臺灣博碩士論文加值系統

在近年報導中指出，以transient方式轉殖正常p53基因可以提高細胞對藥物的反應。 本研究則採用轉殖正常p53方式選出穩定表現p53的細胞，再作細胞對藥物反應的研究。
將建構有正常p53與neo基因的質體轉殖入無p53蛋白表現的null-p53 H1299的細胞，挑選到能持續表現p53蛋白的兩株細胞 (S16以及S40)。 其中有較多量p53調控的下游基因p21以及Mdm2蛋白質表現的S40細胞，選定作為對應null-p53 H1299細胞，作為後續細胞毒性研究。
分別處理cisplatin、X-ray、或亞砷酸鈉 (SA) 再分別以： (A) SRB (sulforhodamine B) 染色細胞方式與 (B) 流式細胞技術比較p53-H1299-S40細胞及null-p53 H1299細胞的細胞毒性結果。 在SRB細胞毒性結果中，以50%抑制生長濃度值 (IC50) 為基準，在cisplatin處理組，轉殖p53的H1299細胞與null-p53 H1299細胞之IC50各為0.8μM和1.8μM，在敏感程度上，前者為後者的2.3倍；在X-ray處理組，ID50分別為2.2Gy與3.8Gy，有轉殖p53的細胞與無轉殖者敏感程度比約為1.7倍；在SA處理組，IC50分別為5.5μM與12μM，有p53轉殖的H1299細胞敏感程度較無轉殖的細胞株高2.2倍。
使用Hoechst33258與PI螢光染色細胞樣品，再以流式細胞技術分析結果獲得：有轉殖p53的H1299細胞在分別處理上述的三種試劑後，所得凋亡細胞的百分比都明顯增加。 處理4μM cisplatin組有22%；照射X-ray 6Gy組有28%；而處理20μM亞砷酸鈉組有27%。
分別處理上述三種試劑後，檢查參與細胞程序性死亡相關的蛋白質p53、Bak、caspase3表現方面，結果如下：處理任何一種試劑，轉殖p53的H1299細胞之p53表現都有隨著處理劑量提高而增加；在低濃度處理下有明顯的caspase 3的表現。 相同的處理方式下，對應null-p53 H1299細胞則完全沒有caspase 3表現。 至於，Bak蛋白質表現方面，三種處理組，所得結果各有不同：在處理X-ray或亞砷酸鈉組中，轉殖p53的細胞，Bak的表現量皆隨著提高劑量而明顯增加。 至於cisplatin處理組，在有無轉殖p53的H1299細胞中，Bak的表現並無不同。
本研究成功轉殖正常p53於原來無p53表現的null-p53 H1299細胞中並建立一支含有穩定p53表現的H1299-S40細胞株，以SRB細胞毒性與流式細胞技術測度，此細胞對cisplatin、X-ray、與SA的敏感度以及細胞凋亡程度都比null-p53 H1299高。 與細胞程序性死亡有關的蛋白分子p53、Bak (除了處理cisplatin組)、caspase 3也都有顯著的表現。 因此，轉殖p53的H1299細胞株，在分別處理cisplatin、X-ray、及亞砷酸鈉後，所造成的敏感度提高，可能是透過活化p53調控之細胞程序性死亡路徑所致。 此外，cisplatin處理組造成的細胞凋亡可能與Bak調控的路徑無關。 未來，進一步可使用本實驗所建立的細胞株模式，可提供在測試其他藥物及試劑的反應或p53分子機制相關的基礎研究上。

To determine whether p53 plays a role as a modulator of chemo- and radio- therapeutic agents in NSCLC, the stable transfection of wild-type p53 into H1299 cells were performed. In this study, the role of both wild-type and null p53 in modulating the sensitivity of H1299 cells to three therapeutic agents, cisplatin, X-ray, and sodium arsenite, is examined.
The sensitivity to the three agents was tested in the sulforhodamine B (SRB) cell viability assay. The data showed that H1299 cells transfected with wild-type p53 gene had an increase in susceptibility to the three agents (2.3-fold to cisplatin, 1.7-fold to X-ray, and 2.2-fold to sodium arsenite) in comparison with parental or neo-transfected H1299 cells.
Furthermore, the apoptotic cell fraction was also determined by dual-parameter flow cytometry after treatment with the three agents. By growing cells in the presence of cisplatin, X-ray, and sodium arsenite, respectively, the apoptotic fractions of H1299-S40 cells increased to 22%, 28%, and 27%, respectively.
The expressions of p53, Bak (pro-apoptotic protein) and caspase3 (apoptotic proteolytic enzyme) were examined by Western blot. In p53-transfected H1299-S40 cells p53 proteins were obviously increased after treated individually with the three agents. The results showed similar Bak protein expression was found in both p53-transfected H1299-S40 and null-H1299 cells following cisplatin treatment, but not sodium arsenite treatment or X-ray exposure. In comparison, the active patterns of caspase 3 protein in H1299-S40 cells after above mentioned three agents were dramatically expressed but not in parental H1299 cells. These indicated that the p53 enhanced sensitivities to above three agents in cells through Bak-dependent or Bak-independent (cisplatin) apoptotic pathway.
These observations suggest that the increase in chemo- or radio-sensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In the future, the model cell line system could be used in other drugs investigation or p53 molecule related research.

中文摘要 1
Abstract 3
Introduction 5
The purpose of this study 14
Materials and methods 15
Results 23
Discussion 30
References 35
Figures 39
Appendix 1 56
Appendix 2 57

Baker SJ, Markowitz S, Fearon ER, Willson JK, Vogelstein B (1990): Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249:912-5.
Bradford CR, Zhu S, Ogawa H, Ogawa T, Ubell M, Narayan A, Johnson G, Wolf GT, Fisher SG, Carey TE (2003): P53 mutation correlates with cisplatin sensitivity in head and neck squamous cell carcinoma lines. Head Neck 25:654-61.
Brognard J, Dennis PA (2002): Variable apoptotic response of NSCLC cells to inhibition of the MEK/ERK pathway by small molecules or dominant negative mutants. Cell Death Differ 9:893-904.
Bunz F, Hwang PM, Torrance C, Waldman T, Zhang Y, Dillehay L, Williams J, Lengauer C, Kinzler KW, Vogelstein B (1999): Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest 104:263-9.
Canman CE, Gilmer TM, Coutts SB, Kastan MB (1995): Growth factor modulation of p53-mediated growth arrest versus apoptosis. Genes Dev 9:600-11.
Choi JH, Ahn KS, Kim J, Hong YS (2000): Enhanced induction of Bax gene expression in H460 and H1299 cells with the combined treatment of cisplatin and adenovirus mediated wt-p53 gene transfer. Exp Mol Med 32:23-8.
Chou RH, Huang H (2002): Restoration of p53 tumor suppressor pathway in human cervical carcinoma cells by sodium arsenite. Biochem Biophys Res Commun 293:298-306.
Cohen SM, Lippard SJ (2001): Cisplatin: from DNA damage to cancer chemotherapy. Prog Nucleic Acid Res Mol Biol 67:93-130.
Degenhardt K, Chen G, Lindsten T, White E (2002): BAX and BAK mediate p53-independent suppression of tumorigenesis. Cancer Cell 2:193-203.
Fan S, el-Deiry WS, Bae I, Freeman J, Jondle D, Bhatia K, Fornace AJ, Jr., Magrath I, Kohn KW, O'Connor PM (1994): p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res 54:5824-30.
Filippova M, Duerksen-Hughes PJ (2003): Inorganic and dimethylated arsenic species induce cellular p53. Chem Res Toxicol 16:423-31.
Friedlander P, Legros Y, Soussi T, Prives C (1996): Regulation of mutant p53 temperature-sensitive DNA binding. J Biol Chem 271:25468-78.
Fujita T, Kiyama M, Tomizawa Y, Kohno T, Yokota J (1999): Comprehensive analysis of p53 gene mutation characteristics in lung carcinoma with special reference to histological subtypes. Int J Oncol 15:927-34.
Fukazawa T, Walter B, Owen-Schaub LB (2003): Adenoviral Bid overexpression induces caspase-dependent cleavage of truncated Bid and p53-independent apoptosis in human non-small cell lung cancers. J Biol Chem 278:25428-34.
Gebel T (1997): Arsenic and antimony: comparative approach on mechanistic toxicology. Chem Biol Interact 107:131-44.
Gjerset RA, Lebedeva S, Haghighi A, Turla ST, Mercola D (1999): Inhibition of the Jun kinase pathway blocks DNA repair, enhances p53-mediated apoptosis and promotes gene amplification. Cell Growth Differ 10:545-54.
Herr I, Debatin KM (2001): Cellular stress response and apoptosis in cancer therapy. Blood 98:2603-14.
Huang H, Huang CF, Wu DR, Jinn CM, Jan KY (1993): Glutathione as a cellular defence against arsenite toxicity in cultured Chinese hamster ovary cells. Toxicology 79:195-204.
Huang H, Huang SY, Chen TT, Chen JC, Chiou CL, Huang TM (2004): Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells. J Cell Biochem 91:756-65.
Jing Y, Dai J, Chalmers-Redman RM, Tatton WG, Waxman S (1999): Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood 94:2102-11.
Johnstone RW, Ruefli AA, Lowe SW (2002): Apoptosis: a link between cancer genetics and chemotherapy. Cell 108:153-64.
Kapahi P, Takahashi T, Natoli G, Adams SR, Chen Y, Tsien RY, Karin M (2000): Inhibition of NF-kappa B activation by arsenite through reaction with a critical cysteine in the activation loop of Ikappa B kinase. J Biol Chem 275:36062-6.
Karasuyama H, Tohyama N, Tada T (1989): Autocrine growth and tumorigenicity of interleukin 2-dependent helper T cells transfected with IL-2 gene. J Exp Med 169:13-25.
Kawasaki M, Nakanishi Y, Kuwano K, Yatsunami J, Takayama K, Hara N (1997): The utility of p53 immunostaining of transbronchial biopsy specimens of lung cancer: p53 overexpression predicts poor prognosis and chemoresistance in advanced non-small cell lung cancer. Clin Cancer Res 3:1195-200.
Kim SO, Ono K, Han J (2001): Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages. Am J Physiol Lung Cell Mol Physiol 281:L1095-105.
Koivusalo R, Krausz E, Ruotsalainen P, Helenius H, Hietanen S (2002): Chemoradiation of cervical cancer cells: targeting human papillomavirus E6 and p53 leads to either augmented or attenuated apoptosis depending on the platinum carrier ligand. Cancer Res 62:7364-71.
Lai SL, Perng RP, Hwang J (2000): p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells. J Biomed Sci 7:64-70.
Lee TC, Wei ML, Chang WJ, Ho IC, Lo JF, Jan KY, Huang H (1989): Elevation of glutathione levels and glutathione S-transferase activity in arsenic-resistant Chinese hamster ovary cells. In Vitro Cell Dev Biol 25:442-8.
Levine AJ (1997): p53, the cellular gatekeeper for growth and division. Cell 88:323-31.
Lowe SW (1995): Cancer therapy and p53. Curr Opin Oncol 7:547-53.
Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE, Jacks T (1994): p53 status and the efficacy of cancer therapy in vivo. Science 266:807-10.
Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T (1993): p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362:847-9.
Maeda H, Hori S, Nishitoh H, Ichijo H, Ogawa O, Kakehi Y, Kakizuka A (2001): Tumor growth inhibition by arsenic trioxide (As2O3) in the orthotopic metastasis model of androgen-independent prostate cancer. Cancer Res 61:5432-40.
Maltzman W, Czyzyk L (1984): UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol Cell Biol 4:1689-94.
Mello JA, Acharya S, Fishel R, Essigmann JM (1996): The mismatch-repair protein hMSH2 binds selectively to DNA adducts of the anticancer drug cisplatin. Chem Biol 3:579-89.
Michael D, Oren M (2003): The p53-Mdm2 module and the ubiquitin system. Semin Cancer Biol 13:49-58.
Nemunaitis J, Swisher SG, Timmons T, Connors D, Mack M, Doerksen L, Weill D, Wait J, Lawrence DD, Kemp BL, Fossella F, Glisson BS, Hong WK, Khuri FR, Kurie JM, Lee JJ, Lee JS, Nguyen DM, Nesbitt JC, Perez-Soler R, Pisters KM, Putnam JB, Richli WR, Shin DM, Walsh GL, Merritt J, Roth J (2000): Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J Clin Oncol 18:609-22.
Nishio K, Nakamura T, Koh Y, Suzuki T, Fukumoto H, Saijo N (1999): Drug resistance in lung cancer. Curr Opin Oncol 11:109-15.
Oren M (1999): Regulation of the p53 tumor suppressor protein. J Biol Chem 274:36031-4.
Osaki S, Nakanishi Y, Takayama K, Pei XH, Ueno H, Hara N (2000): Alteration of drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells. Cancer Gene Ther 7:300-7.
Pataer A, Fang B, Yu R, Kagawa S, Hunt KK, McDonnell TJ, Roth JA, Swisher SG (2000): Adenoviral Bak overexpression mediates caspase-dependent tumor killing. Cancer Res 60:788-92.
Pearson AS, Spitz FR, Swisher SG, Kataoka M, Sarkiss MG, Meyn RE, McDonnell TJ, Cristiano RJ, Roth JA (2000): Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells. Clin Cancer Res 6:887-90.
Reed JC (1994): Bcl-2 and the regulation of programmed cell death. J Cell Biol 124:1-6.
Roth JA, Swisher SG, Merritt JA, Lawrence DD, Kemp BL, Carrasco CH, El-Naggar AK, Fossella FV, Glisson BS, Hong WK, Khurl FR, Kurie JM, Nesbitt JC, Pisters K, Putnam JB, Schrump DS, Shin DM, Walsh GL (1998): Gene therapy for non-small cell lung cancer: a preliminary report of a phase I trial of adenoviral p53 gene replacement. Semin Oncol 25:33-7.
Shieh SY, Ikeda M, Taya Y, Prives C (1997): DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91:325-34.
Spitz FR, Nguyen D, Skibber JM, Meyn RE, Cristiano RJ, Roth JA (1996): Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation. Clin Cancer Res 2:1665-71.
Swisher SG, Roth JA (2002): Clinical update of Ad-p53 gene therapy for lung cancer. Surg Oncol Clin N Am 11:521-35.
Ullman KS, Powers MA, Forbes DJ (1997): Nuclear export receptors: from importin to exportin. Cell 90:967-70.
Viksman MY, Liu MC, Schleimer RP, Bochner BS (1994): Application of a flow cytometric method using autofluorescence and a tandem fluorescent dye to analyze human alveolar macrophage surface markers. J Immunol Methods 172:17-24.
Vogelstein B, Kinzler KW (1992): p53 function and dysfunction. Cell 70:523-6.
Vogt U, Striehn E, Bosse U, Klinke F, Falkiewicz B (1999): Lack of squamous cell lung carcinoma in vitro chemosensitivity to various drug regimens in the adenosine triphosphate cell viability chemosensitivity assay. Acta Biochim Pol 46:299-302.
Vogt U, Zaczek A, Klinke F, Granetzny A, Bielawski K, Falkiewicz B (2002): p53 Gene status in relation to ex vivo chemosensitivity of non-small cell lung cancer. J Cancer Res Clin Oncol 128:141-7.
Weinrib L, Li JH, Donovan J, Huang D, Liu FF (2001): Cisplatin chemotherapy plus adenoviral p53 gene therapy in EBV-positive and -negative nasopharyngeal carcinoma. Cancer Gene Ther 8:352-60.
Zhang WW, Fang X, Mazur W, French BA, Georges RN, Roth JA (1994): High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther 1:5-13.