轉殖p53突變基因對化學抗藥性的變異

Correlation of topoiosomerase II inhibitor chemosensitivity and p53 variants in human non-small cell lung cancer cells

林佳薇(Chai-Wei Lin)

指導教授 ： 方剛

國立臺灣師範大學/理學院/生命科學研究所/碩士(2006年)

摘要

癌細胞Topoisomerase II活性一般皆高於正常細胞，利用抑制細胞DNA topoisomerase II也是抗癌藥物治療的一個方向，而兩種抗癌藥物Etoposide(VP-16)及Ellipticine都是topoisomerase II的抑制劑，他們都可以抑制癌細胞生長。此外，p53也扮演調控癌細胞的生長週期的重要角色，已知50%以上的肺癌細胞內都有突變的p53。有鑑於此，實驗主要目的就是評估VP-16及Ellipticine對轉殖不同形式p53突變基因細胞株及母細胞株H1299的生長抑制效應與相關機制。另外實驗也嘗試暸解Ellipticine處理含外源性p21Cip1/WAF1的H1437(p53突變)細胞株的生長調控機制。本論文共分為為四部份：第一部份實驗內容為建立穩定p53基因型肺癌細胞株，結果顯示這些轉殖的肺癌細胞株都能持續表現突變形式的p53，只是蛋白質的代謝速率並不一樣。第二部份則是以VP-16處理不同這些轉殖p53基因型肺癌細胞株，結果顯示VP-16處理p53缺失的H1299細胞株，有明顯抑制細胞生長的效果，主要原因是由於降解磷酸化pAkt-Ser473。第三部份的實驗內容是以ellipticine處理不同p53基因型肺癌細胞株。發現ellipticine影響細胞表型變化，與轉殖之p53基因型無關，反而是藉著調控磷酸化pAkt-Ser473，影響細胞存活率及誘導細胞凋亡。第四部份實驗則是轉殖p53所調控的下游基因p21Cip1/WAF1至突變p53肺癌細胞株H1437，再以Ellipticine處理之，結果顯示對p53突變細胞的敏感率，可以藉外源性p21Cip1/WAF1基因而增加。

關鍵字

p53 ； p21 ； Akt ； VP-16 ； 玫瑰樹鹼 ； 細胞凋亡

並列摘要

Since topoisomerase II are active in cancer cells, the two inhibitors, ellipticine and etoposide (VP-16), are commonly used in cancer therapy by interrupting DNA replication during mitosis. It is also known that tumor suppressor p53 plays critical role in cell cycle regulation and is mutated in more 50% of the lung cancer. The purpose of the thesis is to learn how the two drugs regulate cell growth in p53- deficient non-small cell lung cancers, H1299, with stable expression of various p53 genotypes. The work also included H1437 cells p53(R267P) with ectopic expression of p21Cip1/WAF1. The work in the thesis contained: first, the establishment of stable NSCLC cells with mutated p53 genotypes using H1299 cells; second, treatment of VP-16 in the stable H1299 clones expressing wild-type and mutated p53 and their association with cell growth suppression through dephosphorylation of pAkt-Ser473; third,ellipticine induced apoptosis differently in the various p53 clones; and, finally, ellipticine induced apoptosis in H1437 cells with ectopic expression of p21Cip1/WAF1 that is absent in the parental cells. The results indicated that the sensitivity of ellipticine in p53-deficient NSCLC cells can be enhanced through introduction of downstream of p53 regulators.

並列關鍵字

p53 ； p21 ； Akt ； VP-16 ； ellipticine ； apoptosis

參考文獻

Acton, E. M., Narayanan, V. L., Risbood, P. A., Shoemaker, R. H., Vistica, D. T., Boyd, M. R. Anticancer specificity of some ellipticinium salts against human brain tumors in vitro. J Med Chem. (1994) 37: 2185–9.

Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat. Cell Biol. (2001) 3: 245-252.

Bosken, C. H.,Wei, Q., Amos, C. I., and Spitz, M. R. An analysis of DNA repair as a determinant of survival in patients with non-small-cell lung cancer.J Natl Cancer Inst.(2002) 94: 1091-1099.

Boulares, A. H., Zoltoski, A. J., Yakovlev, A., Xu, M., and Smulson, M. E. Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis. J Biol Chem. (2001) 276: 38185-38192.

Chiu, C. C., Lin, C.H., Fang, K. Etoposide (VP-16) sensitizes p53- deficient human non-small cell lung cancer cells to caspase-7-mediated apoptosis. Apoptosis (2005) 10:643-50.