Autophagy induced by farnesyltransferase inhibitors in cancer cells

[Cancer Biology & Therapy 7:10, 1679-1684; October 2008]; ©2008 Landes Bioscience

Research Paper

Autophagy induced by farnesyltransferase inhibitors in cancer cells Jingxuan Pan,1,* Bo Chen,2-4 Chun-Hui Su,3 Ruiying Zhao,3 Zhi-Xiang Xu,5 Lily Sun,6 Mong-Hong Lee3 and Sai-Ching Jim Yeung4,6,* 1Department

of Pathophysiology; Sun Yat-Sen University Medical School; Guangzhou, Guangdong P.R. China; 2Department of Surgical Oncology, The First Affiliated Hospital; China Medical University; Shenyang, Liaoning People’s Republic of China; Departments of 3Molecular and Cellular Oncology; 4General Internal Medicine; Ambulatory Treatment and Emergency Care; 5Molecular Therapeutics; 6Endocrine Neoplasia and Hormonal Disorders; and The University of Texas M.D. Anderson Cancer Center; Houston, Texas USA

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Abbreviations: FTI, farnesyltransferase inhibitor; Rheb, Ras homologue enriched in brain; mTOR, mammalian target of rapamycin; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; LC3, light chain 3; Z-VAD.fmk, N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate

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for malignancies is promising. Researchers originally developed FTIs to inhibit the mitogenic function of ras oncogenes, but their mechanisms of action most probably involve multiple farnesylated proteins.3 The mechanisms of action of FTIs involve Ras homologue enriched in brain (Rheb) and the phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway.3 Rheb is modified by farnesylation, is not a substrate for alternative prenylation by geranylgeranyltransferase, and plays a significant role in the antineoplastic activity of the FTI lonafarnib (SCH66336).4 Rheb acts downstream of tuberous sclerosis complex 1 (TSC1)/ TSC2 and upstream of mTOR to regulate cell growth5 and activates S6 kinase 1 during amino acid deprivation via mTOR6-8 Membrane localization of Rheb is important for its biological function because a Rheb mutant altered at the farnesylation site stimulates S6 kinase 1 less efficiently than wild-type Rheb.9 Furthermore, mTOR occupies a key position in regulation of autophagy.10 Collectively, the literature suggests that FTIs very likely induce autophagy. However, no reported studies have looked at this effect of FTIs on this cellular process relevant to cancer biology. We hypothesize that FTIs can induce autophagy in cancer cells and investigate our hypothesis using the FTIs manumycin A, FTI-276, and lonafarnib. Manumycin A, a natural product of Streptomyces parvulus,11 inhibits farnesyltransferase by competing with the farnesyl pyrophosphate group.12,13 FTI-276 is a peptidomimetic of the CAAX amino acid motif in the C-terminal region of Ras proteins.14 Lonafarnib is an orally active tricyclic peptidomimetic FTI that has shown promising antineoplastic activity in various clinical trials.15,16 Here we report the evidence for autophagy induced by FTIs in two cancer cell lines.

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The mechanisms of action of farnesyltransferase inhibitors (FTIs) involve Rheb and the phosphatidylinositide 3-kinase/ Akt/mammalian target of rapamycin (mTOR) pathway. mTOR in particular plays a key role in the regulation of autophagy. Collectively, the literature suggests that FTIs very likely induce autophagy, but thus far there have been no reports that FTIs affect this process relevant to cancer cell biology. We hypothesized that FTIs can induce autophagy. In this study, we found that the FTIs manumycin A, FTI-276, and lonafarnib induced autophagy in two human cancer cell lines. We also found that neither inhibition of apoptosis with a pan-caspase inhibitor nor inhibition of autophagy increased the number of clones of lonafarnib-treated U2OS osteosarcoma cells that formed in soft agar. Although whether autophagy is a cell death or cell survival mechanism after FTI treatment remains unresolved, our data show that cancer cells apparently can shift between apoptosis and autophagy once they are committed to die after FTI treatment.

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Key words: autophagy, farnesyltransferase, lonafarnib, manumycin A, pancreatic cancer, osteosarcoma, apoptosis

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Introduction

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Like geranylgeranylation, farnesylation is a posttranslational modification of proteins in which farnesyltransferase catalyzes the attachment of the isoprenoid group from farnesyl pyrophosphate to a cysteine residue at the C-terminus of proteins bearing the CAAX amino acid motif. Farnesyltransferase inhibitors (FTIs) are anticancer drugs that are under clinical development.1,2 Although phase III trials of FTIs administered as single agents have shown no improvement in overall survival durations, use of combinations of FTIs with other therapies *Correspondence to: Sai-Ching Jim Yeung; Department of General Internal Medicine; Ambulatory Treatment and Emergency Care; Unit 437; The University of Texas M.D. Anderson Cancer Center; 1515 Holcombe Boulevard; Unit 1465; Houston, Texas 77030 USA; Tel.: 713.745.4516; Fax: 713.563.4491; Email: syeung@ mdanderson.org or Jingxuan Pan; Department of Pathophysiology; Sun Yat-Sen University Medical School; 74 Zhongshan Rd II; Guangzhou 510089 P.R. China; Tel.: 86.20.87332750; Fax: 86.20.87332750; Email: [email protected] Submitted: 05/14/08; Revised: 07/18/08; Accepted: 07/21/08 Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/article/6661 www.landesbioscience.com

Results FTIs induce autophagy in cancer cells. We have shown that manumycin A induces apoptosis in anaplastic thyroid carcinoma cells.17,18 When we examined the effects of FTIs on the ultrastructural morphology of other cancer cell lines, we discovered that manumycin A induced ultrastructural changes in the human pancreatic cell line Panc-1 that were characteristic of autophagy (Fig. 1).

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Figure 1. Treatment with manumycin A induced autophagic features in Panc-1 cells. (A) Control cells showing a lack of vacuoles. (B) Manumycin A-treated Panc-1 cells showing short ribbons of condensed chromatin in a perinuclear distribution, blebbing on the cell surface with loss of microvilli, and formation of autophagic vacuoles (same magnification as in A). (C) Detailed image of the autophagic vacuoles labeled A in (B). (D) Detailed image of a lysosome (L) abutting and possibly fusing with a mitochondrion (arrowheads) (same magnification as in C).

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We treated Panc-1 cells with 54 μM manumycin A for 6 h and control cells with a culture medium containing 0.1% dimethyl sulfoxide (DMSO; v/v) for the same duration. After this treatment, we fixed the cells and processed them for transmission electron microscopy as described previously.19 We observed no vacuoles in the control cells (Fig. 1A). However, manumycin A induced formation of short ribbons of condensed chromatin in a perinuclear distribution, blebbing on the cell surface with loss of microvilli, and formation of autophagic vacuoles in Panc-1 cells (Fig. 1B). Figure 1C shows more details of the autophagic vacuoles at a high magnification, and Figure 1D shows a lysosome abutting and perhaps fusing with a mitochondrion. Researchers have used a plasmid expressing green fluorescent protein (GFP)-microtubule-associated protein-1 light chain 3 (LC3) chimeric protein (GFP-LC3) in the investigation of autophagy.20 Lipidation of LC3, as LC3 coats autophagosomes during autophagy, changes the electrophoretic mobility of LC3. We stably transfected the human osteosarcoma cell line U2OS with a plasmid expressing GFP-LC3 (U2OS/GFP-LC3) to allow observation of autophagosomes by fluorescent microscopy. Treatment of the transfected cells with 20 μM lonafarnib for 24 hours induced formation of autophagosomes which were seen as punctate green fluorescence in these cells (Fig. 2A). Also, the occurrence of autophagy was indicated by the formation of a lipidated second gel band of LC3 (GFP-LC3-II). The percentage of U2OS/GFP-LC3 cells with punctate green fluorescence was significantly higher in lonafarnib-treated cells than in untreated control cells (two-tailed t-test; p < 0.001) (Fig. 2B). Because we did not have access to a specific antibody against LC3, we used an antibody against GFP to detect chimeric GFP-LC3 in immunoblotting and demonstrated that 30 μM lonafarnib induced electrophoretic mobility shift of GFP-LC3 leading to the appearance of a second gel band (GFP-LC3-II) (Fig. 2C). We also found that three FTIs (manumycin A, lonafarnib, and FTI-276) induced autophagy using this mobility shift assay (Fig. 2D). The degree of autophagy according to the ratio of the integrated optical density of GFP-LC3-II to that of GFP-LC3-I depended on the concentration of manumycin A and lonafarnib (Fig. 2E). In the same cancer cell line, lonafarnib inhibited colony formation in soft-agar in a dosedependent manner (Fig. 2F). Lonafarnib-induced autophagy is inhibited by 3-methyladenine and enhanced by N-benzyloxycabonyl-Val-AlaAsp-fluoromethylketone. We further investigated this novel effect of FTIs using inhibitors of autophagy and apoptosis. We treated U2OS cells with a DMSO control culture medium, 5 mM 3-methyladenine (3-MeA; an autophagy inhibitor), 20 μM lonafarnib, and a combination of 3-MeA and lonafarnib for 24 hours. Transmission electron microscopy revealed that 3-MeA inhibited the ultrastructural features of autophagy but induced ultrastructural features consistent with apoptosis (vacuoles and mitochondrial condensation) in cells treated with both 3-MeA and lonafarnib (Fig. 3A). We also treated U2OS/ GFP-LC3 cells with DMSO control culture medium, 50 μM N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk; a pan-caspase inhibitor), 20 μM lonafarnib, and a combination of Z-VAD.fmk and lonafarnib for 24 hours. Transmission electron microscopy showed that Z-VAD.fmk increased the autophagosomes in cells treated with both Z-VAD.fmk and lonafarnib (Fig. 3B). In addition, we lysed cells treated as aforementioned in RIPA

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buffer for immunoblotting with an antibody against GFP to detect GFP-LC3, which confirmed the degree of autophagy in these cells. Caspase inhibition minimally induced autophagy as compared with control cells [LC3-II—the lower band on the immunoblot, Fig. 3B (above the electron micrographs)]. Lonafarnib increased LC3-II, and the combination of Z-VAD.fmk and lonafarnib further increased LC3-II. These results suggested that lonafarnib induced autophagic cell death and that inhibition of caspases further increased the lonafarnib-induced autophagic cell death. Lonafarnib inhibits U2OS anchorage-independent clonal growth and inhibits mTOR signaling. If autophagy promotes cancer cell survival, inhibition of autophagy should decrease the number of cancer cells that survive lonafarnib treatment. On the other hand, if autophagy mediates cell death, inhibition of autophagy should increase the number of surviving cancer cells. Based on this argument, we counted the U2OS cells capable of anchorage-independent clonal expansion after treatment with lonafarnib in the presence or absence of 3-MeA to determine whether lonafarnib-induced autophagy is a cellular survival or cell death (type II programmed cell death) mechanism. Inhibition of autophagy by 3-MeA did not change (neither increase nor decrease) the number of clones in soft agar (Fig. 4A). If autophagy promotes cancer cell survival and inhibition of apoptosis by a pan-caspase inhibitor Z-VAD.fmk increased autophagy (see above & Fig. 3B), inhibition of apoptosis should decrease the number of cancer cells that survive lonafarnib treatment. On the other hand, if autophagy mediates cell death and


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the appearance of punctate green fluorescence when GFP-LC3-II coated the autophagosomes (Fig. 4D). Similarly, transfection with siRNA to suppress RHEB expression resulted in appearance of autophagosomes in these U2OS cells transfected with GFP-LC3, but there was no increase in autophagosomes in the cells transfected with control siRNA (Fig. 4D). Considered together, these results are consistent with the notion that FTIs induce autophagy by interfering with the function of Rheb.

Discussion

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In this study, we obtained results confirming the hypothesis that FTIs can induce autophagy in cancer cells. Specifically, we observed that FTIs induce autophagy in a dose-dependent manner. The evidence for autophagy reported here includes visualization of autophagosomes coated with GFP-LC3-II under a fluorescence microscope, electrophoretic mobility shift assay of LC3 showing post-translational modification of LC3 in autophagy, and identification of autophagosomes using transmission electron microscopy (the gold standard method to detect autophagy). We observed similar results of autophagy for the three FTIs tested, which have different chemical structures. Therefore, the induction of autophagy is very likely Figure 2. FTIs induced autophagy in osteosarcoma cells. (A) Green fluorescent micrographs a pharmacologic class effect of inhibition of farnesylof control and lonafarnib-treated U2OS/GFP-LC3 cells (same magnification). (B) Plot of the transferase. percentage of U2OS/GFP-LC3 cells with punctate green fluorescence (autophagic cells). The Although a recent paper by Zeng and Kinsella21 error bars represent 95% confidence intervals. (C) Immunoblots using antibodies against GFP. reported that rapamycin co-treatment inhibited The arrows on the right indicate LC3-(I) and lipidated autophagosome-bound LC3-(II). An 6-thioguanine-induced autophagy in HCT116 immunoblot against actin was included as a loading control. (D) Immunoblots against GFP in U2OS/GFP-LC3 cells treated with the three FTIs at various concentrations. (E) The degree of (MLH1(+)) and HT29 cells, mTOR inhibits autophagy in U2OS/GFP-LC3 cells as measured by the ratio of the integrated optical density autophagy in the majority of cases,10,22,23 which is (OD) of the LC3-II band to that of LC3-I band, which is plotted for the concentrations of the consistent with our results. Specifically, we found that three FTIs. (F) Anchorage-independent growth of lonafarnib-treated cells (relative to that of control cells) plotted for the FTI concentrations used. The error bars represent 95% confidence rapamycin induced autophagy in U2OS cells (Fig. 4). When bound to the 12-kDa FK506-binding protein intervals. (FKBP12), rapamycin interacts with and inhibits Z-VAD.fmk increased autophagy (Fig. 3B), inhibition of apoptosis the kinase activity of mTORC1 (the complex composed of mTOR, may not increase the number of surviving cancer cells. Inhibition mLST8, and Raptor). mTORC2 (the complex composed of mTOR, of caspases by Z-VAD.fmk did not change (neither increase nor mLST8, and Rictor), which is not inhibited by rapamycin, is the decrease) the number of clones in soft agar (Fig. 4A). Therefore, one Ser-473 kinase that activates Akt which inhibits autophagy.24,25 possible explanation is that lonafarnib-induced autophagy mediates Also, prolonged rapamycin treatment can decrease the assembly of cell death. However, these results are still not sufficient to declare that mTORC2,25 leading to suppression of Akt. Besides mTOR, there autophagy is always a cell death pathway after FTI treatment. are other regulators of autophagy such as beclin-1 and DRAM. FTIs are well known to interfere with the function of Rheb, Elucidation of the mechanism behind the unexpected response which regulates mTOR,4 an established regulator of autophagy.10 to rapamycin obtained by Zeng and Kinsella21 in the context of To confirm the predicted downregulation of signaling through 6-thioguanine treatment may provide new insight in the regulamTOR by an FTI, we examined the phosphorylation status of tion of autophagy. Nevertheless, induction of autophagy by FTIs is mTOR in U2OS cells using immunoblot analysis. Immunoblots consistent with previous findings by others that FTIs inhibit Rheb against total mTOR and phosphospecific mTOR in U2OS cells farnesylation and mTOR signaling4 and that autophagy is negatively showed that lonafarnib decreased phosphorylation (i.e., activation) controlled by mTOR. of mTOR in a dose-dependent manner (Fig. 4B). Lonafarnib 20 Cross-talk exists between the molecular machinery that reguμM for 24 hoursours decreased the level of phospho-S6 kinase lates apoptosis and the machinery that regulates autophagy. The (S6K) (a signaling molecule downstream of mTOR) in U2OS cells mammalian apoptosis-specific proteins (ASP) and human autophagy (Fig. 4C). Using U2OS cells transfected with the plasmid expressing five homologue (Apg5) share sequence homology,26 suggesting GFP-LC3, a 24-hour treatment with an inhibitor of mTORC1 evolutionary conservation in the degradative processes of apoptosis function, rapamycin 1 μM, induced autophagy as evidenced by and autophagy. Beclin-1, a protein that interacts with Bcl-2,27 www.landesbioscience.com


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Figure 3. Influence of inhibitors of autophagy or apoptosis on FTI-induced autophagy. (A) Treatment with 3-MeA decreased lonafarnib-induced autophagy in U2OS cells. Stably transfected GFP-LC3-overexpressing U2OS cells were treated with 20 μM lonafarnib in the absence or presence of 50 μM 2-MeA for 24 h. The labeled images show the ultrastructural features of the cell samples at the same magnification. (B) Treatment with a pan-caspase inhibitor increased lonafarnib-induced autophagy in U2OS cells. Stably transfected GFP-LC3-overexpressing U2OS cells were treated with 20 μM lonafarnib in the absence or presence of 50 μM Z-VAD.fmk for 24 h. The upper panels show immunoblots with the antigen labeled on the left whereas the lower panels show the ultrastructural features of the cell samples at the same magnification.

promotes autophagy in MCF-7 cells, which normally do not have detectable levels of Beclin-1 expression.28 Bax/Bak double-knockout cells can undergo autophagic cell death, which depends on APG5 and Beclin-1 and is modulated by BCL-x(L).29 The Bcl-2 family of proteins may play important roles in coordinating the two pathways of programmed cell death (apoptosis and autophagy).30 Experimental evidence supports a role for autophagy in both development and suppression of cancer.31 Whether autophagy 1682

protects or kills cancer cells when they are exposed to chemotherapeutic drugs is a source of controversy. In the case of FTI-induced autophagy, we found that neither inhibition of autophagy by 3-MeA nor inhibition of caspases by Z-VAD.fmk significantly changed the number of cancer cells capable of undergoing clone formation in soft agar. These findings are not sufficient to declare autophagy as a cell death pathway in the context of lonafarnib treatment, but they do suggest that autophagy and apoptosis may be alternative cell death


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Materials and Methods

accelerating voltage of 80 kV. Whole cells in the sections were imaged at a magnification of x6000. Changes in cytoplasmic organellar morphology were observed at a magnification of x27,000. Sulforhodamine B protein biomass assay. A colorimetric sulforhodamine B protein biomass assay was performed for protein biomass as described previously.19 siRNA interference of RHEB. U2OS/GFP-LC3 cells were transfected with either human RHEB ON-TARGET plus SMART pool (Dharmacon, L-009692-00) or On-Target plus SiControl Non-target pool siRNA (Dharmacon, D-00181010-20) using the DharmaFECT1 transfection reagent (Dharmacon, T-2001-03). Cells were photographed or collected after 72 h of transfection. Fluorescent microscopy. Live-cell images were recorded and evaluated for the presence of autophagosomes using an OLYMPUS IX70 fluorescent microscope. Preparation of cell lysates. Control or drug-treated cells were rinsed with phosphate-buffered saline (PBS) and then lysed with RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) with added protease and phosphatase inhibitors (10 mM β-glycerophosphate, 1 mM Na3VO4, 10 μg/mL pepstatin A, 5 μg/mL aprotinin, 10 μg/mL leupeptin, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride). The DNA in the lysates was sheared using sonication in eight 1-s bursts at medium power. SDS-polyacrylamide gel electrophoresis and immunoblot analysis. SDS-polyacrylamide gel electrophoresis and immunoblot analysis of cell lysates were performed using standard methods as described previously.17,18 The protein concentrations in samples were measured using a modified Lowry method (DC Protein Assay; Bio-Rad Laboratories, Hercules, CA). Equal amounts of total protein from each sample were loaded onto SDS-polyacrylamide gels. Kaleidoscope prestained standards (Bio-Rad Laboratories) were used for molecular weight calibration. Immunoblotting (Western blotting) was performed using polyvinylidene fluoride membranes. Kodak XAR film (Eastman Kodak, Rochester, NY) was used to record images generated using enhanced chemiluminescence with an ECL kit (Amersham Biosciences, Piscataway, NJ).

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pathways once death of an FTI-treated cancer cell is committed. Future studies will elucidate mechanistic details regulating the decision for cell death and the choice of cell death pathway between apoptosis and autophagy.

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Figure 4. Lonafarnib induced autophagic cell death and downregulated signaling through the mTOR signaling pathway. (A) The bar chart shows anchorage-independent growth of lonafarnib-treated cells (relative to that of control cells) in the presence (+) or absence (-) of 3-MeA or Z-VAD.fmk plotted for the concentration of lonafarnib, 3-MeA, and Z-VAD.fmk. The error bars represent 95% confidence intervals. (B) Immunoblots against total mTOR and phosphospecific mTOR (P-mTOR) in U2OS cells treated with lonafarnib at different concentrations are shown. EGF, epidermal growth factor. (C) Immunoblot using phospho-specific antibody against S6 kinase in U2OS cells treated with lonafarnib (+) and untreated control (-) is shown. The actin immunoblot controls for gel loading. (D) Fluorescent photomicrographs of U2OS cells transfected with GFP-LC3 are shown. The left upper panel shows untreated control cells; the left lower panel shows cells treated with rapamycin; the right upper panel shows cells transfected with control siRNA; the right lower panel shows cells transfected with siRNA against RHEB.

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Cell culture. The human pancreatic cancer cell line Panc-1 and the human osteosarcoma cell line U2OS were obtained from the American Type Culture Collection (Manassas, VA). The plasmid expressing GFP-LC3 was provided by Dr. Mizushima.20 Antibodies. Antibodies against mTOR, phosphorylated mTOR, p70 S6 kinase 1, S6 ribosomal protein, and GFP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), Cell Signaling Technology (Beverly, MA) and Upstate (Charlottesville, VA). A monoclonal anti-actin antibody, clone AC-15, was obtained from Sigma (St. Louis, MO). Drugs, chemicals and hormones. The FTIs manumycin A and FTI-276 were purchased from Sigma. Lonafarnib was provided by Schering-Plough (Kenilworth, NJ). The FTIs were dissolved in tissue culture-grade DMSO (Sigma) at appropriate concentrations prior to dilution in tissue culture medium to a final concentration of less than 0.1% DMSO (v/v). Z-VAD.fmk was obtained from BD Pharmingen (San Diego, CA). Recombinant human epidermal growth factor was purchased from Promega (Madison, WI). Rapamycin was purchased from Sigma. Transmission electron microscopy. Cancer cells (grown as adherent culture on glass cover slips in Petri dishes) were fixed in 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for one hour as described previously.19 The samples were then washed with 1% cacodylate-buffered tannic acid, postfixed in 1% buffered osmium tetroxide for one hour, and stained en bloc with 1% uranyl acetate before dehydration in ethanol, embedment in Spurr’s low-viscosity embedding medium, and polymerization at 60°C for two days. Ultra-thin sections of the samples were stained with uranyl acetate and lead citrate and examined under a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan) at an www.landesbioscience.com


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This research was partially supported by a grant from Abbott Laboratories (Thyroid Research Advisory Council; to S.-C.J.Y.), by a generous donation from Mr. Robert Brochstein (to S.-C.J.Y.), NIHR01CA 089266 (to M.H.L.), Directed Medical Research (DOD SIDA 8C062166)(to S.-C.J.Y. & M.H.L.), Major REsearch Plan of National Science Fund of China (90713036 to J.P.), National High Technology Research and Development Program of China (863 program grant 2007AA02Z490 to J.P.), and National Basic Research Program of China (973 Program grant 2009CB825506 to J.P.). The Research Animal Support Facility and Veterinary Histopathology Core at The University of Texas M.D. Anderson Cancer Center are partially supported by the Cancer Center Support (Core) Grant (CA16672). We thank Dr. Corazon D. Bucana and Mr. Kenneth Dunner, Jr. (High Resolution Electron Microscopy Facility, The University of Texas M.D. Anderson Cancer Center) for their expert technical assistance.

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Statistical analysis. Statistical analysis was performed using the SPSS software program (version 12.0; SPSS Inc., Chicago, IL). The significance of statistical differences among multiple groups was assessed using analysis of variance. When the data sets failed the normality test, Kruskal-Willis one-way analysis of variance on ranks was used. Post-hoc pairwise comparison of groups was tested for significance using Dunn’s method. The null hypothesis was accepted if the p value was less than 0.05.

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2008; Vol. 7 Issue 10