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Clinical Cancer Research 13, 2577-2583, May 1, 2007. doi: 10.1158/1078-0432.CCR-06-2062
© 2007 American Association for Cancer Research
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Human Cancer Biology

Expression of a Peroxisome Proliferator-Activated Receptor {gamma}1 Splice Variant that Was Identified in Human Lung Cancers Suppresses Cell Death Induced by Cisplatin and Oxidative Stress

Hyo Jung Kim1, Jin-Yong Hwang2, Hyun Jun Kim3, Wan Sung Choi3, Jae Heun Lee1, Hye Jung Kim1, Ki Churl Chang1, Toru Nishinaka4, Chihiro Yabe-Nishimura4 and Han Geuk Seo1

Authors' Affiliations: Departments of 1 Pharmacology, 2 Internal Medicine, and 3 Anatomy, Gyeongsang Institute of Health Science, College of Medicine, Gyeongsang National University, Jinju, Korea and 4 Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto, Japan

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{at}gnu.ac.kr.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
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Purpose: The activation of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) 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 PPAR{gamma}1 (hPPAR{gamma}1) 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 hPPAR{gamma}1 (hPPAR{gamma}1tr) in primary human lung cancer tissues.

Experimental Design: The expression and localization of hPPAR{gamma}1tr was surveyed in human primary lung cancer tissues using immunohistochemistry and Western blot analysis. Using transfectants stably expressing wild-type hPPAR{gamma}1 (hPPAR{gamma}1wt) and hPPAR{gamma}1tr, we also analyzed the pathophysiologic roles of hPPAR{gamma}1tr.

Results: We showed that PPAR{gamma} 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 PPAR{gamma}1tr in primary lung SCC tissue but not in nontumorous tissue. Expression of PPAR{gamma}1tr 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 PPAR{gamma}1tr with small interfering RNA.

Conclusions: hPPAR{gamma}1tr 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 PPAR{gamma}1tr-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 identified: PPAR{alpha} (NR1C1), PPAR{delta} (NR1C2, also known as PPARß, FAAR, and NUC1), and PPAR{gamma} (NR1C3; refs. 5, 6). PPAR{gamma} 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 PPAR{gamma} (hPPAR{gamma}) gene has nine exons, resulting in distinct mRNAs (PPAR{gamma}1, PPAR{gamma}2, PPAR{gamma}3, and PPAR{gamma}4) 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 PPAR{gamma} 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 PPAR{gamma} 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 PPAR{gamma} expression (16, 17). In several tumor-derived cell lines, PPAR{gamma} ligands have been implicated in the induction of growth arrest and morphologic changes associated with differentiation and apoptosis (12, 18, 19). Furthermore, activation of PPAR{gamma} inhibits tumor progression via induction of differentiation and apoptosis in several lung cancers and lung cancer–derived cell lines (12). In contrast, mutations or alternative splicing of transcripts that result in impaired PPAR{gamma} 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 PPAR{gamma} ligands in cancer chemotherapy. Although the therapeutic effectiveness of PPAR{gamma} 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 hPPAR{gamma}1 in human tumor-derived cell lines (PPAR{gamma}1tr) 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 PPAR{gamma}1tr lacks part of the hinge region, as well as the entire ligand binding domain. Because PPAR{gamma}1tr interferes with the transcriptional activity of wild-type PPAR{gamma}1 (PPAR{gamma}1wt), its expression in cancer cells seems to antagonize the antitumor activity of PPAR{gamma}1wt. Therefore, these findings suggest a potential role for PPAR{gamma}1tr as a key molecule in the variations in response to PPAR{gamma} ligands that are observed in cancer chemotherapy. To elucidate the role of PPAR{gamma}1tr in the therapeutic effectiveness of PPAR{gamma} agonists, we investigated its expression levels in tumorous and nontumorous tissues of primary human lung cancer. We also examined the effects of PPAR{gamma}1tr 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 hPPAR{gamma}1 (hPPAR{gamma}1tr) was detected in primary lung squamous cell carcinoma (SCC) tissue but not in nontumorous tissue. In primary human lung SCC, PPAR{gamma} was expressed predominantly in the nucleus of nontumorous tissues, whereas hPPAR{gamma}1tr was present in both the nucleus and the cytoplasm of tumorous tissues. Furthermore, CHO cells expressing PPAR{gamma}1tr were more resistant to chemotherapeutic drug- or chemical-induced cell death, through the regulation of expression of several apoptotic-related proteins.


   Materials and Methods
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Cell culture. CHO cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin G, and 100 µg/mL streptomycin at 37°C and 5% CO2.

Plasmid construction. Mammalian expression vectors were constructed by PCR amplification of fragments from pSG5-hPPAR{gamma}1wt and pSG5-hPPAR{gamma}1tr (21) using the primers 5'-GGGGTACCTGGCCGCAGAAATGACCAT-3' and 5'-CCGCTCGAGCTAGTACAAGTACAAGTCCTT-3'. These fragments were digested with KpnI/XhoI and ligated into the similarly digested mammalian expression vector pcDNA3.1/Hygro(+) (Stratagene), generating pcDNA3.1-hPPAR{gamma}1wt and pcDNA3.1-hPPAR{gamma}1tr, respectively. All recombinant plasmids were sequenced.

Generation of monoclonal antibody specific to hPPAR{gamma}1tr. The hPPAR{gamma}1tr-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 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-diamidino-2-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 x 105 per well) seeded into six-well tissue culture plates for 24 h before transfection were transfected with 0.5 µg pSV ß-gal (SV40 ß-galactosidase expression vector; Promega) in the presence or absence of 0.5 µg PPRE3-tk-luc, 1 µg pGL2-aP2, 1 µg pSG5-hPPAR{gamma}1wt, and 1 µg pSG5-hPPAR{gamma}1tr 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 hPPAR{gamma}1 (hPPAR{gamma}1wt) or hPPAR{gamma}1tr were generated by transfection with pcDNA3.1-hPPAR{gamma}1wt or pcDNA3.1-hPPAR{gamma}1tr followed by screening with 600 µg/mL hygromycin. Stably transfected cells cultured to 80% confluence were treated with 2 mmol/L H2O2 or 30 µmol/L cisplatin for 9 or 12 h, respectively. Fixed cells were stained with DAPI (2 µg/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 PPAR{gamma}1wt or PPAR{gamma}1tr were transfected with 80 nmol/L control small interfering RNA (siRNA) or PPAR{gamma} siRNA (Ambion) using Welfect-Q (WelGENE). After 72 h, cells were treated with 2 mmol/L H2O2 or 30 µmol/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 ± SE.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Differential expression of PPAR{gamma}1tr in human lung SCC tissues. The expression patterns of hPPAR{gamma}1tr and hPPAR{gamma}1wt were compared between primary SCC and surrounding normal tissues. Immunohistochemical analysis indicated that hPPAR{gamma} was present at higher levels in tumor tissues than in surrounding normal tissues (Fig. 1A ). To localize hPPAR{gamma} in the tumor and surrounding normal regions, we used anti-hPPAR{gamma} 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 hPPAR{gamma} expression was much higher in tumor tissue (Fig. 1B, e and f). Broadly stained cells were observed using the anti-hPPAR{gamma} NH2-terminal antibody (Fig. 1B, e and i), whereas the anti-hPPAR{gamma} 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).


Figure 1
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  		Fig. 1. Immunohistochemical analysis of PPAR{gamma} expression in nontumorous and tumorous regions of primary lung SCC tissues. A, representative 3,3'-diaminobenzidine staining of PPAR{gamma} in paraffin-embedded cancer tissue (a) and adjacent normal lung tissue (b) using an anti-hPPAR{gamma}1wt NH2-terminal antibody. Bars, = 50 µm. B, localization of hPPAR{gamma} in normal lung tissue (top) and in cancer tissue (middle). High-resolution photomicrographs of cancer tissue (bottom). Immunopositive signals were obtained for hPPAR{gamma} using an anti-hPPAR{gamma} NH2-terminal antibody (red) and anti-hPPAR{gamma} 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 µm (d and h) and 30 µm (l). Immunohistochemistry was done at least thrice using different sections and similar results were obtained each time.

 
To investigate the expression of hPPAR{gamma}1tr in primary human lung cancer, we examined tissue from 11 patients with non–small cell lung carcinoma with anti-hPPAR{gamma} NH2-terminal antibody. We identified the band corresponding to hPPAR{gamma}1tr in nine samples from tumorous tissue regions of SCC but not in any of the surrounding normal tissues (Fig. 2A ).


Figure 2
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  		Fig. 2. Western blot analysis of PPAR{gamma} expression in nontumorous and tumorous regions of primary lung SCC tissues. N and T, adjacent nontumorous tissues and tumorous tissues, respectively. A, PPAR{gamma} expression detected with an NH2-terminal–specific antibody. Seventy micrograms of total protein were analyzed. WT, hPPAR{gamma}1wt; TR, hPPAR{gamma}1tr; NS, nonspecific. B, detection of PPAR{gamma}1 expression. A duplicate membrane containing the same protein was probed with the antibodies indicated. C, detection of hPPAR{gamma}1tr in nontumorous and tumorous regions of primary lung SCC using an anti-hPPAR{gamma}1tr 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. hPPAR{gamma}1tr was visualized using a CyIII-conjugated secondary antibody (red). Nuclei were stained with DAPI. f, merged image of (d/e). Bars, 50 µm (c) and 30 µm (f). Immunohistochemistry was done at least thrice using different sections and similar results were obtained each time.

 
Detection of PPAR{gamma}1tr using a mAb specific to PPAR{gamma}1tr in human lung SCC tissues. To confirm whether hPPAR{gamma}1tr is the protein that is not recognized by the anti-hPPAR{gamma} COOH-terminal–specific antibody, we raised a mAb against a COOH-terminal hPPAR{gamma}1tr–specific amino acid sequence. This antibody cross-reacted with the smaller band recognized by the anti-hPPAR{gamma} NH2-terminal antibody, confirming specificity (Fig. 2B). We then did an immunohistochemical analysis of the lung SCC tissue samples using anti-hPPAR{gamma}1tr–specific mAb. Consistent with earlier results, hPPAR{gamma}1tr 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 hPPAR{gamma}1tr is expressed in both the cytoplasm and the nucleus of lung SCC.

Stable expression of hPPAR{gamma}1wt and hPPAR{gamma}1tr in CHO cells. To analyze the pathophysiologic roles of hPPAR{gamma}1tr, stable transfectants expressing hPPAR{gamma}1wt or hPPAR{gamma}1tr were selected in CHO cells. Western blot analysis showed a clear increase in hPPAR{gamma}1wt and hPPAR{gamma}1tr expression in each transfectant (Fig. 3A ).


Figure 3
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  		Fig. 3. Isolation and characterization of stable transfectants expressing pcDNA3.1 (CTR), pcDNA3.1-hPPAR{gamma}1wt (WT), or pcDNA3.1-hPPAR{gamma}1tr (TR). A, protein expression. Total lysates were analyzed by Western blotting with an NH2-terminal–specific anti-PPAR{gamma} or ß-actin antibody. Similar results were obtained in three independent experiments. B, effects of troglitazone on the transcriptional activity of hPPAR{gamma}1wt or hPPAR{gamma}1tr. 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 hPPAR{gamma}1tr on the transcriptional activity of hPPAR{gamma}1wt. CHO cells were transfected with increasing amounts of pSG5-hPPAR{gamma}1tr (0.01, 0.1, and 1.0 µg) along with a constant amount of pSG5-hPPAR{gamma}1wt (1.0 µg) and the luciferase reporter construct PPRE3-tk-luc (0.5 µg). 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 by Western blotting with an NH2-terminal–specific anti-PPAR{gamma} or ß-actin antibody. Columns, mean of four to six independent transfections; bars, SE.

 
Because binding to PPAR{gamma} 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 hPPAR{gamma}1wt-expressing cells but not in hPPAR{gamma}1tr-expressing cells (Fig. 3B). We then tested whether hPPAR{gamma}1wt or hPPAR{gamma}1tr affects transcriptional activation of a target gene. In hPPAR{gamma}1wt-expressing 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 hPPAR{gamma}1wt in the presence of troglitazone significantly increased the luciferase activity. On the other hand, cotransfection of increasing amounts of hPPAR{gamma}1tr with constant amount of hPPAR{gamma}1wt dose dependently suppressed the reporter activity (Fig. 3C). These results suggest that hPPAR{gamma}1tr interacts with the wild-type in a dominant-negative manner.

Effects of hPPAR{gamma}1tr overexpression on apoptotic cell death induced by multiple stimuli. We treated stably transfected CHO cells expressing pcDNA 3.1, hPPAR{gamma}1tr, or hPPAR{gamma}1wt with 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 hPPAR{gamma}1wt than for those expressing hPPAR{gamma}1tr (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, hPPAR{gamma}1wt-expressing cells, and hPPAR{gamma}1tr-expressing cells, respectively. Similar results were also obtained for cisplatin-treated cells (Fig. 4B).


Figure 4
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  		Fig. 4. Effects of hPPAR{gamma}1wt or hPPAR{gamma}1tr overexpression on apoptotic cell death and apoptosis-related proteins. A and B, detection of apoptotic cells. CHO cells stably expressing the pcDNA3.1, hPPAR{gamma}1wt, or hPPAR{gamma}1tr 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 a TUNEL-positive reaction (n = 100 each). Columns, mean of three separate determinations; bars, SE. {dagger}, P < 0.01, compared with pcDNA 3.1 treated with H2O2; {dagger}{dagger}, P < 0.01, compared with hPPAR{gamma}1wt treated with H2O2; #, P < 0.01, compared with pcDNA 3.1 treated with cisplatin; ##, P < 0.01, compared with hPPAR{gamma}1wt treated with cisplatin; {ddagger}, P < 0.05, compared with pcDNA 3.1 treated with H2O2; {ddagger}{ddagger}, P < 0.05, compared with hPPAR{gamma}1wt treated with H2O2; *, P < 0.05, compared with pcDNA 3.1 treated with cisplatin; **, P < 0.05, compared with hPPAR{gamma}1wt treated with cisplatin. C, expression of apoptosis-related proteins induced by cisplatin. Total protein was extracted from CHO cells treated with 30 µmol/L cisplatin for 12 h and analyzed by Western blotting using specific antibodies against the proteins indicated.

 
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 hPPAR{gamma}1wt. In contrast, expression of the apoptotic proteins p53, Bax, cytochrome c, caspase-3, and caspase-9 markedly decreased in hPPAR{gamma}1tr-expressing 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 hPPAR{gamma} on apoptotic cell death. To verify the role of hPPAR{gamma}1tr in resistance to apoptotic cell death induced by H2O2 or cisplatin, effects of siRNA against hPPAR{gamma} were examined. The levels of hPPAR{gamma}1 and hPPAR{gamma}1tr in CHO cells stably expressing hPPAR{gamma}1wt or hPPAR{gamma}1tr were significantly reduced when cells were transfected with hPPAR{gamma} siRNA but not with control siRNA consisting of a pool of nonspecific sequence (Fig. 5A ). The down-regulation of either hPPAR{gamma}1wt or hPPAR{gamma}1tr with siRNA against hPPAR{gamma} 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 hPPAR{gamma}1wt compared with hPPAR{gamma}1tr-expressing cells. Whereas the population of apoptotic cells was reduced in hPPAR{gamma}1wt-expressing cells transfected with siRNA against hPPAR{gamma}, resistance to cell death was almost abolished in hPPAR{gamma}1tr-expressing cells transfected with hPPAR{gamma} siRNA (Fig. 5C). Similar results were observed for cisplatin-induced apoptosis. Accordingly, overexpression of hPPAR{gamma}1tr was suggested to confer resistance to cell death through expressional regulation of apoptotic proteins.


Figure 5
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  		Fig. 5. Effects of siRNA against hPPAR{gamma} on apoptotic cell death. A, CHO cells stably expressing hPPAR{gamma}1wt or hPPAR{gamma}1tr were transfected with hPPAR{gamma} 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-ß-actin antibody. B, expression of apoptosis-related proteins induced by cisplatin. At 72 h after the transfection of hPPAR{gamma}1wt- or hPPAR{gamma}1tr- expressing cells with hPPAR{gamma} siRNA or control siRNA, cells were treated with cisplatin. C, cell cycle analysis of cells expressing hPPAR{gamma}1wt or hPPAR{gamma}1tr with or without hPPAR{gamma} 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.

 

   Discussion
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References
 
We have shown that PPAR{gamma} is expressed in primary human lung SCC tissues and that PPAR{gamma}1tr, which exhibits dominant-negative 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 PPAR{gamma} is elevated relative to normal tissues (24, 25). In contrast, PPAR{gamma} 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 PPAR{gamma} 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 PPAR{gamma} reported in esophageal cancer (26). Immunohistochemical examinations using anti-PPAR{gamma} NH2-terminal and COOH-terminal antibodies and anti-PPAR{gamma}1tr antibodies also revealed a significant difference in PPAR{gamma} expression between nontumorous and tumorous regions of primary lung SCC tissues. Immunoreactive signals of wild-type hPPAR{gamma} (hPPAR{gamma}wt) were detected in the nuclei of tumorous and nontumorous tissue, whereas those of hPPAR{gamma}1tr were limited to the cytoplasm of tumorous tissue. Western blot analysis also confirmed the presence of PPAR{gamma}1tr in primary lung SCC. These results support the finding that expression of PPAR{gamma} 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 PPAR{gamma} (16, 26) is associated with differences in the localization of PPAR{gamma} in cellular compartments.

We have shown the presence of hPPAR{gamma}1tr in human primary lung SCC and it seems to exert a dominant-negative effect, repressing activity of hPPAR{gamma}wt. Most notably, the expression of hPPAR{gamma}1tr 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-hPPAR{gamma}1wt was antagonized by a 10-fold lower level of pSG5-hPPAR{gamma}1tr (21). These results suggested that even a small amount of hPPAR{gamma}1tr may interfere with the signaling pathways and expression of genes governed by hPPAR{gamma}1wt. Thiazolidinedione (a PPAR{gamma}-specific ligand) induces growth arrest in human lung cancer cells through the induction of apoptosis and differentiation (12, 28). As hPPAR{gamma}1tr lacks a ligand binding domain, specific ligands may be unable to activate hPPAR{gamma}1tr and inhibit cancer growth. Further studies will be necessary to determine whether there is any correlation between the expression levels of hPPAR{gamma}1tr and the prognosis of esophageal or lung cancer patients.

Overexpression of hPPAR{gamma}1tr seemed to induce resistance to the cellular responses invoked by exogenous apoptotic stimuli. Loss of function by somatic mutations or alternative splicing of hPPAR{gamma} 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 hPPAR{gamma}1tr by alternative splicing may represent a general mechanism for regulation of PPAR activity under different pathophysiologic conditions. Whether it is the presence of hPPAR{gamma}1tr that causes tumor development or that the formation of a tumor evokes the generation of hPPAR{gamma}1tr remains unclear. However, our findings suggest that expression of hPPAR{gamma}1tr in cancerous tissues may disrupt the cellular apoptotic signaling pathways.

In conclusion, the present study suggests the participation of the novel isoform of PPAR{gamma}1 in the development of lung cancer. It is of particular interest that the growth arrest caused by PPAR{gamma} ligands might be alleviated in cells expressing truncated products of the PPAR{gamma} gene. Expression of hPPAR{gamma}1tr may also affect the apoptotic signaling pathways of cancer cells. In this context, the presence of hPPAR{gamma}1tr 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 PPAR{gamma}1 isoform in cancer cells may aid in the development of new therapeutic strategies in cancer treatment.


   Footnotes
 
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.

Received 8/17/06; revised 2/14/07; accepted 2/20/07.


   References
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 Discussion
 References
 

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