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J. Biol. Chem., Vol. 277, Issue 41, 38930-38938, October 11, 2002
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From the
Received for publication, June 14, 2002, and in revised form, July 18, 2002
The anticancer effects of
N-(4-hydroxyphenyl)retinamide (4HPR), a potential
chemopreventive or chemotherapeutic retinamide, are thought to be
derived from its ability to induce apoptosis. However, the
mechanism of apoptosis induced by 4HPR remains unclear. Thus, this
study was designed to identify the gene(s) responsible for induction of
apoptosis by 4HPR. Apoptosis was effectively induced by
4HPR in human hepatoma cells. Using the differential display-PCR
method, a gene involved in the response to 4HPR was identified, and
cells in which the expression of that gene was modulated were analyzed
for survival, induction of apoptosis, and cell cycle.
GADD153, a gene involved in growth arrest and apoptosis,
was preferentially expressed in human hepatoma cells as well as in
other cancer cells during 4HPR-induced apoptosis. 4HPR regulates
GADD153 expression at the post-transcriptional level in Hep
3B cells and at the transcriptional and post-transcriptional levels in
SK-HEP-1 cells, when assayed by in vitro transfection and
mRNA stability experiments. To determine the role of the GADD153 protein overexpression that is induced by 4HPR, Hep 3B cells with ectopic overexpression of GADD153 were found to be growth-arrested (at
G1) and readily underwent apoptosis following
treatment with 4HPR or even when they reached confluence.
N-Acetyl-L-cysteine or GADD153
antisense significantly protected the cells from 4HPR-induced apoptosis, accompanying by the inhibition of GADD153
overexpression. Parthenolide-mediated overexpression of GADD153
resulted in enhanced 4HPR-induced apoptosis. These results
suggest that GADD153 overexpression induced by 4HPR may contribute to
the anticancer effects (induction of apoptosis and growth arrest)
of 4HPR on cancer cells.
GADD153, a member of the CCAAT/enhancer-binding protein family of
transcription factors, is transcriptionally activated and is highly
expressed following treatment of cells with a variety of growth arrest
and/or DNA-damaging factors (1, 2), such as calcium ionophore (3),
glucose deprivation (4), oxidative stress (5), reductive stress (6),
endoplasmic reticulum stress (7), or activation of acute phase response
(8). GADD153 has been implicated in the commitment to growth
arrest or cell death. Microinjection of GADD153 induces 3T3
cells to arrest at the G1/S boundary (9), while ectopic
expression of GADD153 causes M1 myeloblast leukemia cells to
undergo apoptosis (10). Chemotherapeutic drugs, including paclitaxel,
cisplatin, and etoposide, were reported to induce GADD153
overexpression, which was associated with cellular injury/apoptosis
(11-13). More recently, GADD153 was reported to sensitize
cells to endoplasmic reticulum stress through mechanisms that involve
down-regulation of Bcl-2 and enhanced oxidant injury (14). However,
studies to date are not sufficient to explain how the anticancer
effects of GADD153 overexpression function in drug-induced apoptosis.
N-(4-Hydroxyphenyl)retinamide
(4HPR),1 a potential
chemotherapeutic or chemopreventive retinamide, seems promising in
terms of its effectiveness relative to toxicity. 4HPR also induces
apoptosis of human neuroblastoma cells (15, 16), cervical (17), breast (18), human ovarian carcinoma cells (19), head and neck (20), lung
(21), and human malignant hematopoietic cells. 4HPR-sensitive cells
include those carrying mutations of the retinoic acid receptor (RAR)
and which are thus unresponsive to all-trans-retinoic acid (22). Furthermore, 4HPR was found to activate RARs and several RAR-specific antagonists partially inhibited 4HPR-induced apoptosis (23). Thus, both RAR-independent and -dependent pathways
are involved in 4HPR-mediated apoptosis. 4HPR stimulates the generation of intracellular free radicals, which appear to play a causative role
in the induction of apoptosis in vitro (24, 25). Increased ceramide levels resulting from 4HPR treatment induce cell death in a
p53- and caspase-independent manner through mixed apoptosis and
necrosis pathways (26). In addition, 4HPR modulates the expression
levels of some apoptosis-related genes, such as p21, c-myc, and c-jun (27), and activates c-Jun
N-terminal kinase-mediated apoptotic signaling (28). However, the
underlying mechanism(s) by which 4HPR induces apoptosis remains
unclear. Quite recently, we observed that 4HPR effectively induces
apoptosis in Fas-defective hepatoma cells through caspase-8 activation
(29), and 4HPR-induced apoptosis was effectively modulated by
alteration of mitochondrial membrane potential (30). Thus, to elucidate
the molecular and cellular mechanism(s) by which 4HPR induces
apoptosis, we undertook to identify the gene(s) involved in this
drug-induced apoptosis. Using the differential-display PCR (DD-PCR)
method, we identified genes that were differentially expressed between
untreated cells and 4HPR-treated cells. One of them,
GADD153, which was preferentially expressed in 4HPR-treated
hepatoma cells, seemed to be responsible for the induction of apoptosis
and growth arrest. Thus, in this study, we address the role of GADD153
overexpression in the antitumor effects of 4HPR.
Cell Lines and Reagents--
The hepatoma cell lines, including
Hep 3B (deletion of p53) and SK-HEP-1 (wild-type
p53) (31), and other cancer cell lines were obtained from
the American Tissue Culture Collection (ATCC, Manassas, VA). 4HPR was
kindly supplied by the Johnson Pharmaceutical Research Institute
(Spring House, PA) and was dissolved in absolute ethanol at a
concentration of 10 Cell Culture and Detection of Apoptotic Cells--
Cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum in air containing 5% CO2. Viable cells
were determined by trypan blue dye exclusion. The percentage of
apoptotic cells was evaluated by DAPI staining. Briefly, Hep 3B and
SK-HEP-1 cells were treated with the indicated concentrations of 4HPR,
or with equitoxic concentrations of 10 µM 4HPR or 15 µM 4HPR, which caused about 40% cell death,
respectively, for 3 days in 6-well dishes. Cells were fixed in ice-cold
methanol for 10 min and were then stained with the DAPI reagent (1 µg/ml). Fluorescence microscopy was performed using a Nikon diaphot
microscope. Apoptotic cells were recognized as condensed, fragmented,
degraded nuclei, or ghosts that stained only faintly. At least 200 cells were counted for each time point, and all counting was done in a
blinded fashion.
Detection of DNA Fragmentation--
Cells (1 × 106) were seeded in 6-cm Petri dishes and allowed to settle
and attach. Cells were treated with 10 µM 4HPR for
48 h or 72 h. For analysis of genomic DNA, cells were
harvested and combined with nonattached cells in the supernatant. Cells
were resuspended in 0.5 ml of lysis buffer (50 mM Tris-HCl,
100 mM EDTA, 0.5% SDS, pH 8.0) containing 0.1 mg/ml RNase
A. After incubation at 37 °C for 30 min, extracts were treated with
1 mg/ml proteinase K for an additional 16 h at 37 °C. DNA was
extracted with phenol/chloroform, then with chloroform, and was finally
precipitated with ethanol and sodium acetate. 20 µl of each extract
(dissolved in 50 µl of H2O) was then loaded on a 1.5%
agarose gel and separated in the presence of 0.5 µg/ml ethidium bromide.
Flow Cytometric Analysis of Apoptotic Quantitation and Cell
Cycle--
Measurements of apoptotic cells and cell cycle distribution
were performed using a modification of the technique described previously (32). Apoptotic cells were quantified by staining with
FITC-conjugated Annexin V (Clontech, Palo Alto,
CA). Cells (1 × 106) were collected at 72 h for
flow cytometric (FCM) measurement and stained with FITC-conjugated
annexin V and PI as instructed by the manufacturer, and then analyzed
by flow cytometry using a FACScan (BD Biosciences, San Jose, CA) with
an argon laser set to excite at 488 nm. In addition, cells were
harvested at each time point and were fixed in 70% ethanol and stored
at 4 °C. Cell cycle distribution was performed at the indicated
intervals following 4HPR treatment. PI (40 µg/100 µl PBS) was added
to 1 ×106 cells suspended in 800 µl of PBS together with
100 µl of RNase A (1 µg/ml), and was incubated at 37 °C for 30 min before FCM analysis of 2 × 104 cells. Red
fluorescence due to propidium-bound DNA was measured using a 630 nm-long bandpass filter. Data were analyzed as single-parameter frequency histograms in an SFIT model.
DD-PCR--
Modified DD-PCR was performed using a GeneHunter RNA
image kit (GeneHunter Corp, Nashville, TN), based on an improved method described previously (33). 4 µg of total RNAs from treated or untreated cells were reverse-transcribed with 200 units of SuperScript II RT enzyme (Invitrogen) in the presence of 1 µmol/liter 1-base anchored oligo(dT) primers for 1 h at 42 °C in a total volume of 10 µl. The reaction was terminated by incubation at 75 °C for 10 min. 2 µl of the reaction mixture was PCR-amplified with Dynazyme (Finnzyme OY, Epsoo, Finland) in 1 µmol/liter H-AP, a 13-mer (5'-end primers) and oligo(dT)15 primers (3'-end primers), using a
GeneAmp PCR system 9600 (PerkinElmer, Norwalk, CT). PCR reactions and reamplification of cDNAs of interest were carried out as previously described (34). After cloning into the pGEM-T vector using the TA
cloning system (Promega, Madison, WI), the sequenced cDNAs were
analyzed via the BLAST program for matches in the GenBankTM
data base and were compared with each other via FASTA analysis.
Northern Blot Analysis--
Cells were cultured in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum until they
reached 60% confluence and were then treated with 10 µM
or 15 µM 4HPR for times as indicated in the text. Total
RNAs were extracted from treated or untreated cells using phenol and
guanidine thiocyanate solution (Tri Reagent; Molecular Research Center,
Inc. Cincinnati, OH). RNAs were then fractionated by electrophoresis on
1.0% agarose gels containing formaldehyde and were transferred to
membranes. Blots were hybridized overnight in 2 × 106
cpm/ml cDNA probe, labeled with [P32]dCTP
(PerkinElmer Life Sciences) by random-priming, washed, then exposed to
X-Omat AR film (Kodak) at Luciferase Assay--
pGADD-LUC, a hamster GADD153
promoter-driven luciferase reporter construct was a gift from Dr. Nikki
J. Holbrook (National Institute on Aging, Baltimore, MD) (36). The
cells were transfected with the pGADD-LUC construct using Lipofectin
(Invitrogen). Cells were plated at 2 × 104
cells per well in 24-well plates, and then 18 h later the cells were incubated at 37 °C for 16 h with 500 ng of pGADD153-LUC
plasmid and 50 ng of pRL-TK plasmid (Promega, Madison, WI) as well as Lipofectin. Following the transfection, the cells were replenished with
complete medium and were treated with equitoxic levels of 4HPR. The
cells were lysed in 120 µl of lysis buffer at the indicated time
intervals and were stored at Transfections--
Transfection of the GADD153 gene
into Hep 3B cells was performed using an expression plasmid vector
encoding human GADD153 cDNA or control pcDNA3. The
construct of the GADD153 expression vector was made by
ligating human GADD153 (a gift from Dr. Nikki J. Holbrook)
with pcDNA3 in each BamHI/XhoI site in the
sense orientation. For the generation of the antisense
GADD153 expression vector, human GADD153 was
PCR-amplified with the forward primer containing the XbaI
restriction enzyme site (5'-GCTCTAGAGGGCTGCAGAGATGGC-3') and the
reverse primer containing the EcoRI restriction enzyme site
(5'-GGAATTCGGGACTGATGCTCCCA-3'). The PCR-amplified GADD153 double-strand DNA was ligated to pcDNA3 in
XbaI/EcoRI site in the antisense orientation.
Transfections were carried out by Lipofectin (Invitrogen) according to
the manufacturer's protocol. Sequence-verified GADD153-transfected and neo-transfected cells were selected
in the presence of 600 µg/ml G418 for 2-3 weeks. Finally, individual colonies were isolated using cloning rings, expanded, and assayed for
expression of the transfected gene by Northern analysis and Western analysis.
Western Immunoblotting--
Cells were washed twice with cold
PBS on ice and were harvested by scraping with a rubber policeman.
Cells were sedimented by centrifugation at 4 °C and were resuspended
directly into Laemmli sample buffer containing 62.5 mM
Tris-HCl, pH 6.8, 2% SDS (w/v), 12% glycerol (w/v), and 5%
Statistical Analysis--
All data were entered into Microsoft
Excel 5.0, and GraphPad Software was used to perform two-tailed
Student's t tests. All p values of less than
0.05 were considered to be statistically significant.
4HPR-induced Apoptosis in Hepatoma Cells--
We observed that
4HPR effectively induced cell death in a
concentration-dependent manner after 3 days in culture. Hep
3B cells, which have a defect in p53 function, rounded up, shrank, and
detached during 3 days of exposure to 10 µM 4HPR. In
contrast, SK-HEP-1 cells, which have normal p53 function, were less
susceptible to 4HPR-induced cell death. The IC50 for Hep 3B
and for SK-HEP-1 cells was 12.5 and 17.5 µM, respectively
(Fig. 1A). Thus, we used equitoxic concentrations, 10 and 15 µM, that caused 40%
cell death of Hep 3B and SK-HEP-1 cells, respectively. Cells exposed to
those equitoxic concentrations of 4HPR for 3 days were stained with Annexin V and PI to prove apoptotic cell death. 4HPR effectively induced apoptosis in up to 40% of those hepatoma cells, as shown by
their positive staining for Annexin V as assessed by FCM (Fig. 1B). The percent of cells in the sub-G1 fraction
that were stained with PI showed similar results (data not shown). In
support of those findings, staining with the DNA-binding dye DAPI
revealed condensed chromatin and fragmented nuclear morphologies
characteristic of apoptosis in Hep 3B and SK-HEP-1 hepatoma cells. The
percentage of apoptotic cells measured using DAPI was the same as the
apoptotic population measured by FCM in each cell line (data not
shown).
Induction of Endogenous GADD153 by 4HPR--
We identified the
GADD153 gene, which is preferentially expressed in
apoptotic Hep 3B cells, by the DD-PCR method (Fig. 1C) and confirmed that equitoxic levels of 4HPR caused the prominent induction of GADD153 mRNA in Hep 3B and SK-HEP1 cells in
a time-dependent manner (Fig. 1D, upper
panels). Next we determined whether the GADD153 protein expression
correlates with GADD153 mRNA induction during
4HPR-induced apoptosis. 4HPR induced the expression of GADD 153 protein, which relates to GADD153 mRNA expression. (Fig. 1D, lower panels). In addition, to check whether
GADD153 is also induced in other types of cancer cells by 4HPR, we
choose colon (HCT116 and HT29), lung (A549 and NCI-H522), and breast
(MCF7 and MDA-MB-231) cancer cell lines, according to p53 function
(wild-type and mutant type, respectively), and kidney cancer cells with
wild-type p53 (A498 and ACHN) (37). Each cell line was treated with 10 µM 4HPR for 48 h. The apoptotic cell death was
counted by staining with DAPI and ranged from 21 to 51% (Fig.
2A) while less than 7% of
untreated control cells were apoptotic. 4HPR also induced GADD153
overexpression in all cell lines, independent of their functional p53
status (Fig. 2B). Therefore, GADD153
overexpression by 4HPR seems to be general, not cell specific.
4HPR Stabilizes GADD153 mRNA--
To determine the effect of
4HPR on activation of the GADD153 promoter, Hep 3B and
SK-HEP-1 cells were transiently transfected with pGADD-LUC, which
contains the hamster GADD153 promoter coupled to the
luciferase reporter gene (11). Those transfected cells were exposed to
10 or 15 µM 4HPR, respectively, for 48 h (Fig. 3A). The lack of change in
luciferase activity (relative to the level in untreated control cells)
indicated that 4HPR did not increase GADD153 promoter
activation in transfected Hep 3B cells. In contrast, in SK-HEP-1 cells,
4HPR increased GADD153 promoter activation in a
time-dependent manner that corresponded well with its
effect on the level of endogenous GADD153 mRNA (over
4-fold induction of GADD153 mRNA after 48 h
treatment with 4HPR). These results suggest that 4HPR increases
GADD153 mRNA levels at the post-transcriptional level in
Hep 3B cells, whereas it occurs at the transcriptional level in
SK-HEP-1 cells. To further investigate the post-transcriptional
regulation by 4HPR, we investigated whether 4HPR might affect the
stability of GADD153 mRNA. The decline of GADD153 mRNA levels in 4HPR-treated cells was examined
following 4HPR withdrawal and/or the addition of a inhibitor of
transcription, actinomycin D. Subconfluent Hep 3B cells or SK-HEP-1
cells were treated with 10 or 15 µM 4HPR, respectively,
for 48 h to bring about the accumulation of GADD153
mRNA levels. Cells treated with 4HPR were then fed with 4HPR-free
medium and to which 4HPR alone, actinomycin D (Act D, 5 µg/ml)+4HPR, or Act D+vehicle was added, respectively (13). This time
is considered 0 h. RNA was extracted at subsequent time points
from each group, and relative mRNA levels were calculated by
comparison of GAPDH-normalized values with the level
observed in cells at time 0 (Fig. 3B). 4HPR sustained the
increase of GADD153 mRNA following treatment for 12 h in both Hep 3B cells and SK-HEP-1 cells. In the presence of
actinomycin D, 4HPR did not change the GADD153 mRNA
level in Hep 3B cells, whereas 4HPR slowly decreased the
GADD153 mRNA level in SK-HEP-1 cells. The half-life
(t1/2) of GADD153 mRNA is ~5 h. The
treatment with vehicle progressively decreased the GADD153
mRNA level in both cell lines. However, the rate of decrease of
GADD153 mRNA was higher in SK-HEP-1 cells
(t1/2 ~1 h) than in Hep 3B cells
(t1/2 ~2 h). These results imply that 4HPR
regulates GADD153 mRNA levels post-transcriptionally in
Hep 3B cells and at the transcriptional level in SK-HEP 1 cells, in
accordance with results from the in vitro transfection
experiment. In contrast, in the presence of actinomycin D, the rate of
decrease of GADD153 mRNA was higher during vehicle
treatment than during 4HPR treatment in SK-HEP-1 cells, suggesting that
some post-transcriptional regulation of the GADD153 mRNA
concurrently exists. Thus, 4HPR may regulate GADD153
mRNA expression post-transcriptionally and/or transcriptionally in a cell specific manner.
Effect of Ectopic GADD153 Overexpression on Apoptosis--
To
determine the role of GADD153 protein overexpression, we introduced the
GADD153 expression system into Hep 3B cells because these cells
expressed less constitutive GADD153. Two types of Hep 3B
cells (3B-G1 and 3B-G5), which stably expressed different levels of
human GADD153 protein, were isolated (Fig.
4A) and then treated with 10 µM 4HPR. In the cells transfected with
GADD153, the level of expression was compared with that
observed in 4HPR-treated cells. Northern blot analysis revealed that
the expression of GADD153 mRNA in the transfectants was
lower than that seen in the treated cells, thus it does not seem to be
sufficient to induce apoptosis on its own. Compared with control cells
transfected with the empty vector alone (3B-C1 and 3B-C2), the 3B-G1
and the 3B-G5 cells became readily apoptotic within 48 h of
treatment (p < 0.01). 3B-G1 cells, which express less
ectopic GADD153, were up to 70% apoptotic while 3B-G5
cells, which express more ectopic GADD153, were up to 80% apoptotic
(Fig. 4B). The proportion of apoptotic cells seems to be
related to the expression level of the GADD153 protein. Furthermore,
both types of cells were apoptotic (27.0 or 52.5%, respectively,
p < 0.01) at confluence after 72 h of culture
even without any drug treatment (Fig. 4C), suggesting that
confluence or nutrient depletion also sensitizes both types of cells to
apoptosis. This was confirmed since serum deprivation resulted in
similar proportions of apoptotic cells, which displayed disintegrated
nuclei and nonrandom DNA fragmentation, as assessed by agarose gel
electrophoresis of genomic DNA (Fig. 4C, lower left). Similarly, the apoptotic sub-G1 fraction of the
cells was increased by confluence using FCM analysis (Fig.
4C, lower right). These results suggest that
apoptotic cell execution induced by 4HPR or by confluence/nutrient
depletion is higher in GADD153 transfectants than in vector
control cells.
Effect of Ectopic GADD153 Overexpression on the Cell
Cycle--
Hep 3B cells that stably overexpress GADD153 protein showed
lower cell numbers than the vector control cells during culture. Thus,
we investigated whether GADD153 overexpression is associated with
inhibition of cell growth. Thymidine uptake assays for DNA synthesis
were significantly inhibited i.e. 70 and 53% of the control
in 3B-G1 and 3B-G5 cells, respectively (p < 0.01)
(Fig. 5A). We next analyzed
the impact of GADD153 overexpression on the cell cycle of Hep 3B cells.
In asynchronized cells, GADD153 overexpression increased the
G1 fraction of cells from 45.9 to 59.7%, compared with
vector control cells after 12 h in culture (Fig. 5B).
Thus, we further studied the cell cycle inhibition in synchronized
cells. For synchronization, Hep 3B cells were arrested in M phase with
0.5 µg/ml nocodazole. Cells were then analyzed at 3-h intervals
following release from the M block. Cells that stably overexpressed
GADD153 clearly showed delayed cell cycling and G1 arrest
(disappearance of G2/M), and thus, GADD153 overexpression
seems to be responsible for the G1 growth arrest.
Inhibition of GADD153 Overexpression Relates to Inhibition of
Apoptosis--
We examined whether prevention of GADD153
overexpression might lead to the inhibition of 4HPR-induced apoptosis.
The generation of reactive oxygen species (ROS) is responsible for
4HPR-induced apoptosis in some tumor cells (24, 25). Accordingly, we
observed that an antioxidant NAC effectively inhibited 4HPR-induced
apoptosis in hepatoma cells, although not completely (29). NAC (2 mM) inhibited 4HPR-induced apoptosis by about 41% (23.5 versus 39.7%) (p < 0.05) (Fig.
6A). Thus we examined whether
this inhibition of apoptosis by NAC correlates with the inhibition of
GADD153 overexpression. Interestingly, NAC inhibited the overexpression of GADD153 that is induced by 4HPR, which is proportional to
inhibition of apoptosis (Fig. 6B). For another approach to
examine the down-regulation of expression of GADD153, we used
transfection of an antisense expression plasmid of GADD153.
During 4HPR-induced apoptosis, the GADD153 protein overexpression was
inhibited in Hep3B cells transfected with the antisense
GADD153 cDNA (3B-AS3 and 3B-AS14) (Fig. 6C).
3B-AS3 and 3B-AS14 cells were significantly resistant to 4HPR,
e.g. about 27 and 22.2% of transfectants were apoptotic (p < 0.05 and p < 0.01, respectively), compared with 44.3 and 39.3% of vector control cells
(3B-C3 and 3B-C4) (Fig. 6D). These results suggest that the
inhibition of GADD153 expression correlates positively with cell
survival in hepatoma cells.
Parthenolide-mediated GADD153 Overexpression Enhances 4HPR-induced
Apoptosis--
Recently, we observed that an NF- Previously we observed that 4HPR effectively induced
apoptosis and/or inhibited cell growth (G1 arrest) in
hepatoma cells in a cell-specific manner (30). However, little is
known about the molecular and cellular mechanism(s) by which treatment
with 4HPR has these effects. Thus, the aims of this study were to
identify the gene(s) responsible for 4HPR-induced growth inhibition and apoptosis and to clarify its functional role(s). Human hepatomas are
resistant to chemotherapy and radiation and are often associated with
mutations of p53 (40). p53-independent cell growth arrest or
apoptosis pathways are known to respond to varying chemotherapeutic drugs and stress in tumor cells, and it is also known that 4HPR induces
p53-independent apoptosis (41). Using DD-PCR we found that
the GADD153 gene was preferentially expressed
during 4HPR-induced apoptosis in hepatoma cells. The induction
of the GADD153 gene by anticancer agents has been
reported to occur in a p53-independent fashion (42). We first
found that GADD153 overexpression was associated with the 4HPR-mediated
apoptosis in hepatoma cells as well as in other types of cancer cells,
regardless of the state of their p53 function. Furthermore, there was a
good correlation between increases in GADD153 mRNA
levels and the chemo-response rates of cisplatin in head and neck
cancers (43). Introduction of the GADD153 gene into gastric
cancer cells increased their sensitivity to anticancer drugs (44).
Thus, the sum of these results suggests that the GADD153
gene may be an important target gene for apoptotic cell death induced
by anticancer drugs.
The ability of 4HPR to activate the transfected GADD153
promoter is barely detectable in Hep 3B cells although it stabilized the GADD153 mRNA. Therefore, post-transcriptional
stabilization may play a major role in increasing GADD153
mRNA levels in response to this drug. However, in addition to the
increase in GADD153 mRNA stability induced by 4HPR, the
magnitude of the activation of the GADD153 promoter
substantially correlated with changes elicited in endogenous mRNA
levels in response to treatment with 4HPR in SK-HEP-1 cells. Thus, 4HPR
transcriptionally and post-transcriptionally regulates
GADD153 mRNA levels in SK-HEP-1 cells. The mRNA
levels of GADD genes have been shown to be regulated both at
the transcriptional and at the post-transcriptional levels.
Transcriptional mechanisms accounted for the induction of
GADD153 genes in cells treated with DNA-damaging agents. In
contrast, signals responsible for withdrawal from an active
proliferative state have been shown to stabilize GADD153
transcripts (45, 46). Treatment with methyl methanesulfonate or with UV
radiation stabilizes GADD153 mRNA in actively
proliferating cells, an effect that was not observed in growth-arrested
cells (47). Etoposide regulates GADD153 mRNA mainly at
the transcriptional level (37). In contrast, 4HPR regulates
GADD153 mRNA levels at the transcriptional and/or the post-transcriptional levels in a cell-specific manner.
The GADD153-transfected cells had increased sensitivity to
4HPR-mediated cytotoxicity, which was associated with cell death and
the formation of internucleosomal DNA ladders. Even confluence/nutrient depletion alone readily triggered apoptosis in those cells without any drug treatment. Other groups had previously reported that induction
of GADD153 correlated with the onset of apoptosis (37, 48). To date,
however, few studies have addressed the mechanistic link between
expression of GADD153 and cell death. Recently, overexpression of
GADD153 sensitized cells to ER stress through the down-regulation of
Bcl-2 expression. This down-regulation of Bcl-2 expression enhanced
oxidant injuries, e.g. depletion of cellular glutathione and
exaggerated production of ROS (14). Our study previously revealed that
4HPR did not distinctly change Bcl-2 protein levels in SK-HEP-1 cells,
and furthermore, that Hep 3B cells were defective in Bcl-2 expression
(29). Thus, the down-regulation of Bcl-2 by GADD153 overexpression did
not seem to be a causative role in hepatoma cells. Confluent culture or
nutrient depletion readily induces apoptosis in cells stably
transfected with GADD153. In support of this concept, the
overexpression of GADD153 by platelet-derived growth factor-BB in
vascular smooth muscle cells was reported to significantly reduce cell
viability and to induce apoptosis, when 100% confluency is reached
(49). GADD153 was found to be induced during serum starvation or
glutamine deprivation (40, 50). Thus, cell-cell contact or nutrient
depletion may critically trigger apoptosis in cells overexpressing GADD153.
3B-G1 and 3B-G5 cells showed inhibited cell growth, particularly at
G1 arrest, which is in accordance with a previous report wherein microinjection of GADD153 expression plasmids into NIH-3T3 cells blocked the cells from progressing from G1 to S
phase. However, a mechanistic link between the overexpression of
GADD153 and G1 arrest remains largely undefined. GADD153 is
a transcription factor that plays an important role in regulating the
expression of various genes (2). Thus, GADD153 overexpression could
activate downstream pathways of signal transduction G1
arrest in cancer cells. Previously, the tumor suppressor retinoblastoma
protein pRb, a central regulator of the G1/S phase
transition (51), was found to be highly up-regulated in 4HPR-treated
breast cancer cells (52), suggesting a link between the
anti-proliferative activity of the retinoid and pRb regulation.
Recently, the antiproliferative activity of 4HPR was reported to arise
from its capacity to maintain pRb in a de-phosphorylated growth-suppressive status in S-G2/M, possibly through
cyclin D1 mRNA down-regulation and inhibition of pRb-targeting Cdk2
and Cdk4 kinase activities against pRb (53). Thus, it should be further
elucidated whether pRb and Cdk regulation by 4HPR is mediated through
the overexpression of GADD153.
Treatment with 4HPR generates intracellular ROS (29). These free
radicals play a key role in the apoptotic response to 4HPR. Antioxidants effectively block the formation of these 4HPR-induced free
radicals and markedly suppress the apoptotic effects of the retinamide. In the present study we observed that NAC inhibits the
overexpression of GADD153 as much as it inhibits 4HPR-induced apoptosis, imply that ROS generation is the upstream pathway in the
overexpression of GADD153. Similarly, NAC prevents induction of the
GADD153 gene and accompanying apoptosis by etoposide (13). In agreement with this, in transfection experiments with an antisense expression plasmid of GADD153, the down-regulation of
GADD153 increased resistance to 4HPR-induced apoptosis in cells stably transfected with antisense GADD153.
Recently, NF- In summary, it is not currently known how overexpression of the
GADD153 gene connects to the downstream signal transduction pathway, which leads to apoptosis and cell growth arrest. However, our
data reveal that the higher expression of GADD153 correlates with the
greater induction of apoptosis and cell growth inhibition in
4HPR-treated cells and that modulation of GADD153 expression can change
the sensitivity of cancer cells to 4HPR. Thus, oxidative stress-mediated GADD153 overexpression appears to contribute to the
anticancer effects of 4HPR, and these anticancer effects may be
potentiated by parthenolide through the enhancement of GADD153 overexpression. Thus, further elucidation of the downstream signaling pathway(s) involved in 4HPR-induced apoptosis and cell growth arrest
will advance chemotherapeutic or chemopreventive strategies that use
4HPR to treat various cancer diseases.
We thank the R. W. Johnson Pharmaceutical
Research Institute for providing 4HPR and Dr. Nikki J. Holbrook for
providing plasmids.
*
This work was supported by the 21C Frontier Human Genome
Project grant.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Division of
Gastroenterology and Hepatology, Dept. of Internal Medicine, The
Research Inst. of Clinical Medicine, Chonbuk National University
Medical School and Hospital, 634-18 Keumam-dong, Dukjin-ku, Chonju,
Chonbuk 561-172, South Korea. Tel.: 82-63-250-1681; Fax:
82-63-254-1609; E-mail: daeghon@moak.chonbuk.ac.kr.
Published, JBC Papers in Press, July 22, 2002, DOI 10.1074/jbc.M205941200
The abbreviations used are:
4HPR, N-(4-hydroxyphenyl)retinamide;
RAR, retinoic acid
receptor;
DD-PCR, differential display-PCR;
DAPI, 4',6'-diamidino-z-phenylindole, NAC,
N-acetyl-L-cysteine;
PI, propidium iodide;
FCM, flow cytometry;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
ROS, reactive oxygen species;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate.
GADD153-mediated Anticancer Effects of
N-(4-Hydroxyphenyl)retinamide on Human Hepatoma Cells*
§,,, Division of Gastroenterology and Hepatology,
Department of Internal Medicine, Institute for Molecular Biology and
Genetics, Chonbuk National University Medical School and Hospital,
Chonju, Chonbuk 561-712 and the ¶ ICLAS Monitoring Subcenter
Korea, Korea Research Institute of Bioscience and Biotechnology, Taejon
305-600, South Korea
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 M and was stored in
aliquots at 
20 °C for a maximum of 2 weeks. 4',6'-diamidino-z-phenylindole (DAPI),
N-acetyl-L-cysteine (NAC), and propidium iodide
(PI) were purchased from Sigma. Parthenolide was obtained from
Calbiochem (San Diego, CA).
70 °C, as described previously (35).
As a loading control, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA was detected with a
GAPDH cDNA probe.
20 °C until assayed. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System
(Promega) as per the manufacturer's instructions and was normalized by
Renilla luciferase activity.
mercaptoethanol (v/v). Extracted proteins were resolved by 13%
SDS-PAGE and were transferred to nitrocellulose membranes. Membranes
were incubated overnight at 4 °C in the primary antibody and were
then incubated for 45 min in the secondary antibody. Following
incubation in the secondary antibody, blots were washed three times
with PBS/0.1% Tween, then developed using a commercial
chemiluminescence detection kit (Amersham Biosciences). GADD153
polyclonal antibody (F168) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Concentration-dependent effects
of 4HPR on apoptotic cell death of hepatoma cells and the induction of
endogenous GADD153 mRNA levels. A,
Hep 3B and SK-HEP cells (1 × 106) were treated with
the indicated concentrations of 4HPR for 72 h. Cells were
harvested and stained with trypan blue dye to determine cell viability.
Each point represents the mean ± S.E. of quadruplicate
determinations. B, Hep 3B and SK-HEP cells (1 × 106) were treated with equitoxic concentrations of 10 and
15 µM 4HPR, respectively, and were collected at 72 h
for FCM. The cells were stained with FITC-conjugated annexin V and PI
according to the manufacturer's instructions and were then analyzed by
FCM using a FACScan. The apoptotic fraction was estimated by gating
Annexin V-FITC positive cells. C, differentially
displayed bands from DD-PCR analysis between vehicle-treated cells
(Control) and 4HPR-treated cells (4HPR) using
1-base anchored oligo-dT primer
(H-T11C) in combination with
arbitrary 13 mers (5'-AAGCTTATGAAGG-3', H-AP56). Duplicate
samples were displayed on a denaturing 6% polyacrylamide gel. The
arrows indicate bands of GADD153 cDNA
specifically expressed in 4HPR-treated cells. D, total
RNAs from Hep 3B and SK-HEP-1 cells treated with 10 and 15 µM 4HPR, respectively, were fractionated by
electrophoresis on 1.0% agarose gels containing formaldehyde and were
then transferred to membranes. Blots were hybridized overnight with
2 × 106 cpm/ml GADD153 cDNA probe
labeled with [P32]dCTP by random-priming, washed, then
exposed to X-Omat AR film (Kodak) at 70 °C. The blots were
stripped and sequentially hybridized with a probe for GAPDH
cDNA as a loading control (upper panels). For
immunoblotting of GADD153, 30 µg of extracted proteins were resolved
by 13% SDS-PAGE and were transferred to the membrane. The blot was
probed with a polyclonal antibody to GADD153 (F168) and was then
stripped and reprobed with a monoclonal antibody to actin as a loading
control (lower panels).
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Fig. 2.
Effect of 4HPR on the induction
GADD153 mRNA levels in various types of cancer
cells. A, colon (HCT116 and HT29), lung (A549 and
NCI-H522), and breast (MCF7 and MDA-MB-231) cancer cells, according to
p53 function (wild-type and mutant type, respectively), and kidney
cancer cells with wild-type p53 (A498 and ACHN) were treated with
vehicle or 10 µM 4HPR for 48 h. Apoptotic cell death
was counted by staining with DAPI. Vertical bars represent
the means ± S.E. of three experiments performed in duplicate.
B, total RNAs from the cells treated with vehicle or 10 µM 4HPR were fractionated by electrophoresis on 1.0%
agarose gels containing formaldehyde and were then transferred to
membranes. The Northern blot was prepared and hybridized with the
GADD153 cDNA probe. The blot was stripped and
sequentially hybridized with a probe for GAPDH cDNA as a
loading control.
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Fig. 3.
Transcriptional and post-transcriptional
regulation of GADD153 mRNA in 4HPR-treated
hepatoma cells. A, luciferase activity measured in
Hep 3B and SK-HEP-1 cells transiently transfected with pGADD-LUC. Hep
3B and SK-HEP-1 cells were treated with 10 and 15 µM
4HPR, respectively, for the indicated time-intervals. Vertical
bars represent the means ± S.E. of three experiments
performed in duplicate. B, the decay of
GADD153 transcript in Hep 3B cells or SK-HEP-1 cells. The
cells were treated with 10 or 15 µM 4HPR, respectively,
for 48 h, and were then fed with 4HPR-free medium to which 4HPR
alone, actinomycin D (Act D, 5 µg/ml)+4HPR, or Act
D+vehicle was added, respectively. Total RNA was extracted from the
three sets at subsequent time point (upper). Autoradiograms
were read using densitometric scanning and were normalized against
GAPDH mRNA levels (lower).
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Fig. 4.
Effect of ectopic GADD153
overexpression on apoptosis induced by 4HPR treatment or by growth to
confluence. A, total RNAs from cells transfected
with the GADD153 expression plasmid (3B-G1 and 3B-G5) or the
vector control (3B-C1 and 3B-C2) and Hep 3B cells treated with 10 µM 4HPR or vehicle for 72 h, respectively, were
fractionated by electrophoresis on 1.0% agarose gels containing
formaldehyde, and were then transferred to membranes. The Northern blot
was prepared and hybridized with the GADD153 cDNA probe.
The blot was stripped and sequentially hybridized with a probe for
GAPDH cDNA as a loading control (upper
panels). Western blot analysis of GADD153 in cells transfected
with the GADD153 expression plasmid (3B-G1 and 3B-G5) or the
vector control (3B-C1 and 3B-C2) (lower panels).
B, effect of treatment with vehicle or 10 µM 4HPR for 72 h on cell death of Hep 3B cells
stably overexpressing GADD153. Apoptotic cell death was determined by
DAPI staining. Vertical bars represent the means ± S.E. of two experiments in duplicate (**, significantly different from
vector controls at p < 0.01). C,
effect of confluent culture on Hep 3B cells stably overexpressing
GADD153. Apoptotic cell death was determined by DAPI staining
(upper), agarose gel analysis of the formation of
internucleosomal DNA ladders (M, marker) (lower
left), and FCM analysis of the sub-G1 fraction from
the 3B-G5 cells stably overexpressing GADD153 after reaching confluence
(M1, apoptotic fraction) (lower
right). Vertical bars or values represent the
means ± S.E. of two experiments in duplicate (**, significantly
different from vector controls at p < 0.01).
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Fig. 5.
Inhibition of the cell cycle by ectopic
GADD153 overexpression. A, GADD153 overexpressing
(3B-G1, 3B-G5) and control (3B-C2) cells were plated in 96-well
microtiter plates at a density of 1 × 103 cells/well
and were incubated for 72 h. 24 h prior to harvest, 1 µCi
of [3H]-thymidine was added to each well, and the
incubation was continued for an additional 24 h. Cellular uptake
of [3H]thymidine was determined by liquid scintillation
counting. Assays were performed in triplicate, and the experiment was
repeated at least two times with similar results. Vertical
bars represent the means ± S.E. of three experiments
performed in duplicate (**, significantly different from vector control
at p < 0.01). B, in asynchronized
cells, the cell cycle distribution of the cells was performed 12 h
after seeding using a FACScan (as described under "Experimental
Procedures"). For synchronization, 3B-G5 and 3B-C2 cells were
arrested in M phase by treatment with 0.5 µg/ml nocodazole. Cells
were analyzed at 3-h intervals following release from the M block. Data
were analyzed as single-parameter frequency histograms
in the SFIT model. M1 represents a fraction of apoptotic
sub-G1 (Asyn, asynchronized; Syn,
synchronized).
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Fig. 6.
NAC or antisense GADD153
inhibits 4HPR-induced apoptosis. A, Hep
3B cells were treated with 10 µM 4HPR in the presence or
absence of 2 mM NAC for 72 h and apoptotic cell death
was determined by DAPI staining. Each bar represents the
mean ± S.E. of quadruplicate determinations (*, significantly
different from treatment with 4HPR alone at p < 0.05).
B, Western blot analysis of GADD153 expression. Cell
lysates were prepared from Hep 3B cells treated with 10 µM 4HPR in the presence (+) or absence ( ) of 2 mM NAC for 72 h. 30 µg of proteins were extracted
and resolved by 13% SDS-PAGE and were transferred to the membrane. The
blot was probed with a polyclonal antibody to GADD153 (F168) and was
then stripped and reprobed with a monoclonal antibody to actin as a
loading control. C, GADD153 expression in
Hep 3B cells transfected with antisense GADD153 cDNA or
empty vector. Cell lysates were prepared from Hep 3B cells treated with
10 µM 4HPR for 72 h. 30 µg of proteins were
extracted and resolved by 13% SDS-PAGE and were transferred to the
membrane. The blot was probed with a polyclonal antibody to GADD153
(F168) and was then stripped and reprobed with a monoclonal antibody to
actin as a loading control. D, Hep 3B cells transfected
with antisense GADD153 cDNA were treated with 10 µM 4HPR for 72 h. Apoptotic cell death was
determined by DAPI staining. Vertical bars represent the
means ± S.E. of quadruplicate experiments (* and **,
significantly different from vector controls at p < 0.05 and p < 0.01, respectively).
B
inhibitor, parthenolide, enhanced the GADD153 mRNA
overexpression induced by 4HPR as described previously (39). Therefore,
we determined the role of parthenolide on the apoptotic cell death
induced by 4HPR. Non-cytotoxic dose of parthenolide (4 µM) effectively enhanced 4HPR-induced GADD153 overexpression by about 2-fold in Hep 3B and in SK-HEP-1 cells (Fig.
7A), which in turn enhanced
4HPR-induced apoptosis by about 2-fold in Hep 3B and in SK-HEP-1 cells
(55.5 and 53.0%, respectively) (p < 0.01), compared
with cells treated with 4HPR alone (25.5 and 29.0%, respectively)
(Fig. 7B). Similarly, FCM analysis revealed that
parthenolide enhanced 4HPR-induced apoptosis by 2-fold
(p < 0.01) (Fig. 7C). The degree of
induction of GADD153 seemed to correlate with the increase of apoptotic
cell death. These results suggest that GADD153 overexpression modulates
the cell execution response to 4HPR in cancer cells.
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Fig. 7.
Parthenolide enhances
4HPR-induced apoptosis. A, cell lysates were
prepared from Hep 3B and SK-HEP-1 cells treated with 10 µM 4HPR in the presence (+) or absence ( ) of 4 µM parthenolide for 72 h. 30 µg of proteins were
extracted and resolved by 13% SDS-PAGE and were transferred to the
membrane. The blot was probed with a polyclonal antibody to GADD153
(F168) and was then stripped and reprobed with a monoclonal antibody to
actin as a loading control (lower) (P,
parthenolide). B, apoptotic cell death was
determined by DAPI staining in Hep 3B and SK-HEP-1 cells treated with
10 µM 4HPR in the presence (+) or absence () of 4 µM parthenolide for 48 h. Vertical bars
represent the means ± S.E. of quadruplicate experiments (**,
significantly different from treatment with 4HPR alone at
p < 0.01). C, quantitation of the
apoptotic fraction by FCM analysis in Hep 3B cells treated with 10 µM 4HPR in the presence (+) or absence () of 4 µM parthenolide for 48 h. The sub-G1
fraction was estimated by gating hypodiploid cells in the histogram
using the LYSIS II program. DNA contents are plotted on the
linear abscissa (M1, apoptotic
fraction). Each value represents the mean ± S.E. of triplicate
experiments (**, significantly different from treatment with 4HPR alone
at p < 0.01).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B was reported to repress GADD153 activation
and parthenolide was shown to sensitize breast cancer cells to ER
stress (39). Furthermore, parthenolide and IB
increase paclitaxel-induced apoptosis through inhibition of a distinct NF-B regulated cell survival pathway (38). Similarly, we
observed that NF-B was activated during 4-HPR-induced
apoptosis and that its inhibitor parthenolide enhanced the
overexpression of GADD153, which in turn correlated with enhanced
4HPR-induced apoptosis. However, parthenolide can have other biological
functions beyond NF-B inhibition because it is a small
chemical molecule. Thus, it should be further elucidated that
NF-B activation suppresses 4HPR-induced apoptosis through
inhibition of GADD153 expression. Thus, these results suggest that
modulation of GADD153 expression can affect 4HPR-induced apoptosis.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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