請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/22976
標題: Cdk5對於不同侵犯能力之肺癌細胞在生長與移動的影響
Effects of Cdk5 on the proliferation and migration of lung cancer cells with different invasiveness
作者: 江明憬
Chiang, Ming-Ching
關鍵字: Cdk5
lung cancer cells
出版社: 生命科學系所
引用: 1. Khuder SA. Effect of cigarette smoking on major histological types of lung cancer: a meta-analysis. Lung Cancer 2001;31:139-48. 2. Wistuba, II, Berry J, Behrens C, et al. Molecular changes in the bronchial epithelium of patients with small cell lung cancer. Clin Cancer Res 2000;6:2604-10. 3. Rusch V, Klimstra D, Venkatraman E, Pisters PW, Langenfeld J, Dmitrovsky E. Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer but does not predict tumor progression. Clin Cancer Res 1997;3:515-22. 4. Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers--a different disease. Nat Rev Cancer 2007;7:778-90. 5. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003;284:31-53. 6. West KA, Linnoila IR, Belinsky SA, Harris CC, Dennis PA. Tobacco carcinogen-induced cellular transformation increases activation of the phosphatidylinositol 3''-kinase/Akt pathway in vitro and in vivo. Cancer Res 2004;64:446-51. 7. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008;359:1367-80. 8. Malumbres M, Pellicer A. RAS pathways to cell cycle control and cell transformation. Front Biosci 1998;3:d887-912. 9. Sears RC, Nevins JR. Signaling networks that link cell proliferation and cell fate. J Biol Chem 2002;277:11617-20. 10. Calo V, Migliavacca M, Bazan V, et al. STAT proteins: from normal control of cellular events to tumorigenesis. J Cell Physiol 2003;197:157-68. 11. Martino A, Holmes JHt, Lord JD, Moon JJ, Nelson BH. Stat5 and Sp1 regulate transcription of the cyclin D2 gene in response to IL-2. J Immunol 2001;166:1723-9. 12. Sinibaldi D, Wharton W, Turkson J, Bowman T, Pledger WJ, Jove R. Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling. Oncogene 2000;19:5419-27. 13. Norbury C, Nurse P. Animal cell cycles and their control. Annu Rev Biochem 1992;61:441-70. 14. Malumbres M, Barbacid M. Mammalian cyclin-dependent kinases. Trends Biochem Sci 2005;30:630-41. 15. Satyanarayana A, Berthet C, Lopez-Molina J, Coppola V, Tessarollo L, Kaldis P. Genetic substitution of Cdk1 by Cdk2 leads to embryonic lethality and loss of meiotic function of Cdk2. Development 2008;135:3389-400. 16. Mainprize TG, Taylor MD, Rutka JT, Dirks PB. Cip/Kip cell-cycle inhibitors: a neuro-oncological perspective. J Neurooncol 2001;51:205-18. 17. Nakayama K. Cip/Kip cyclin-dependent kinase inhibitors: brakes of the cell cycle engine during development. Bioessays 1998;20:1020-9. 18. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149-63. 19. Zhang H, Xiong Y, Beach D. Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes. Mol Biol Cell 1993;4:897-906. 20. Polyak K, Lee MH, Erdjument-Bromage H, et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 1994;78:59-66. 21. Lew J, Beaudette K, Litwin CM, Wang JH. Purification and characterization of a novel proline-directed protein kinase from bovine brain. J Biol Chem 1992;267:13383-90. 22. Tsai LH, Takahashi T, Caviness VS, Jr., Harlow E. Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system. Development 1993;119:1029-40. 23. Meyerson M, Enders GH, Wu CL, et al. A family of human cdc2-related protein kinases. EMBO J 1992;11:2909-17. 24. Malumbres M, Harlow E, Hunt T, et al. Cyclin-dependent kinases: a family portrait. Nat Cell Biol 2009;11:1275-6. 25. van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 1993;262:2050-4. 26. Lew J, Huang QQ, Qi Z, et al. A brain-specific activator of cyclin-dependent kinase 5. Nature 1994;371:423-6. 27. Tsai LH, Delalle I, Caviness VS, Jr., Chae T, Harlow E. p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 1994;371:419-23. 28. Tang D, Yeung J, Lee KY, et al. An isoform of the neuronal cyclin-dependent kinase 5 (Cdk5) activator. J Biol Chem 1995;270:26897-903. 29. Ko J, Humbert S, Bronson RT, et al. p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment. J Neurosci 2001;21:6758-71. 30. Patrick GN, Zhou P, Kwon YT, Howley PM, Tsai LH. p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) is degraded by the ubiquitin-proteasome pathway. J Biol Chem 1998;273:24057-64. 31. Harada T, Morooka T, Ogawa S, Nishida E. ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1. Nat Cell Biol 2001;3:453-9. 32. Gu Y, Rosenblatt J, Morgan DO. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J 1992;11:3995-4005. 33. Zukerberg LR, Patrick GN, Nikolic M, et al. Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth. Neuron 2000;26:633-46. 34. Matsuura I, Wang JH. Demonstration of cyclin-dependent kinase inhibitory serine/threonine kinase in bovine thymus. J Biol Chem 1996;271:5443-50. 35. Lee MH, Nikolic M, Baptista CA, Lai E, Tsai LH, Massague J. The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons. Proc Natl Acad Sci U S A 1996;93:3259-63. 36. Patrick GN, Zukerberg L, Nikolic M, de la Monte S, Dikkes P, Tsai LH. Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 1999;402:615-22. 37. Tarricone C, Dhavan R, Peng J, Areces LB, Tsai LH, Musacchio A. Structure and regulation of the CDK5-p25(nck5a) complex. Mol Cell 2001;8:657-69. 38. Kusakawa G, Saito T, Onuki R, Ishiguro K, Kishimoto T, Hisanaga S. Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25. J Biol Chem 2000;275:17166-72. 39. Lee MS, Kwon YT, Li M, Peng J, Friedlander RM, Tsai LH. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 2000;405:360-4. 40. Ahlijanian MK, Barrezueta NX, Williams RD, et al. Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5. Proc Natl Acad Sci U S A 2000;97:2910-5. 41. Selkoe DJ. Translating cell biology into therapeutic advances in Alzheimer''s disease. Nature 1999;399:A23-31. 42. Nguyen MD, Lariviere RC, Julien JP. Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron 2001;30:135-47. 43. Upadhyay AK, Ajay AK, Singh S, Bhat MK. Cell cycle regulatory protein 5 (Cdk5) is a novel downstream target of ERK in carboplatin induced death of breast cancer cells. Curr Cancer Drug Targets 2008;8:741-52. 44. Goodyear S, Sharma MC. Roscovitine regulates invasive breast cancer cell (MDA-MB231) proliferation and survival through cell cycle regulatory protein cdk5. Exp Mol Pathol 2007;82:25-32. 45. Kuo HS, Hsu FN, Chiang MC, et al. The role of Cdk5 in retinoic acid-induced apoptosis of cervical cancer cell line. Chin J Physiol 2009;52:23-30. 46. Strock CJ, Park JI, Nakakura EK, et al. Cyclin-dependent kinase 5 activity controls cell motility and metastatic potential of prostate cancer cells. Cancer Res 2006;66:7509-15. 47. Lin H, Juang JL, Wang PS. Involvement of Cdk5/p25 in digoxin-triggered prostate cancer cell apoptosis. J Biol Chem 2004;279:29302-7. 48. Selvendiran K, Koga H, Ueno T, et al. Luteolin promotes degradation in signal transducer and activator of transcription 3 in human hepatoma cells: an implication for the antitumor potential of flavonoids. Cancer Res 2006;66:4826-34. 49. Ohshima T, Gilmore EC, Longenecker G, et al. Migration defects of cdk5(-/-) neurons in the developing cerebellum is cell autonomous. J Neurosci 1999;19:6017-26. 50. Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB, Herrup K. Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex. J Neurosci 1998;18:6370-7. 51. Ohshima T, Ward JM, Huh CG, et al. Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc Natl Acad Sci U S A 1996;93:11173-8. 52. Li BS, Zhang L, Takahashi S, et al. Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3. EMBO J 2002;21:324-33. 53. Li BS, Ma W, Jaffe H, et al. Cyclin-dependent kinase-5 is involved in neuregulin-dependent activation of phosphatidylinositol 3-kinase and Akt activity mediating neuronal survival. J Biol Chem 2003;278:35702-9. 54. Rosales JL, Lee KY. Extraneuronal roles of cyclin-dependent kinase 5. Bioessays 2006;28:1023-34. 55. Lin H, Chen MC, Chiu CY, Song YM, Lin SY. Cdk5 regulates STAT3 activation and cell proliferation in medullary thyroid carcinoma cells. J Biol Chem 2007;282:2776-84. 56. Abercrombie M, Dunn GA, Heath JP. The shape and movement of fibroblasts in culture. Soc Gen Physiol Ser 1977;32:57-70. 57. Adams JC. Cell-matrix contact structures. Cell Mol Life Sci 2001;58:371-92. 58. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110:673-87. 59. Mueller SC, Ghersi G, Akiyama SK, et al. A novel protease-docking function of integrin at invadopodia. J Biol Chem 1999;274:24947-52. 60. Katoh K, Kano Y, Amano M, Onishi H, Kaibuchi K, Fujiwara K. Rho-kinase--mediated contraction of isolated stress fibers. J Cell Biol 2001;153:569-84. 61. Orlichenko LS, Radisky DC. Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development. Clin Exp Metastasis 2008;25:593-600. 62. Mazar AP. Urokinase plasminogen activator receptor choreographs multiple ligand interactions: implications for tumor progression and therapy. Clin Cancer Res 2008;14:5649-55. 63. Rudolph-Owen LA, Chan R, Muller WJ, Matrisian LM. The matrix metalloproteinase matrilysin influences early-stage mammary tumorigenesis. Cancer Res 1998;58:5500-6. 64. Koblinski JE, Ahram M, Sloane BF. Unraveling the role of proteases in cancer. Clin Chim Acta 2000;291:113-35. 65. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2002;2:442-54. 66. Kwon YT, Gupta A, Zhou Y, Nikolic M, Tsai LH. Regulation of N-cadherin-mediated adhesion by the p35-Cdk5 kinase. Curr Biol 2000;10:363-72. 67. Nakano N, Nakao A, Ishidoh K, et al. CDK5 regulates cell-cell and cell-matrix adhesion in human keratinocytes. Br J Dermatol 2005;153:37-45. 68. Gao C, Negash S, Guo HT, Ledee D, Wang HS, Zelenka P. CDK5 regulates cell adhesion and migration in corneal epithelial cells. Mol Cancer Res 2002;1:12-24. 69. Kawauchi T, Chihama K, Nabeshima Y, Hoshino M. Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration. Nat Cell Biol 2006;8:17-26. 70. Hou Z, Li Q, He L, et al. Microtubule association of the neuronal p35 activator of Cdk5. J Biol Chem 2007;282:18666-70. 71. He L, Hou Z, Qi RZ. Calmodulin binding and Cdk5 phosphorylation of p35 regulate its effect on microtubules. J Biol Chem 2008;283:13252-60. 72. Haesslein JL, Jullian N. Recent advances in cyclin-dependent kinase inhibition. Purine-based derivatives as anti-cancer agents. Roles and perspectives for the future. Curr Top Med Chem 2002;2:1037-50. 73. Havlicek L, Hanus J, Vesely J, et al. Cytokinin-derived cyclin-dependent kinase inhibitors: synthesis and cdc2 inhibitory activity of olomoucine and related compounds. J Med Chem 1997;40:408-12. 74. Meijer L, Borgne A, Mulner O, et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem 1997;243:527-36. 75. Wesierska-Gadek J, Borza A, Komina O, Maurer M. Impact of roscovitine, a selective CDK inhibitor, on cancer cells: bi-functionality increases its therapeutic potential. Acta Biochim Pol 2009;56:495-501. 76. Maurer M, Komina O, Wesierska-Gadek J. Roscovitine differentially affects asynchronously growing and synchronized human MCF-7 breast cancer cells. Ann N Y Acad Sci 2009;1171:250-6. 77. Cappellini A, Chiarini F, Ognibene A, McCubrey JA, Martelli AM. The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage: implications for therapy of drug resistant breast cancers. Cell Cycle 2009;8:1421-5. 78. Wesierska-Gadek J, Wandl S, Kramer MP, Pickem C, Krystof V, Hajek SB. Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells. J Cell Biochem 2008;105:1161-71. 79. Fleming IN, Hogben M, Frame S, McClue SJ, Green SR. Synergistic inhibition of ErbB signaling by combined treatment with seliciclib and ErbB-targeting agents. Clin Cancer Res 2008;14:4326-35. 80. Wesierska-Gadek J, Borza A, Walzi E, et al. Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment. J Cell Biochem 2009;106:937-55. 81. Hsieh WS, Soo R, Peh BK, et al. Pharmacodynamic effects of seliciclib, an orally administered cell cycle modulator, in undifferentiated nasopharyngeal cancer. Clin Cancer Res 2009;15:1435-42. 82. Hui AB, Yue S, Shi W, et al. Therapeutic efficacy of seliciclib in combination with ionizing radiation for human nasopharyngeal carcinoma. Clin Cancer Res 2009;15:3716-24. 83. Raynaud FI, Whittaker SR, Fischer PM, et al. In vitro and in vivo pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine and CYC202. Clin Cancer Res 2005;11:4875-87. 84. McClue SJ, Blake D, Clarke R, et al. In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine). Int J Cancer 2002;102:463-8. 85. Swede H, Dong Y, Reid M, Marshall J, Ip C. Cell cycle arrest biomarkers in human lung cancer cells after treatment with selenium in culture. Cancer Epidemiol Biomarkers Prev 2003;12:1248-52. 86. Lockwood WW, Chari R, Coe BP, et al. DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers. Oncogene 2008;27:4615-24. 87. Choi HS, Lee Y, Park KH, et al. Single-nucleotide polymorphisms in the promoter of the CDK5 gene and lung cancer risk in a Korean population. J Hum Genet 2009;54:298-303. 88. Chu YW, Yang PC, Yang SC, et al. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 1997;17:353-60. 89. Zhang J, Cicero SA, Wang L, Romito-Digiacomo RR, Yang Y, Herrup K. Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons. Proc Natl Acad Sci U S A 2008;105:8772-7. 90. Xie Z, Tsai LH. Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration. Cell Cycle 2004;3:108-10. 91. Humbert S, Dhavan R, Tsai L. p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton. J Cell Sci 2000;113 ( Pt 6):975-83. 92. Lin S, Wang J, Ye Z, Ip NY, Lin SC. CDK5 activator p35 downregulates E-cadherin precursor independently of CDK5. FEBS Lett 2008;582:1197-202. 93. Munoz JP, Huichalaf CH, Orellana D, Maccioni RB. cdk5 modulates beta- and delta-catenin/Pin1 interactions in neuronal cells. J Cell Biochem 2007;100:738-49.
摘要: 肺癌為台灣及歐美國家癌症死因之首,每年罹患肺癌的病患人數有逐漸攀升的跡象。根據病理組織的特徵做分類,肺癌主要可分為兩大類:小細胞肺癌以及非小細胞肺癌,其中又以非小細胞肺癌的病患佔多數,但是非小細胞肺癌的形成機制尚未明確的被指出。因此試著找出可能造成非小細胞肺癌的形成機轉成為目前研究的課題之一。根據我們先前所發表的實驗結果指出,Cyclin-dependent kinase 5, Cdk5 可能參與由STAT3調控的甲狀腺癌細胞的增生,另有研究學者指出,Cdk5亦會調控攝護腺癌細胞的移動與轉移潛力。但是在肺癌方面,只有幾篇報導指出Cdk5在肺癌中其基因表現量有增加的情形,而對於生物功能的調控沒有進一步的深入探討。因此本篇論文的目的在於探討Cdk5在三株侵犯能力不同的肺癌細胞株之中,對於生長和移動所受到的影響。在此我們利用了MTT assay以及生長曲線的計算,分析了Cdk5對於肺癌細胞株的生長影響。發現在侵犯能力較低的A549以及CL1-0細胞株中,增加Cdk5的表現量可以刺激細胞的增生,抑制Cdk5的活性或是表現量則抑制細胞的增生,而在侵犯能力較強的CL1-5細胞中則得到相反的結果,增加Cdk5的表現會抑制細胞的增生,抑制Cdk5的表現則促進細胞的增生。我們同時利用了傷口癒合實驗以及transwell的方式分析了Cdk5對於細胞移動的影響。根據結果顯示,抑制Cdk5的活性或是表現會抑制A549細胞的移動,但是會促進CL1-5的移動。接著我們利用西方墨點法以及免疫組織化學染色法進一步分析了臨床肺癌的檢體,並且比較在正常的組織以及癌化的組織中Cdk5和p35的表現情形。發現Cdk5的表現量在正常組織與癌組織中,並沒有明顯的不同,反而是p35的表現量有46%的情況是癌組織高於正常組織的。此外,也發現Grade 1 和Grade 2的病人其p35的表現量較高,而Grade 3的病人其p35表現量會下降。若進一步比較TNM分類與Cdk5、p35表現量的關係,可以發現在TNM分類為N0的14位病人中,有8位 (57.14%)病人具有高的p35表現量。但是在N1及N2的30位病人中,有16位(43.3%)病人其p35表現量較低。配合之前細胞株的實驗結果可以發現,Cdk5的活性對於不具侵犯性的癌細胞具有刺激的效果,但是當癌細胞具有侵犯能力時,Cdk5的重要性似乎便下降了。綜合上述結果,本實驗首次證明Cdk5對於肺癌細胞的影響以及Cdk5/p35蛋白在臨床檢體中的表現。我們期許未來可以利用p35的表現量作為肺癌的診斷指標,而Cdk5則可作為肺癌治療目標。
Lung cancer is the leading cause of cancer death in Taiwan and worldwide. According to the pathological characteristics, lung cancer could be divided into two major groups, small cell lung cancer and non-small cell lung cancer. Most lung cancer patients are non-small cell lung cancer; however, the tumorogenic mechanism is still unclear. It is important to figure out the possible mechanism of non-small cell lung cancer tumorigenesis. Our previous studies showed Cyclin-dependent kinase 5 (Cdk5) involves STAT3-regulated thyroid cancer proliferation. Other research indicates Cdk5 controls prostate cancer cell migration and metastasis potential. However, in lung cancer, there are only several articles indicate Cdk5 gene expression increased. There is still no evidence show the regulation of Cdk5 on the biological function of lung cancer. In this research, three lung cancer cell lines with different invasiveness were used to verify the effects of Cdk5 on cell proliferation and cell migration. MTT assay and growth curve were used to identify the effect of Cdk5 on cell proliferation. The results of low invasiveness lung cancer cell lines A549 and CL1-0 showed that increased-Cdk5 expression stimulated cell proliferation and knockdown Cdk5 expression inhibited cell proliferation. However, in high invasiveness lung cancer cell line, CL1-5, showed adverse results to A549 and CL1-0 cells. Wound closure assay and transwell migration assay were used to verify the effects of Cdk5 on cell migration. As the results showed, inhibition of Cdk5 activity or knockdown Cdk5 expression inhibit A549 cell migration, however, stimulate CL1-5 migration. Clinical samples of lung cancer were further investigate for the protein expression level by western blotting and immunohistochemistry. There is no significant difference for Cdk5 expression between normal lung tissue and lung tumor tissue. In addition to Cdk5, p35 expressed more in 46% of tumor tissue than in normal tissue. Furthermore, the expression level of p35 was higher in grade 1 and grade 2 patients than in grade 3 patients. The relationship of Cdk5/p35 expression and TNM classification were further investigated. The results showed 8 out of 14 (57.14%) N0 patients had high p35 expression, but 13 of 30 (43.3%) N1 and N2 patients express lower p35. Accompanied with in vitro results, Cdk5 activity supporting non-invasive cancer cells proliferation and migration, however, the importance of Cdk5 activity for supporting proliferation and migration decreased in invasive lung cancer cells. In conclusion, this is the first time I demonstrate the effects of Cdk5 on lung cancer cells and expression level of Cdk5/p35 on lung cancer samples. Judging from the above results, p35 might be a diagnostic marker and Cdk5 might be a target for treatment.
URI: http://hdl.handle.net/11455/22976
其他識別: U0005-2501201023105500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2501201023105500


在 DSpace 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。