Human Cancer Biology
Expression of a Peroxisome Proliferator-Activated Receptor ;1
Splice Variant that Was Identified in Human Lung Cancers
Suppresses Cell Death Induced by Cisplatin
and Oxidative Stress
Hyo Jung Kim,1 Jin-Yong Hwang,2 Hyun Jun Kim,3 Wan Sung Choi,3 Jae Heun Lee,1 Hye Jung Kim,1
Ki Churl Chang,1 Toru Nishinaka,4 ChihiroYabe-Nishimura,4 and Han Geuk Seo1
Abstract
Purpose: The activation of peroxisome proliferator-activated receptor g (PPARg) has been
implicated in the inhibition of tumor progression in lung cancers through the induction of differentiation and apoptosis. Recently, we identified a novel splice variant of human PPARg1
(hPPARg1) that exhibits dominant-negative activity in human tumor-derived cell lines. This study
aimed to examine the expression and pathophysiologic roles of a truncated splice variant of
hPPARg1 (hPPARg1tr) in primary human lung cancer tissues.
Experimental Design: The expression and localization of hPPARg1tr was surveyed in human
primary lung cancer tissues using immunohistochemistry and Western blot analysis. Using transfectants stably expressing wild-type hPPARg1 (hPPARg1wt) and hPPARg1tr, we also analyzed the
pathophysiologic roles of hPPARg1tr.
Results: We showed that PPARg is expressed predominantly in the nucleus of nontumorous
tissues, whereas it is present in both the nucleus and the cytoplasm of tumorous tissues in
squamous cell carcinoma (SCC) of the lung. Western blot analysis confirmed the presence of
PPARg1tr in primary lung SCC tissue but not in nontumorous tissue. Expression of PPARg1tr in
Chinese hamster ovary cells attenuated their susceptibility to cell death induced by oxidative
stress or cisplatin, whereas their susceptibility was completely recovered by down-regulation of
PPARg1tr with small interfering RNA.
Conclusions: hPPARg1tr is expressed strongly in tumorous tissues of primary human lung SCC
and its overexpression renders transfected cells more resistant to chemotherapeutic drug- and
chemical-induced cell death. These data suggest that the decreased drug sensitivity of
PPARg1tr-expressing cells may be associated with increased tumor aggressiveness and poor
clinical prognosis of patients.
Peroxisome proliferator-activated receptors (PPAR) are members of a superfamily of nuclear hormone receptors involved in
a wide range of physiologic roles, such as lipid homeostasis,
energy metabolism, inflammation, and cellular differentiation
and proliferation (1 – 4). Three different isoforms have been
Authors’ Affiliations: Departments of 1Pharmacology, 2Internal Medicine, and
3
Anatomy, Gyeongsang Institute of Health Science, College of Medicine,
Gyeongsang National University, Jinju, Korea and 4Department of Pharmacology,
Kyoto Prefectural University of Medicine, Kyoto, Japan
Received 8/17/06; revised 2/14/07; accepted 2/20/07.
Grant support: The Korean Science and Engineering Foundation grant funded by
the Korea government grant R13-2005-012-01003-0 (H.G. Seo and W.S. Choi);
Korea Research Foundation grant KRF-2006-005-J04202 (H.G. Seo); and
Technology Development Program for Agriculture and Forestry, Ministry of
Agriculture and Forestry, Republic of Korea (H.G. Seo).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Han Geuk Seo, Department of Pharmacology, College of
Medicine, Gyeongsang National University, 92 Chilam-Dong, Jinju 660-751, Korea.
Phone: 82-55-751-8773; Fax: 82-55-759-0609; E-mail: hgseo@ gnu.ac.kr.
F 2007 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-06-2062
www.aacrjournals.org
identified: PPARa (NR1C1), PPARy (NR1C2, also known as
PPARh, FAAR, and NUC1), and PPARg (NR1C3; refs. 5, 6).
PPARg is the most intensively studied isoform, participating in
a wide variety of biological pathways and disease conditions,
such as insulin sensitivity, type 2 diabetes, atherosclerosis, and
cancer (1). The human PPARc (hPPARg) gene has nine exons,
resulting in distinct mRNAs (PPARc1, PPARc2, PPARc3, and
PPARc4) that differ through alternative promoter usage and
splicing (7). Other splice variants have been identified in
macrophages and sporadic colorectal cancers (8, 9). The
expression of PPARg is observed in a variety of human tumors
and tumor-derived cell lines, including those from lung, breast,
prostate, gastric, and colon cancers (10 – 15). In lung cancers,
the expression of PPARc mRNA was found to be significantly
lower in tumorous tissue than in adjacent nontumorous tissues,
and there was a correlation between lower survival rates and
decreased PPARg expression (16, 17). In several tumor-derived
cell lines, PPARg ligands have been implicated in the induction
of growth arrest and morphologic changes associated with
differentiation and apoptosis (12, 18, 19). Furthermore,
activation of PPARg inhibits tumor progression via induction
of differentiation and apoptosis in several lung cancers and
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Human Cancer Biology
lung cancer – derived cell lines (12). In contrast, mutations or
alternative splicing of transcripts that result in impaired PPARg
ligand binding have been identified in sporadic colon cancers
(9, 20). Naturally occurring somatic mutations and splicing
variants may alter the efficacy of treatment with PPARg ligands
in cancer chemotherapy. Although the therapeutic effectiveness
of PPARg agonists has been shown for several cancer cell lines,
variations in response to these agonists have not been fully
elucidated and the molecular mechanisms underlying their
therapeutic effects remain unclear.
We identified recently a novel splicing variant of hPPARg1 in
human tumor-derived cell lines (PPARg1tr) that was generated
from insertion of a novel exon 3¶ (21). Insertion of this exon
results in the introduction of a premature stop codon and thus
PPARg1tr lacks part of the hinge region, as well as the entire
ligand binding domain. Because PPARg1tr interferes with the
transcriptional activity of wild-type PPARg1 (PPARg1wt), its
expression in cancer cells seems to antagonize the antitumor
activity of PPARg1wt. Therefore, these findings suggest a
potential role for PPARg1tr as a key molecule in the variations
in response to PPARg ligands that are observed in cancer
chemotherapy. To elucidate the role of PPARg1tr in the
therapeutic effectiveness of PPARg agonists, we investigated
its expression levels in tumorous and nontumorous tissues of
primary human lung cancer. We also examined the effects of
PPARg1tr overexpression on the susceptibility of Chinese
hamster ovary (CHO) cells to apoptosis induced by oxidative
stress and cisplatin.
Here, we report that the truncated splice variant of hPPARg1
(hPPARg1tr) was detected in primary lung squamous cell
carcinoma (SCC) tissue but not in nontumorous tissue. In
primary human lung SCC, PPARg was expressed predominantly in the nucleus of nontumorous tissues, whereas hPPARg1tr
was present in both the nucleus and the cytoplasm of tumorous
tissues. Furthermore, CHO cells expressing PPARg1tr were more
resistant to chemotherapeutic drug- or chemical-induced cell
death, through the regulation of expression of several apoptotic-related proteins.
Materials and Methods
Cell culture. CHO cells were maintained in DMEM supplemented
with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin
G, and 100 Ag/mL streptomycin at 37jC and 5% CO2.
Plasmid construction. Mammalian expression vectors were constructed by PCR amplification of fragments from pSG5-hPPARg1wt and
pSG5-hPPARg1tr (21) using the primers 5¶-GGGGTACCTGGCCGCAG A A A T G A C C A T - 3 ¶ a n d 5 ¶- C C G C T C G A G C T A G T A C A A G T A CAAGTCCTT-3¶. These fragments were digested with KpnI/XhoI and
ligated into the similarly digested mammalian expression vector
pcDNA3.1/Hygro(+) (Stratagene), generating pcDNA3.1-hPPARg1wt
and pcDNA3.1-hPPARg1tr, respectively. All recombinant plasmids were
sequenced.
Generation of monoclonal antibody specific to hPPARg1tr. The
hPPARg1tr-specific peptide (EELQKDSY) was synthesized with a
terminal cysteine; monoclonal antibody (mAb) was generated as
described previously (21).
Preparation and immunohistochemistry of tumor tissue samples. Tumor
and adjacent nontumor tissue samples were obtained from 11 patients
with non – small cell lung carcinoma. Patients who underwent surgical
resection at the Department of Thoracic Surgery, Gyeongsang National
University Hospital (Jinju, Korea) provided informed consent. This study
Clin Cancer Res 2007;13(9) May 1, 2007
was approved by the Ethics Committee of the medical school of
Gyeongsang National University. All tumor (10 SCCs and 1 adenocarcinoma) and nontumor tissue specimens were verified by histologic
examination. Staining was done using the manufacturer’s protocol
(avidin-biotin complex method kit, Vector Laboratories). Immunofluorescent histochemistry was done using CyII- or CyIII-conjugated
secondary antibodies and observed by fluorescence microscopy (Olympus). Nuclei were identified using Hoechst 33258 or 4¶,6-diamidino2-phenylindole (DAPI) counterstaining.
Western blot analysis. All cells and tissues were washed in ice-cold
PBS and lysed in PRO-PREP Protein Extraction Solution (iNtRON
Biotechnology). Supernatants were separated using 10% SDS-PAGE and
transferred to a Hybond-P+ polyvinylidene difluoride membrane
(Amersham Biosciences). Membranes were probed with specific antibodies as described previously (22).
Reporter gene assay. The PPRE3-tk-luc and pGL2-aP2 luciferase
reporter vectors were generously provided by Dr. Frank J. Gonzalez
(National Cancer Institute, NIH, Bethesda, MD) and Dr. Hiroshi
Wakao (Helix Research Institute, Chiba, Japan), respectively. CHO cells
(1 105 per well) seeded into six-well tissue culture plates for
24 h before transfection were transfected with 0.5 Ag pSV h-gal (SV40
h-galactosidase expression vector; Promega) in the presence or absence
of 0.5 Ag PPRE3-tk-luc, 1 Ag pGL2-aP2, 1 Ag pSG5-hPPARg1wt, and 1 Ag
pSG5-hPPARg1tr using SuperFect reagent (Qiagen). After incubation for
4 h, cells were provided with fresh medium and incubated for 24 h.
Cells were incubated for a further 24 h in medium containing
troglitazone (50 nmol/L) or DMSO. Luciferase activity was determined
as described previously (23).
Identification of apoptosis by DAPI staining and fluorescence-activated
cell sorting. Stable CHO transfectants expressing wild-type hPPARg1
(hPPARg1wt) or hPPARg1tr were generated by transfection with
pcDNA3.1-hPPARg1wt or pcDNA3.1-hPPARg1tr followed by screening
with 600 Ag/mL hygromycin. Stably transfected cells cultured to 80%
confluence were treated with 2 mmol/L H2O2 or 30 Amol/L cisplatin for
9 or 12 h, respectively. Fixed cells were stained with DAPI (2 Ag/mL) for
30 min at room temperature, washed, and then visualized using an
Olympus JP/1X71 fluorescence microscope. Apoptotic cells were
counted by an independent observer over four individual low-power
fields. For analysis of apoptosis using fluorescence-activated cell sorting,
cells treated with H2O2 or cisplatin as above were fixed and
resuspended in propidium iodide staining solution. Following incubation in the dark for 30 min at room temperature, cell cycle profiles were
determined using a FACSCalibur (Becton Dickinson Biosciences).
At least 20,000 cells were analyzed from each sample.
Terminal deoxynucleotidyl transferase – mediated dUTP nick end
labeling assay. Apoptotic cells were identified by terminal deoxynucleotidyl transferase – mediated dUTP nick end labeling (TUNEL) using
an In situ Cell Death Detection kit (Roche Applied Science) and a
confocal laser scanning microscope (Olympus). TUNEL-positive cells
were counted from at least four randomly chosen fields.
Small interfering RNA studies. Stably transfected cells expressing
PPARg1wt or PPARg1tr were transfected with 80 nmol/L control small
interfering RNA (siRNA) or PPARg siRNA (Ambion) using Welfect-Q
(WelGENE). After 72 h, cells were treated with 2 mmol/L H2O2 or
30 Amol/L cisplatin for 9 or 12 h, respectively.
Statistical analysis. The Student’s t test was used to compare means.
All data are expressed as means F SE.
Results
Differential expression of PPARg1tr in human lung SCC
tissues. The expression patterns of hPPARg1tr and hPPARg1wt
were compared between primary SCC and surrounding normal
tissues. Immunohistochemical analysis indicated that hPPARg
was present at higher levels in tumor tissues than in surrounding normal tissues (Fig. 1A). To localize hPPARg in the
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Expression of a PPARg1SpliceVariant in Lung Cancer
Fig. 1. Immunohistochemical analysis
of PPARg expression in nontumorous
and tumorous regions of primary
lung SCC tissues. A, representative
3,3¶-diaminobenzidine staining of PPARg in
paraffin-embedded cancer tissue (a) and
adjacent normal lung tissue (b) using an
anti-hPPARg1wt NH2-terminal antibody.
Bars, = 50 Am. B, localization of hPPARg in
normal lung tissue (top) and in cancer tissue
(middle). High-resolution photomicrographs
of cancer tissue (bottom). Immunopositive
signals were obtained for hPPARg using an
anti-hPPARg NH2-terminal antibody (red)
and anti-hPPARg COOH-terminal
antibody (green). c, g, and k, merged
images of (a/b), (e/f), and (i/j), respectively.
d, h, and l, Hoechst 33258 ^ stained cells.
Bars, 50 Am (d and h) and 30 Am (l).
Immunohistochemistry was done at least
thrice using different sections and similar
results were obtained each time.
tumor and surrounding normal regions, we used anti-hPPARg
NH2- and COOH-terminal – specific antibodies conjugated
with CyIII fluorescence (red) and CyII fluorescence (green),
respectively. In normal tissue, the individual and merged
images indicate that both antibodies hybridized to the nuclei
(Fig. 1B, a-c); this finding was corroborated by the overlap with
Hoechst 33258 staining (Fig. 1B, d). In contrast, the level of
hPPARg expression was much higher in tumor tissue (Fig. 1B,
e and f). Broadly stained cells were observed using the antihPPARg NH2-terminal antibody (Fig. 1B, e and i), whereas the
anti-hPPARg COOH-terminal antibody predominantly stained
nuclei (Fig. 1B, f and j). In the merged images, the yellow color
was found in foci corresponding to nuclei (Fig. 1B, g and k),
a finding supported by overlap with Hoechst 33258 staining
(Fig. 1B, h and l).
To investigate the expression of hPPARg1tr in primary human
lung cancer, we examined tissue from 11 patients with non –
small cell lung carcinoma with anti-hPPARg NH2-terminal
antibody. We identified the band corresponding to hPPARg1tr
in nine samples from tumorous tissue regions of SCC but not in
any of the surrounding normal tissues (Fig. 2A).
Detection of PPARg1tr using a mAb specific to PPARg1tr in
human lung SCC tissues. To confirm whether hPPARg1tr is
the protein that is not recognized by the anti-hPPARg
COOH-terminal – specific antibody, we raised a mAb against a
COOH-terminal hPPARg1tr – specific amino acid sequence.
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This antibody cross-reacted with the smaller band recognized
by the anti-hPPARg NH2-terminal antibody, confirming
specificity (Fig. 2B). We then did an immunohistochemical
analysis of the lung SCC tissue samples using anti-hPPARg1tr –
specific mAb. Consistent with earlier results, hPPARg1tr was
expressed strongly in tumorous regions relative to the
surrounding normal lung tissues (Fig. 2C, a and b). In addition, it was detected in both nucleic and cytoplasmic compartments of SCC, as confirmed by merging with DAPI-stained
images (Fig. 2C, d-f). These data strongly suggest that
hPPARg1tr is expressed in both the cytoplasm and the nucleus
of lung SCC.
Stable expression of hPPARg1wt and hPPARg1tr in CHO
cells. To analyze the pathophysiologic roles of hPPARg1tr,
stable transfectants expressing hPPARg1wt or hPPARg1tr were
selected in CHO cells. Western blot analysis showed a clear
increase in hPPARg1wt and hPPARg1tr expression in each
transfectant (Fig. 3A).
Because binding to PPARg on the PPAR response element
(PPRE) is a common event following troglitazone stimulus,
we tested the effects of troglitazone on activity of a reporter
construct (PPRE3-tk-luc) containing three copies of PPRE
originating from rat ACOX. Troglitazone treatment of stable
transfectants resulted in increased luciferase activity in hPPARg1wtexpressing cells but not in hPPARg1tr-expressing cells (Fig. 3B).
We then tested whether hPPARg1wt or hPPARg1tr affects
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Fig. 2. Western blot analysis of PPARg
expression in nontumorous and tumorous
regions of primary lung SCC tissues.
N and T, adjacent nontumorous tissues
and tumorous tissues, respectively.
A, PPARg expression detected with an
NH2-terminal ^ specific antibody. Seventy
micrograms of total protein were analyzed.
WT, hPPARg1wt ; TR, hPPARg1tr ; NS,
nonspecific. B, detection of PPARg1
expression. A duplicate membrane
containing the same protein was probed
with the antibodies indicated. C, detection
of hPPARg1tr in nontumorous and tumorous
regions of primary lung SCC using an
anti-hPPARg1tr monoclonal antibody.
a, nontumorous region; b, tumorous region;
c, negative control; a tumorous region was
stained without primary antibody. d to f,
high-resolution photomicrographs.
hPPARg1tr was visualized using a
CyIII-conjugated secondary antibody (red).
Nuclei were stained with DAPI. f, merged
image of (d/e). Bars, 50 Am (c) and 30 Am
(f). Immunohistochemistry was done at
least thrice using different sections and
similar results were obtained each time.
transcriptional activation of a target gene. In hPPARg1wtexpressing cells, troglitazone treatment enhanced the luciferase
activity of an aP2 gene (reporter construct contains -1 to -5.4 kb
of the aP2 promoter; Fig. 3B).
As shown in Fig. 3B and C, cotransfection of PPRE3-tk-luc
with hPPARg1wt in the presence of troglitazone significantly
increased the luciferase activity. On the other hand, cotrans-
fection of increasing amounts of hPPARg1tr with constant
amount of hPPARg1wt dose dependently suppressed the
reporter activity (Fig. 3C). These results suggest that hPPARg1tr
interacts with the wild-type in a dominant-negative manner.
Effects of hPPARg1tr overexpression on apoptotic cell death
induced by multiple stimuli. We treated stably transfected CHO
cells expressing pcDNA 3.1, hPPARg1tr, or hPPARg1wt with
Fig. 3. Isolation and characterization of
stable transfectants expressing pcDNA3.1
(CTR), pcDNA3.1-hPPARg1wt (WT), or
pcDNA3.1-hPPARg1tr (TR). A, protein
expression.Total lysates were analyzed by
Western blotting with an NH2-terminal ^
specific anti-PPARg or h-actin antibody.
Similar results were obtained in three
independent experiments. B, effects of
troglitazone on the transcriptional activity of
hPPARg1wt or hPPARg1tr. CHO cells
transfected with luciferase reporter
constructs, pGL2-aP2 or PPRE3-tk-luc, were
treated for 24 h with either troglitazone or
DMSO.The activities of the reporter plasmid
are indicated as the ratio to the activity of
pcDNA3.1-expressing cells transfected with
pGL2-aP2 or PPRE3-tk-luc. C, top, effects of
increasing amounts of hPPARg1tr on the
transcriptional activity of hPPARg1wt. CHO
cells were transfected with increasing
amounts of pSG5-hPPARg1tr (0.01, 0.1, and
1.0 Ag) along with a constant amount of
pSG5-hPPARg1wt (1.0 Ag) and the luciferase
reporter construct PPRE3-tk-luc (0.5 Ag).
Cells were treated for 24 h with either
troglitazone (black columns) or DMSO
(white columns).The activity of the reporter
plasmid is indicated as the ratio to the activity
of cells transfected with pSG5 vector alone.
Bottom, in parallel, total lysates from
transfected cells were analyzed byWestern
blotting with an NH2-terminal ^ specific
anti-PPARg or h-actin antibody. Columns,
mean of four to six independent
transfections; bars, SE.
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Expression of a PPARg1SpliceVariant in Lung Cancer
apoptotic stimuli (H2O2 and cisplatin). Using DAPI staining to
identify punctuate nuclei, a hallmark of apoptotic cells, we
observed a higher incidence of apoptosis for cells expressing
pcDNA 3.1 or hPPARg1wt than for those expressing hPPARg1tr
(Fig. 4A). This difference in sensitivity to apoptotic stimuli was
supported by TUNEL analysis. H2O2 induced 26%, 32%, and
12% TUNEL-positive cells in pcDNA 3.1-transfected cells,
hPPARg1wt-expressing cells, and hPPARg1tr-expressing cells,
respectively. Similar results were also obtained for cisplatintreated cells (Fig. 4B).
We examined the expression patterns of key proteins
regulating cell proliferation and apoptosis in CHO cells.
Following treatment with cisplatin, there was little difference
between cells expressing pcDNA 3.1 or hPPARg1wt. In contrast,
expression of the apoptotic proteins p53, Bax, cytochrome c,
caspase-3, and caspase-9 markedly decreased in hPPARg1trexpressing cells, whereas expression of the antiapoptotic
proteins Bcl-xL and Bcl-2 increased slightly and expression of
the cyclin-dependent kinase inhibitor p21cip1/waf1 remained
unchanged (Fig. 4C).
Effects of siRNA against hPPARg on apoptotic cell death. To
verify the role of hPPARg1tr in resistance to apoptotic cell
death induced by H2O2 or cisplatin, effects of siRNA against
hPPARg were examined. The levels of hPPARg1 and hPPARg1tr
in CHO cells stably expressing hPPARg1wt or hPPARg1tr were
significantly reduced when cells were transfected with hPPARg
siRNA but not with control siRNA consisting of a pool of nonspecific sequence (Fig. 5A). The down-regulation of either
hPPARg1wt or hPPARg1tr with siRNA against hPPARg counteracted the expression pattern of apoptosis-related proteins
(Fig. 5B). Fluorescence-activated cell sorting analysis indicated
that H2O2 or cisplatin caused a marked apoptosis in cells expressing pcDNA 3.1 or hPPARg1wt compared with hPPARg1tr-
expressing cells. Whereas the population of apoptotic cells was
reduced in hPPARg1wt-expressing cells transfected with siRNA
against hPPARg, resistance to cell death was almost abolished
in hPPARg1tr-expressing cells transfected with hPPARg siRNA
(Fig. 5C). Similar results were observed for cisplatin-induced
apoptosis. Accordingly, overexpression of hPPARg1tr was
suggested to confer resistance to cell death through expressional
regulation of apoptotic proteins.
Discussion
We have shown that PPARg is expressed in primary human
lung SCC tissues and that PPARg1tr, which exhibits dominantnegative activity on the wild-type isoform, is expressed
predominantly in tumorous regions of SCC tissues but not in
nontumorous tissues. In colon and pancreatic cancers, the
expression level of PPARg is elevated relative to normal tissues
(24, 25). In contrast, PPARg expression has been reported
to be down-regulated in esophageal and lung cancers, and
this decreased expression correlates with a poor prognosis
(16, 26). We presently found an increase in the expression of
PPARg in primary human lung SCC tissues compared with
adjacent normal tissues. Although our data are consistent
with previous studies in primary tumor from non – small cell
lung carcinoma (27), they are at variance with the mRNA
levels of PPARc reported in esophageal cancer (26). Immunohistochemical examinations using anti-PPARg NH2-terminal
and COOH-terminal antibodies and anti-PPARg1tr antibodies
also revealed a significant difference in PPARg expression
between nontumorous and tumorous regions of primary lung
SCC tissues. Immunoreactive signals of wild-type hPPARg
(hPPARgwt) were detected in the nuclei of tumorous and
nontumorous tissue, whereas those of hPPARg1tr were limited
Fig. 4. Effects of hPPARg1wt or hPPARg1tr
overexpression on apoptotic cell death and
apoptosis-related proteins. A and B,
detection of apoptotic cells. CHO cells
stably expressing the pcDNA3.1,
hPPARg1wt, or hPPARg1tr were treated with
H2O2 or cisplatin. Following DAPI (A) or
TUNEL (B) staining, cells were chosen from
at least four randomly selected fields and
scored for condensed and fragmented
nuclei or aTUNEL-positive reaction
(n = 100 each). Columns, mean of three
separate determinations; bars, SE.
c, P < 0.01, compared with pcDNA 3.1
treated with H2O2; cc, P < 0.01, compared
with hPPARg1wt treated with H2O2;
#, P < 0.01, compared with pcDNA 3.1
treated with cisplatin; ##, P < 0.01,
compared with hPPARg1wt treated with
cisplatin; b, P < 0.05, compared with
pcDNA 3.1treated with H2O2; bb, P < 0.05,
compared with hPPARg1wt treated with
H2O2; *, P < 0.05, compared with pcDNA
3.1treated with cisplatin; **, P < 0.05,
compared with hPPARg1wt treated with
cisplatin. C, expression of apoptosis-related
proteins induced by cisplatin. Total protein
was extracted from CHO cells treated with
30 Amol/L cisplatin for 12 h and analyzed by
Western blotting using specific antibodies
against the proteins indicated.
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Fig. 5. Effects of siRNA against hPPARg on
apoptotic cell death. A, CHO cells stably
expressing hPPARg1wt or hPPARg1tr were
transfected with hPPARg siRNA or
nonspecific control siRNA. Cells were
harvested at 72 h after transfection
and proteins from cell extracts were
immunoblotted with an NH2-terminal ^
specific antibody or an anti-h-actin
antibody. B, expression of apoptosis-related
proteins induced by cisplatin. At 72 h
after the transfection of hPPARg1wt- or
hPPARg1tr- expressing cells with hPPARg
siRNA or control siRNA, cells were treated
with cisplatin. C, cell cycle analysis of cells
expressing hPPARg1wt or hPPARg1tr with or
without hPPARg siRNA or control siRNA.
Cells treated with H2O2 or cisplatin were
stained with propidium iodide. Percentages
of cells with sub-G1 DNA. Representative
histograms from three independent
experiments.
to the cytoplasm of tumorous tissue. Western blot analysis
also confirmed the presence of PPARg1tr in primary lung SCC.
These results support the finding that expression of PPARg
is located in nuclei of nontumorous tissues, whereas it is
present in both the nuclei and the cytoplasm of lung cancer
tissues (17). It remains to be investigated whether the poor
prognosis for patients with esophageal and lung cancers
that exhibit low levels of PPARg (16, 26) is associated with
differences in the localization of PPARg in cellular compartments.
We have shown the presence of hPPARg1tr in human
primary lung SCC and it seems to exert a dominant-negative
effect, repressing activity of hPPARgwt. Most notably, the
expression of hPPARg1tr in CHO cells markedly affected
susceptibility to apoptosis through regulation of the expression of several apoptosis-related proteins. Previously, we
showed that the activity of pSG5-hPPARg1wt was antagonized by a 10-fold lower level of pSG5-hPPARg1tr (21). These
results suggested that even a small amount of hPPARg1tr may
interfere with the signaling pathways and expression of
genes governed by hPPARg1wt. Thiazolidinedione (a PPARgspecific ligand) induces growth arrest in human lung cancer
cells through the induction of apoptosis and differentiation
(12, 28). As hPPARg1tr lacks a ligand binding domain, specific
ligands may be unable to activate hPPARg1tr and inhibit cancer
growth. Further studies will be necessary to determine whether
there is any correlation between the expression levels of
hPPARg1tr and the prognosis of esophageal or lung cancer
patients.
Clin Cancer Res 2007;13(9) May 1, 2007
Overexpression of hPPARg1tr seemed to induce resistance to
the cellular responses invoked by exogenous apoptotic stimuli.
Loss of function by somatic mutations or alternative splicing of
hPPARc transcripts have been identified in primary sporadic
colon cancers (9, 20) and in type 2 diabetic patients with severe
insulin resistance (29). A growing number of studies have
highlighted the role of dysregulated alternative splicing in the
progression of human diseases (30) and genomic analysis
suggests the presence of several cancer-related splice isoforms
(31). Thus, the generation of hPPARg1tr by alternative splicing
may represent a general mechanism for regulation of PPAR
activity under different pathophysiologic conditions. Whether
it is the presence of hPPARg1tr that causes tumor development
or that the formation of a tumor evokes the generation of
hPPARg1tr remains unclear. However, our findings suggest that
expression of hPPARg1tr in cancerous tissues may disrupt the
cellular apoptotic signaling pathways.
In conclusion, the present study suggests the participation of
the novel isoform of PPARg1 in the development of lung
cancer. It is of particular interest that the growth arrest
caused by PPARg ligands might be alleviated in cells expressing
truncated products of the PPARc gene. Expression of hPPARg1tr
may also affect the apoptotic signaling pathways of cancer cells.
In this context, the presence of hPPARg1tr in tissues adjacent to
the tumor region could serve as a diagnostic marker and predict
resistance to such chemotherapeutic agents as cisplatin.
Identification of the PPARg1 isoform in cancer cells may aid
in the development of new therapeutic strategies in cancer
treatment.
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Expression of a PPARg1SpliceVariant in Lung Cancer
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Clin Cancer Res 2007;13(9) May 1, 2007
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Research.
Expression of a Peroxisome Proliferator-Activated Receptor
γ1 Splice Variant that Was Identified in Human Lung Cancers
Suppresses Cell Death Induced by Cisplatin and Oxidative
Stress
Hyo Jung Kim, Jin-Yong Hwang, Hyun Jun Kim, et al.
Clin Cancer Res 2007;13:2577-2583.
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