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MOLECULAR CARCINOGENESIS 37:39–50 (2003)
Molecular Mechanisms of G0/G1 Cell-Cycle
Arrest and Apoptosis Induced by
Terfenadine in Human Cancer Cells
Jean-Dean Liu,1 Ying-Jan Wang,2 Chien-Ho Chen,3 Cheng-Fei Yu,3 Li-Ching Chen,4 Jen-Kun Lin,5
Yu-Chih Liang,1 Shyr-Yi Lin,1 and Yuan-Soon Ho3*
1
Department of Internal Medicine, School of Medicine, Taipei Medical University and Hospital, Taipei, Taiwan
Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan
3
Institute of Biomedical Technology, Taipei Medical University, Taipei, Taiwan
4
Department of Nursing, TzuChi University, Hualien, Taiwan
5
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan
2
Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central
nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant
growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G0/G1
phase cell-cycle arrest. The minimal dose of TF induced significant G0 /G1 arrest in these cells was 1–3 mM. The protein
levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin-dependent
kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF-treated cells. On the other hand, significant
apoptosis, but not G0 /G1 arrest, was induced in the HL 60 (p53-null) or Hep 3B (with deleted p53) cells when treated
with TF (3–5 mM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G0 /G1 arrest
and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and
p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis.
Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF-treated
group was clearly attenuated by pretreatment with p27/Kip1–specific ODNs. These results indicated that p27/Kip1
(but not p21/Cip1) protein indeed played a critical role in the TF-induced apoptosis. We also demonstrated that the
TF-induced G0 /G1 cell-cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least
7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF-treated normal human
fibroblast, even at a dose as high as 25 mM. Our study showed the molecular mechanisms for TF-induced cell growth
inhibition and the occurrence of apoptosis in human cancer cells. ß 2003 Wiley-Liss, Inc.
Key words: apoptosis; G0 /G1 cell-cycle arrest; terfenadine; p53; p21/Cip1; p27/Kip1
INTRODUCTION
The occurrence of gastrointestinal (GI) cancers has
increased strikingly during the last decade. For instance, colorectal cancer is the second leading cause
of cancer mortality in Western societies [1] and one
of the world’s most common malignancies [2,3].
Hepatocellular carcinoma (HCC) is one of the most
frequent malignancies worldwide [4], showing the
highest prevalence in Asia and Africa [5,6]; it is the
second most common form of lethal cancer in China
after gastric cancer [7,8]. The incidence of HCC is
generally low in Western Europe and the United
States. However, following an increase in hepatitis C
virus infection rates, it is now occurring with increasing frequency in the United States [9]. Furthermore,
HCC and colorectal cancer are the second and third
causes of all cancer deaths respectively in Taiwan
[10–13]. Therefore, fighting against GI cancer is an
important global issue. To our knowledge, the ability
of chemotherapeutic agents to inhibit cancer cell
growth and to initiate apoptosis is an important
ß 2003 WILEY-LISS, INC.
determinant of their therapeutic response. Previously, our in vitro and in vivo studies demonstrated that
antifungal agents, including miconazole, ketoconazole (KT), and griseofulvin, exert antitumor effects in
various types of human GI cancers cell lines through
the induction of apoptosis and cell-cycle arrest [14–
17]. In addition, our recent study also demonstrated
*Correspondence to: Graduate Institute of Biomedical Technology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110,
Taiwan.
Received 12 September 2002; Revised 16 January 2003; Accepted
18 March 2003
Abbreviations: GI, gastrointestinal; HCC, hepatocellular carcinoma; KT, ketoconazole; TF, terfenadine; CYP 3A4, cytochrome
p450-3A; ATCC, American Type Culture Collection; FCS, fetal calf
serum; DMSO, dimethyl-sulfoxide; SDS-PAGE, sodium dodesyl
sulfate-polyacrylamide gel electrophoresis; CDK, cyclin-dependent
kinase; ODNs, oligodeoxynucleotides.
DOI 10.1002/mc.10118
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LIU ET AL.
that significant apoptosis is easily induced by terfenadine (TF) in human colon and liver cancer cells
[18].
TF was first reported by Kinsolving et al. in 1975
[19]. It appears to be the first highly potent H1
histamine receptor antagonist that in clinically
effective doses lacks side effects, such as sedation,
impaired psychomotor performance, and excessive
mucosal drying [19–21]. The metabolisms of TF to its
desalkyl and hydroxymetabolites are demonstrated
to be mediated by human liver microsomal enzymes
cytochrome p450-3A (CYP 3A4) isoforms [22,23]. KT,
an antifungal drug, is a potent and selective inhibitor
of the CYP 3A4 enzyme [24,25]. Our recent study
demonstrated that TF-induced apoptosis is significantly potentiated by KT through the inhibition of
CYP 3A4 in the HCC cell line [18]. In this study, we
demonstrated for the first time that TF induced G0/
G1 cell-cycle arrest in human cancer cells, including
Hep G2 (wild type p53), HT 29 (mutated p53), and
COLO 205 (wild type p53) cells. In contrast,
apoptosis, but not G0/G1 arrest, was induced in
HL 60 (p53-null) or Hep 3B (with deleted p53) cells
treated with the same dose of TF. The proteins that
regulate the G0/G1 phase cell cycle and apoptosis
induced by TF were determined in this study. Our
results provided direct evidence that additional
cytotoxic mechanisms (induction of apoptosis and
cell growth inhibition) induced by TF were found in
some types of human cancer cell lines.
MATERIALS AND METHODS
Chemicals
TF was purchased from Sigma Chemical Co. (St.
Louis, MO). The protein assay kit was purchased from
Bio-Rad Co. (Bio-Rad Labs, Hercules, CA).
Cell Lines and Cell Culture
The HT 29 and COLO 205 cell lines were isolated
from human colon adenocarcinoma (American Type
Culture Collection [ATCC, Manassas, VA] HTB-38
and CCL-222) [26]. Hep 3B and Hep G2 cell lines were
derived from human HCC (ATCC HB-8064 and HB8065) [27,28]. The HL 60 cell line was derived from
human myeloid leukemia cells (ATCC 59170). The
cell line CCD-922SK (ATCC CRL 1828) was derived
from normal human fibroblasts. The p53 gene in the
COLO 205, CCD-922SK, and Hep G2 cells was wildtype [29,30]. In contrast, the p53 gene is mutated in
codon 273 in HT 29 cells [31]. The p53 gene was
found to be partially deleted (7 kb) in Hep 3B [28] and
null in HL 60 cells [32]. Cell lines were grown at
378C in 5% carbon dioxide atmosphere in Eagle’s
minimal essential medium for CCD-922SK, Hep 3B,
and Hep G2 cells, and in RPMI 1640 for COLO 205,
HT 29, and HL 60 cell, supplemented with 10% fetal
calf serum (FCS), 50 mg/mL gentamycin, and 0.3 mg/
mL glutamine.
Determination of Cell Growth Curve
Human cancer (1 104) and fibroblast (10 104)
cells were plated in 35-mm Petri dishes. The next
day, the medium was changed and TF (0.1–5 mM)
was added. Control cells were treated with dimethylsulfoxide (DMSO) in a final concentration of
0.05% (v/v). The incubation medium was renewed
every day during the experiment. At the end of
incubation, cells were harvested for cell count with a
hemocytometer.
Cell Synchronization, Drug Treatment,
and Flow Cytometry Analysis
Twenty-four hours after plating of cells, the
medium was removed. Cells were washed three
times with medium alone, and then incubated with
medium containing 0.04% FCS for 24 h. Under such
conditions, cells were arrested in G0/G1, as determined by flow cytometry analysis. The low-serum
medium was removed, and the cells were then
stimulated by the addition of medium containing
10% FCS. TF solutions were prepared by dissolving
this compound in a final concentration of 0.05%
(v/v) DMSO. The stages of cell cycle in the TF- and
mock-treated groups were measured by flow cytometry analysis.
Western Analysis
Treated and untreated cells were rinsed three times
with ice-cold phosphate-buffered saline, then lysed
in 500 mL of freshly prepared extraction buffer
(10 mM Tris-HCl, pH 7; 140 mM sodium chloride;
3 mM magnesium chloride; 0.5% [v/v] NP-40; 2 mM
phenylmethylsulfonyl fluoride; 1% [w/v] aprotinin;
and 5 mM dithiothreitol) for 20 min on ice. The
extracts were centrifuged for 30 min at 10 000 g.
Proteins were loaded at 100 mg/lane on 12.5% [w/v]
sodium dodesyl sulfate (SDS)-polyacrylamide gel,
blotted, and probed with antibodies including cyclin
E (Santa Cruz, Biotechnology, Inc., Santa Cruz, CA),
p53, p21/CIP1, p27/Kip1, cyclin A, cyclin D1, cyclin
D3, PCNA, cyclin B1, cyclin-dependent kinase 2
(CDK2), CDK4, and GAPDH (Transduction Laboratories, Lexington, KY). Immunoreactive bands were
visualized by incubating with the colorigenic substrates, nitro blue tetrazolium, and 5-bromo-4chloro-3-indolyl-phosphate (Sigma).
Immunoprecipitation and CDK Kinase Activity Assay
CDK2-associated histone H1 kinase activity was
determined as described by Wu et al. [33]. Briefly,
with anti-p21/Cip1 antibody (2 mg) and protein A
agarose beads (20 mL), the p21/Cip1-associated CDK2
was precipitated from 200 mg of protein lysate per
sample as described above. Beads were washed three
times with lysis buffer and then once with kinase
assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
and 1 mM DTT). Phosphorylation of histone H1 was
TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS
measured by incubating the beads with 40 mL of
‘‘hot’’ kinase solution (0.25 mL [2.5 mg] of histone
H1, 0.5 mL of [g-32P] ATP, 0.5 mL of 0.1 mM ATP, and
38.75 mL of kinase buffer) for 30 min at 378C. The
reactions were stopped by boiling the samples in SDS
sample buffer for 5 min. The samples were analyzed
by 12% SDS-PAGE, and the gel was dried and subjected to autoradiography.
Similarly, CDK4-Rb kinase activity was also determined as described by Wu et al. [33] with some
modifications. Briefly, TF-treated cells were lysed in
Rb lysis buffer (50 mM HEPES-KOH, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM
DTT, 0.1% Tween-20, 10% glycerol, 80 mM b-glycerophosphate, 1 mM sodium fluoride, 0.1 mM
sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/mL leupetin and aprotinin),
and immunoprecipited with anti-p21/Cip1 antibody (2 mg) and protein A agarose beads (20 mL).
The p21/Cip1-associated CDK4 in beads were
washed twice with Rb lysis buffer and then once
with Rb kinase assay buffer (50 mM HEPES-KOH, pH
7.5, containing 2.5 mM EGTA, 10 mM b-glycerophosphate, 1 mM sodium fluoride, 0.1 mM sodium
orthovanadate, 10 mM MgCl2, and 1 mM DTT).
Phosphorylation of Rb was measured by incubating
the beads with 40 mL of hot Rb kinase solution
(0.25 mL [2 mg] of Rb-GST fusion protein, 0.5 mL of
[g-32P] ATP, 0.5 mL of 0.1 mM ATP, and 38.75 mL of
Rb kinase buffer) for 30 min at 378C. The reaction
was stopped by boiling the samples in SDS sample
buffer for 5 min. The samples were analyzed by
12.5% SDS-PAGE, and the gel was dried and subjected to autoradiography.
Determination of Apoptosis
As in our previous studies, apoptosis was judged by
four methods: (a) observation of the morphological
changes in cells as described previously [30]; (b)
translocation of phosphotidyl serine to the cell
surface detected by an Annexin V-FITC apoptosis
detection kit (Calbiochem, Bad Soden, Germany)
[34]; (c) the presence of sub-G1 peak detected by flow
cytometry on a FACSCalibur (Becton Dickinson,
Heidelberg, Germany) [35]; and (d) the appearance
of DNA fragmentation analyzed by the methods
described previously [16].
Antisense Oligodeoxynucleotide (ODN)
Transfection Procedures
The p27/Kip1-specific antisense ODN sequence
used in the experiments was (50 -TCTTCTGTTCTGTTGGCCCT-30 ); and sense ODN sequence was (50 CGTGAGAGTGTCTAACGGGAG-30 ) [36]. The p21/
Cip1-specific antisense ODNs (50 -UCCGCGCCCAGCUCC-30 ); and sense ODNs (50 -UCCGCCCGCAGUCCC-30 ) [37] phosphothioates (S-oligos) were
synthesized and purified with high-performance
liquid chromatography by Genset. Antisense or
41
sense ODNs were added to the HL 60 cells at a final
concentration of 20 mM 16 h before the cells were
challenged with 10% FCS and TF (10 mM) treatment
for additional 15 h. The methods for transfection
of the ODNs were the same as in our previous
report [14].
RESULTS
TF Induces G0 /G1 Phase Cell-Cycle Arrest in
Human Cancer Cells
In this study, we demonstrated that TF induced
significant growth inhibition of human cancer cells
including hepatoma (Hep G2 and Hep 3B), colon
cancer (HT 29 and COLO 205), and leukemia (HL 60)
cells (Figure 1A and E). Furthermore, similar results
were also observed in the human normal fibroblast
treated with higher doses of TF (1–5 mM) (Figure 1F),
indicating that TF-induced cell growth inhibition
and cytotoxic effects were dose- and cell line–
dependent. In order to determine whether TF could
induce cell-cycle arrest in human cells, HT 29 and
COLO 205 cells were synchronized at the G0/G1
phase by 0.04% serum starvation for 24 h and then
treated with complete medium (with 10% FCS)
containing TF (5 mM). Flow cytometry analysis was
determined at the indicative time points. Our results
showed that TF (5 mM) treatment caused an apparent
G0/G1 phase arrest in COLO 205 (85.3%) and HT 29
cells (more than 90%) (Figure 2D and H). Normal
cell-cycle progression was observed in DMSO-treated
cells after being challenged with complete medium
without TF.
Figure 2D and H demonstrates that the most
significant difference of the G0/G1 phase cell population between the TF- and DMSO-treated groups is at
15 h after restimulation with complete medium.
Accordingly, this time point (15 h) was selected for
studying the dose-dependent effect of TF-induced
G0/G1 arrest in various types of human cancer and
fibroblast cells (Figure 3A–F). Our results revealed that G0/G1 arrest was induced significantly in
the COLO 205 and the Hep G2 cells treated with TF
(1–3 mM). However, apoptosis occurred when these
two types of cells were treated with a higher dose of
TF (>15 mM) (Figure 3B and C). Interestingly, G0/G1
cell-cycle arrest, but not apoptosis, was observed in
the HT 29 cells, even with a higher dose of TF (30 mM).
In contrast, apoptosis was more significantly induced in the Hep 3B and the HL 60 cells treated with
TF (3 mM) (Figure 3D and E). Importantly, neither
apoptosis nor cell growth cycle arrest was observed in
human normal fibroblast, even with a higher dose of
TF (25 mM) (Figure 3F). From these data, we found
that induction of the G0/G1 phase cell-cycle arrest
and apoptosis were observed in the p53-expression
(COLO 205, Hep G2, and HT 29) cells, while the
occurrence of apoptosis was more easily induced in
the p53-deficient (Hep 3B and HL 60) cells when
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LIU ET AL.
Figure 1. Viability of human cancer and fibroblast cells treated with TF. COLO 205 (A), HT 29 (B), Hep G2 (C),
Hep 3B (D), HL 60 (E), and fibroblast (CCD 922SK) (F) cells were treated with TF (0.1–5 mM) at the indicated time
points. The viability was then determined by trypan blue exclusion assay as described in Materials and Methods.
Results were the mean of three independent experiments.
exposed to TF. The TF-induced G0/G1 cell-cycle arrest
was not reversed by TF removal, and this growth
inhibition lasted for at least 7 d (data not shown).
TF-Induced Apoptosis in Various Types of
Human Cancer Cell Lines
Our recent study [35] demonstrated that apoptosis
can be induced in Hep G2 and COLO 205 cells that
have suffered a higher dose of TF (>10 mM) treatment. In this study, the cells treated with TF (15 mM)
exhibited morphological changes (data not shown)
as well as progressive internucleosomal degradation
of DNA, yielding a ladder of DNA fragments (Figure
4). Interestingly, at such a high dose (15 mM) of TF,
apoptosis was more easily induced in the p53-null
(HL 60 and Hep 3B) cells compared to the p53 wild
type (Hep G2 and COLO 205) cells (Figures 3 and 4).
Our data demonstrated that either apoptosis or G0/
G1 cell-cycle arrest effects induced by TF depended
on TF dosage and cell type.
Molecular Mechanisms of TF-Induced G0/G1 Arrest
As shown in Figure 3, significant G0/G1 arrest was
induced by TF (>5 mM) in both HT 29 and COLO 205
cells. COLO 205 (with wild type p53) cells were then
selected to investigate the molecular mechanisms
of TF-induced G0/G1 cell-cycle arrest. The COLO 205
cells were synchronized at the G0/G1 phase as described previously [17]. The cells were then exposed to
5 mM TF and immunoblotting analysis was performed time-dependently. The time points that we
selected for drug treatment were, as in our previous
paper [17], reported as: 0 h (representing the G0/G1
phase), 15 h (representing the S phase), 18 h (representing the G2/M phase), and 24 h (representing
the second G0/G1 phase). The expression of p21/
Cip1, a key regulator of cell entry into mitosis, was
monitored by immunoblotting analysis. Our results
revealed that the p21/Cip1 protein was induced
initially at 15 h and persisted for at least 24 h by TF
TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS
43
Figure 2. Time-dependent response of TF-induced G0 /G1 phase
arrest in COLO 205 and HT 29 cells. COLO 205 (A–D), and HT 29 (E–
H) cells were synchronized with 0.04% FCS for 24 h as described in
Materials and Methods. After synchronization, cells were then
released into complete medium (10% FCS) containing DMSO
(0.05%), and TF (5 mM). The DNA content distribution histograms
of FACS analysis were measured by flow cytometry at the indicated
time points (0 and 18 h) after TF treatment. Different phases of the
cell cycle were determined with established CellFIT DNA analysis
software. Each blot is representative of three similar experiments.
Significance was accepted at **P < 0.01.
treatment (Figure 5). Our results also showed that the
level of cyclin D1, cyclin D3, cyclin E, PCNA, CDK2,
and CDK4 in TF-treated cells was not significantly
changed at 15 h by TF-treatment (Figure 5).
p27/Kip1, which inhibits CDK2 kinase activity,
was also analyzed for its expression level. Our results
indicated that the p27/Kip1 protein level was
significantly elevated at 15 h after TF-treatment. In
contrast, cyclin A and cyclin B, which promote cell
entry from G0/G1 into S and from S into the G2/M
phase, respectively, were downregulated in TFtreated cells (Figure 5). Previous studies demon-
strated that the p53 protein is a potent transcription
factor, activated and accumulated in response to
DNA-damaging agents [38–40], leading to cell-cycle
arrest, or apoptosis [41,42]. Our results in Figures 2B
and 5 demonstrated that G0/G1 cell-cycle arrest and
induction of the p53 protein expression were observed concomitantly in COLO 205 cells after 5 mM
TF treatment. These findings suggested that the p53signaling pathway was involved in the regulation of
TF-induced G0/G1 cell-cycle arrest.
To further scrutinize such observations, human
colon cancer cell lines with wild-type (COLO 205) or
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LIU ET AL.
Figure 3. TF-induced G0 /G1 phase cell-cycle arrest and apoptosis
in human cancer cells. HT 29 (A), COLO 205 (B), Hep G2 (C), Hep 3B
(D), HL 60 (E), and human fibroblast (CCD 922SK) (F) cells were
treated by TF dose-dependently. FACS analysis of DNA content was
performed at 15 h after release from quiescence by incubation in
culture media supplemented with 10% FCS and various concentrations of TF (0.1–30 mM) in 0.05% DMSO. Percentages of cells in the
Sub-G1, G0 /G1, S, and G2/M phases of the cell cycle were determined
with established CellFIT DNA analysis software. Three samples were
analyzed in each group, and values represent the mean SE.
mutated p53 (HT 29) status, and human leukemia
cell line (HL 60) with nulled p53 were selected
to verify the role of p53 in response to TF treatment.
All cells within each category were treated with TF
(0.1–10 mM) dose-dependently for 15 h, then the
cells were harvested and subjected to Western blot
analysis with antibodies as illustrated in Figure 6. The
p53 and p21/Cip1 protein levels were elevated significantly in the HT 29 and COLO 205 cells (Figure 6).
Interestingly, the cyclin D3 and CDK4 protein
expression in all these cells were downregulated
dose-dependently. As shown in Figure 6, induction
TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS
Figure 4. DNA fragmentation analysis in human cancer cells
undergoing TF-induced apoptosis. Human cancer cells, including
HT 29 (lane 1), COLO 205 (lane 2), Hep G2 (lane 3), HL 60 (lane 4),
Hep 3B (lane 5), and normal fibroblast (lane 6) cells were treated with
TF (15 mM). DNA fragmentation analysis was examined 24-h later
(upper panel). Cells in the lower panel received mock treatment as
controls.
of p21/Cip1 was only observed in the HL 60 cells
treated with a higher dose of TF (>10 mM). Importantly, at such a dose of TF (>10 mM), significant
apoptosis instead of G0/G1 arrest was observed in HL
60 cells (Figure 3E).
As shown in Figure 7, protein levels of p53, p27/
Kip1, CDK2-associated p21/Cip1, and CDK4-associated p21/Cip1 were induced in the TF (5 mM)treated HT 29 and COLO 205 cells (Figure 7A). Kinase
assays revealed that the activities of CDK2 and CDK4
in the TF-treated COLO 205 and HT 29 cells were
inhibited (Figure 7B). As described previously, p21/
Cip1 was a potent inhibitor of CDK2/4. Our data
revealed that CDK2 and CDK4 kinase activities were
inhibited concomitantly with the increased binding
of p21/Cip1 to CDK2 and CDK4 in cells treated with
TF (Figure 7). Collectively, our data indicated that
G0/G1 arrest induced by TF was due to decreased
activity of CDKs mediated by an increase of p21/
45
Figure 5. Time-dependent effect of TF on cell-cycle regulatory
protein levels in COLO 205 cells. The COLO 205 cells were
synchronized with 0.04% FCS for 24 h as described in Materials
and Methods. After synchronization, cells were then released into
complete medium (10% FCS) containing TF (5 mM) and DMSO
(0.05%, v/v) for the indicated time points. Protein extracts (100 mg/
lane) were separated by SDS-PAGE, probed with specific antibodies,
and detected with the NBT/BCIP system.
Cip1-CDKs association. We further investigated the
phosphorylation status of the pRb and the association of pRb with E2F in response to TF. Figure 7A
shows that pRb was remarkably dephosphorylated at
15 h after TF treatment, and the hypophosphorylated pRb-E2F complexes were increased at 15 h after
TF treatment in the COLO 205 and HT 29 cells. In
contrast, the levels of the pRb hypophosphorylation
and the pRb-E2F complexes were not changed
significantly in the TF-treated HL 60 cells.
As shown in Figure 3D and E, apoptotic cells were
detected in the Hep 3B and HL 60 cells treated with a
lower dose of TF (3–5 mM). Such effects implied that
some regulatory proteins were involved in apoptosis
induction in response to TF treatment. Previous
studies indicated that the p27/Kip1 protein is activated during the apoptosis process induced by different agents [43–47]. The p27/Kip1 expression was
easily induced by TF treatment in both of the HL 60
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LIU ET AL.
Figure 6. Dose-dependent effect of TF on the cell-cycle regulatory
protein levels in human cancer cells. Human cancer cells with
different p53 status, including COLO 205 (wild type p53), HT 29
(mutated p53), and HL 60 (p53-null) cells were rendered quiescent by
incubation for 24 h in the cultured media containing 0.04% FCS.
Cells were then challenged with 10% FCS and treated with various
concentrations of TF (0.1–10 mM) for additional 15 h. Protein
extracts (100 mg/lane) were separated by SDS-PAGE, probed with
specific antibodies, and detected with the NBT/BCIP system.
Membranes were also probed with anti-GAPDH antibody to correct
for differences in protein loading.
and COLO 205 cells (Figures 6 and 7). In contrast,
significant induction of the p27/Kip1 was detected in
HT 29 cells treated with a higher dose of TF (>10 mM)
(Figure 6). Such results implied that p27/Kip1 might
play an important role in the TF-induced apoptosis
observed in the COLO 205 and HL 60 cells.
protein expression levels were monitored by immunoblotting analysis (Figure 8). Figure 3E demonstrated that apoptosis was induced by TF in the HL 60
cells dose-dependently. HL 60 cells were selected for
further clarifying the roles of p27/Kip1 involved in
TF-induced apoptosis. The HL 60 cells were treated
with 20 mM of p27/Kip1-specific antisense ODNs for
16 h and then exposed to TF (15 mM) for another 15 h.
Our results revealed that the TF-induced p27/Kip1
protein expression was attenuated by the p27/Kip1specific antisense ODNs (Figure 8A, lane 3), but not
by its sense ODNs (Figure 8A, lane 5). To examine
further whether the TF-induced apoptosis would be
reduced by the p27/Kip1-specific antisense ODNs,
the apoptotic cells were detected by annexin V/PI
analysis. Our data demonstrated that the percentage
of TF-induced apoptotic-cell population clearly
decreased when pretreated with the p27/Kip1-specific ODNs (Figure 8A, bar 3).
To confirm such observations, similar experiments
were performed and the p27/Kip1-(Figure 8B) or p21/
Cip1-specific (Figure 8C) antisense ODNs (20 mM)
were added to COLO 205 cells for 16 h and then
exposed to TF (15 mM) for another 15 h. Our results
p27/Kip1, Not p21/Cip1, Plays a Major Role
in TF-Induced Apoptosis in Human HL 60
and COLO 205 Cells
As described previously, p21/Cip1 and p27/kip1
were demonstrated as negative regulators of cyclin
and CDK activity and appear to be both essential and
sufficient to arrest cells before the late G1 restriction
point [48,49]. However, previous study demonstrated that adenovirus-mediated p27/Kip1 overexpression leads to apoptosis in human cancer
cells. In sharp contrast, a similar overexpression of
p21/Cip1 results in G1-S arrest but minimum
cytotoxicity [50]. To clarify the roles of the p21/
Cip1 and the p27/Kip1 protein expression that was
involved in G0/G1 arrest and apoptosis induced by TF
in human cancer cells, antisense ODNs specific to the
p21/Cip1 and the p27/Kip1 were used, and the
TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS
47
Figure 7. The G0 /G1 phase regulatory proteins were involved in
TF-induced human cancer cells growth inhibition. (A) Human cancer
cells with different p53 status, including HT 29 (p53 mutant), COLO
205 (p53 wild type), and HL 60 (p53-null) cells, were treated with
5 mM TF (þ) or 0.05% (v/v) DMSO () for 15 h after release from
quiescence. Protein extracts (100 mg/lane) were separated by SDSPAGE, probed with specific antibodies, and detected with the NBT/
BCIP system. (B) CDK2 and CDK4 kinase activity were determined as
described in Materials and Methods.
revealed that TF-induced apoptosis in the COLO 205
cells was significantly attenuated by the p27/Kip1specific antisense ODNs (Figure 8B, lane 3, and bar 3).
In contrast, the TF-induced apoptosis in the COLO
205 cells was not inhibited by the p21/Cip1-specific
ODNs (Figure 8C, lane 3, and bar 3). Our results
demonstrated that the p27/Kip1 protein might play
an important role in the TF-induced apoptosis in
both COLO 205 and HL 60 cells.
DISCUSSION
TF-Induced Human Cancer Cell-Cycle Arrest Was
Through the p53-Dependent and p53-Independent
Signaling Pathways
The p53 tumor suppressor is a predominantly
nuclear transcription factor, activated by various
stresses including chemotherapeutic and chemopreventive agents [51]. Although analysis of a large
number of human tumor-derived cell lines has
suggested a link between p53-status and drug-
Figure 8. p27/Kip1 protein expression plays a critical role in TFinduced apoptosis in COLO 205 and HL 60 cells. Antisense or sense
ODNs specific to p27/Kip1 and p21/Cip1 were added to HL 60 (A)
and COLO 205 (B and C) cells at a final concentration of 20 mM at 16
h before the cell was challenged with 10% FCS and 15 mM TF
treatment for additional 15 h. The levels of p27/Kip1, p21/Cip1, and
GAPDH proteins were determined by Western blot analysis.
Percentage of apoptotic cells was determined by with annexin V/PI
analysis shown in the bottom chart. M, size marker; AS, antisense
oligonucleotide; S, sense oligonucleotide.
sensitivity [52], the role of p53 involved in the
response of human tumor cells to chemotherapeutic
agents is controversial [53]. In this study, human
cancer cells (Hep G2 and COLO 205) with wild-type
p53 status were more sensitive to TF-induced G0/G1
cell-cycle arrest. Our recent studies demonstrated
that G0/G1 cell-cycle arrest is induced by antifungal
agents (such as KT and miconazole) in the COLO 205
48
LIU ET AL.
cells [14,17]. All of these agents induce p53, p21/
Cip1, and p27/Kip1 protein expression in the COLO
205 cells via a p53-dependent signaling pathway
[14,17]. In this study, we further demonstrated that
significant G0/G1 cell-cycle arrest was also induced
by TF in HT 29 (p53 His273 mutant) cells (Figure 2H).
We demonstrated that p21/Cip1 protein was markedly induced in p53-mutated (HT 29) cells (Figure 6).
Such results implied that p21/Cip1 protein expression in HT 29 cells was induced by TF in a p53independent signaling pathway. Our observations
were similar to those of previous studies that
indicated that p21/Cip1 protein expression can be
stimulated by a variety of transcriptional activators
other than p53 that are often associated with growth
arrest and differentiation (such as MyoD7, STAT18,
and BRCA19) [54–56]. Direct evidence of p53regulated TF-induced p21/Cip1 protein expression
and its association with G0/G1 arrest will be investigated in our further study by the p53-specific
antisense ODNs as described recently [14].
TF-Induced Apoptosis Tended to Occur in HL 60
and Hep 3B Cells
Our recent report [35] and this study (Figures 3B
and C) demonstrated that apoptosis can be induced
in Hep G2 and COLO 205 cells with a higher dose of
TF (>10 mM). Importantly, when the COLO 205 and
Hep G2 cells were exposed to TF at a lower dose (0.1–
5 mM), G0/G1 cell-cycle arrest instead of apoptosis
was induced within 24 h. To avoid the occurrence of
apoptosis and specifically investigate the TF-induced
G0/G1 cell-cycle arrest in COLO 205 and Hep G2
cells, the dose of TF was limited to a narrow range of
0.1–5 mM. Moreover, we also demonstrated that
apoptosis tended to be induced by TF in the p53-null
cells (HL 60 and Hep 3B) at this dosage range. Our
data demonstrated that either apoptosis or G0/G1
cell-cycle arrest induced by TF was a dose- and cell
type–dependent effect. Such results implied that the
p53-signaling pathway might play an important role
in G0/G1 cell-cycle arrest as a response to TF treatment instead of apoptosis.
To our knowledge, the p21/Cip1 is a negative
regulator of cell-cycle check point and is a transcriptional target of p53 [57,58]. However, previous
studies have established a role for p21/Cip1 as a
survival factor by demonstrating that defective p21/
Cip1 expression can lead to human colon cancer
cells’ (HCT116) undergoing apoptosis instead of cellcycle arrest [59,60]. In this study, significant G0/G1
phase cell-cycle arrest instead of apoptosis was found
in HT 29 cells after a higher dose of TF (30 mM). Our
results suggest that p21/Cip1 protein induction
might play a key regulatory role in TF-induced protection of apoptosis in HT 29 cells. Additional
experiments may be needed to clarify the role of
p21/Cip1 protein expression involved in TF-induced
G0/G1 arrest in human HT 29 cells.
p27/Kip1 Protein Was the Key Regulator in
TF-Induced Apoptosis in Human Cancer Cells
As described above, apoptosis was significantly
induced by a lower dose of TF (3–5 mM) in HL 60 cells.
However, p21/Cip1 protein induction in the HL
60 cells was only observed at a higher dose of TF
(>10 mM). Such results suggested that p21/Cip1
protein was not playing a role in the prevention of
TF-induced apoptosis in HL 60 cells. Additional
regulatory proteins may be involved in TF-induced
apoptosis. Previous studies demonstrated that p27/
Kip1 protein plays an important role in human
cancer cell apoptosis induced by various stimuli [61–
64]. To further scrutinize the role of p21/Cip1 and
p27/Kip1 involved in TF-induced apoptosis, antisense ODNs specific to the p21/Cip1 and p27/Kip1
were added in cultured HL 60 cells treated with
higher dose of TF (10 mM). The induction of apoptosis
is significantly attenuated by the p27/Kip1-specific
ODNs, whereas the p21/Cip1-specific ODNs are not.
Such observations demonstrated that p27/Kip1 is
involved in TF-induced apoptosis in HL 60 cells. As
described previously [59,60,65], p21/Cip1 induction
is involved in the prevention of apoptosis in human
colon carcinoma cells. However, our study demonstrated that induction of p21/Cip1 was not involved
in TF-induced apoptosis in HL 60 cells, as evidenced
by the antisense ODNs experiments (data not
shown). The role of increased p21/Cip1 expression
in response to TF-induced G0/G1 cell-cycle arrest and
apoptosis in HT 29 and HL 60 cells might be cell
type–specific and needs to be further clarified.
TF-Induced G0 /G1 Arrest Was Irreversible
As shown in Figure 3A, G0/G1 arrest but not
apoptosis was observed in HT 29 cells in response
to a higher dose (30 mM) of TF. Such results implied
that HT 29 cells could be used as a candidate to
illustrate the molecular mechanisms of TF-induced
G0/G1 cell-cycle arrest. In this study, we demonstrated that TF-induced G0/G1 arrest was irreversible
in the HT 29 cells. The antiproliferation effect of
many anticancer drugs used clinically was reversible.
Therefore, once the drug treatment was terminated,
the growth of the tumor cell rebounded. Lately,
scientists and clinicians have been searching for
some new drugs that will exert an irreversible antiproliferative effect. Moreover, a drug with an irreversible effect can be applied in a lower dose for a
longer period of time to achieve the same therapeutic
effect as given by a higher dose of another drug, but
with fewer side effects. Accordingly, we attempted to
test the irreversibility of the antiproliferative effect of
TF on tumor cell growth. More experiments must be
performed in our further study to support this
hypothesis. Our results showed the molecular basis
of TF-induced cancer cell growth inhibition in vitro,
and further animal experiments will be important
TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS
to demonstrate the potential anticancer effect of TF
in vivo.
ACKNOWLEDGMENTS
This study was supported by the National Science
Council grant NSC 90-2320-B-038-033, NSC
90-2320-B-006-086, and by the Jin Lung Yen
Foundation.
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