Mol. Cells, Vol. 16, No. 3, pp. 331-337
M olecules
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Cells
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KSMCB 2003
Induction of Apoptosis and Expression of Cell Cycle Regulatory
Proteins in Response to a Phytosphingosine Derivative in
HaCaT Human Keratinocyte Cells
Hye Jung Kim†, Ho Jin Kim1,†, Sung Cil Lim2, Sang Hoon Kim1,*, and Tae-Yoon Kim*
Department of Dermato-Immunology, The Catholic University of Korea, Seoul 137-701, Korea;
1
Department of Biology, Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Korea;
2
College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea.
(Received June 25, 2003; Accepted July 21, 2003)
Ceramide, a compound derived from sphingomyelin, a
sphingolipid precursor, affects cell functions such as
growth, differentiation, cell division and apoptosis. We
have shown that the phytosphingosine derivative, tetraacetyl phytosphingosine (TAPS), inhibits the growth of
HaCaT cells mainly by inducing apoptosis. In this study,
we investigated its effect on the cell cycle and on cell
cycle regulatory proteins. We showed by flow cytometry
and staining for BrdU and phosphorylated histone H3
that the cells accumulated in S phase and arrested in G2
phase and did not divide before undergoing apoptosis.
The level of the pro-apoptotic regulator Bax peaked
after 6 h and then returned to normal, whereas the level
of the anti-apoptotic regulator Bcl-xL, which is presumably induced in order to inhibit apoptosis, started
to increase at 6 h, and remained high for 24 h. Phosphorylation of Cdc2 on Tyr-15 greatly increased while
p21 rose to a plateau at 8 h. Levels of p53 and Mad2
proteins were unchanged. Our observations suggest that
TAPS induces apoptosis of the HaCaT cells at least in
part via transient G2 arrest.
Keywords: Apoptosis; Bax; Bcl-XL; G2 Phase; Mad2;
p21; TAPS.
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Introduction
Apoptosis plays a significant role in embryonic development, tissue homeostasis and neurodegeneration (Cohen,
†
1993; Gehri et al., 1996; Jacobson et al., 1997; Kerr et al.,
1994; Kusiak et al., 1996). Inappropriate apoptosis is involved in many disorders, including AIDS, cancer, autoimmune diseases and Alzheimer’s disease (Barr and
Tomei, 1994; Carson and Ribeiro, 1993; Kusiak et al.,
1996). Therefore, it is important to understand its molecular mechanisms.
Sphingolipid metabolites such as ceramides and sphingosines can influence cellular activities, and may act as
second messengers in some signaling pathways (Choo et
al., 2001; Merrill et al., 1997). Ceramides regulate cell
differentiation, cell cycle arrest, proliferation and apoptosis (Mathias et al., 1998; Perry and Hannun, 1998).
Sphingosine also induces apoptosis and growth inhibition
in various cell types as an intracellular signal mediator
(Spiegel and Merrill, 1996). Naturally occurring ceramides consist of a long-chain sphingoid base with an amide-linked fatty acid substituent (typically with acyl chain
lengths of 16−24 carbon atoms). Phytosphingosine (PS) is
abundant in fungi and plants (Dickson, 1998) and is present in animals including humans (Schurer et al., 1991).
Both phytosphingosine and N-acetyl phytosphingosine
(NAPS) are involved in the heat stress response of Saccharomyces cerevisiae (Jenkins et al., 1997; Wells, 1998),
and cause the death of CHO cells (Lee et al., 2001b).
However, little is known about their cellular functions.
Ceramide induces cell cycle arrest and apoptosis in
many cell lines and its downstream target is thought to be
the Rb gene product (Dbaibo et al., 1995; McConkey et
al., 1996). It induces hypophosphorylation of Rb leading
to arrest in G1. Exposure of human diploid fibroblasts and
These first two authors contributed equally to this work.
* To whom correspondence should be addressed.
Tel: 82-2-590-2626; Fax: 82-2-3482-8261
E-mail: [email protected]/ [email protected]
Abbreviations: BrdU, 5-bromo-2′-deoxy-uridine; PBS, phosphatebuffered saline; PI, propidium iodide; TAPS, tetraacetyl phytosphingosine; TUNEL, TdT-mediated dUTP nick end labeling.
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Induction of Apoptosis by TAPS
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vascular endothelial cells to ceramide results in G1 phase
arrest after downregulation of cyclin A (Lee et al., 2000;
Spyridopoulos et al., 2001). However, in NIH-3T3 cells,
ceramide causes the accumulation of cells in G2/M phase
(Rani et al., 1995). The G2/M transition is mediated by a
signaling cascade regulating phosphorylation of the cyclin
B/Cdc2 complex.
Extracellular agents also inhibit division of human
keratinocytes and induce apoptosis. For example, ultraviolet B induces G1 or G2/M arrest of immortalized skin
keratinocytes by downregulating the cyclinD-CDK4 complex or inactivating the cyclin B-Cdc2 complex (Athar et
al., 2000; Kim et al., 2002). Ceramide, tumor necrosis
factor α and 1α, 25-dihydroxyvitamin D3 induce apoptosis of HaCaT keratinocytes (Muller-Wieprecht et al.,
2000), and an apoptosis signal initiated by ceramide may
play a role in proliferative epidermal disorders (Geilen et
al., 1997; Nardo et al., 2000).
In a previous study, we showed that the phytosphingosine derivatives N-acetyl phytosphingosine (NAPS) and
tetraacetyl phytosphingosine (TAPS) were cytotoxic for
HaCaT human keratinocyte cells (Kim et al., 2003). In the
present study, we investigated a potential effect of TAPS
on cell cycle progression and apoptosis in HaCaT cells.
Materials and Methods
Materials Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS) and antibiotics (penicillin/streptomycin)
were obtained from Gibco-BRL (Rockville, MD). 5-Bromo-2′deoxyuridine (BrdU) was obtained from Boehringer Mannheim
(Mannheim, Germany). Anti-Bcl-xL (#2762), anti-Bax (#2772),
and anti-tyrosine 15 phosphate antibodies that recognize phospho-cdc2, were obtained from Cell Signaling Technology (USA).
Anti-p21 was obtained from Upstate Biotechnology (Lake
Placid, USA), and anti-β-tubulin and anti-p53 were purchased
from Santa Cruz Biotechnology (Santa Cruz, USA). Anti-mad2
antibody was obtained from Covance (USA), and FITCconjugated anti-BrdU was obtained from Becton Dickinson.
RNaseA and propidium iodide were from Sigma (USA), antiphosphorylated histone H3 rabbit antibody was from Upstate
Biotechnology (USA), and the phytosphingosine derivative,
tetra-acetyl phytosphingosine (TAPS) was from Doosan Biotech
(Korea) (Fig. 1).
Cell culture The differentiated human keratinocyte cell line,
HaCaT, was kindly provided by Professor N. Fuseng (German
Cancer Research, Germany). The cells were maintained as
monolayer cultures at 37°C in DMEM supplemented with 10%
FBS, 100 units/ml penicillin and 100 µg/ml streptomycin in a
humidified atmosphere with 5% CO2. They were seeded at 1 ×
106 cells/100-mm dishes. After 48 h, they were washed with
serum-free medium and incubated in medium without FBS for
12 h prior to TAPS treatment.
Fig. 1. The structure of TAPS.
Cell viability The cell viability was determined colorimetrically
using the MTT reagent. The MTT assay was performed as previously described (Wilson and Spier, 1987). Cell proliferation
was measured by using trypan blue exclusion method. After
drug treatment, supernatant and adherent cells were collected
and incubated with 0.4% trypan blue in phosphate-buffered
saline (PBS) for 5 min at 37°C and the number of stained (nonviable) and unstained (viable) cells were counted. Cell viability
was expressed as the percent ratio of unstained cells vs. the total
number of cells.
Western blot analysis Cells were washed in PBS, and suspended
in lysis buffer containing 50 mM Tris pH 6.8, 100 mM DTT, 2%
SDS, 0.1% bromophenol blue, and 10% glycerol. The lysates
were resolved on 12% SDS-polyacrylamide gels and transferred
to nitrocellulose membranes (PROTRAN, Schleicher & Schuell
Co., Germany), which were first incubated with primary antibodies then with horseradish peroxidase-conjugated secondary
antibodies (Santa Cruz Biotechnology, USA). The blots were
developed by the enhanced chemoluminescence detection
method (Santa Cruz Biotechnology, USA).
Flow cytometry Cells were pelleted at 1500 rpm and washed
once with 10 ml of ice-cold phosphate-buffered saline (PBS).
The resulting pellets were resuspended in 1 ml of cold PBS, and
ethanol (80%), pre-chilled at –20°C, was added with periodic
vortexing. The resulting mixture was kept on ice for 60 min, and
the cells were permeabilized in 0.5% Triton X-100, 230 µg/ml
RNaseA and 50 µg/ml propidium iodide in PBS. The samples
were kept at 37°C for 30 min followed by flow cytometric
analysis (Becton Dickinson FACScan) using the CellQuest program. Cells with a DNA content less than the G0/G1 amount of
the untreated cells were considered to be apoptotic.
Analysis of BrdU pulse-chase experiments Cells were pulselabeled with 30 µM 5-bromo-2′-deoxyuridine (BrdU) for 30 min
and chased for 18 h with BrdU-free medium in the presence of
TAPS, then harvested and fixed in 70% ethanol. The fixed cells
were washed with PBS and exposed to 2 N HCl for 30 min at
room temperature to denature the DNA. They were then neutralized with 0.1 M sodium tetraborate (pH 8.5), washed and incubated with FITC-conjugated anti-BrdU antibody for 30 min.
Their DNA was stained with 5 µg/ml of propidium iodide (PI),
and green (FITC) and red (PI) fluorescence in response to 488
nm laser excitation was recorded in a FACScan (Becton Dickson) and plotted as a two-parameter histogram.
Staining of phosphorylated histone H3 Cells were harvested
and fixed in 70% ethanol, then washed and incubated with anti-
Hye Jung Kim et al.
333
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A
B
A
B
Fig. 2. TAPS is toxic to cells. Viability was determined by the
MTT (A) and trypan blue exclusion (B) assays. A. HaCaT cells
were incubated with 1, 3, 10 or 30 µM of TAPS for 24 h. Each
data point was measured in triplicate. B. HaCaT cells incubated
with 30 µM TAPS for the indicated times. Data are representative of three independent experiments and error bars represent
SEM (* p < 0.05).
phosphorylated histone H3 rabbit antibody followed by FITCconjugated goat anti-rabbit immunoglobulin G antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:30 in PBS. DNA was stained with PI, and green and red
fluorescence determined by flow cytometry.
Indirect immunofluorescence Cells were placed on glass coverslips. After TAPS treatment, they were washed and fixed in
5% paraformaldehyde (Sigma, USA) in PBS. They were then
permeabilized with 0.1% NP-40 (Calbiochem, USA) in PBS for
15 min, and blocked for 30 min with 10% serum in PBS. After
exposure to 10 µg/ml primary anti-phosphorylated-histone H3
antibody for 3 h they were incubated for 30 min with FITCconjugated goat anti-rabbit IgG (Zymed Laboratories Inc., USA),
then stained with DAPI and mounted in Gel/Mount (Biomeda
Corp. USA). Positive cells were scored with a fluorescence microscope.
Statistical analysis Data are expressed as means ± SEM where
applicable. Differences between groups were analyzed using an
unpaired 2-sided t-test. All experiments were repeated at least
three times.
Results
Inhibition of cell proliferation by TAPS We examined
the effect of TAPS on the viability of HaCaT cells by
means of MTT (Fig. 2A) and trypan blue exclusion assays
(Fig. 2B). Incubation of the HaCaT cells in the presence
of 1−30 µM TAPS for 24 h reduced cell viability in a
concentration-dependent manner. These results confirm
that it is highly cytotoxic.
TAPS-induced apoptosis occurs via caspase activation
In a previous study we showed that TAPS was more cytotoxic than C2-ceramide, and the TUNEL assay demon-
Fig. 3. A. Immunodetection of cleaved caspase-3 (active form)
in TAPS-treated cells. Thirty µg of total protein were electrophoresed on 12% polyacrylamide gels and immunoblotted with
anti-cleaved caspase-3 (cpp20) polyclonal antibodies. The results were confirmed in three independent experiments. B. HaCaT cells were treated with 30 µM TAPS with or without
zVAD-fmk (100 µM) for 24 h. After treatment, the cells were
harvested and stained with propidium iodide, and their cell cycle
distribution analyzed by FACS. The M1 cell population represents the apoptotic cells in each sample. The data are means of
from three independent experiments.
strated that it caused apoptosis (Kim et al., 2003). We
confirmed the induction of apoptosis by TAPS by Western
blot analysis measuring the production of the cleaved
form of caspase-3 (CPP20) (Fig. 3A). In order to show
that TAPS induces apoptosis via a caspase pathway, cells
were exposed to 30 µM TAPS together with the general
caspase inhibitor, zVAD-fmk. Figure 3B shows that apoptosis was efficiently blocked by the caspase inhibitor.
TAPS causes accumulation of HaCaT cells in S phase
Cells were briefly pulse-labeled with BrdU, TAPS was
added and samples were harvested at the indicated times
for flow cytometric analysis. The uptake of BrdU identifies cells that were in S phase at the time of TAPS treatment. After 18 h of chase, the majority of the BrdUlabeled control cells were in G1 and G2/M, whereas the
BrdU-labeled TAPS treated cells remained in S phase
(Figs. 4A and 4B).
TAPS induces transient G2 arrest preceding apoptosis
On the basis of previous results, the apoptotic cells induced by TAPS may have escaped from G2/M phase due
to collapsing G2/M phase following the 18 h TAPS treatment.
In order to distinguish cell cycle exit within G2/M
phase entering apoptosis in the TAPS treated HaCaT cells,
we performed immunofluorescence analysis with antiphosphorylated histone H3.
Three-fold fewer histone H3-phosphoryated cells were
found in the TAPS-treated culture than in the control cells
(Fig. 5), and the TAPS-treated cells had fewer condensed
chromosomes. Taken together, these results suggest that
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Induction of Apoptosis by TAPS
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A
B
Fig. 4. The distribution of BrdU-positive cells in the TAPStreated HaCaT cells. A. Cells were pulsed-labeled for 30 min
with 30 µM BrdU followed by TAPS, and immediately fixed (0
h) or chased for 18 h in BrdU-free medium (18 h). The population of BrdU-positive cells was measured by flow cytometric
analysis. B. the proportion of BrdU-positive cells in each cell
cycle phase was calculated in the TAPS-treated cells as in (A).
A
B
Fig. 5. Morphological detection and phosphohistone H3 staining
of cells. HaCaT cells were treated with 30 µM of TAPS. A.
Morphological changes in the TAPS-treated cells. B. Phosphohistone H3 positive cells estimated by fluorescence microscopy
and quantified as a percentage (± SEM) of total cells (* p <
0.001). Scale bars, 20 µm.
A
HaCaT cells undergo caspase-dependent apoptosis after
exposure to TAPS in part due to transient G2 arrest.
Expression of cell cycle regulatory proteins in the
TAPS-treated HaCaT cells The Bcl-2 family of proteins,
the anti-apoptotic proteins Bcl-2 and Bcl-xL, and the proapoptotic proteins Bax, regulate apoptosis by controlling
mitochondrial permeability. A variety of stimuli regulate
apoptosis by affecting the expression of Bcl-2 and Bax, or
the Bcl-2/Bax ratio. Gajate et al. (2000) reported that BclxL blocked the signaling pathway between G2-M arrest
and triggering of apoptosis. To test whether TAPS affects
the expression of Bcl-xL and Bax, we performed a Western blot analysis. As shown in Fig. 6A, HaCaT cells expressed only a low level of the pro-apoptotic Bax, and this
increased slightly from 3 to 6 h after TAPS treatment before dropping back to basal level. In contrast, antiapoptotic Bcl-xL increased dramatically between 6 h and
24 h after treatment. Phosphorylation of Cdc2 on Tyr-15
increased substantially indicating that the inactive form of
Cdc2 predominates in the TAPS-treated cells. In addition
the level of p21 increased gradually to a maximum at 8 h
whereas levels of p53 and Mad2 did not change.
In summary, the increased phosphorylation of Cdc2 and
elevated level of p21, together with the cell cycle data
showing accumulation of cells in S phase and the occurrence of G2 arrest preceding induction of apoptosis, suggest that TAPS induces apoptosis in HaCaT cells at least
in part by inducting proteins related to G2 arrest.
Discussion
In our previous study (Kim et al., 2003) the detection of
high levels of cleaved caspase-8 indicated the involvement of the extrinsic pathway in the induction of apoptosis by TAPS. We detected only low levels of the full
length and cleaved forms of caspase-9, indicating that
B
Fig. 6. Expression of cell cycle regulatory proteins in TAPStreated HaCaT cells. A. TAPS alters the ratio Bcl-xL / Bax.
Thirty µg of protein from TAPS-treated HaCaT cells was separated on 12% polyacrylamide gels and an immunoblot analysis
was carried out using anti-Bax and anti-Bcl-XL antibodies. The
results were confirmed in duplicate experiments. B. Cells were
treated with 30 µM of TAPS for the indicated times, proteins
were extracted and the levels of the indicated proteins determined by Western blotting.
caspase-9 has only a minor role in the TAPS-induced
apoptosis. In the present study, we focused on the potential link between cell cycle progression and apoptosis, and
the expression of cell cycle regulatory protein, in response
to TAPS.
The poor expression of Bax confirmed that there is little involvement of the intrinsic pathway in TAPS-induced
apoptosis. As wild-type p53 is known to upregulate Bax
expression (Miyashita et al., 1994), the absence of functional p53 in HaCaT cells could be responsible for the
low level of expression of Bax. The dramatic induction of
the anti-apoptotic Bcl-xL in the late phase of apoptosis
may be part of an adaptive response designed to over-
Hye Jung Kim et al.
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come the strong apoptotic signal initiated by TAPS.
It has been reported that activation of caspases induces
cell cycle arrest (Emanuele et al., 2002; Liu et al., 2003;
Park et al., 2003). Our cell cycle analysis revealed a
marked accumulation of cells in S and G2 phases after
exposure to TAPS. This agrees with a previous report that
ceramide arrests human colon cancer cells in the G2/M
phase and causes accumulation of cells in S phase (Ahn
and Schroeder, 2002). Therefore, we propose that TAPS
affects cell cycle progression in human keratinocytes as
well as tumor cells.
Genotoxic stresses such as UV-irradiation arrest human
keratinocyte in different phases of the cell cycle depending on their p53 status (Athar et al., 2000; Herzinger et al.,
1995). Normal keratinocytes exhibit a p53-dependent delay in G1 whereas HaCaT cells, which are defective in
p53, exhibit G2/M arrest due to rapid inhibition of cyclin
B-associated Cdc2 kinase activity and increased tyrosine
phosphorylation of Cdc2. This probably explains the ability of cells in G2 to progress through the G2-M transition.
Consistent with previous results, we showed that TAPSinduced G2/M arrest in HaCaT cells was associated with
the phosphorylation of Cdc2 protein. p53 induces transcriptional activation of p21 in response to DNA-damaging
agents, and this leads to arrest in G1 or G2/M depending
on cell type (Agarwal et al., 1995; Hunter and Pines,
1991; Sherr and Roberts, 1995). On the other hand, p21
can also be upregulated by a p53-independent pathway
(Kim et al., 2000; Lee et al., 2001a; Michieli et al., 1994).
In the present study, we also found that expression of p21
increased following TAPS treatment. Since HaCaT cells
are homozygous for a defective p53 (Lehman et al., 1993),
expression of p21 must be induced by a p53-independent
pathway. p21 has a broad range of specificity for cell cycle regulatory proteins and is able to inhibit all the cyclinCDK kinases including the cyclinB-Cdc2 complex (Harper
et al., 1995; Polyak et al., 1994). Therefore, it is likely
that the p21 inhibits the kinase activity of Cdc2, and that
this at least partially inhibits the cells from entering M
phase. However, we cannot exclude the possibility that
apoptosis is induced in the S or G1 phases, because the
cell cycle checkpoints in S or G1 phase may still be active.
We will focus future studies on these checkpoints.
Psoriasis, a common skin disease, is caused by hyperproliferation of the epidermal skin layer. The level of endogenous ceramide is very low in psoriasis patients
(Motta et al., 1994; Wertz et al., 1989), and it has been
suggested that this may be implicated in the uncontrolled
cell division. A ceramide-induced apoptosis signal could
play an important role in the mechanism of proliferative
epidermal disorders (Geilen et al., 1997; Nardo et al.,
2000). Therefore, the study of TAPS-induced apoptosis in
human keratinocyte cells may provide useful insights into
treatments for hyperproliferative skin diseases such as
psoriasis.
335
Acknowledgments This work was supported by National Research Laboratory (NRL) Grant (M1-0104-00-0266) from the
Korean Ministry of Science and Technology and by a grant from
the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (HMP-00-PT-21100-0004) to T-Y.K.
This work also was supported in part by research grants from
the Basic Research Program of the Korean Science and Engineering Foundation (R01-2000-000-00089-0) to S.H.K.
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