(Received for publication, July 15, 1996, and in revised form, September 16, 1996)
From the Department of Hematology and Oncology,
Clinical Sciences for Pathological Organs, Graduate School of Medicine,
Kyoto University, 54 Shogoin-kawaramachi, Sakyo-ku, Kyoto 606, Japan,
the § Department of Medicine, Osaka Dental University,
1-5-31 Otemae, Chuo-ku, Osaka 540, Japan, and
Pharmaceutical
Basic Research Laboratories, Japan Tobacco Inc., 13-2 Fukuura, 1-chome
Kanazawa-ku, Yokohama, Kanagawa 236, Japan
Ceramide is now recognized as an intracellular
lipid signal mediator, which induces various kinds of cell functions
including apoptosis. Ceramide-induced apoptosis was reported to be
blocked by 12-O-tetradecanoylphorbol 13-acetate, a protein
kinase C (PKC) activator, but its mechanism remained unclear.
Therefore, we investigated whether ceramide has any effects on PKC in
the induction of apoptosis. We here report that
N-acetylsphingosine (synthetic membrane-permeable ceramide)
induced translocation of PKC- and -
isozymes from the membrane to
the cytosol within 5 min in human leukemia cell lines. Treatment with
sphingomyelinase, tumor necrosis factor-
, or anti-Fas antibody, all
of which can induce apoptosis by generating natural ceramide, similarly
induced cytosolic translocation of PKC-
and -
. In Fas-resistant
cells anti-Fas antibody did not induce cytosolic translocation of
PKC-
and -
because of no generation of ceramide,
whereas N-acetylsphingosine induced apoptosis with cytosolic translocation of PKC-
and -
. Furthermore, both
12-O-tetradecanoylphorbol 13-acetate and a nonspecific
kinase inhibitor, staurosporine, prevented ceramide-induced apoptosis
by inhibiting cytosolic translocation of PKC-
and -
. These data
suggest that cytosolic translocation of PKC-
and -
plays an
important role in ceramide-mediated apoptosis.
Sphingolipids have recently emerged as intracellular signal
mediators in a variety of cell functions (1). Hannun et al. (2, 3) reported that sphingosine, the backbone of sphingolipids, and
lysosphingolipids inhibit PKC1 in
vitro and in vivo. Thereafter, the "sphingomyelin
cycle," transient hydrolysis of sphingomyelin and concomitant
generation of ceramide, was discovered in the early phase of monocytic
differentiation of human leukemia HL-60 cells induced by
1,25-dihydroxy-vitamin D3 (4, 5). Many reports have
supported the idea that ceramide is an intracellular signal mediator
transducing the effects of various extracellular stimulants including
tumor necrosis factor-
(TNF-
) (6, 7),
-interferon (6),
interleukin-1 (8), and nerve growth factor (9). Recent studies have
shown that ceramide plays an important role in apoptosis (10). Besides TNF-
, apoptosis-inducing stimuli such as cross-linking of Fas (11,
12), ionizing radiation (13), glucocorticoid (14), anti-immunoglobulin
antibody (15), anti-cancer drugs (16, 17), and serum deprivation (18)
have been reported to induce the sphingomyelin hydrolysis and/or
generation of ceramide.
PKC is a family of serine/threonine kinases that takes part in various
cellular responses (19). Molecular cloning and biochemical studies have
revealed the presence of at least 10 PKC isozymes that can be
classified into three subgroups. The classical PKC members (,
I,
II, and
) are activated by Ca2+, phosphatidylserine,
and diacylglycerol (DAG) or phorbol esters. The novel PKC members (
,
,
/L, and
), which lack the C2 region, are
activated by phosphatidylserine and DAG or phorbol esters without
Ca2+. The atypical PKC members (
and
), which have
only one cysteine-rich region, are dependent on phosphatidylserine but
are not affected by DAG, phorbol esters, or Ca2+. The role
of different PKC isozymes in cellular functions remains unclear.
It has been reported that ceramide has no effect on PKC activity
in vitro (2), but it remains unclear whether ceramide has
any effect on PKC in vivo. It is known that activation of PKC by DAG or phorbol esters induces the translocation of PKC from the
cytosol to the membrane fraction (20) and inhibits ceramide-induced
apoptosis (10, 18, 21). Although it was recently reported that ceramide
inhibited the membranous translocation of PKC induced by PKC-activating
stimuli (22, 23) and inactivated PKC- (24), the mechanisms by which
PKC activators inhibit ceramide-induced apoptosis are still not known.
We therefore investigated the change of the subcellular distribution of
each PKC isozyme in the apoptosis-inducing process by ceramide and
ceramide-generating signals. We here show that
N-acetylsphingosine (C2-ceramide),
membrane-permeable synthetic ceramide, induced the translocation of
PKC-
and -
from the membrane to the cytosol fraction in three
human leukemia cells (HL-60, U-937, and HPB-ALL cells). Induction of
apoptosis by neutral bacterial sphingomyelinase, TNF-
, or anti-Fas
antibody, all of which generate natural ceramide, induced cytosolic
translocation of PKC-
and -
as well. Furthermore, we show the
following lines of evidence demonstrating how ceramide-induced
cytosolic translocation of PKC-
and -
is indispensable in
leukemic cell apoptosis: 1) HPB-
FR cells are resistant to anti-Fas
antibody-inducing apoptosis because of no generation of ceramide by
anti-Fas antibody and the consequent failure of cytosolic translocation
of PKC-
and -
; 2) ceramide can induce both apoptosis and
cytosolic translocation of PKC-
and -
in HPB-
FR cells; 3)
translocation of PKC-
and -
to the membrane by
12-O-tetradecanoylphorbol 13-acetate (TPA) or
staurosporine inhibits ceramide-induced apoptosis as a consequence of
prevention of cytosolic translocation of PKC-
and -
. These data
suggest that ceramide-induced cytosolic translocation of PKC-
and
-
plays an important role in the signaling pathway leading to
apoptosis. We also discuss the topological meanings of PKC
translocation in apoptotic signals.
D-erythro-C2-ceramide was purchased from Matreya, Inc. D-erythro-N-Acetyldihydrosphingosine (D-erythro-C2-dihydroceramide) was kindly provided by Dr. Y. A. Hannun (Duke University). p-Amidinophenyl methanesulfonyl fluoride hydrochloride was purchased from Wako (Osaka, Japan). Recombinant neutral bacterial sphingomyelinase was purchased from Higeta-Shoyu (Choshi, Japan). Other chemicals were obtained from Sigma.
Cell CultureHuman myelogenous leukemia HL-60 cells, human
monoblastic leukemia U-937 cells, and human T-lymphoblastic leukemia
HPB-ALL cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. HL-60 and
U-937 cells in exponentially growing phase were washed once in RPMI
1640 medium containing 5 µg/ml transferrin and 5 µg/ml insulin
instead of serum, resuspended in the serum-free medium at a
concentration of 5 × 105 cells/ml overnight, and then
used for experiments. HPB-ALL cells were treated in serum-containing
medium. The HPB-FR subline, resistant to anti-Fas antibody, was
obtained by continuous culture of HPB-ALL cells with anti-Fas
antibody.
Subcellular fractionation was performed as described (25, 26) with modifications. The cells were washed once with ice-cold phosphate-buffered saline and lysed in buffer A (20 mM Tris/HCl, pH 7.4, 10 mM EDTA, 5 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 mM p-amidinophenyl methanesulfonyl fluoride hydrochloride, 100 µg/ml leupeptin, 0.15 unit/ml aprotinin) by passing through a 27-gauge needle. Complete cell lysis was confirmed by microscopy. The lysate was centrifuged at 800 × g for 5 min at 4 °C in order to remove nuclei, and the supernatant was centrifuged at 100,000 × g for 20 min at 4 °C in a Beckman TL-100s ultracentrifuge. The supernatant was collected and used as the cytosol fraction. The membrane pellet was solubilized in buffer A containing 1% Triton X-100 by bath sonication and centrifuged at 12,000 × g for 10 min at 4 °C in a microcentrifuge, and the supernatant was used as the membrane fraction. Protein concentration was determined by using the Protein Assay kit (Bio-Rad).
Western Blot AnalysisThe samples (50 µg) were denatured by boiling in Laemmli sample buffer for 5 min, subjected to SDS-polyacrylamide gel electrophoresis using a 7.5% running gel, and electroblotted to Immobilon-P transfer membrane (Millipore Corp.) as described (27). Nonspecific binding was blocked by incubation of the membrane with PBS containing 5% skim milk and 0.1% Tween 20 for more than 1 h. Then the membrane was washed in PBS containing 0.1% Tween 20 (PBS-T) for 15 and 5 min and incubated with a 1:200 dilution of rabbit anti-PKC isozymes (Santa Cruz Biotechnology) in PBS-T for 1 h. The membrane was washed in PBS-T for 15 and 5 min and incubated with a 1:4000 dilution of goat anti-rabbit immunoglobulin peroxidase conjugate (Tago) in PBS-T for 1 h. After washing the membrane for 3 × 5 min in PBS-T, detection of PKC isozymes was performed using ECL Western blotting detection reagents (Amersham Corp.) according to the manufacturer's protocol. Recombinant human PKC isozymes were kindly provided by Dr. D. J. Burns and used as positive controls.
Analysis of DNA FragmentationDNA extraction was performed
using the G NOME DNA isolation kit (BIO 101) according to the
manufacturer's protocol with modification. Briefly, the cells (1 × 106) were washed once in PBS and resuspended in 185 µl
of cell suspension solution. After the addition of 5 µl of RNase Mixx
and 10 µl of cell lysis/denaturing solution, the cell lysate was
incubated at 55 °C for 15 min. Then 3 µl of Protease Mixx was
added to the lysate, and the mixture was further incubated for 2 h. After the addition of 50 µl of Salt Out mixture, the mixture was
cooled on ice for 10 min and centrifuged at 100,000 × g for 10 min at 4 °C in a Beckman TL-100s
ultracentrifuge. Then 1 ml of 80% ethanol diluted with TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) was added to the
supernatant, stored at 20 °C for 2 h, and centrifuged at
12,000 × g for 15 min at 4 °C in a microcentrifuge.
The DNA pellet was dissolved in TE buffer (pH 8.0). The concentration of DNA was calculated by determining the absorbance at 260 nm. Electrophoresis was carried out through 3% NuSieve-agarose (FMC BioProducts) minigel in 1 × TAE buffer (0.04 M Tris
acetate, 0.001 M EDTA) at 50 V for 1.5 h. DNA was
visualized under UV light after staining with ethidium bromide.
Extraction of cellular lipids and ceramide measurement using DAG kinase were performed as described (4). The solvent system to separate ceramide phosphate and phosphatidic acid was chloroform/acetone/methanol/acetic acid/H2O (10:4:3:2:1).
Because
treatment with 10 µM C2-ceramide induced
apoptosis (internucleosomal DNA fragmentation) of human myelogenous
leukemia HL-60 cells within 2 h (data not shown), we examined the
effect of 10 µM C2-ceramide on the
cellular distribution of PKC isozymes within 1 h before the cells
showed apoptotic characteristics. As shown in Fig. 1,
A and B, PKC- and -
, which initially
existed more abundantly in the membrane than in the cytosol fraction, showed translocation from the membrane to the cytosol fraction, reaching a maximum within 2-5 min by treatment with
C2-ceramide. The increased levels of PKC-
and -
in
the cytosol fraction subsequently decreased near the control levels
after 30 min, whereas the levels of PKC-
and -
in the membrane
fraction once recovered after 15 min but decreased after 30 min (Fig.
1B). The reason for the transient nature of cytosolic
translocation of PKC-
and -
may not be due to the metabolism of
ceramide to other inactive compounds because the metabolism of
C2-ceramide is not so rapid in HL-60 cells as we reported
before (5), whereas another report showed the extensive metabolism of
C2-ceramide in Chinese hamster ovary cells (28). Although
it remains to be elucidated why PKC-
and -
disappeared from the
cytosol, one possibility is that PKC-
and -
may be degraded after
translocation to the cytosol by some proteases as mentioned in a recent
report (29). In terms of the brief reappearance of PKC-
and -
in
the membrane, the increase of DAG by treatment with ceramide might be
the cause, but it is at present unclear. The classical PKC (
,
II,
and
) and the atypical PKC-
did not show significant changes in
the subcellular distribution by treatment with C2-ceramide
(Fig. 1A). Further experiments were performed to determine
whether ceramide translocated PKC-
and -
to the nucleus, but no
significant changes of PKC-
and -
were detected in the nuclear
fraction (data not shown).
C2-ceramide induced the cytosolic translocation of PKC-
and -
in a dose-dependent manner (Fig. 1C).
The increase of PKC-
and -
in the cytosol fraction was detected
at the concentration as low as 1 µM
C2-ceramide, which began to inhibit HL-60 cell growth (5).
Because ceramide induced apoptosis in monoblastic leukemia U-937 cells
and T-lymphoblastic leukemia HPB-ALL cells as well (data not shown), we
investigated whether ceramide could induce translocation of PKC-
and
-
to the cytosol fraction in these cell lines. Translocation of
PKC-
and -
from the membrane to the cytosol was also detected in
these cell lines (Fig. 1, D and E), suggesting
that this translocation of PKC-
and -
might be a general
phenomenon in ceramide-induced leukemic cell apoptosis.
C2-dihydroceramide, which has almost the
same chemical structure as C2-ceramide except for lacking
the C4-C5 double bond of sphingoid backbone, did not induce apoptosis
in HL-60 cells in contrast to C2-ceramide (30).
C2-dihydroceramide did not induce significant translocation
of PKC- and -
(Fig. 2A), demonstrating the biological specificity of ceramide effects on the cytosolic translocation of PKC-
and -
. The effects of natural ceramide on translocation of PKC-
and -
to the cytosol were investigated by treating HL-60 cells with exogenous neutral bacterial
sphingomyelinase (SMase). Because SMase purified from bacteria may
contain some other phospholipases, we used recombinant neutral SMase
(Higeta-Shoyu, Japan) (31, 32). The cytosolic translocation of PKC-
and -
was detected 60 min after treatment with 100 milliunits/ml SMase (Fig. 2B), which induced DNA fragmentation within
3 h in HL-60 cells (data not shown). SMase-induced translocation
occurred later and more weakly than C2-ceramide-induced
translocation, presumably due to insufficient action of ceramide
generated in the outer membrane by exogenous SMase. In order to examine
the specificity of SMase action, we checked the effects of other
phospholipases on the subcellular localization of PKC-
and -
(Fig. 2B). Treatment with the phospholipases other than
SMase did not induce apoptosis within at least 6 h in HL-60 cells,
whereas 30-50% of the cells showed apoptotic changes by treatment
with SMase in 6 h. PKC-
in the cytosol fraction was decreased
within 5 min of treatment with phosphatidylinositol-specific
phospholipase C, which generates DAG and inositol trisphosphate from
phosphatidylinositol, and remained under the control until 60 min after
treatment. It was likely that translocation of PKC from the cytosol to
the membrane was induced by phosphatidylinositol-specific phospholipase
C-generated DAG. Neither phospholipase D, which generates phosphatidic
acid mainly from phosphatidylcholine, nor phospholipase A2,
which generates arachidonic acid and lysophosphatidylcholine, induced
significant changes in the subcellular localization of PKC-
. No
significant changes of translocation of PKC-
were detected by
phosphatidylinositol-specific phospholipase C, phospholipase D, or
phospholipase A2. These results suggest that natural
ceramide generated by SMase was specifically involved in translocation
of PKC-
and -
to the cytosol in contrast to other lipid second
messengers including DAG, phosphoinositide, phosphatidic acid, and
arachidonic acid.
TNF-
Generation of ceramide has been reported to be
induced by various stimuli including TNF- (6, 7) and anti-Fas
antibody (11, 12). We investigated whether these biological stimuli translocated PKC-
and -
to the cytosol in the process of
apoptosis. As shown in Fig. 3, TNF-
in U-937 cells
induced cytosolic translocation of PKC-
and -
more evidently than
in HL-60 cells, probably because U-937 cells were more susceptible to
the apoptosis-inducing effect of TNF-
than HL-60 cells (data not
shown). Cytosolic translocation of PKC-
and -
was observed within
5-15 min after treatment with TNF-
in U-937 cells. Because HL-60
and U-937 cells were not very sensitive to anti-Fas antibody (data not
shown), we used HPB-ALL cells, highly sensitive to anti-Fas antibody,
to examine its effect on PKC translocation. Treatment with anti-Fas
antibody induced cytosolic translocation of PKC-
and -
within
15-60 min (Fig. 4A). In order to confirm the
role of translocation to the cytosol in induction of apoptosis, we used
a Fas-resistant HPB-ALL subline (HPB-
FR) in which anti-Fas antibody
hardly induced apoptosis (Fig. 4B) and did not generate
ceramide at least within 3 h, whereas in HPB-ALL cells anti-Fas
antibody induced ceramide generation (Fig. 4C). In HPB-
FR
cells, treatment with anti-Fas antibody did not induce cytosolic
translocation of PKC-
and -
(Fig. 4A), whereas
treatment with C2-ceramide translocated PKC-
and -
from the membrane to the cytosol and subsequently induced apoptosis (Figs. 1E and 4B). These results suggest that
physiological apoptotic stimuli including TNF-
and anti-Fas antibody
may require ceramide-mediated cytosolic translocation of PKC-
and
-
.
Inhibition of Both Ceramide-induced DNA Fragmentation and Cytosolic Translocation of PKC-
Previous reports have shown that phorbol esters
inhibited ceramide-induced apoptosis (10, 18). We confirmed that TPA
inhibited DNA fragmentation induced by C2-ceramide in a
dose-dependent manner in HL-60 cells (Fig.
5A). The cytosolic translocation of PKC
induced by C2-ceramide was also inhibited by treatment with
TPA at the concentrations that blocked ceramide-induced apoptosis (Fig.
5B). Because higher concentrations of TPA induced rapid
down-regulation of PKC-, the amount of PKC-
in the membrane
fraction was decreased within 5 min by treatment with
C2-ceramide and 10 nM TPA and remained down-regulated at least after 30 min. PKC-
was also down-regulated within 15 min of treatment with TPA (data not shown). Conversely TPA-induced translocation of PKC-
and -
from the cytosol to the
membrane was competed by C2-ceramide
dose-dependently (Fig. 5C), and higher
concentrations of C2-ceramide induced apoptosis against
treatment with TPA (Fig. 5D). Furthermore, we investigated the effects of a synthetic DAG analogue,
1,2-dioctanoyl-sn-glycerol (diC8) on the
ceramide-induced translocation of PKC-
and -
to the cytosol,
because DAG is a critical physiological activator of PKC. We found that
diC8 inhibited the translocation of PKC-
and -
to the
cytosol by C2-ceramide as well as TPA did (Fig. 5E). Although it was reported that diC8
inhibited ceramide-induced apoptosis in HL-60 cells (18, 21), in our
hands it did not inhibit ceramide-induced DNA fragmentation, presumably
because of the short duration of action due to its rapid metabolism
compared with TPA and because a higher concentration (20 µM) of diC8 induced DNA fragmentation itself
(data not shown).
Because staurosporine, a nonspecific kinase inhibitor, was reported to
induce the translocation of PKC- from the cytosol to the membrane in
SH-SY5Y human neuroblastoma cells (33), we examined its effect on the
ceramide-induced cytosolic translocation of PKC-
and -
. We found
that staurosporine translocated PKC-
as well as PKC-
in HL-60
cells and inhibited the cytosolic translocation of PKC-
and -
induced by C2-ceramide (Fig. 6A).
Furthermore, we investigated the effect of staurosporine on
ceramide-induced apoptosis and found that ceramide-induced DNA
fragmentation was suppressed by staurosporine (Fig. 6B).
These results demonstrated that ceramide-induced apoptosis and
cytosolic translocation of PKC-
and -
was inhibited by both PKC
activator and non-PKC activator, which translocated PKC-
and -
from the cytosol to the membrane, and therefore membranous
translocation (rather than activation) of PKC-
and -
might be
necessary to inhibit ceramide-induced apoptosis. In other words, it is
suggested that cytosolic translocation of PKC-
and -
plays an
important role in ceramide-mediated apoptosis.
Although it is known that sphingosine or lysosphingolipids inhibit
PKC activity (2, 3), ceramide has been considered to have no direct
effect on PKC. Recent studies have shown that treatment with
C2-ceramide (or sphingomyelinase) inhibits membranous translocation of PKC by PKC-activating stimuli (22, 23) and inactivates
PKC- (24), but there have been no reports that demonstrate the
biological effect of ceramide on PKC in induction of apoptosis. We here
showed that treatment with C2-ceramide translocated PKC-
and -
from the membrane to the cytosol fraction in three different
human leukemia cells (promyelocytic HL-60, monoblastic U-937, and T
cell HPB-ALL cells). Neither treatment with dihydroceramide lacking the
C4-C5 double bond of ceramide structure nor exogenous phospholipases
(PLC, phospholipase D, and phospholipase A2 except SMase)
induced cytosolic translocation of PKC-
and -
, demonstrating the
specificity of ceramide effect on the cytosolic translocation of
PKC-
and -
. Furthermore, treatment with TNF-
or anti-Fas antibody, which induced apoptosis in the consequence of generating ceramide by hydrolysis of sphingomyelin (6, 7, 11, 12), translocated
PKC-
and -
to the cytosol as well as ceramide. These data suggest
that cytosolic translocation of PKC-
and -
may be closely related
to the induction of apoptosis by ceramide and ceramide-generating
stimuli including TNF-
and anti-Fas antibody.
In order to confirm that ceramide and ceramide-generating stimuli
required the translocation of PKC- and -
to the cytosol for
completing apoptotic signals, we investigated 1) whether cytosolic translocation of PKC-
and -
was blocked by the failure of
ceramide generation and 2) whether apoptosis was affected by the
inhibition of ceramide-induced cytosolic translocation of PKC-
and
-
. The results showed that in HPB-
FR cells neither cytosolic
translocation of PKC-
and -
nor apoptosis was induced because
of no generation of ceramide by the anti-Fas antibody and that the
resistance to apoptosis in HPB-
FR cells was overcome by ceramide
treatment accompanied by cytosolic translocation of PKC-
and -
.
We also showed that ceramide-induced apoptosis was suppressed by
competitive inhibition of cytosolic translocation of PKC-
and -
with TPA or a nonspecific kinase inhibitor staurosporine, both of which induced translocation to the membrane of PKC-
and -
. Taken
together, cytosolic translocation of PKC-
and -
seemed to be
indispensable to ceramide-mediated apoptosis in leukemia cells.
It has been reported that phorbol esters and DAG analogues, both
activators of PKC, inhibited apoptosis induced by ceramide or
ceramide-generating stimuli (10, 18, 21). As shown in Fig. 5,
ceramide-induced apoptosis and cytosolic translocation of PKC- and
-
were overcome by TPA, and increasing doses of ceramide induced
apoptosis and cytosolic translocation of PKC-
and -
against TPA.
These data suggested that TPA and ceramide performed competitively in
terms of the translocation of PKC-
and -
between the cytosol and
the membrane. Although diC8 inhibited ceramide-induced
cytosolic translocation of PKC-
and -
, apoptosis was not
suppressed by diC8, probably due to its rapid metabolism and because higher concentrations of diC8 induced apoptosis
with DNA fragmentation. On the other hand, we here investigated the effects of staurosporine on the ceramide-induced cytosolic
translocation of PKC-
and -
because Jalava et al. (33)
showed translocation to the membrane of PKC-
by staurosporine.
Surprisingly, staurosporine suppressed both ceramide-induced cytosolic
translocation of PKC-
and -
and DNA fragmentation. Whereas higher
concentrations of staurosporine (100 nM or more) were
reported to induce apoptosis, conceivably due to nonspecific inhibition
of various kinases (34, 35), at the concentration (3 nM)
used in our experiments staurosporine might inhibit PKC more
effectively than cyclic AMP-dependent kinase or tyrosine
kinase, judging from IC50 values (2.7, 8.2, and 6.4 nM, respectively). These results suggest that translocation
of PKC-
and -
to the membrane may be more strongly related to the inhibition of ceramide-induced apoptosis than to the activation of PKC.
More complex modulation of PKC activity related to its topological
changes may be critical to dissect the signal transduction between
apoptosis and proliferation. In other words, the increase (possibly
activation) of cytosolic PKC-
and -
may be the decisive signal to
ceramide-mediated apoptosis as discussed below.
It therefore remains to be elucidated how PKC- and -
translocated
to the cytosol are involved in the signal transduction pathway leading
to apoptosis. It was reported that overexpression of PKC-
induced
morphological change and growth inhibition by treatment with TPA in
Chinese hamster ovary cells and NIH 3T3 cells, whereas overexpression
of PKC-
increased the growth rate in NIH 3T3 cells and induced
malignant transformation in Rat 6 fibroblasts (36-38). Although these
data suggest that activation of PKC-
may inhibit cell growth or cell
cycle progression, whereas that of PKC-
may have the opposite
effects, neither the mechanism of topological changes of PKC between
the cytosol and the membrane nor the relation between the activity of
each PKC isozyme and the induction of apoptosis is known. Recently,
proteolytic activation of PKC-
to a cytosolic 40-kDa fragment in
apoptotic cells treated with radiation, TNF-
, or anti-Fas antibody
was reported (29). These findings may be a clue to clarifying the
biological meanings of ceramide-induced cytosolic translocation of
PKC-
and -
in induction of apoptosis, because we found that
treatment with TNF-
or anti-Fas antibody degraded not only PKC-
but also PKC-
in the cytosol fraction.2
These suggest that ceramide-induced cytosolic translocation of PKC-
and -
may be a prerequisite for their proteolytic activation in
TNF-
or anti-Fas antibody-induced apoptosis. The more precise implications of ceramide-induced cytosolic translocation of PKC-
and
-
in apoptosis will be defined by the further biochemical and
biological investigations on the relation between cytosolic translocation and the activation of PKC-
and -
consequent to their degradation in the cytosol.