predominantly located in the cytosolic
fraction of apoptotic PCT cells and was translocated to the particulate
fractions when apoptotic PCT cells were treated with rescuing levels of
IL-6. Furthermore, PKC levels decreased in both the cytosolic and
particulate fractions of apoptotic PCT cells. Similarly, confocal
microscopy studies of normal human tonsilar epithelium showed that
cytosolic PKC levels are greater in the apoptotic superficial layer
of epithelium when compared with the nonapoptotic basal and suprabasal
layers of epithelium.44 Others have reported comparable
alterations in the levels and subcellular distribution of PKC
and/or PKC in other cell types.25,45,46
Because it is the current model that PKC is translocated upon
activation from the cytosolic to the particulate fraction and because
PKC was rapidly translocated to the cytosolic fraction after
irradiation in TF-1 cells not rescued from apoptosis by GM-CSF, these
data suggest that PKC is inactivated after irradiation in unrescued
cells and that this inactivation is associated with apoptosis induction
in TF-1 cells. These data further implicate a role for ceramide in
mediating these alterations in PKC distribution during apoptosis and
GM-CSF rescue. Ionizing radiation acts on cell membranes to generate
ceramide and commence apoptosis.35 Several studies show
that ceramide affects PKC activity and/or subcellular
distribution. C2-ceramide blocks bradykinin-induced translocation of PKC to the particulate fraction in the murine epidermal (HEL-37) and human skin fibroblast (SF 3155) cell
lines.47 C2-ceramide treatment causes a
decrease in PKC levels in the particulate fraction in the human
promyelocytic leukemia cell line HL-60.48 Additionally, Lee
et al40 have shown that both C2 and
C6-ceramide inhibit PKC activity in Molt-4 human
leukemia cells by inhibiting basal and induced phosphorylation
(although these studies did not find C6-ceramide-induced
changes in PKC translocation). Therefore, it is likely that the
translocation of PKC to the particulate fraction of TF-1 cells
during the early stages of apoptosis is the result of ceramide-induced
changes in PKC location. It is also likely that this translocation
corresponds to PKC inactivation and that this inactivation is
necessary for apoptosis induction in TF-1 cells. It is therefore our
hypothesis that PKC mediates a long-term survival or proliferative
signal. This hypothesis is supported by the fact that PKC inhibition by pretreatment with Gö 6976 abrogated the GM-CSF rescue
phenomenon (Table 2).
It should be noted that the proposed PKC-mediated survival signal is
probably a function of a signaling pathway distinct from that of the
GM-CSF proliferative pathway. PKC has been shown to mediate the
IL-3/GM-CSF proliferative signal in TF-1 cells49 and in NIH
3T3 cells.50 However, PKC levels and subcellular distribution did not change in apoptotic TF-1 cells or in GM-CSF rescued TF-1 cells (data not shown). That the proposed survival signal
is mediated by PKC, and not PKC, is further supported by our
finding that GM-CSF rescue was blocked by the cPKC isoform inhibitor,
Gö 6976 (Table 2). While others have reported a role for PKC
during apoptosis in other cells types,30,51 our data do not
support such a role in TF-1 cells. Because TF-1 cells express both
PKC and 2 and because Gö 6976 inhibits the activity of both
isoforms, the possibility that PKC2 also plays a role in mediating
GM-CSF rescue cannot be completely ruled out, however, a significant
role for PKC2 in mediating long-term survival appears unlikely. We
did not find alterations in PKC2 subcellular location or levels in
TF-1 cells treated with apoptosis-inducing levels of ionizing radiation
either in the presence or absence of GM-CSF rescue (data not shown).
Additionally, overexpression of PKC did not lead to increased
clonogenic survival or radioresistance in C3H 10 T1/2 cells, despite
the fact that this cell line showed increased PKC expression after
exposure to ionizing radiation.29 Further exclusion of a
role for PKC, or other PKC isoforms, in mediating a cell survival
signal in TF-1 cells awaits successful genetic manipulation of PKC
expression and/or development of a method to monitor or
manipulate PKC isoform-specific activity at the cellular level.
A mechanism by which PKC might mediate long-term survival/growth
factor rescue from apoptosis in TF-1 cells is through alterations in
the levels of the antiapoptosis protein Bcl-2 (and possibly other
members of the Bcl-2 family). This hypothesis is supported by the
observation that Bcl-2 levels decrease in TF-1 cells undergoing apoptosis and in TF-1 cells treated with the PKC inhibitors calphostin and H-7.39 It has been suggested that IL-3 suppression of
apoptosis in NFS/N1 H-7 cells is a result of cPKC-mediated alterations
in Bcl-2 levels.31 Furthermore, the effect of PKC on Bcl-2
levels determines p21ras-mediated entry into pathways for
cell growth or apoptosis in Jurkat cells.52 We have shown
that rescue from apoptosis by GM-CSF in TF-1 cells corresponded with
increased Bcl-2 expression (Fig 4).
Because PKC was degraded in irradiated TF-1 cells in either the
presence or absence of growth factor rescue, our data tend to support a
hypothesis that the changes in PKC levels are a response to irradiation. Immediately after cellular insult and genotoxic stress,
cells must sense the insult, affect cell cycle arrest, and repair DNA
and cellular damage, if possible; or enter apoptosis, if not. PKC
may play a role in one or several of these events. A role for PKC in
cell cycle control has been implicated in proliferation and
differentiation control in the absence of cellular insult in other
types of cells.8,53-56 Additionally, PKC has been shown to
activate the growth inhibitory protein p53.57 However, the
exact role of PKC in cell cycle control is unclear at present.
Sawai et al30 have reported that PKC translocation from
the particulate to cytosolic fraction is essential to ceramide-induced apoptosis in three leukemia cell lines: the HL-60 promyelocyte, U937
monoblast, and HPB-A11 T cell lines. That finding is in contrast to our
data in that, in those studies, PKC translocation was blocked during
rescue by TPA, and we did not see significant differences in PKC
translocation between apoptotic TF-1 cells and TF-1 cells rescued from
apoptosis by GM-CSF addition. However, the feature that is common
between both sets of data is that the removal of PKC from the
particulate fraction is associated with the induction of apoptosis.
Sawai et al30 also reported that PKC and were degraded in cytosolic fractions of leukemia cells when apoptosis was
induced by tumor necrosis factor (TNF) or anti-Fas antibody. Others
have reported PKC proteolysis by an interleukin-1 converting enzyme (ICE)-like protease to an active 40 kD cytosolic
fragment during apoptosis induced by radiation, TNF, or anti-Fas
antibody stimulation in U937 cells.58 Those studies suggest
that PKC may be translocated from the particulate fraction to the
cytosolic fraction, where it is subsequently degraded during apoptosis
by certain types of stimuli. Such an event would appear as a decrease in full size (78 kD) PKC in immunoblots of particulate and cytosolic fractions. Degradation in the cytosol of PKC by an ICE-like protease might explain the our data, however, a lower molecular weight immunoreactive protein was not detected by the methods used here in
either whole cell lysates or the particulate, cytosolic, or nuclear
fractions of apoptotic TF-1 cells.
The results we report offer a possible explanation for the functional
diversity of two PKC isoforms in mediating cellular responses to
ionizing radiation and in apoptosis. These data implicate a possible
role for PKC in cell cycle regulation and/or sensing genotoxic stress. Furthermore, these data imply that PKC mediates a
long-term cell survival signal in TF-1 cells and that this survival signal may counteract the sphingomyelin pathway. Because it has been
suggested that PKC may modulate Bcl-2 expression31,39,52 and because Bcl-2 levels increased in TF-1 cells rescued from apoptosis
with GM-CSF, it is likely that PKC regulates its proposed cell
survival signal through alterations in Bcl-2 expression. While
additional studies are required to confirm this hypothesis, it does
allow us to speculate that therapeutic blockade of this function may
induce apoptosis and/or enhance cell death by radiotherapy.
| |
FOOTNOTES |
Submitted September 24, 1997;
accepted March 2, 1998.
Supported by grants from the Cancer Association of Greater New Orleans
to M.L.K. and Department of Defense, Tulane/Xavier Center for
Bioenvironmental Research Grant to S.C. and B.S.B.
Address reprint requests to Barbara S. Beckman, PhD,
Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Alan Miller (Tulane University, New Orleans, LA)
for his generous gift of TF-1 cells and Dr John C. Reed (Burnham
Institute, La Jolla, CA) for his generous gift of Bcl-2 antibodies.
| |
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Abstract