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研究生:張宏彰
研究生(外文):Hung-Chang Chang
論文名稱:c-Myc過度表現的老鼠乳癌細胞中抗藥性機轉的研究
論文名稱(外文):The mechanism of drug resistance in c-Myc-overexpressing mouse mammary carcinoma cell line
指導教授:王正康
指導教授(外文):Jehng-Kang Wang
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:101
中文關鍵詞:c-Mycp53抗藥性
外文關鍵詞:c-Myc、p53、drug resistance
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c-myc為一原致癌基因,在許多的癌症中皆會發現其有過度表現之情形且常伴隨著較差的預後,而抗藥性的產生也是造成預後差的主要原因之一,一般已知抗藥性的產生大部分是因為多重抗藥蛋白 (MDR) 過度表現而將化療藥物排放出去所造成的,但MDR之機轉卻無法解釋為何這些具有抗藥性之病人對於放射治療也具有抵抗性,推測應該是有其他調控因子,文獻中得知p53可能扮演此一關鍵性角色。由之前實驗室研究發現: PD153035 (上皮生長因子接受器酪胺酸激酶活性抑制劑) 及anisomycin (p38 MAPK、JNK活化劑) 會造成Myc83 (c-Myc過度表現且具有野生型 p53之老鼠乳癌上皮細胞株) 有大量細胞凋亡之情形且p53蛋白表現降低,因此我們想利用Myc83細胞株培養出具有抗藥性之細胞株,進一步地去觀察其產生抗藥性及抗放射線之主因且和p53之間的相關性為何?在利用了PD153035及anisomycin重覆地對Myc83加藥處理約八代後,我們成功地篩選出分別對PD153035及anisomycin具有抗藥性同時對放射線及傳統化療藥物 (5-FU)有抵抗性之細胞株 (PD8及AN8) 。本論文主要焦點放在PD8細胞株上,分別以下面三大部分來探討: 1、 PD8本身抗藥性之機轉; 2、PD8抗放射線之機轉; 3、PD8之抗5-FU之機轉。實驗結果中發現: 1.PD8抗藥性細胞株中p53蛋白的表現量沒有顯著之變化。 2.隨著加藥篩選代數之增加,細胞凋亡有顯著減少之現象,但p53蛋白的表現卻遞增且p38 MAPK蛋白的活化遞減,推測p38 MAPK蛋白的不活化可能和PD8細胞株產生抗藥性具有正相關性。 3.當處理PD153035時發現PD8細胞株相較於Myc83其p38 MAPK蛋白不活化、p53蛋白高度表現及Akt及Erk1/2蛋白有較為活化之情形,推測PD8細胞株可能是經由這些因子的調控而具有抗藥性。 4.處理5-FU造成PD8細胞株相較於Myc83,其p38 MAPK蛋白的表現有明顯不活化的現象,推測可能和PD8細胞株對5-FU產生抗藥性具有正相關性。 5.以5-FU或放射線處理皆造成這PD8細胞株之p53絲胺酸15位置的磷酸化,推測可能和抗藥性之機轉具有相關性,但仍需進一步去探討。 6.這二株抗藥性細胞株在處理MG132 (蛋白質降解體抑制劑) 後,會使得p53蛋白的表現有回升的現象,推測此二抗藥性細胞株中MG132可以抑制p53蛋白之降解。
The proto-oncogene c-Myc is implicated in various physiological processes: cell growth and apoptosis. Overexpression of c-Myc is associated with many human cancers and further with poor prognosis. In general, acquired drug resistance of tumor cells is frequently observed in cancer patients with the treatment of chemotherapy. Chemotherapy- induced drug resistance is driven by MDR, which could be irrelevant for radiation resistance. However, certain p53 mutants may enhance drug resistance in cancer cells. Myc83 cell line is derived from a tumor of MMTV-c-Myc transgenic mice, thus Myc83 is a c-Myc-overexpressing mouse mammary carcinoma cell line with wild type p53. Myc83 cells were utilized to study the possible relationship between c-Myc and drug resistance. In the current study, we utilized PD153035 (an inhibitor of EGFR tyrosine kinase) and anisomycin (an activator of p38 MAPK and JNK) as useful tools. We found that PD153035 and anisomycin can trigger the down-regulation of p53. We further utilize Myc83 cells to develop drug resistance cells to study the possible mechanism. We hypothesize that p53 has been influenced in Myc83-derived resistant cells. After a serious of treatments, we have selected two independent drug resistant cell clones by repeated treatment with PD153035 and anisomycin. We named these drug resistant cells are PD8 and AN8, respectively. Furthermore, we found these resistant cells are also resistant to IR and 5-FU. In this study, we focus on PD8 cells. Therefore, we tried to find the resistant mechanism of PD8. Our results have demonstrated that: 1. The expression of p53 is not dramatic altered in PD8 cell. 2. In the process to obtain resistant cells, we found the decrease of p53 degradation and the decrease of p38 MAPK activation along with the decrease of apoptosis. Therefore p53 and p38 MAPK could play important roles in the process. 3. When PD8 cells were treated with PD153035, we found the phosphorylation of p38 MAPK is decreased, the degradation of p53 is decreased, and the phosphorylation of Akt and Erk1/2 is increased. Therefore Akt and Erk1/2 could be important in the drug resistant mechanism of PD8. 4. When PD8 cells were treated with 5-FU, the phosphorylation of p38 MAPK is decreased. 5. When PD8 cells were treated with 5-FU and IR, p53 is highly phosphorylated at serine-15. 6. When PD8 cells were treated with MG132 (proteasome inhibitor), p53 degradation is decreased. Our results have revealed some possible factors in drug resistant mechanism, however further experiments need to be performed.
目錄
頁次
圖目錄--------------------------------------------- VIII
縮寫表----------------------------------------------XII
中文摘要-------------------------------------------- XIV
英文摘要---------------------------------------------XVI
第一章 緒論-------------------------------------------1
第一節 癌症 (惡性腫瘤) 的形成-----------------------1
第二節 腫瘤的分類-----------------------------------2
第三節 致癌基因 (oncogene) -------------------------3
第四節 原致癌基因 (proto-oncogene) : c-Myc ----------4
第五節 抑癌基因 (tumor suppressor gene: p53) ---------5
第六節 p53絲胺酸第15號位置之磷酸化------------------7
第七節 細胞凋亡 (apoptosis) ------------------------7
第八節 Bcl-2 家族---------------------------------- 10
第九節 caspases ------------------------------------11
第十節 MAPK 超級家族------------------------------- 12
第十一節 上皮生長因子接受器所調控之細胞存活訊號傳遞路徑-----------------------------------------13
第十二節 多重抗藥蛋白: MDR ------------------------- 14
第十三節 實驗目的---------------------------------- 15
第二章 實驗材料與方法-------------------------------- 16
第一節 實驗材料----------------------------------17
第二節 實驗方法-----------------------------------22
第三章 實驗結果-------------------------------------- 28
第一節 c-Myc引起Myc83細胞株進行細胞凋亡作用--------28
第二節 利用Myc83細胞株篩選出具有抗藥性之細胞株-----29
第三節 篩選之抗藥性細胞株對於放射治療及傳統化療藥物誘
導之細胞凋亡皆具有抵抗性-------------------- 31
第四節 Myc83及抗藥性細胞株中p53、c-Myc蛋白表現之影響-------------------------------------------- 32
第五節 加藥篩選之不同代數細胞株中p53、p38 MAPK蛋白之影響-----------------------------------------33
第六節 以PD153035及anisomycin加藥處理後之Myc83及抗
藥性細胞株其細胞凋亡情形及p53、p38 MAPK、
p-p38 MAPK蛋白之影響------------------------ 34
第七節 以PD153035及anisomycin加藥處理後之Myc83及抗
藥性細胞株中細胞存活訊號傳遞路徑蛋白Akt、
p-Akt (Ser-473) 、Erk1/2、p-Erk1/2活化之影響---------------------------------------------37
第八節 以PD153035及anisomycin加藥處理後Myc83及抗藥
性細胞株中細胞凋亡相關蛋白 caspase-3、
PARP之影響----------------------------------38
第九節 PD98059及LY294002引起Myc83及抗藥性細胞株之細
胞凋亡率------------------------------------ 40
第十節 蛋白質降解體抑制劑 (MG132) 對於抗藥性細胞株:
PD8及AN8中p53蛋白表現之影響--------------- 41
第十一節 放射線對於抗藥性細胞株PD8及AN8 中p53、
p-p53 (Ser-15)、MDM2、c-Myc蛋白之影響------- 42
第十二節 放射線對於抗藥性細胞株PD8及AN8中細胞存活訊號傳遞路徑蛋白Akt、p-Akt (Ser-473)、Erk1/2、p-Erk1/2活化之影響------------------------ 43
第十三節 放射線對於抗藥性細胞株PD8及AN8中p38 MAPK、p-p38 MAPK蛋白活化之影響-------------------43
第十四節 放射線對於抗藥性細胞株PD8及AN8中Bak、Bax、Bcl-xL蛋白表現之影響-----------------------44
第十五節 5-FU對於抗藥性細胞株PD8及AN8中p53、
p-p53 (Ser-15) 、MDM2及c-Myc蛋白之影響-----45
第十六節 5-FU對於抗藥性細胞株PD8及AN8中細胞存活訊號傳遞路徑蛋白Akt、p-Akt (Ser-473)、Erk1/2、p-Erk1/2活化之影響------------------------ 46
第十七節 5-FU對於抗藥性細胞株PD8及AN8中p38 MAPK、p-p38 MAPK蛋白活化之影響-------------------46
第十八節 5-FU對於抗藥性細胞株PD8及AN8中Bak、Bax蛋白表現之影響---------------------------------47
第四章 討論------------------------------------------ 48
第一節 由c-Myc過度表現之乳癌上皮細胞成功地篩選出具有抗藥特性之細胞株-----------------------------48
第二節 利用PD153035所篩選出不同代數抗藥性細胞株可能是經由p38 MAPK 蛋白的不活化而造成有抗藥性之機轉---------------------------------------------49
第三節 PD153035造成PD8 產生抗藥性之機轉可能部分經由
p38 MAPK蛋白之不活化或經由Akt及Erk1/2之活化所
造成-----------------------------------------51
第四節 處理PD153035引起PD8產生抗藥性對於Bcl-2家族相
關蛋白之影響---------------------------------51
第五節 Myc83及抗藥性細胞株之PI3K/Akt、Erk1/2訊號傳遞
路徑之影響-----------------------------------52
第六節 放射線引起抗藥性細胞株細胞凋亡相關蛋白之比較53
第七節 5-FU引起抗藥性細胞株細胞凋亡相關蛋白之比較--54
第八節 抗藥性細胞中處理放射線或5-FU之p53絲胺酸第15
號位置磷酸化之探討---------------------------55
第九節 蛋白質降解體抑制劑 (MG132) 使得抗藥性細胞株之
p53蛋白表現回升----------------------------- 55
第十節 結論---------------------------------------- 56
圖--------------------------------------------------- 57
參考文獻--------------------------------------------- 94
附錄--------------------------------------------- 101




圖目錄
頁次
圖1 PD153035及anisomycin引起Myc83細胞株之細胞凋亡率及對於p53蛋白之影響-----------------------------57
圖2 利用PD153035及anisomycin所篩選出不同代數抗藥性細胞株之細胞凋亡率------------------------------- 58
圖3 放射線 (Radiation) 處理後之細胞凋亡率----------- 59
圖4 化療藥物 (5-FU) 處理後之細胞凋亡率-------------- 60
圖5 Myc83及抗藥性細胞株中p53蛋白之影響-------------61
圖6 Myc83及抗藥性細胞株中c-Myc蛋白之影響-----------62
圖7 篩選之不同代數細胞中加藥處理後p53蛋白之影響-----63
圖8 篩選之不同代數細胞中加藥處理後p38 MAPK蛋白之活化 ------------------------------------------------64
圖9 處理PD153035及anisomycin後Myc83及抗藥性細胞株之細胞凋亡率------------------------------------- 65
圖10 處理PD153035及anisomycin後Myc83及抗藥性細胞株
p53蛋白之影響--------------------------------- 66
圖11 處理PD153035及anisomycin後Myc83及抗藥性細胞株
p38 MAPK蛋白之活化---------------------------- 67
圖12 處理PD153035及anisomycin後Myc83及抗藥性細胞株
Akt蛋白之活化--------------------------------- 68
圖13 處理PD153035及anisomycin後Myc83及抗藥性細胞株
Erk1/2蛋白之活化------------------------------ 69
圖14 處理PD153035及anisomycin後Myc83及抗藥性細胞株
caspase-3蛋白之影響--------------------------- 70
圖15 處理PD153035及anisomycin後Myc83及抗藥性細胞株
PARP蛋白之影響-------------------------------- 71
圖16 處理PD98059及LY294002後Myc83及抗藥性細胞株
之細胞凋亡率---------------------------------- 72
圖17 Proteasome抑制劑 (MG132) 處理後p53蛋白之影響--73
圖18 放射線處理後p53蛋白之影響--------------------- 74
圖19 放射線處理後p-p53 (Ser-15) 蛋白之影響----------75
圖20 放射線處理後MDM2蛋白之影響---------------------76
圖21 放射線處理後c-Myc蛋白之影響--------------------77
圖22 放射線處理後Akt蛋白之活化----------------------78
圖23 放射線處理後Erk1/2蛋白之活化-------------------79
圖24 放射線處理後p38 MAPK蛋白之活化-----------------80
圖25 放射線處理後Bak蛋白之影響----------------------81
圖26 放射線處理後Bax蛋白之影響----------------------82
圖27 放射線處理後Bcl-xL蛋白之影響-------------------83
圖28 5-FU處理後p53蛋白之影響-----------------------84
圖29 5-FU處理後p-p53 (Ser-15) 蛋白之影響------------85
圖30 5-FU處理後MDM2蛋白之影響----------------------86
圖31 5-FU處理後c-Myc蛋白之影響---------------------87
圖32 5-FU處理後Akt蛋白之活化-----------------------88
圖33 5-FU處理後Erk1/2蛋白之活化--------------------89
圖34 5-FU處理後p38 MAPK蛋白之活化------------------90
圖35 5-FU處理後Bak蛋白之影響-----------------------91
圖36 5-FU處理後Bax蛋白之影響-----------------------92
圖37 PD8抗藥性細胞株中以PD153035、IR、5-FU處理時相關蛋白表現之示意圖-------------------------------93
附錄目錄
頁次
附錄1 MAPK及PI3K訊號傳遞路徑示意圖---------------- 101
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