[Chinese Journal of Cancer 27:8, 130-5; 130-134; August 2008]; 2008 Sun Sun Yat-Sen University Cancer Center Basic Research Paper Overexpressing exogenous S100A13 gene and its effect on proliferation of human thyroid cancer cell line TT Ren-Xian Cao, Li-Na Tian, Fang Wen, Xing Liu, Jing Zhong and Ge-Bo Wen* Institute of Clinical Research; First Affiliated Hospital; University of South China; Hengyang, Hunan, P.R. China Key words: S100A13 gene, thyroid neoplasm, TT cell, gene transfection, cell proliferation, cell cycle Background and Objective: S100A13 is involved in tumor formation, and is highly expressed in human thyroid gland. This study was to investigate the effect of exogenous S100A13 overexpression on the proliferation of human thyroid cancer cell line TT. Methods: The eukaryotic expression plasmid pcdna3.1/nt-gfp-s100a13 and empty vector pcdna3.1/nt-gfp were transfected into TT cells. The cells were selected by G418. The expression of green fluorescent protein (GFP) was observed under laser scanning microscope, and the expression of S100A13 mrna and protein was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The effects of S100A13 on cell proliferation and cell cycle progression were measured by cell growth curve and flow cytometry. Results: TT-S100A13-GFP and TT-GFP cells, which separately expressed S100A13 and pcdna3.1/nt-gfp, were constructed successfully. TT-S100A13-GFP cells grew faster than TT-GFP and TT cells [(2.30 ± 0.24) 10 5 vs. (1.40 ± 0.25) 10 5 and (1.50 ± 0.22) 10 5 at the seventh day of cell culture, p < 0.05]; both S phase proportion and G 2 /M phase proportion were significantly higher in TT-S100A13-GFP cells than in TT-GFP and TT cells [(6.47 ± 0.14)% vs. (5.86 ± 0.23)% and (5.99 ± 0.28)% at S phase, p < 0.05; (50.27 ± 0.66)% vs. (39.39 ± 0.23)% and (39.64 ± 0.64)% at G 2 /M phase, p < 0.05]. Conclusion: Exogenous S100A13 gene overexpression could accelerate cell proliferation, and promote cell cycle progression of TT cells from G 0 /G 1 phase to S and G 2 /M phase. Materials and Methods Materials. Human thyroid cancer cell line TT was purchased from the cell bank of Shanghai Institute of Biological Sciences, *Correspondence to: to: Ge-Bo Wen; Institute of Clinical Research; First Affiliated Hospital; University of South China; Hengyang, Hunan 421001, P.R. China; Tel.: Submitted: 86.734.8279392; XX/XX/XX; Fax: Revised: 86.734.8279008; XX/XX/XX; Accepted: Email: wengb@nhu.edu.cn XX/XX/XX This Submitted: paper 11/21/07; was translated Revised: into English 03/10/08; from its Accepted: original publication 03/10/08in Chinese. Translated by: XXXXXXXXXXXXXXXX on XX/XX/XX. This paper was translated into English from its original publication in Chinese. The Translated original by: Chinese Beijing version Xinglin of Meditrans this paper Center is published and Wei in: Liu Ai on Zheng(Chinese 05/14/08. Journal of Cancer), 27(8); http://www.cjcsysu.cn/cn/article.asp?id=xxxxx The original Chinese version of this paper is published in: Ai Zheng(Chinese Previously Journal of Cancer), published 27(8); online http://www.cjcsysu.cn/cn/article.asp?id=14349 as a Chinese Journal of Cancer E-publication: http://www.landesbioscience.com/journals/cjc/article/xxxx Previously published online as a Chinese Journal of Cancer E-publication: Addendum http://www.landesbioscience.com/journals/cjc/article/6613 to: Chinese Academy of Science. Plasmid pcdna3.1/nt-gfp- S100A13 was stored in our lab. SuperScript TM III cdna reverse transcription kit, Lipofectamine TM 2000, Trizol reagent, primary rabbit anti-human green fluorescent protein (GFP) polyclonal antibodies, and G418 were all purchased from Invitrogen Company. Primary rabbit anti-human GAPDH polyclonal antibodies and horseradish peroxidase-labeled goat anti-rabbit secondary antibodies were purchased from Santa Cruz Company. BCA protein quantization kit, pre-blueranger stained protein molecular standard and protein fluorescent measuring kit were purchased from Hyclone Pierce. Fetal bovine serum and F12K non-serum culture media were purchased from Gibco and Sigma Company respectively. Primers and probes were synthesized by Takara Biotechnology (Dalian) Co., Ltd. (Table 1). Methods. Cell culture and grouping. TT cells were cultured with F12K culture media containing 10% fetal bovine serum, at 37 C incubator in an atmosphere of 5% CO 2. Cells at logarithmic growth phase were collected and divided into experimental group (TT-S100A13-GFP), empty vector group (TT-GFP), and blank group (TT). S100A13 gene transfection. Plasmids pcdna3.1/nt-gfp-s100 A13 expression and pcdna3.1/nt-gfp were respectively transfected into TT cells with Lipofectamine2000 according to the instructions. After 24 hours, complete culture media containing 500 µg/ml G418 were used to screen out positive clones. After four weeks of continuous screening, positive clones were formed. Sub-cellular localization of exogenous S100A13 protein. After amplified culture of positive cell clones, TT cells were washed with PBS twice. Then the cells were fixed with 3% paraformaldehyde at room temperature for 10 min, and washed again with PBS twice. TT cells were stained with DAPI at room temperature for 3 min and washed again with PBS, then observed under a laser scanning confocal microscope. Fluorescent real-time quantitative RT-pCR. GAPDH was used as internal control. Total RNA were extracted from the cells with Trizol reagent, and synthesized into cdna according to the instructions of SuperScript TM III cdna reverse transcription kit. On the Lightcycler2.0 real-time fluorescent quantitative polymerase chain reaction (PCR) equipment, gene was amplified. The reaction system (20 µl) was composed of 10 µl of 2 Premix Ex Taq TM, 0.4 µl of upstream primer and 0.4 µl of downstream primer, 0.8 µl of 130 Chinese Journal of Cancer 2008; Vol. 27 Issue 8
Table 1 The primer and probe sequences of S100A13 and GAPDH used in real-time RT-PCR Figure 1. Subcellular localization of green fluorescent protein (GFP) in human thyroid cancer TT cells at 48 h after transfection ( 400). (A) GFP locates in the whole cell after transfection of pcdna3.1/nt-gfp. (B) GFP locates in cytoplasm of the TT cell after transfection of pcdna3.1/nt-gfp-s100a13. Figure 2. Clones of TT cells after transfection of S100A13-GFP ( 100). (A) Clones of TT cells after transfection of exogenous S100A13-GFP fusion protein. (B) Clones of TT cells observed under spontaneous light. (C) Merged image of (A) and (B). Figure 3. The localization of S100A13-GFP fusion protein in TT cells ( 200). (A) Exogenous S100A13-GFP fusion protein (in green) locates in cytoplasm of TT cells. (B) Nuclei of TT cells were stained with 4',6-diamidino-2-phenylindole (DAPI). (C) Merged image of (A) and (B). fluorescent probe, 2.0 µl of cdna, and 6.4 µl of sterile distilled water. The PCR conditions were as follow: pre-degeneration at 95 C for 10 s, 40 cycles of degeneration at 95 C for 5 s, and annealing and elongation at 60 C for 20 s. Western blot analysis. Total protein were extracted from the cells respectively, and the protein concentration was measured by BCA protein quantization kit. After incubation at 100 C for 10 min, equivalent total protein was used for SDS-PAGE electrophoresis before transferring onto PVDF membrane. The membrane was washed with TBST, then blocked with TBST containing 5% defatted milk powder for 2 h, and incubated with primary rabbit anti-human GFP polyclonal antibody (1:2000) at 4 C overnight. On the second day, primary antibody was removed and the membrane was washed with TBST three times. The proteins www.landesbioscience.com Chinese Journal of Cancer 131
were incubated with secondary horseradish peroxidase-labeled goat anti-rabbit antibody (1:1000) at 37 C for 45 min, then washed with TBST buffer for three times. The expression of S100A13 was detected with protein fluorescent kit. Cell growth measurement. The three groups of cells were respectively seeded into 24-well plates with 1 10 4 cells per well. For each group, cells were seeded into 21 wells, and three wells of cells were counted every day to obtain the averages. Growth curves were drawn seven days later. The analysis of cell cycle. The three groups of cells at logarithmic growth phase were digested by trypsin. After centrifuged at 1000 g for 5 min, cells were washed twice with pre-cooled PBS solution, then fixed with 1 ml of 70% ethanol (700 µl anhydrous ethanol plus 300 µl PBS) at -20 C overnight. On the second day, cells were centrifuged at 1000 g for 3 min, then washed with PBS, digested with RNAse and washed again with PBS. Cells were stained with propidium iodide for 45 min, then cell cycle was analyzed by flow cytometry. Statistical analysis. The experiments described above were all repeated for three times. All data are presented as mean ± standard deviation (SD). Inter-group differences were analyzed by t-test, using SPSS 11.0 software. A p value of less than 0.05 was considered significant. Results S100A13 expression in TT cells after transfection. At 48 h after transfection, green fluorescence was observed in the cytoplasm and nuclei of TT-GFP cells, while green fluorescence was only observed in the cytoplasm of TT-S100A13-GFP cells (Fig. 1). After four weeks of screening by G418, positive clones were obtained and green fluorescence was seen (Fig. 2). With DAPI nuclear staining, exogenous S100A13 protein was localized in the cytoplasm of TT-S100A13-GFP cells (Fig. 3). The mrna level of S100A13 was significantly higher in TT-S100A13-GFP cells than in TT-GFP and TT cells (p < 0.01), while the mrna level of GAPDH showed no significant difference among the three groups (p > 0.05) (Fig. 4). GFP protein was detected both in TT-S100A13-GFP cells and in TT-GFP cells, but it was 11 ku bigger in TT-S100A13-GFP cells than in TT-GFP cells, matching the size of S100A13 protein; no expression of GFP protein was detected in TT cells (Fig. 5). Proliferation and cell cycle of TT cells after S100A13 transfection. The growth rate of TT-S100A13-GFP cells was much faster than those of TT-GFP and TT cells (p < 0.05), and there was no significant difference between TT-GFP and TT cells (p > 0.05) (Fig. 6). The results suggested that of S100A13 gene overexpression could promote the growth of TT cells. The proportion of TT-S100A13-GFP cells was significantly decreased at G 0 /G 1 phase, while increased at G 2 /M phase (p < 0.01); the differences in cell cycle were not significant between TT-GFP and TT cells (p > 0.05) (Table 2). Figure 4. (At right) Detection of S100A13 and GAPDH expression in TT cells with real-time RT-PCR. (A1) and (A2) standard curves (fluorescent light of S100A13 plasmid template multiple diluted); (A3) fluorescent light curve of S100A13 expression; (B1) and (B2) standard curves (fluorescent light of GAPDH plasmid template multiple diluted); (B3) fluorescent light curve of GAPDH expression. Curve 1: TT-S100A13-GFP cells; curve 2: TT cells; curve 3: TT-GFP cells. 132 Chinese Journal of Cancer 2008; Vol. 27 Issue 8
Figure 5. Detection of GFP protein expression in TT cells with Western blot. Lane 1: TT-GFP cells; lane 2: TT cells; lane 3: TT-S100A13-GFP cells. Figure 6. Proliferation of TT cells after transfection of S100A13. Table 2 Discussion The effects of S100A13 on cell cycle of TT cells after transfection All values are presented as mean ± SD of 3 experiments. a p < 0.05, vs. TT cells. S100 protein family is a signal protein superfamily with EF-hands and its biological functions are achieved through regulating calcium ions and interacting with target proteins. 5 Up to date, 21 members have been discovered and many of them are abnormally expressed in tumors, which are related to the invasion and metastasis of tumors. Currently, it was discovered that S100A4 is highly expressed in breast cancer, colorectal cancer, gastric cancer, esophageal squamous cell cancer, osteosarcoma, and so on. 6,7 The expression of S100B is up-regulated in melanoma and prostate tumor, and it could be used as a tumor marker and prognosis predictor for melanoma. 8 In addition, S100A6 is highly expressed in breast cancer, colorectal tumor, thyroid tumor, malignant fibrous histiocytoma, and melanoma, 8 S100A8 and S100A9 are highly expressed in colorectal tumor, 9 while S100P is highly expressed in breast cancer. 10 S100A13, an important member in the S100 protein family, is widely expressed in various tissues, such as heart, skeletal muscle, brain, pancreas, kidney, uterus, small intestine, thyroid gland, thymus, bladder, liver, mammary gland, appendix, lung, trachea, placenta, and others, especially highly expressed in the follicular cells of thyroid. 1 Currently, researches on the functions of S100A13 protein primarily focus on its effect on the nonclassical transmembrane transport of FGF-1 and IL-1α, which lack signal peptides. 11 FGF, a structure-related multi-gene family, is involved in many biological activities. During the fetal development, FGFs could regulate the induction of mesoderm, as well as the neurulation and the formation of skeletal system; they also play important roles in angiogenesis, tissue regeneration, inflammatory reaction, and tumor formation. FGF-1, an extremely powerful angiogenesis factor and mitogen, could stimulate the proliferation and migration of vascular endothelial cells and smooth muscular cells, stimulate endothelial cells to secrete collagenase and proteolytic enzymes, promote the hydrolysis and precipitation of collagen, and induce the formation of tube-like structure, therefore, promote cancer metastasis. 12 It was reported that FGF-2 is associated with the initiation and progression of thyroid tumor, and its expression level is positively correlated to malignant degree and infiltration ability. 13 However, the expression of FGF-1 in thyroid gland and thyroid cancer has not been reported yet. In this study, S100A13 was successfully transfected into thyroid cancer TT cells as verified by real-time RT-PCR and Western blot. The results revealed that the over-expression of S100A13 gene could promote the proliferation of TT cells, push the progression of TT cells from G 1 phase to S and G 2 /M phases. We speculated that the over-expression of S100A13 gene could significantly promote the proliferation of thyroid cancer cells by upregulating the expression of FGF-1, which promotes angiogenesis and inhibits the apoptosis of tumor cells. Acknowledgements Grant: Natural Science Foundation of Hunan Province (No. 06jj5046) References [1] Ridinger K, Schafer BW, Durussel I, et al. S100A13 biochemical characterizeation and subcellular localization in different cell lines [J]. J Biol Chem, 2000,275(12):8686-8694. [2] Brewer GJ, Dick RD, Grover DK, et al. Treatment of metastatic cancer with tetrathiomolybdate, an anticopper, antiangiogenic agent: phase I study [J]. 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Cancer Res, 2000,60(6):1595-1603. [8] Ilg EC, Schafer BW, Heizmann CW. Expression pattern of S100 calcium-binding proteins in human tumors [J]. Int J Cancer, 1996,68(3):325-332. [9] Stulik J, Osterreicher J, Koupilova K, et al. The analysis of S100A9 and S100A8 expression in matched sets of macroscopically normal colon mucosa and colorectal carcinoma: the S100A9 and S100A8 positive cells underlie and invade tumor mass [J]. Electrophoresis, 1999,20(425):1047-1054. www.landesbioscience.com Chinese Journal of Cancer 133
[10] Guerreiro Da Silva ID, Hu YF, Russo IH, et al. S100P calcium-binding protein overexpression is associated with immortalization of human breast epithelial cells in vitro and early stages of breast cancer development in vivo [J]. Int J Oncol, 2000,16(2):231-240. [11] Yang J, Cao RX, Wen GB. Progress of calcium-binding protein S100A13 [J]. Guo Wai Yi Xue Sheng Li, Bing Li Ke Xue Yu Lin Chuang Fen Ce, 2005, 25(2):156-158. [Article in Chinese] [12] Billottet C, Janji B, Thiery JP, et al. Rapid tumor development and potent vascularization are independent events in carcinoma producing FGF-A or FGF-2 [J]. Oncogene, 2002,21(53):8128-8139. [13] Zhao WX, Yang YH, Chen DL. Expression of basic fibroblast growth factor and fibroblast growth factor receptor relate to invasion and metastases in thyroid carcinoma [J]. Fu Jian Yi Ke Da Xue Xue Bao, 2002,36(3):286-289. [Article in Chinese] 134 Chinese Journal of Cancer 2008; Vol. 27 Issue 8