前胸腺素Alpha之細胞內分佈對膀胱癌細胞之影響與機轉之探討

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(1)NSC 97 2314 97 8 1. B 006 051 MY3 100 7 31.

(2) immunohistochemistry (IHC). ,. (urothelial TCC) IHC PTMA. prothymosin –α (PTMA) PTMA negativ (. ). PTMA PTMA PTMA. J82. PTMA. Wide type (. )or deNLS( Lentivirus. cell sorting PTMA cell growth wound healing assay. (1)UREA-PAGE (2) cell sorting In vitro MTT and wound healing assays SCID. PTMA. deNLS(. )PTMA J82 (1)UREA-PAGE (2) MTT assay. cell migration. SCID. PTMA. J82. PTMA )PTMA. PTMA PTMA RNA differential array. PTMA. J82.

(3) Study background: By immunohistochemistry staining, we found that, along the urothelial epithelium of human genitourinary tract including renal pelvis, ureter and urinary bladder, only urothelial cancer but not tumor-adjacent normal uroepithelium is positive for prothymosin-alpha (PTMA) stain. In addition, the intracellular location of PTMA staining is significantly correlated with tumor grading and staging. In order to further understand the significance of intracellular localization of PTMA, we designed this three-year study to investigate the biological behavior change of PTMA gene when it was transfected into a PTMA low-expression human bladder cancer cell line J82. Material and methods: Wide type or deNLS PTMA-expressing J82 transfectants and its control were produced and collected after infected with lentivirus carrying each gene as indicated. We confirmed the expression level of PTMA protein, by (1)UREA-PAGE (2) Immunohistochemical staining for PTMA expression and collected them by cell sorting. Thereafter, we performed MTT assay to compare the difference of cell growth among the transfectants and also wound healing assay to investigate the effect of PTMA expression on cell migration. In Vivo, SCID mice were subcutaneously inoculated with these transfectant tumor cells to compare their tumorigenesis and tumor growth rate in mice. Results and conclusions: We had successfully created the several J82 transfectants with various expression localization of PTMA protein. In vitro MTT and wound healing assays showed there was no difference among these transfectants. However, in vivo tumorigenesis in SCID mice showed J82 cells expressing deNLSPTMA protein had more rapid growth rate and shorter survival than cells with wtPTMA expression or control cells. Such an inconsistence required further investigation. In patients with upper urothelial carcinoma, tumors with cytoplasmic PTMA expression had a shorter recurrence time than other tumors. Such findings were compatible with in vivo tumorigenesis study. Taken together, cytoplasmic PTMA expression or lack of ability of PTMA into the nucleus might play an important role in tumor recurrence of urothelial carcinoma. In future, we will perform the RNA differential array to see the effect of PTMA localization in urotehlial carcinoma. Key words: prothymosin-alpha, urotehlial carcinoma.

(4) - (Prothymosin, PTMA) (MLR) upregulate. [1] IL-2 receptor. Immunomodulating cell proliferation mixed lymphocyte reaction. MHC class II expression IL-2. upregulate NK cell LAK cell IL-2 receptor activities[4,5] antitumor immunity lymphocytes TNF-. [2] T lymphocyte T lymphocyte [3]. expression cytotoxic induce tumor specific CD8+ cytotoxic T. MHC non-restricted antitumor activity[6] PTMA. myeloma. Antisense PTMA oligomer G1 S. [7]. mRNA. [8,9] PTMA mRNA [11]. PTMA. NIH3T3 [10]. PTMA (NSC 92-2314-B-006-135). 238. TCC. 22. , PTMA 151. TCC ROC. specificity 71.8% PTMA ,. myeloma. 60 groups 0.5 ng/ml cutoff value. sensitivity. 64.24.%. Urology 2006, 67:294-299 [12] immunohistochemistry (IHC) (1) IHC. (urothelial TCC) immunohistochemistry(IHC) PTMA. PTMA negativ. PTMA. PTMA. 96 ,. (2) ( 1. N: nuclear expression only, 2.. C: Cytoplasm expression only, 3. CN: mixed but cytoplasm expression dominant, 4. NC: mixed but nuclear expression dominant) 94 PTMA 63.8% urothelial TCC cytoplasm –dominant expression (C+CN), 17.1% nuclear –dominant expression (N+NC), 18.1% negative expression prothymosinnegative cytoplasmic-expression dominant urothelial TCC tumor stage tumor grade PTMA. nuclear-dominant PTMA. TCC(UUT TCC). (p = 0.011 and 0.038, respectively) cytoplasm urothelial TCC. (3) cytoplasmic-expression dominant [13,14] (. +/) cytoplasm –dominant. (upper urothelial PTMA.

(5) (1). PTMA. PTMA PTMA biological behavior. ? 2 up- down-regulate. PTMA RNA differential array. 3 PTMA. PTMA. western blotting T24 TCC-SUP BFTC-905. expression (PTMA. Human TCC cell lines J82. TSGH8301. TCC-SUP PTMA Human TCC cell lines J82. (1) J82 clones with wide type PTMA ) (2) J82 clones NLSPTMA(PTMA (3) J82-GFP clones (as. a control) recombinant DNA NLS prothymosin-. DNA(. ,. encode. MAP). wild type PTMA. DNA. Lentivirus vectors. J82 SCID mice. Experiment 1-Establishment of wide type or NLS prothymosin-alpha-expressing J82 transfectants and its control. Five Lentiviral vectors (pWPI-ProT, pWPI-ProT NLS, pWPXL-GFP-wtProT, pWPXL-GFP-ProTdeNLS, pLVTHM [as empty control]), and package vectors (pMD2.G, pSPAX2) were co-transfected into 293T cells for production of Lentivirus carrying wide type (wt) PTMA, or deNLS-PTMA (loss of nucleus leading sequence), and empty control. The transfection method was calcium phosphate precipitation method with 20 microgram per plasmid. Forty-eight hours later, the medium was collected and stored at -80 . For selecting stably J82 transfectants carrying wtPTMA-GFP, or deNLSPTMA-GFP, J82 cells was infected with virus-containing supernatant medium by aid of polybrene (750 microgram per ml). Two days after infection, cells were subcultured in 1:10 dilution, and observe the colony formation with green fluorescence under fluorescence scope. Cell sorting was performed for collecting the infected cells according to the presence of GFP. Experiment 2 To confirm the expression level of PTMA protein, urea-PAGE, and immunofluoresence staining, and IHC for PTMA expression were done. UREA-PAGE Cells were lysed in 150 ul hot (100 ) lysis buffer (6mM Tris-HCl, pH 6.8, 10% SDS, 2% 2-mercatoethanol) for 3 min at 100 . Then 150 ul (100 ) phenol, saturated with 20mM ammonium acetate (pH 4.5) and 10mM EDTA was added. The mixture was mixed, incubated for 3 min at 100 , then 10 min at 0 . After centrifugation (12000rpm, 5min), the precipitate of cell debris was discarded and 100 ul chloroform was added to the supernatant for phase separation..

(6) The upper aqueous phase was re-extracted with 150 ul saturated phenol and mixed with 20 3 M sodium acetate(pH 5.2). This results in new separation of phases. 3 vol. ethanol were added to the lower phenol phase, the mixture was vortexed, chilled(-20 , 30 min), centrifuged at 12000 rpm for 10 min, the pellet was washed with ethanol, dried and dissolved in 20 ul water. Electrophoresis in 12 polyacrylamide gel containing 7M urea without SDS(150 mm×120 mm×1 mm) was according to the standard technique(40mA, 200-300 V). The gel were stained with 0.2 methylene blue in 50 mM sodium acetate(pH 4.5) for 5 minutes, briefly destained with water(5 times× 5min with shaking), and immediately photographed. Further washing of gel may result in destaining of PTMA band. Immunofluoresence staining and immunohistochemistry (IHC) To characterize the localization of PTMA expression, we performed IHC in these transfectants. In brief, 2×103 cells/well were plating into a 96-well dish. After overnight, fix with 10% formaldehyde for 5 minutes and then treat with the mixture of methanol and acetic acid (1:1) for 5min. Endogenous peroxidases was blocked by incubating with 3 % H2O2 for 5 minutes at room temperature. The cells were be subsequently incubated at 4°C with anti-PTMA (2F11), purchased from Alexis Biochemicals (Lausen, Switzerland), at a dilution of 1:5000 overnight. The cells were then treated with biotin-conjugated second antibody before adding streptavidin-HRP (LSAB 2 system kit; DAKO, Carpinteria, CA, USA). For color reaction, peroxidase was detected with aminoethyl carbazole substrate kit (AEC kit; Zymed laboratories, San Francisco, CA). Monitor the development of brown color, and stop development by washing with PBS three times. Only when the IHC signals are present, the distribution of PTMA protein was recorded. In addition, some cells were stained with DAPI for nuclear staining. Observe and record under immunofluorescence scope. Experiment 3-- Cell proliferation assay-Microculture tetrazolium test (MTT assay) for cell proliferation To compare the difference of cell proliferation between the transfectants, we performed MTT assay. In brief, transfectants were seeded in a 96-well plate at 1000 cells per well overnight with 2% FBS- containing medium. When control wells are near 80-90 % of the confluency, the viable cells were determined with the MTT assays. In Brief, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma, St. Louis, MO, USA) were added to the cells to a final concentration of 0.05 % and incubated 3 h at 37°C. Then, the media were removed and replaced by 50 l DMSO, and the plates were shaken for 3 min. The optical density (OD) of each well was determined using a microplate reader at a wavelength of 570 nm. The percentage of cell viability was calculated by the OD value of treated cells normalized with the OD value of control cells. The growth index was calculated by the formula: (%48h or %72 h / %24h).. . Experiemtn 4--Wound healing test for cell migration-Cell migration assay (in vitro wound-healing assay) To evaluate the effect of PTMA expression on cell migration, we performed wound healing assay. In brief, cells as indicated at a density of 1 × 104 cells per well were seeded onto 24-well plates for overnight. After cells have attached on plates completely, we scraped the cells with 3 horizontal lines per well in a plate by a yellow tip. After scraped, the cells were washed with PBS and were incubated with the medium containing 2% FBS. Each line was photographed at 24-hour intervals with a magnification of 100× with a microscope video system. We measured the width of each line in each well then averaged them. The migration index was calculated by the formula: 1 −(width 24 or 48 or 72 h/width0 h). Experiment 5-Animal studies To explore the tumor formation, growth under the PTMA expression, we measured the probability of tumorigenesis, tumor growth rate in mice. SCID Mice were subcutaneously.

(7) . inoculated with 2 10 6 transfectants as indicated in 100 µl 2% FBS-containing medium. Tumor formation and growth was recorded every other day for at least 60 days according to the 'Guidelines for the Welfare of Animals in Experimental Neoplasia' (1998). Tumor volumes were calculated using the formula: length (width)2 0.45.. . . Statistical analysis Experimental results were analyzed for statistical significance (Student’s t- test) or curve-fit analysis using Prism GraphPad Prism 4.00 forWindows (GraphPad Software, San Diego, CA) and SigmaPlot 8.0 (SPSS, Chicago, IL) software packages. Results presented were representative of at least duplicate experiments, and statistical significance is indicated (* p < 0.05; ** p < 0.01; *** p < 0.001)..

(8) 1. After lentivirus infection, J82 cells carrying GFP, wtPTMA-GFP and deNLSPTMA-GFP gene were collected by cell sorting. After sorting, cells were stained with anti-PTMA (2f11) antibody and DAPI and observed under immunofluorescence scope. UREA-PAGE, IHC, and immunofluorescence staining confirmed the ectopic expression of PTMA and its localization.. 2F11. DAPI. GFP. Merge FCM. UREA-PAGE. 2. MTT assay MTT assay showed there was no significance in cell growth among J82-GFP, J82-wtPTMA-GFP and J82-dePTMA-GFP..

(9) 3. Migration assay The result from wound healing assay showed there was no difference on migration among three transfectants.. 4. Tumor formation in animals Tumoriegensis formation in SCID mice showed J82-deNLSPTMA-GFP cells had more rapid growth rate in vivo and short survival than the other two groups.. 100 J82-GFP J82-wtPTMA-GFP J82-dNLSPTMA-GFP. 2000. 80. Survival (%). Tumor volumn (mm3). 2500. 1500. 1000. 60 40. J82-GFP J82-wtPTMA-GFP. 20. 500. 0. 0 0. 7 10 12 14 17 19 21 24 26 28 31 33 35 38 40 42 45. Days after tumor inoculation. *. *** J82-∆NLSPTMA-GFP 0. 12. 24. 36. 48. 60. Days after tumor cells inoculation.

(10) We had successfully created the several J82 transfectants with various expression localization of PTMA protein. In vitro MTT and wound healing assays showed there was no difference among these transfectants. However, in vivo tumorigenesis in SCID mice showed J82 cells expressing deNLSPTMA protein had more rapid growth rate and shorter survival than cells with wtPTMA expression or control cells. Such an inconsistence required further investigation. In patients with upper urothelial carcinoma, tumors with cytoplasmic PTMA expression had a shorter recurrence time than other tumors. Such findings were compatible with in vivo tumorigenesis study. Taken together, cytoplasmic PTMA expression or lack of ability of PTMA into the nucleus might play an important role in tumor recurrence of urothelial carcinoma. In future, we will perform the RNA differential array to see the effect of PTMA localization in urotehlial carcinoma..

(11) 1. Baxevanis, CN, Reclos GJ, Papamichail M, Tsokos GC. Prothymosin-alpha restores the depressed autologous and allogegeic mixed lymphocyte responses in patients with systemic lupus erythematous. Immunopharmacol. Immunotoxicol. 1987,9:429-440. 2. Baxevanis CN, Thanos D, Reclos GJ, Anastasopoulos E, Tsokos GC, Papamatheakis GJ, Papamichail M Prothymosin-alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation. J Immunol 1992,148:1979-1984. 3. Cordero OJ, Sarandeses C, Lopez JL, Cancio E, Regueiro BJ, and Nogueira M. Prothymosin-alpha enhances interleukin 2 receptor expression in normal human T-lymphocytes. Int J Immunopharmacol 1991,13:1059-1065. 4. Cordero OJ, Sarandeses C, Lopez JL and Nogueira M. Prothymosin-alpha enhances human natural killer cell cytotoxicity: role in mediating signals for NK activity. Lymphokine Cytokine Res. 1992,11:277-285. 5. Lopez-Rodriguez JL, Cordero OJ, Sarandeses C, Vinuela J and Nogueira M. Interleukin-2 killer cells: in vitro evaluation of combination with prothymosin-alpha Lymphkine Cytokine Res. 1994, 13:175-182. 6. Baxevanis CN, Gritxapis AD,Spanakos G, Tsitsilonis OE, and Papamichali M. Induction of tumor-specific T lymphocyte responses in vivo by prothymosin. Cancer Immunol Immunother 1995, 40:410-418. 7. Sburlati AR, Manrow RE, and Berger SL. Prothymosin-alpha antisense oligomers inhibit myeloma cell division. Proc. Natl Acad Sci USA 1991, 88:253-257. 8. Roson E, Gallego R, Garcia-Caballero T, Heimer EP, Felix AM, Dominguez F. Prothymosin alpha expression is associated to cell division in rat testis. Histochemistry 1990, 94:597-599. 9. Bustelo XR, Otero A, Gomez-Marquez J, Freire M. Expression of the rat prothymosin a gene during T-lymphocyte proliferation and liver regeneration. J Biol Chem 1991, 266:1443-1447. 10. Eschenfeldt WH, Berger SL, The human prothymosin alpha gene is polymorphic and induced upon growth stimulation; evidence using a cloned cDNA. Proc. Natl Acad Sci USA 1986, 83:9403-9407. 11. Dominguezs F, Magdalena C, Cancio E, Roson E, Paredes J, Lourdes L, Zalvide J, Fraga M, Forteza J, Regueiro BJ, Puente JL. Tissue concentration of prothymosin alpha: a novel proliferation index of primary breast cancer. Eur J Cancer 1993, 26A:893-897. 12. Tzai, T. S., Tsai, Y. S., Shiau, A. L. et al.: Urine prothymosin-alpha as novel tumor marker for detection and follow-up of bladder cancer. Urology 2006, 67: 294 13. Tsai YS, Jou YC, Lee GF, Chen YC, Shiau AL, Tsai HT, Wu CL, Tzai TS. Aberrant prothymosin-alpha expression in human bladder cancer. Urology 2009 ,73(1):188-192. 14. Jou YC, Dong GL, Tsai YS, Shen CH, Chen SY, Shiau AL, Tsai HT, Wu CL, Tzai TS. Prognostic relevance of prothymosin-alpha expression in human upper urinary tract transitional cell carcinoma. Urology, 2009 (Accepted, in press).

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(14) NSC 97 2314. PTMA. B 006. 051 MY3.

(15) Plasmid map.

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