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研究生(外文):Yuan-Hao Lee
論文名稱(外文):Induction of a Senescence-like Phenomenon by Over-expression of Cofilin
指導教授(外文):Yi-Jang Lee
外文關鍵詞:Cofilinp27Kip1G1 phase arrestsenescence
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細胞老化阻止細胞無限制地生長分裂並促使細胞凋亡,這個現象卻無法在一些缺乏p53 和 p16INK4 抑癌蛋白的癌細胞上自然地發生。在老化的過程中,細胞會變得扁平而寬大,這外觀上的改變提供了我們一個重要訊息— 細胞骨架的改變或許跟細胞老化有關。因此,本篇研究係探討破壞細胞骨架肌動蛋白的組織結構是否會促使癌細胞產生老化現象。
目前已有研究顯示,肌動蛋白單體在細胞核的累積和細胞老化有關。又切絲蛋白負責細胞骨架肌動蛋白的去聚合作用與促進肌動蛋白在細胞核之分佈。因此,我們藉由改變切絲蛋白的表現量來探討細胞老化是否會受到影響。 HCOXP (H1299 with inducible cofilin over-expression) 細胞株帶有tet-on 基因表現的系統,可以被doxycycline誘發而使其切絲蛋白之過度表現。切絲蛋白過度表現造成肌動蛋白累積在細胞核內,其誘發之細胞老化現象則以SA β-gal (senescence-associated β-galactosidase) assay來評估。由實驗結果得知,切絲蛋白大量表現的細胞其β-gal呈色現象較為明顯。並且,切絲蛋白之過度表現引發了p27Kip1-mediated G1 phase arrest。在利用siRNA抑制細胞的p27Kip1表現後,細胞週期停滯現象解除了,且細胞的類老化現象也消失了。更進一步地,我們藉由光學測量法來定量出細胞類老化現象的程度並且發現此現象之發生與p27Kip1之大量表現有關。這些結果顯示了cofilin-p27Kip1之調控路徑影響了SA β-gal所決定的老化現象,且這些結果或許能提供癌症治療一個新的策略。
Cellular senescence can prevent unlimited cell proliferation and may prompt cellular death. However, this phenomenon does not spontaneously occur in tumor cells lacking p53 and p16INK4 tumor suppressor proteins. Cells tend to become flattened and enlarged during senescence. This morphological change implies that actin cytoskeletal alteration may be related to cellular senescence. Thus, this study focused on investigating whether enforcement of actin cytoskeletal disorganization will affect the senescence in tumor cells.
Nuclear accumulation of monomeric actins has been reported to be associated with cellular senescence. In cells, actin deoplymerizing factor (ADF)/cofilin is responsible for increasing actin monomers and promoting actin relocalization to nucleus. Here we explore if altered expression of ADF/cofilin (cofilin hereafter) will affect the occurrence of senescence. HCOXP (H1299 with inducible cofilin over-expression) stable cell line harbors a tet-on gene inducible system that is able to over-express cofilin responding to tetracycline. Over-expression of cofilin led to accumulation of actin in nucleus. The extent of cellular senescence was determined by senescence-associated ��-galactosidase (SA-��-gal) staining, and the results showed that heavy staining of SA-��-gal was visualized in cofilin over-expressing cells. Concomitantly, over-expression of cofilin induced a p27kip1-mediated G1 phase arrest. Knockdown of p27kip1 expression by siRNA in cofilin over-expressing cells rescued the G1 phase arrest and reduced the level of the SA-��-gal staining. Furthermore, the extent of SA-�n��-gal staining was quantified by a spectroscopic system to confirm that over-expressed cofilin may promote a senescence-like phenomenon associating with up-regulation of p27kip1. These results suggest that a cofilin-p27kip1 regulatory pathway may be involved in the senescence phenomenon determined by SA-��-gal biomarker, and it may be an important implication in considering the therapeutic design for cancer treatment.
中文摘要 1
Abstract 2
Purpose 4
Chapter 1 Introduction 5
1.1 Senescence and its theory 5
1.2 Cellular senescence and cancer 6
1.3 Actin depolymerizing factor (ADF), cofilin 7
1.4 Tumor suppressor, p27Kip1 7
1.5 Spectroscopy for senescence-like phenomenon of β-galactosidase 8
Chapter 2 Materials and Methods 10
2.1 Cell cultures 10
2.2 siRNA transfection 10
2.3 Lentiviral infection using shRNA constructs 11
2.4 RT-PCR analysis 12
2.5 Flow cytometry and cell cycle analysis 13
2.6 Generation of cell growth curve 13
2.7 Western blot assay 14
2.8 Protein degradation analysis using cycloheximide 15
2.9 SA ß-gal assay 15
2.10 Spectroscopic measurement 16
2.11 Immunofluorescence microscopy 16
2.12 Trypan blue assay 17
2.13 Colony formation assay 17
2.14 Letiviral infection evaluated by EYFP expression 18
2.15 Statistic method 18
Chapter 3 Results 19
3.1 Cofilin over-expression led actin to accumulate in the nuclei of senescence-like HCOXP cells 19
3.2 Cofilin-induced G1 phase arrest was followed by cell growth retardation and cell survival reduction 20
3.3 P27Kip1 was up-regulated by cofilin over-expression in HCOXP cells and prompted to accumulate in the nuclei 20
3.4 Cofilin-induced G1 phase arrest and senescence-like phenomenon was rescued by p27Kip1 knockdown 21
3.5 Variances of SA ß-gal levels could be discriminated in HCOXP cells using spectroscopy 22
3.6 Cofilin-induced senescence-like phenomenon was not compromised by a lentiviral system 23
Chapter 4 Discussion 25
Chapter 5 Conclusion 28
References 29
Table 37
Figures 38
Appendix 65
1. Plasmid information for lentiviral production 65
2. Matlab codes for spectrometric analysis 67
3. Experimental techniques 72
4. Material information 79
1. Weismann A. (1889) "Essays upon heredity and kindred biological problems." Clarendon Press, Oxford.
2. Schmitt C. A., Fridman J. S., Yang M., Lee S., Baranov E., Hoffman R. M., Lowe S. W. (2002). "A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy." Cell 109, 35-46.
3. Toussaint O., Remacle J., Dierick J-F, Pascal T., Frippiat C., Royer V., Magalhacs J. P., Zdanov S., Chainiaux F. (2002). "Stress-induced premature senescence: from biomarkers to likeliness of in vivo occurrence." Biogerontology 3, 13–17.
4. Chen X., Zhang W., Gao Y. F., Su X. Q., Zhai Z. H. (2002). "Senescence- like changes induced by expression of p21Waf1/Cip1 in NIH3T3 cell line." Cell Research 12, 229-233.
5. Beausejour C. M., Krtolica A., Galimi F., Narita M., Lowe S. W., Yaswen P., Campisi J. (2003). "Reversal of human cellular senescence: roles of the p53 and p16 pathways" The EMBO Journal 22, 212-4222.
6. Pai S. I., Lin Y-Y, Macaes B., Meneshian A., Hung C-F, Wu T-C (2006). "Prospects of RNA interference therapy for cancer." Gene Therapy 13, 464-477.
7. Schmitt E., Paquet C., Beauchemin M., Bertrand R. (2007). "DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis." Journal of Zhejiang University Science B 8, 377-397.
8. Lai S-L, Perng R-P, Hwang J. (2000). "p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells." Journal of Biomedical Science 7, 64-70.
9. Collado M., Gil J., Efeyan A., Guerra C., Schuhmacher A. J., Barradas M., Benguría A., Zaballos A., Flores J. M., Barbacid M., Beach D., Serrano M. (2005). "Senescence in premalignant tumours." Nature 436, 642.
10. Suzuki K., Mori I., Nakayama Y., Miyakoda M., Kodama S., Watanabe M. (2001). "Radiation-induced senescence-like growth arrest requires TP53 function but not telomerase shortening." Radiation Research 155, 248-253.
11. Kwak I. H., Kim H. S., Choi O. R., Ryu M. S., Lim I. K. (2004). "Nuclear accumulation of globular actin as a cellular senescence marker." Cancer Research 64, 572-580.
12. Gohla A., Birkenfeld J., Bokoch G. M. (2005). "Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics." Nature Cell Biology 7, 21-34.
13. Grifth E., Coutts A. S., Black D. M. (2005). "RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation." Cell Motility and the Cytoskeleton 60, 140–152.
14. Vardouli L, Moustakas A., Stournaras C. (2005). "LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-β." The Journal of Biological Chemistry 280, 11448–11457.
15. Quintero-Monzon O., Jonasson E. M., Bertling E., Chaudhry F, Sihvo M, Lappalainen P, Goode BL. (2009). "Reconstitution and dissection of the 600-kDa Srv2/CAP complex: Role for oligomerization and cofilin-actin binding in driving actin turnover." The Journal of Biological Chemistry 284, 10923–10934.
16. Schuler H., Matuschewski K. (2006). "Regulation of apicomplexan microfilament dynamics by a minimal set of actin-binding proteins." Traffic 7, 1433–1439.
17. Liu C-C, Chang P. W-S, Lee Y-J (2007); "The Study of Cofilin on Affecting the Growth of Human Cancer Cells."
18. Wagner M., Hampel B., Hutter E., Pfister G., Krek W., Zwerschke W., Jansen-Dürr P. (2001). "Metabolic stabilization of p27 in senescent fibroblasts correlates with reduced expression of the F-box protein Skp2." Experimental Gerontology 37, 41-55.
19. Huedberg Y., Ljungberg B., Roos G., Landberg G. (2003). "Expression of cyclin D1, D3, E, and p27 in human renal cell carcinoma analysed by tissue microarray." British Journal of Cancer 88, 1417-1423.
20. Rosner M., Freilinger A., Hanneder M., Fujita N., Lubec G., Tsuruo T., Hengstschläger M. (2007). "p27Kip1 localization depends on the tumor suppressor protein tuberin." Human Molecular Genetics 16, 1541– 1556.
21. Besson A., Gurian-West M., Chen X., Kelly-Spratt K. S., Kemp C. J., Roberts J. M. (2006)."A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression." Genes and Development 20, 47-64.
22. Dertsiz L., Özbilim G., Kükner I., Özbudak, I. H. (2005). "Evaluation of cyclin- dependent kinase inhibitor p27 and Bcl-2 protein in non-small cell lung cancer." Turkish Journal of Cancer 4, 166-170.
23. Catzavelos C, Bhattacharya N., Ung Y. C., Wilson J. A., Roncari L., Sandhu C., Shaw P., Yeger H., Morava-Protzner I., Kapusta L., Franssen E., Pritchard K. I., Slingerland J. M. (1997). "Decreased levels of the cell-cycle inhibitor p27KIP1 protein: prognostic implications in primary breast cancer." Nat Med 3, 227–230.
24. Lloyd R. V., Erickson L. A., Jin L., Kulig E., Qian X., Cheville J. C., Scheithauer B. W. (1999). "p27KIP1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers." American Journal Pathology 154, 313-323.
25. Tsihilas J, Kapusta L. R., DeBoer G., Morava-Protzner I., Zbieranowski I., Bhattacharya N., Catzavelos G. C., Klotz L. H., Slingerland J. M. (1998). "Loss of cyclin-dependent kinase inhibitor p27KIP1 is a novel prognostic factor in localized human prostate adenocarcinoma." Cancer Research 58, 542-548.
26. Guan X-X, Chen L-B, Ding G-X, De W., Zhang A. H. (2004). "Transfection of p27kip1 enhances radiosensitivity induced by 60Co γ- irradiation in hepatocellular carcinoma HepG2 cell line." World Journal of Gastroenterology 10, 3103-3106.
27. Schmidt M., Lu Y., Parant M. J., Lozano G., Bacher G., Beckers T., Fan Z. (2001). "Differential roles of p21Waf1 and p27Kip1 in modulating chemosensitivity and their possible application in drug discovery studies." Molecular Pharmacology 60, 900-906.
28. Richards-Kortum R.., Sevick-Muraca E. (1996). "Quantitative optical spectro- scopy for tissue diagnosis." Annual Review of Physical Chemistry 47, 555-606.
29. Lee Y-J, Keng P. C. (2005). "Studying the effect of actin cytoskeletal destabilization on cell cycle by cofilin overexpression." Molecular Bio- technology 31, 1-10.
30. Ustek D., Durali D., Aktas E. (2007). "Efficient Transfection of 293T Cell Line with Lentiviral Vectors Using FuGENE HD Transfection Reagent." Biochemica. No. 3.
31. Vindelov L. L., Christensen I. J., Nissen N. I. (1983). "A Detergent-Trypsin Method for the Preparation of Nuclei for Flow DNA Analysis." Cytometry 3, 323-327.
32. Fitter S. S., Hunt L. D., Fletcher J. S. (1975). "Resistance of plant cell to cycloheximide." Proceedings of the Oklahoma Academy of Science 55, 49-50.
33. Lee B. Y., Han J. A., Im J. S., Morrone A.,Johung K., Goodwin E. C., Kleijer W. J., DiMaio D., Hwang E-S (2006). "Senescence-associated β-galactosidase is lysosomal ß-galactosidase." Aging Cell 5, 187–195.
34. Pieterson R., Thilo L., Chastellier C. d. (2004). "Mycobacterium tuberculosis and Mycobacterium avium modify the composition of the phagosomal membrane in infected macrophages by selective depletion of cell surface-derived glycoconjugates." European Journal of Cell Biology 83, 153-158.
35. Friedberg D., Friedberg I., Shilo M. (1970). "Interaction of Gram-Negative Bacteria with the Lysosomal Fraction of Polymorphonuclear Leukocytes." Infection and Immunity 1, 311-318.
36. Lecanda J., Ganapathy V., D’Aquino-Ardalan C., Evans B., Cadacio C., Ayala A., Gold L. I. (2009). "TGF-β prevents proteasomal degradation of the cyclin-dependent kinase inhibitor p27kip1 for cell cycle arrest." Cell Cycle 8, 1-15.
37. Bloom J., Pagano M. (2003). "Deregulated degradation of the cdk inhibitor p27 and malignant transformation." Seminars in Cancer Biology 13, 41-47.
38. Nevins J. R. (2001). "The Rb/E2F pathway and cancer." Human Molecular Genetics 10, 699-703.
39. Wang Y. Y., Yang Y., Chen Q-Y, Yu J., Hou Y., Han L., He J., Jiao D., Yu H. (2008). "Simultaneous knockdown of p18INK4C, p27Kip1 and MAD1 via RNA interference results in the expansion of long-term culture-initiating cells of murine bone marrow cells in vitro." Acta Biochim Biophys Sin 40, 711-720.
40. Tamamori-Adachi M., Hayashida K., Nobori K., Omizu C., Yamada K., Sakamoto N., Kamura T., Fukuda K., Ogawa S., Nakayama K. I., Kitajima S. (2004). "Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4." The Journal of Biological Chemistry 279, 50429–50436.
41. Lohez O. D., Reynaud C., Borel F., Andreassen P. R. and Margolis R. L. (2003). "Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53." The Journal of Cell Biology 161, 67-77.
42. Wei W., Herbig U., Wei S., Dutriaux A., Sedivy J. M. (2003). "Loss of retinoblastoma but not p16 function allows bypass of replicative senescence in human fibroblasts." European Molecular Biology Organization 4, 1061-1066.
43. Shin I., Yakes F. M., Rojo F., Shin N. Y., Bakin A. V., Baselga J., Arteaga C. L. (2002). "PKB/Akt mediates cell-cycle progression by phosphorylation of p27Kip1 at threonine 157 and modulation of its cellular localization." Nature Medicine 8, 1145-1152.
44. Tsoli E., Gorgoulis V. G., Zacharatos P., Kotsinas A., Mariatos G., Kastrinakis N. G., Kokotas S., Kanavaros P., Asimacopoulos P., Bramis J., Kletsas D., Papavassiliou A. G., Kittas C. (2001). "Low levels of p27 in association with deregulated p53-pRb protein status enhance tumor proliferation and chromosomal instability in non-small cell lung carcinomas." Molecular Medicine 7, 418–429.
45. Stuschke M., Sak A., Wurm R., Sinn B., Wolf G., Stüben G., Budach V. (2002). "Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint." International Journal of Radiation Biology 78, 807-819.
46. Zagorski W. A., Knudsen E. S., Reed M. F. (2007). "Retinoblastoma deficiency increases chemosensitivity in lung cancer. "Cancer Research 67, 8264-8273.
47. Wiggan O’N, Berstein B. W., Bamburg J. R. (2005). "A phosphatase for cofilin to be HAD." Nature Cell Biology 7, 8-9.
48. Zhang W., Bergamaschi D., Jin B., Lu X. (2009). "Posttranslational modications of p27kip1 determine its binding specicity to different cyclins and cyclin-dependent kinases in vivo." Blood 105, 3691-3698.
49. Maurer B. J., Ihnat M. A., Morgan C., Pullman J., O'Brien C., Johnson S. W., Rasey J. S., Cornwell M. M. (1999)."Growth of human tumor cells in macroporous microcarriers results in p53-Independent, decreased cisplatin sensitivity relative to monolayers." Molecular Pharmacology 55, 938-947.
50. Andrei S., Gorbunova V., Falcovitz A., Sigal A., Milyavsky M., Zurer I., Shohat G., Goldfinger N., Rotter V. (2001). "Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53." Molecular and Cellular Biology 21, 1552–1564.
51. Themis M., Waddington S. N., Schmidt M., von Kalle C., Wang Y., Al-Allaf F., Gregory L. G., Nivsarkar M., Themis M., Holder M. V., Buckley S. M., Dighe N., Ruthe A. T., Mistry A., Bigger B., Rahim A., Nguyen T. H., Trono D., Thrasher A. J., Coutelle C. (2005). "Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice." Molecular Therapy 12, 763-771.
52. Yamauchi M., Suzuki K., Kodama S., Watanabe M. (2005). "Abnormal stability of wild-type p53 protein in a human lung carcinoma cell line." Biochemical and Biophysical Research Communications 330, 483-488.
53. Connolly J. B. (2002). "Lentiviruses in gene therapy clinical research." Gene Therapy 9, 1730–1734.
54. Huang S., Chen C. S., Ingber D. E. (1998). "Control of cyclin D1, p27Kip1, and cell cycle progression in human capillary endothelial cells by cell shape and cytoskeletal tension." Molecular Biology of the Cell 9, 3179- 3193.
55. Wu C-H, Riggelen J. V., Yetil A., Fan A. C., Bachireddy P., Felsher D. W. (2007). "Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation." Proceedings of the National Academy of Sciences of the United States of America 104, 13028-13033.
56. Sidani M., Wessels D., Mouneimne G., Ghosh M., Goswami S., Sarmiento C., Wang W., Kuhl S., El-Sibai M., Backer J. M., Eddy R., Soll D., Condeelis J. (2007). "Cofilin determines the migration behavior and turning frequency of metastatic cancer cells." The Journal of Cell Biology 179, 777-791.
57. Lee Y-J, Mazzatti D. J., Yun Z., Keng P. C. (2005). "Inhibition of invasiveness of human lung cancer cell line H1299 by over-expression of colin." Cell Biology International 29, 877-883.
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