Activation of Peroxisome Proliferator-Activated Receptor γ Contributes to the Survival of T Lymphoma Cells by Affecting Cellular Metabolism
2007; Elsevier BV; Volume: 170; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.060651
ISSN1525-2191
AutoresChunyan Yang, Seung-Hee Jo, Balázs Csernus, Elizabeth Hyjek, Yifang Liu, Amy Chadburn, Y. Lynn Wang,
Tópico(s)NF-κB Signaling Pathways
ResumoPeroxisome proliferator-activated receptor γ (PPARγ) is a metabolic regulator involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPARγ induce apoptosis in several types of tumor cells, leading to the proposal that these ligands may be used as antineoplastic agents. However, apoptosis induction requires high doses of ligands, suggesting the effect may not be receptor-dependent. In this report, we show that PPARγ is expressed in human primary T-cell lymphoma tissues and activation of PPARγ with low doses of ligands protects lymphoma cells from serum starvation-induced apoptosis. The prosurvival effect of PPARγ was linked to its actions on cellular metabolic activities. In serum-deprived cells, PPARγ attenuated the decline in ATP, reduced mitochondrial hyperpolarization, and limited the amount of reactive oxygen species (ROS) in favor of cell survival. Moreover, PPARγ regulated ROS through coordinated transcriptional control of a set of proteins and enzymes involved in ROS metabolism. Our study identified cell survival promotion as a novel activity of PPARγ. These findings highlight the need for further investigation into the role of PPARγ in cancer before widespread use of its agonists as anticancer therapeutics. Peroxisome proliferator-activated receptor γ (PPARγ) is a metabolic regulator involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPARγ induce apoptosis in several types of tumor cells, leading to the proposal that these ligands may be used as antineoplastic agents. However, apoptosis induction requires high doses of ligands, suggesting the effect may not be receptor-dependent. In this report, we show that PPARγ is expressed in human primary T-cell lymphoma tissues and activation of PPARγ with low doses of ligands protects lymphoma cells from serum starvation-induced apoptosis. The prosurvival effect of PPARγ was linked to its actions on cellular metabolic activities. In serum-deprived cells, PPARγ attenuated the decline in ATP, reduced mitochondrial hyperpolarization, and limited the amount of reactive oxygen species (ROS) in favor of cell survival. Moreover, PPARγ regulated ROS through coordinated transcriptional control of a set of proteins and enzymes involved in ROS metabolism. Our study identified cell survival promotion as a novel activity of PPARγ. These findings highlight the need for further investigation into the role of PPARγ in cancer before widespread use of its agonists as anticancer therapeutics. Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily. Like other members of this family, they serve as transcription factors and require ligands for activation. In a basal state, the receptors, in dimerization with retinoid X receptor (RXR), bind to promoter regions of their target genes. Ligand binding to PPARs releases corepressors bound to the receptor and recruits coactivators to initiate transcription of target genes. There are three subtypes of PPARs—α, β/δ, and γ—that differ in tissue distribution and ligand requirement. Among them, PPARγ is of particular interest because it plays a role in a variety of human pathological conditions, including diabetes, atherosclerosis, inflammation, and cancer.1Vamecq J Latruffe N Medical significance of peroxisome proliferator-activated receptors.Lancet. 1999; 354: 141-148Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 2Kersten S Desvergne B Wahli W Roles of PPARs in health and disease.Nature. 2000; 405: 421-424Crossref PubMed Scopus (1654) Google Scholar Most studies of PPARγ function focus on its role as a metabolic regulator. The receptor helps maintain lipid and glucose homeostasis in animals and humans.3Knouff C Auwerx J Peroxisome proliferator-activated receptor-gamma calls for activation in moderation: lessons from genetics and pharmacology.Endocr Rev. 2004; 25: 899-918Crossref PubMed Scopus (240) Google Scholar, 4Rangwala SM Lazar MA Peroxisome proliferator-activated receptor gamma in diabetes and metabolism.Trends Pharmacol Sci. 2004; 25: 331-336Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar Whereas systemic knockout of PPARγ causes embryonic lethality,5Barak Y Nelson MC Ong ES Jones YZ Ruiz-Lozano P Chien KR Koder A Evans RM PPAR gamma is required for placental, cardiac, and adipose tissue development.Mol Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1636) Google Scholar, 6Rosen ED Sarraf P Troy AE Bradwin G Moore K Milstone DS Spiegelman BM Mortensen RM PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro.Mol Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1637) Google Scholar disruption of the receptor in insulin target tissues such as fat, liver, or skeletal muscle results in insulin resistance.7Burstein HJ Demetri GD Mueller E Sarraf P Spiegelman BM Winer EP Use of the peroxisome proliferator-activated receptor (PPAR) gamma ligand troglitazone as treatment for refractory breast cancer: a phase II study.Breast Cancer Res Treat. 2003; 79: 391-397Crossref PubMed Scopus (197) Google Scholar, 8Hevener AL He W Barak Y Le J Bandyopadhyay G Olson P Wilkes J Evans RM Olefsky J Muscle-specific Pparg deletion causes insulin resistance.Nat Med. 2003; 9: 1491-1497Crossref PubMed Scopus (414) Google Scholar Potential physiological ligands for PPARγ are lipophilic molecules that are derived from nutrition or metabolic pathways, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), polyunsaturated fatty acids and their oxidized forms, oxidized phospholipids, and triterpenoids. Thiazolidinediones (TZD, glitazones), potent synthetic ligands of PPARγ, are currently a mainstay therapy for type 2 diabetes. The drugs, by activating the receptor, increase tissue sensitivity to insulin and alleviate hyperglycemia. PPARγ regulates whole body lipid and glucose metabolism by its actions on the transcription of many metabolic genes at the cellular level. In adipose tissue, where it is most abundantly expressed, the receptor regulates genes of enzymes and transporters that increase uptake and reduce release of free fatty acid and glucose from cells to circulation.4Rangwala SM Lazar MA Peroxisome proliferator-activated receptor gamma in diabetes and metabolism.Trends Pharmacol Sci. 2004; 25: 331-336Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar In addition to its role in metabolism, PPARγ has also been implicated in the development of cancer. Despite active research in the past few years, it remains debatable whether PPARγ is pro- or antineoplastic. PPARγ is highly expressed in several cancers, including carcinomas of the colon,9DuBois RN Gupta R Brockman J Reddy BS Krakow SL Lazar MA The nuclear eicosanoid receptor, PPARgamma, is aberrantly expressed in colonic cancers.Carcinogenesis. 1998; 19: 49-53Crossref PubMed Scopus (235) Google Scholar, 10Sarraf P Mueller E Jones D King FJ DeAngelo DJ Partridge JB Holden SA Chen LB Singer S Fletcher C Spiegelman BM Differentiation and reversal of malignant changes in colon cancer through PPARgamma.Nat Med. 1998; 4: 1046-1052Crossref PubMed Scopus (926) Google Scholar breast,11Mueller E Sarraf P Tontonoz P Evans RM Martin KJ Zhang M Fletcher C Singer S Spiegelman BM Terminal differentiation of human breast cancer through PPAR gamma.Mol Cell. 1998; 1: 465-470Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar and prostate12Kubota T Koshizuka K Williamson EA Asou H Said JW Holden S Miyoshi I Koeffler HP Ligand for peroxisome proliferator-activated receptor gamma (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo.Cancer Res. 1998; 58: 3344-3352PubMed Google Scholar and liposarcoma.13Tontonoz P Singer S Forman BM Sarraf P Fletcher JA Fletcher CD Brun RP Mueller E Altiok S Oppenheim H Evans RM Spiegelman BM Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor gamma and the retinoid X receptor.Proc Natl Acad Sci USA. 1997; 94: 237-241Crossref PubMed Scopus (617) Google Scholar In addition, PAX8-PPARγ1 fusion has been identified in cases of human follicular thyroid carcinomas.14Kroll TG Sarraf P Pecciarini L Chen C-J Mueller E Spiegelman BM Fletcher JA PAX8-PPARgamma 1 fusion oncogene in human thyroid carcinoma.Science. 2000; 289: 1357-1360Crossref PubMed Scopus (738) Google Scholar Functionally, most of the in vitro and xenograft studies have shown that synthetic ligands of PPARγ inhibit proliferation and induce differentiation and apoptosis in tumor cells, suggesting that PPARγ is antineoplastic.15Grommes C Landreth GE Heneka MT Antineoplastic effects of peroxisome proliferator-activated receptor gamma agonists.Lancet Oncol. 2004; 5: 419-429Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 16Michalik L Desvergne B Wahli W Peroxisome-proliferator-activated receptors and cancers: complex stories.Nat Rev Cancer. 2004; 4: 61-70Crossref PubMed Scopus (507) Google Scholar However, in animals that are genetically predisposed to colon and mammary gland cancer, activation of PPARγ exacerbates tumor formation and growth.17Lefebvre AM Chen I Desreumaux P Najib J Fruchart JC Geboes K Briggs M Heyman R Auwerx J Activation of the peroxisome proliferator-activated receptor gamma promotes the development of colon tumors in C57BL/6J-APCMin/+ mice.Nat Med. 1998; 4: 1053-1057Crossref PubMed Scopus (575) Google Scholar, 18Saez E Tontonoz P Nelson MC Alvarez JG Ming UT Baird SM Thomazy VA Evans RM Activators of the nuclear receptor PPARgamma enhance colon polyp formation.Nat Med. 1998; 4: 1058-1061Crossref PubMed Scopus (549) Google Scholar, 19Saez E Rosenfeld J Livolsi A Olson P Lombardo E Nelson M Banayo E Cardiff RD Izpisua-Belmonte JC Evans RM PPAR gamma signaling exacerbates mammary gland tumor development.Genes Dev. 2004; 18: 528-540Crossref PubMed Scopus (166) Google Scholar In humans, the receptor itself and several of its target genes are up-regulated in PAX8-PPARγ1-positive follicular thyroid carcinomas in comparison to the tumors lacking the fusion, demonstrating that increased PPARγ transcriptional activity contributes to carcinogenesis.20Lacroix L Lazar V Michiels S Ripoche H Dessen P Talbot M Caillou B Levillain JP Schlumberger M Bidart JM Follicular thyroid tumors with the PAX8-PPARγ1 rearrangement display characteristic genetic alterations.Am J Pathol. 2005; 167: 223-231Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar It is unclear whether the observed in vitro effects of PPARγ ligands are mediated through the receptor or result from nonspecific activities of the drugs.16Michalik L Desvergne B Wahli W Peroxisome-proliferator-activated receptors and cancers: complex stories.Nat Rev Cancer. 2004; 4: 61-70Crossref PubMed Scopus (507) Google Scholar To induce apoptosis of tumor cells, high concentrations of ligands are often required. In addition, antagonists that inhibit PPARγ's activities also show antineoplastic properties by inducing apoptosis, suggesting that the effect may not act through the receptor.21Seargent JM Yates EA Gill JH GW9662, a potent antagonist of PPARgamma, inhibits growth of breast tumour cells and promotes the anticancer effects of the PPARgamma agonist rosiglitazone, independently of PPARgamma activation.Br J Pharmacol. 2004; 143: 933-937Crossref PubMed Scopus (149) Google Scholar, 22Schaefer KL Wada K Takahashi H Matsuhashi N Ohnishi S Wolfe MM Turner JR Nakajima A Borkan SC Saubermann LJ Peroxisome proliferator-activated receptor γ inhibition prevents adhesion to the extracellular matrix and induces anoikis in hepatocellular carcinoma cells.Cancer Res. 2005; 65: 2251-2259Crossref PubMed Scopus (109) Google Scholar In a previous report, we found that PPARγ promotes survival under the condition of growth factor deprivation.23Wang YL Frauwirth KA Rangwala SM Lazar MA Thompson CB Thiazolidinedione activation of peroxisome proliferator-activated receptor γ can enhance mitochondrial potential and promote cell survival.J Biol Chem. 2002; 277: 31781-31788Crossref PubMed Scopus (96) Google Scholar Using cells containing or lacking PPARγ, we showed that the survival-enhancing effect occurs via a receptor-dependent mechanism. Furthermore, we demonstrated that PPARγ promotes cell survival by enhancing the ability of cells to maintain mitochondrial membrane potential. Growth factor-independent survival is characteristic of tumor cells. Under normal conditions, growth factor withdrawal induces a series of metabolic changes leading to apoptotic cell death, such as decreased glucose uptake, impaired glycolysis, depolarization of mitochondrial potential, ATP depletion, hyperpolarization of mitochondrial inner membrane, matrix swelling, rupture of mitochondrial outer membrane, and release of cytochrome c and other proteins from the intermembrane space. These are then followed by caspase activation and eventually cell death. One of the strategies tumor cells use to resist death under growth factor or nutrient limitation is through up-regulation of the expression or activity of antiapoptotic proteins such as BCL-xL and AKT, which suppress apoptosis through their effects on cellular metabolic activities. One of the key functions of BCL-xL is to promote efficient mitochondrial ATP/ADP exchange through voltage-dependent anion channel/adenine nucleotide translocase (VDAC/ANT) complex to sustain coupled respiration in the face of decreased mitochondrial membrane potential,24Vander Heiden MG Chandel NS Schumacker PT Thompson CB Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange.Mol Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar whereas AKT promotes glucose uptake and glycolysis that provide fuel to maintain mitochondrial potential and ATP production. Reactive oxygen species (ROS) generation is one of the early events in several forms of cell death. Although it is still controversial whether ROS production during apoptosis precedes or follows mitochondrial damage,25Gottlieb E Vander Heiden MG Thompson CB Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis.Mol Cell Biol. 2000; 20: 5680-5689Crossref PubMed Scopus (308) Google Scholar, 26Ricci JE Gottlieb RA Green DR Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.J Cell Biol. 2003; 160: 65-75Crossref PubMed Scopus (419) Google Scholar scavenging of ROS blocks or attenuates apoptosis in several systems,27Tan S Sagara Y Liu Y Maher P Schubert D The regulation of reactive oxygen species production during programmed cell death.J Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (643) Google Scholar and BCL2 has been shown to inhibit apoptosis by preventing ROS generation.28Hockenbery D Oltvai Z Yin X Milliman C Korsmeyer S Bcl-2 functions in an antioxidant pathway to prevent apoptosis.Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3286) Google Scholar, 29Kane DJ Sarafian TA Anton R Hahn H Gralla EB Valentine JS Ord T Bredesen DE Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species.Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1613) Google Scholar As a known factor affecting metabolism, PPARγ may impact tumor cell survival through its regulation of cellular metabolism similar to other antiapoptotic proteins. In the current study, we investigated the role of PPARγ in the survival of tumor cells using anaplastic large cell lymphoma (ALCL) as a model. ALCL is a type of non-Hodgkin's T-cell lymphoma characterized by large malignant cells with pleomorphic nuclei and abundant cytoplasm. It represents ∼2% of all types of lymphoma, ∼10% of pediatric lymphomas, and ∼50% of large cell lymphomas in the pediatric population. At the molecular level, ALCL is not a homogeneous entity; about 40 to 60% of cases carry a characteristic chromosomal translocation t(2;5), which generates a fusion between anaplastic lymphoma kinase (ALK) and nucleophosmin (NPM) gene. We found that PPARγ is highly expressed in several primary tissues of ALCL cases. Under the condition of serum starvation, PPARγ agonists promote survival of an ALCL cell line that highly expresses the receptor but not of an ALCL line that lacks PPARγ. Moreover, decreasing the amount of PPARγ by RNA interference reduces the rate of cell survival. Given the role of PPARγ in metabolism, we investigated whether its prosurvival effects are linked to its regulation of cellular metabolic activities. We demonstrated that PPARγ activation leads to higher ATP and lower ROS levels that favor survival in serum-deprived cells. Moreover, PPARγ limits amount of ROS through concerted transcriptional regulation of several proteins and enzymes that control cellular ROS. Last, transfection of PPARγ into the PPARγ-null T-cell lymphoma cell line imparts increased survival and reduced ROS accumulation. The finding that PPARγ promotes survival is compatible with the observations that expression of PPARγ is increased in many cancers and suggests that PPARγ may confer a survival advantage on the malignant cells, allowing them to survive in an adverse environment. 15d-PGJ2, rosiglitazone, and WY14643 were purchased from Cayman Chemical (Ann Arbor, MI). Paraffin-embedded tissues from nine cases of ALCL were obtained from the archives of the Department of Pathology and Laboratory Medicine at Weill Cornell Medical College and studied following Institutional Review Board review and approval. The anaplastic large cell lymphoma lines Karpas 299 and SUP-M2 have been described previously30Ho L Aytac U Stephens LC Ohnuma K Mills GB McKee KS Neumann C LaPushin R Cabanillas F Abbruzzese JL Morimoto C Dang NH In vitro and in vivo antitumor effect of the anti-CD26 monoclonal antibody 1F7 on human CD30+ anaplastic large cell T-cell lymphoma Karpas 299.Clin Cancer Res. 2001; 7: 2031-2040PubMed Google Scholar, 31Morris SW Kirstein MN Valentine MB Dittmer KG Shapiro DN Saltman DL Look AT Fusion of a kinase gene ALK, to a nucleolar protein gene NPM, in non-Hodgkin's lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1942) Google Scholar, 32Popovic M Sarin PS Robert-Gurroff M Kalyanaraman VS Mann D Minowada J Gallo RC Isolation and transmission of human retrovirus (human T-cell leukemia virus).Science. 1983; 219: 856-859Crossref PubMed Scopus (385) Google Scholar and were maintained at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/streptomycin. SUP-M2 cells were transfected with pcDNA3.1-hPPARγ1 with TransFectin lipid reagent from Bio-Rad Laboratories (Hercules, CA) according to the manufacturer's instructions. To perform serum withdrawal, cells were washed three times with RPMI 1640, resuspended, and cultured in serum-free media. Cell viability was determined by cellular exclusion of 2 μg/ml propidium iodide followed by flow cytometric analysis of 10,000 events as described previously.23Wang YL Frauwirth KA Rangwala SM Lazar MA Thompson CB Thiazolidinedione activation of peroxisome proliferator-activated receptor γ can enhance mitochondrial potential and promote cell survival.J Biol Chem. 2002; 277: 31781-31788Crossref PubMed Scopus (96) Google Scholar Various drug treatments of the cells are described in detail in figure legends. Caspase-3 activity was assayed using CaspGLOW fluorescein active caspase-3 staining kit (BioVision, Mountain View, CA). Serum-starved cells (3 × 105 in 0.3 ml) were treated with 1 μl of caspase-3 substrates and incubated for 45 minutes in a 37°C incubator with 5% CO2. Cells were then washed with the wash buffer, and 10,000 events were analyzed by flow cytometry using FL-1. Five micrograms of acylCoAx3-TK-LUC, a Firefly luciferase reporter construct containing three copies of PPRE, and 0.5 μg of Renilla expression plasmid were cotransfected into 5 × 106 Karpas 299 cells using the Amaxa Nucleofector instrument (Amaxa Biosystems, BioCampus, Cologne, Germany) in 100 μl of solution V using the A30 program. Rosiglitazone (2 μmol/L) or DMSO vehicle control was added 16 hours after transfection. Cells were then harvested 24 hours after drug addition. Firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer's instructions. Luminescence was measured using MLX microplate luminometer (Dynex Technologies Inc., Chantilly, VA). PPARγ transcriptional activity was expressed as firefly luciferase activity normalized to Renilla luciferase activity. Immunohistochemical staining for PPARγ (E-8; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and CD30 (clone BerH2; DakoCytomation, Carpinteria, CA) was performed on formalin-fixed, paraffin-embedded tissue sections using the TechMate 500 automated immunostainer (Ventana Medical Systems Inc., Tucson, AZ). Immunostaining for PPARγ was performed following antigen retrieval in an autoclave using Target Retrieval Solution, High pH (DakoCytomation). Immunoreactivity for PPARγ was identified using HRP-labeled mouse Envision Plus detection system (DakoCytomation) and DAB liquid chromogen (DakoCytomation). The sections were then retrieved in the autoclave with Target Retrieval Solution, Citrate pH 6 (DakoCytomation), and immunostained for CD30 using ChemMate alkaline phosphatase detection system (Ventana Medical Systems). The alkaline phosphatase reaction was developed by BT Red reagent substrate provided in the kit. Sections were counterstained with hematoxylin, dehydrated, and mounted in Cytoseal-XYL (Richard-Allan Scientific, Kalamazoo, MI). Small interfering RNA (siRNA) against PPARγ and scrambled double-stranded RNA controls were purchased from Dharmacon Inc. (Boulder, CO) in the form of SMART pool. The delivery of siRNA pools into Karpas 299 cells was performed using nucleofection technology with the Nucleofector instrument (Amaxa Biosystems). A total of 3 μg of the siRNA pools were delivered into 2 × 106 cells suspended in 100 μl of solution V using the A30 nucleofection program. Two micrograms of pmaxGFP vector were cotransfected to monitor transfection efficiency. Intracellular ATP levels were determined using the ATP bioluminescence assay kit HS II (Roche Applied Science, Indianapolis, IN) following the manufacturer's instructions. In brief, 25 μl of lysis reagent was added to 25 μl of cells (1 × 105 cells/ml), incubated for 5 minutes at room temperature, and 50 μl of luciferase reagent was then added. Luminescence was quantified using a MLX microtiter plate luminometer (Dynex Technologies). At various time points following serum withdrawal, 2 × 105 to 4 × 105 cells were stained with 20 nmol/L tetramethylrhodamine ethyl ester in a 37°C CO2 incubator for 30 minutes. Cells were then analyzed by flow cytometry using FL-2. Intracellular ROS were detected with carboxy-H2DCFDA (DCF; Molecular Probes, Eugene, OR). Three hundred thousand cells were washed once, resuspended in 500 μl of PBS, and loaded with 10 μmol/L carboxy-H2DCFDA for 30 minutes at 37°C. DCF fluorescence of 10,000 events was measured by flow cytometry using FL-1. Total RNA was isolated from cells using RNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The amounts of total RNA were quantified using spectrophotometric measurements. RNA was reverse-transcribed into cDNA using a reverse transcription system (Promega) according to the manufacturer's protocol. Real-time PCR was conducted in an ABI PRISM 7000 Sequence Detection System [Applied Biosystems (ABI), Foster City, CA]. cDNA made from 100 ng of total RNA was added to a 20 μl 1× Taqman Universal Master Mix (ABI). The PCR reactions were conducted at 50°C for 2 minutes, 95°C for 10 minutes, followed by 50 cycles of 95°C for 15 se-conds and 60°C for 60 seconds. Primers and probe were purchased from ABI for detection of PPARγ (Hs00234592_m1), catalase (Hs00156308_m1), Cu, Zn superoxide dismutase (CuZn-SOD, Hs00166575_m1), p67 (Hs00166416_m1), and UCP2 (Hs00163349_m1); sequences are not provided by the manufacturer. Manganese SOD (Mn-SOD) was detected using SYBR Green method. Primer sequences are: forward, 5′-AGCATGTTGAGCCGGGCAGT-3′ and reverse 5′-AGGTTGTTCACGTAGGCCGC-3′. Real-time PCR results were analyzed with ABI PRISM 7000 SDS software. Autothresholds and autobaselines determined by the software were applied to generate values of corresponding threshold cycles (Ct). Ct values of various genes were normalized to human β-actin that was purchased from ABI (Hs 99999903_m1). The analyses were conducted as described previously.23Wang YL Frauwirth KA Rangwala SM Lazar MA Thompson CB Thiazolidinedione activation of peroxisome proliferator-activated receptor γ can enhance mitochondrial potential and promote cell survival.J Biol Chem. 2002; 277: 31781-31788Crossref PubMed Scopus (96) Google Scholar Antibodies against PPARγ, UCP2, and p67 were purchased from Santa Cruz Biotechnology, Alpha Diagnostic International (San Antonio, TX), and BD Biosciences (Franklin Lakes, NJ), respectively. The activity of the enzyme was assayed in whole-cell extracts using a superoxide dismutase assay kit purchased from Cayman Chemical. The assays were performed according to the manufacturer's instructions. To determine selectively the activity of Mn-SOD, cell lysates were treated with 3 mmol/L KCN to inactivate CuZn-SOD before performing the assay. Student's t-test was used to perform the statistical analyses. P values are indicated in each figure. Although expression of PPARγ has been found in mouse and human lymphoma cell lines, expression of PPARγ in human primary lymphoma tissues has not been demonstrated. We examined nine cases of ALCL for PPARγ expression by immunohistochemistry. To ensure that PPARγ is expressed in the malignant cells, we performed staining for PPARγ as well as CD30, which specifically marks the malignant large cells in ALCL tissues. Positive PPARγ staining was found in six of nine cases, and a pair of positive and negative cases is shown in Figure 1A. In a typical positive case, the malignant anaplastic large cells that are marked with cytoplasmic CD30 (pink) were stained positive for PPARγ (brown) in nuclei (Figure 1A, left panel), whereas a typical negative case lacks PPARγ staining in CD30-marked tumor cells (Figure 1A, right panel). The admixed positive and negative results among nine cases are not unexpected, because ALCL cases are not homogeneous at the molecular level. To facilitate studies of the function of PPARγ in lymphoma cell survival, two human ALCL lines, Karpas 299 and SUP-M2, were used.30Ho L Aytac U Stephens LC Ohnuma K Mills GB McKee KS Neumann C LaPushin R Cabanillas F Abbruzzese JL Morimoto C Dang NH In vitro and in vivo antitumor effect of the anti-CD26 monoclonal antibody 1F7 on human CD30+ anaplastic large cell T-cell lymphoma Karpas 299.Clin Cancer Res. 2001; 7: 2031-2040PubMed Google Scholar, 31Morris SW Kirstein MN Valentine MB Dittmer KG Shapiro DN Saltman DL Look AT Fusion of a kinase gene ALK, to a nucleolar protein gene NPM, in non-Hodgkin's lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1942) Google Scholar Karpas 299 cells express a high level of PPARγ revealed by both real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, whereas SUP-M2 shows little expression.33Jo S Yang C Miao Q Marzec M Wasik MA Lu P Wang YL PPARγ promotes lymphocyte survival through its actions on cellular metabolic activities.J Immunol. 2006; 177: 3737-3745Crossref PubMed Scopus (33) Google Scholar Immunohistochemical staining on cell blocks made from these two cell lines confirmed the presence of the receptor in the nuclei of Karpas 299 cells and the absence of the receptor in SUP-M2 cells (Figure 1B). Next, we examined whether high levels of PPARγ confer a survival advantage on the lymphoma cells under stress conditions. Serum starvation was performed with Karpas 299 and SUP-M2 cells treated with PPARγ ligand rosiglitazone or drug vehicle, and survival of the cells was determined by propidium iodide exclusion. Although survival of both Karpas 299 and SUP-M2 cells decreased with time, death of Karpas 299 cells was attenuated by rosiglitazone treatment, whereas survival of SUP-M2 cells was not influenced (Figure 1C). The cell death is apoptotic in nature, since caspase-3 was activated in serum-starved Karpas 299 cells (Figure 1D, DMSO). In addition, rosiglitazone treatment of the cells suppressed caspase-3 activation, supporting that the PPARγ agonist is prosurvival in serum-starved cells (Figure 1D, Rosi versus DMSO). A dose titration of rosiglitazone revealed that the survival effect in Karpas 299 cells could be observed at a concentration as low as 0.5 μmol/L and reached maximum at 2 μmol/L (Figure 1E). These concentrations largely overlap with pharmacologically achievable serum concentrations of rosiglitazone in diabetic patients (0.16 to 1.26 μmol/L) (Rosiglitazone Maleate, , 2006). In comparison, in the same dose range, rosiglitazone did not show any effects on the survival of SUP-M2 cells (Figure 1E). Two micromolar was chosen for the subsequent experiments. At this concentration, PPARγ was activated as a transcriptional factor as shown by a luciferase reporter assay (Figure 1F). To ensure that the survival effect is mediated via PPARγ rather than a special property of rosiglitazone, other PPARγ agonists were tested for their activities in serum withdrawal-induced apoptosis. GW7845, a tyrosine analogue that is
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