欢迎访问《解剖学报》官方网站！今天是
English
转录因子环磷酸腺苷反应元件结合蛋白在紫杉醇诱导HeLa 细胞周期阻滞中的作用及其机制
黄帅帅 王雪 庄海慧 王宇多 周细武 王萍*
解剖学报 ›› 2014, Vol. 45 ›› Issue (4) : 485-492.
转录因子环磷酸腺苷反应元件结合蛋白在紫杉醇诱导HeLa 细胞周期阻滞中的作用及其机制
Effects of transcription factor cAMP response element binding protein on taxolinduced HeLa cell-cycle arrest
目的 探讨转录因子环磷酸腺苷反应元件结合蛋白（CREB）在紫杉醇诱导宫颈腺癌HeLa细胞周期阻滞中的作用及其分子机制。方法 MTT法确定紫杉醇的最佳浓度和处理时间；PCR技术构建pCI neo/CREB（PN）重组质粒及pCI neo/CREB-M（PM）定点突变质粒；流式细胞术检测细胞周期，Western blotting检测磷酸化CREB（pCREB）、CREB、cyclins以及CDKs蛋白表达。 结果 紫杉醇抑制HeLa细胞增殖的有效条件为0.1μmol/L处理24 h。0.1μmol/L紫杉醇诱导G2/M细胞数量增加，并呈时间依赖性；cyclin A表达量下调，cyclin B1、D1和pCREB表达量上调。此外，紫杉醇的处理对cyclin E、CDK1、CDK2、CDK4和CREB表达量并无显著性改变。然而，PM联合紫杉醇处理后显著性地反转了紫杉醇单独处理引起的cyclin A下调、cyclin B1和cyclin D1的上调，并且细胞周期G2/M期阻滞显著性减少。 结论 转录因子CREB介导的靶向细胞周期蛋白表达在紫杉醇诱导的细胞周期阻滞过程中扮演重要角色。
Objective To explore the effects of cAMP response element binding protein (CREB) on taxol-induced cell cycle arrest in HeLa cells. Methods MTT assay was used to determine the optimal concentration and treatment time. PCR method was used to construct the recombinant plasmid pCI neo/CREB（PN）and site-directed mutagenesis recombinant plasmid pCI neo/CREB-M（PM）. Cell cycle was assayed by flow cytometry. Expressions of pCREB, CREB, cyclins and CDKs were assayed by Western blotting. Results The effective conditions of taxol treatment on HeLa cells were 0.1μmol/L for 24 hours. After cells were treated with 0.1μmol/L taxol, G2/M phase was arrested in a time-dependent manner, accomplished with the decrease of cyclin A, a significant increase of cyclin B1, D1 and phosphorylated CREB(pCREB) protein expression, whereas, no marked changes were observed in cyclin E, CDK1, CDK2, CDK4 and CREB expressions. However, combinantion of PM and taxol treatment significantly reduced taxol-induced G2/M phase arrest, and reversed the effect of taxol-decreased cyclin A, increased cyclin B1 and D1 expression. Conclusion Tanscription factor CREB-mediated specific cyclins play a pivotal role in taxol-induced G2/M arrest in HeLa cells.
宫颈癌 / 环磷酸腺苷反应元件结合蛋白 / 细胞周期 / 流式细胞术 / 免疫印迹法
Cervical cancer / Response element binding protein / Cell cycle / Flow cytometry / Western blotting
［1］Montminy MR, Bilezikjian LM. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene ［J］. Nature, 1987, 328 (6126):175-178.
［2］Deng L, Li Y, Huang JM, et al. Effects of p-CREB-1 on transforming growth factor-beta3 auto-regulation in hepatic stellate cells ［J］. J Cell Biochem, 2011, 112 (4):1046-1054.
［3］Kitagawa`K, Sasaki T, Terasaki Y, et al. CREB activation is a key player for ischemic tolerance in the brain ［J］. Rinsho Shinkeigaku, 2012, 52 (11):904-907.
［4］Klinz FJ, Korkmaz Y, Cho B, et al. Transcription factor CREB is phosphorylated in human molar odontoblasts and cementoblasts in vivo ［J］. Histochem Cell Biol, 2013, 139 (4):615-620.［5］Wang P, Xu J, Zhang C. CREB, a possible upstream regulator of Bcl-2 in trichosanthin-induced HeLa cell apoptosis ［J］. Mol Biol Rep, 2010, 37 (4):1891-1896.
［6］Wang P, Huang S, Wang F, et al. Cyclic AMP-response element regulated cell cycle arrests in cancer cells ［J］. PloS One, 2013, 8 (6):e65661.
［7］Wang WL, Wang P, Wang F, et al. Effects of RhoA/ROCKI signal pathway on the taxol-induced cell-cycle arrest of cervical cancer cells ［J］. Acta Anatomica Sinica, 2013, 44 (4):485-491. (in Chinese)
王维莉, 王萍, 王峰, 等. RhoA/ROCK信号通路在紫杉醇诱导宫颈细胞周期阻滞中的作用 ［J］. 解剖学报, 2013, 44 (4):485-491.
［8］Shu CH, Yang WK, Shih YL, et al. Cell cycle G2/M arrest and activation of cyclin-dependent kinases associated with low-dose paclitaxel-induced sub-G1 apoptosis ［J］. Apoptosis, 1997, 2 (5):463-470.
［9］Park JE, Woo SR, Kang CM, et al. Paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres: implication in multinucleation and chemosensitization ［J］. Biochem Biophys Res Commun, 2011, 404 (2):615-621.
［10］Yang J, Kornbluth S. All aboard the cyclin train: subcellular trafficking of cyclins and their CDK partners ［J］. Trends Cell Biol, 1999, 9 (6):207-210.
［11］John PC, Mews M, Moore R. Cyclin/Cdk complexes: their involvement in cell cycle progression and mitotic division ［J］. Protoplasma, 2001, 216 (3-4):119-142.
［12］Canavese M, Santo L, Raje N. Cyclin dependent kinases in cancer: potential for therapeutic intervention ［J］. Cancer Biol Ther, 2012, 13 (7):451-457.
［13］Kahl CR, Means AR. Regulation of cell cycle progression by calcium/calmodulin-dependent pathways ［J］. Endocr Rev, 2003, 24 (6):719-736.
［14］Farras R, Baldin V, Gallach S, et al. JunB breakdown in mid-/late G2 is required for down-regulation of cyclin A2 levels and proper mitosis ［J］. Mol Cell Biol, 2008, 28 (12):4173-4187.
［15］Khandelwal P, Padala MK, Cox J, et al. The N-terminal domain of y-box binding protein-1 induces cell cycle arrest in G2/M phase by binding to cyclin D1 ［J］. Int J Cell Biol, 2009, 2009:243532.
［16］Ling YH, Consoli U, Tornos C, et al. Accumulation of cyclin B1, activation of cyclin B1-dependent kinase and induction of programmed cell death in human epidermoid carcinoma KB cells treated with taxol ［J］. Int J Cancer, 1998, 75 (6):925-932.
［17］Shi K, Jiang Q, Li Z, et al. Sodium selenite alters microtubule assembly and induces apoptosis in vitro and in vivo ［J］. J Hematol Oncoly, 2013, 6:7.
［18］Xiao X, Li BX, Mitton B, et al. Targeting CREB for cancer therapy: friend or foe ［J］. Curr Cancer Drug Targets, 2010, 10 (4):384-391.
［19］Tan X, Wang S, Zhu L, et al. cAMP response element-binding protein promotes gliomagenesis by modulating the expression of oncogenic microRNA-23a ［J］. Proc Nat Acad Sci USA, 2012, 109 (39):15805-15810.
［20］Li D, Jin L, Alesi GN, et al. The prometastatic ribosomal S6 kinase 2-cAMP response element-binding protein (RSK2-CREB) signaling pathway up-regulates the actin-binding protein fascin-1 to promote tumor metastasis ［J］. J Biol Chem, 2013, 288 (45):32528-32538.
［21］Nakayama K. cAMP-response element-binding protein (CREB) and NF-kappaB transcription factors are activated during prolonged hypoxia and cooperatively regulate the induction of matrix metalloproteinase MMP1 ［J］. J Biol Chem, 2013, 288 (31):22584-22595.
［22］Kinjo K, Sandoval S, Sakamoto KM, et al. The role of CREB as a proto-oncogene in hematopoiesis ［J］. Cell Cycle, 2005, 4 (9):1134-1135.
［23］Abramovitch R, Tavor E, Jacob-Hirsch J, et al. A pivotal role of cyclic AMP-responsive element binding protein in tumor progression ［J］. Cancer Res, 2004, 64 (4):1338-1346.
［24］Dworet JH, Meinkoth JL. Interference with 3’,5’-cyclic adenosine monophosphate response element binding protein stimulates apoptosis through aberrant cell cycle progression and checkpoint activation ［J］. Mol Endocrinol, 2006, 20 (5):1112-1120.
［25］Kamiya K, Sakakibara K, Ryer EJ, et al. Phosphorylation of the cyclic AMP response element binding protein mediates transforming growth factor beta-induced downregulation of cyclin A in vascular smooth muscle cells ［J］. Mol Cell Biol, 2007, 27 (9):3489-3498.
［26］Bottazzi ME, Buzzai M, Zhu X, et al. Distinct effects of mitogens and the actin cytoskeleton on CREB and pocket protein phosphorylation control the extent and timing of cyclin A promoter activity ［J］. Mol Cell Biol, 2001, 21 (22):7607-7616.
［27］Andreatta CP, Nahreini P, Hanson AJ, et al. Regulated expression of VP16CREB in neuroblastoma cells: analysis of differentiation and apoptosis ［J］. J Neurosci Res, 2004, 78 (4):570-579.
［28］Rozenberg J, Rishi V, Orosz A, et al. Inhibition of CREB function in mouse epidermis reduces papilloma formation ［J］. Mol Cancer Res, 2009, 7 (5):654-664.
［29］An Q, Zhang YH, Liu X, et al. Paclitaxel’s effect on lung cancer A549 cells and its changes on luciferase value of nuclear transcription factors ［J］. Journal of Nanjing University of Traditional Chinese Medicine, 2011, 27 (3):254-256. (in Chinese)
安青, 章永红, 刘旭, 等. 紫杉醇作用于肺癌A549细胞核转录因子荧光素酶值变化的意义 ［J］. 南京中医药大学学报，2011, 27 (3):254-256.
细胞骨架结合蛋白通过转录因子CREB调控肿瘤发生发展的分子机制研究. 国家自然科学基金（主持）;转录因子CREB介导的细胞凋亡调控网络在宫颈上皮癌发生发展中的作用及其靶位治疗的研究. 国家自然科学基金（主持）;RhoA介导的细胞骨架调控网络在肿瘤细胞凋亡过程中的作用及其分子机制研究. 浙江省自然科学基金（主持）
/
| 〈 |
|
〉 |
