突變K-ras 增加類固醇賀爾蒙生合成分子機轉的探討

Investigation on the molecular mechanism of steroidogenesis increased by the activated mutant K-ras

吳佳鴻(Chia-Hung Wu)

指導教授 ： 林?茹

高雄醫學大學/醫學院/醫學研究所/博士(2003年)

摘要

中文摘要
腎上腺是個附著在兩側腎臟上方的小腺體，卻是人類生存不可或缺的重要內分泌器官。然而當腎上腺產生腫瘤時，由於其易於造成腎上腺機能亢進，使患者因內分泌失調而罹病。因此、在癌症研究領域，其雖非惡性腫瘤具有高致死率而急需處理，但其功能性的異常卻造成患者的類固醇賀爾蒙分泌過度而需有效治療。本實驗室先前一系列針對腎上腺皮質功能性腫瘤的分析，証實在所篩檢的致癌基因及腫瘤抑制基因中，K-ras具有高達44%的突變率。進一步探討發現突變K-ras不但生化活性確實受影響，且在牛正常皮質細胞中可產生類固醇賀爾蒙受誘發增加的現象。
延續上述研究成果，吾人研究重點主要在探討人類腎上腺皮質腫瘤生成及類固醇分泌能力增加之分子機轉。而且為了排除物種間可能存在的差異，吾等取自自願捐贈者的腎上腺皮質,進行正常人類腎上腺皮質細胞的培養。而實驗結果證實，吾等培養的正常人類腎上腺皮質細胞具有正常合成及分泌類固醇荷爾蒙cortisol的能力。故另外建立源自於人類腎上腺皮質腫瘤發現的突變 K-ras 致癌基因IPTG誘導表現質體 pK1516MRSV(突變在胺基酸序列第15及16位置)，pK568MRSV(突變在胺基酸序列第15、16、18及31位置)和pK60MRSV(突變在胺基酸序列第60位置)送入具有荷爾蒙分泌能力的正常人類腎上腺皮質細胞內，建立突變 K-ras 基因穩定轉染之人類腎上腺皮質細胞。實驗結果證實，針對不同突變K-ras轉染成功的細胞株經IPTG刺激36小時培養，由轉染後的細胞生長速率的結果可知pK568MRSV穩定轉染的腎上腺皮質細胞的生長速度最快,其次是pK60MRSV及pK1516MRSV穩定轉染的腎上腺皮質細胞，其變化分別為正常腎上腺皮質細胞的10倍、6倍及3倍。同時在對血清的需求分析pK568MRSV， pK60MRSV及pK1516MRSV穩定轉染的腎上腺皮質細胞均呈現顯著的下降，而pKWTRSV穩定轉染的細胞則與正常細胞沒有顯著的差異。同時發現IPTG誘導36小時後，突變K-ras致癌基因(pK568MRSV表現質體)具有使腎上腺皮質細胞外部型態改變的能力(morphologic change) ，但沒有任何一種突變的K-ras穩定轉染的細胞可生長在soft agar上。
針對IPTG誘導36小時後不同突變K-ras表現質體穩定轉染的人類腎上腺皮質細胞,測量其分泌cortisol的量，其中pK60MRSV與pK568MRSV轉染細胞分別是控制組pKWTRSV的16倍及22倍。pK1516MRSV穩定轉染細胞則是控制組pKWTRSV的1.3倍。其中參與腎上腺皮質荷爾蒙生合成相關的酵素包括P450SCC (CYP11A)、P45017α(CYP17) 和3βHSD等均有表現增加的現象存在。由上述的分析得知pK568MRSV轉染腎上腺皮質細胞最適合作為後續研究的細胞株。因此、進一步，吾等用lovastatin經由處理IPTG誘導36小時pK568MRSV轉染的腎上腺皮質細胞，同樣證實會抑制cortisol分泌及P450scc、P450c17及3βHSD mRNA的下降，映証了先前對突變K-ras的推測。綜合上述的結果,可證實突變的K-ras致癌基因不只是促進細胞增生，同時K-ras 致癌基因與腎上腺皮質細胞類固醇荷爾蒙的分泌特性確實有相當直接的關聯性存在。
另一方面、在其分子機制的探討中，發現經pK568MRSV穩定轉染的細胞株經IPTG誘導，RasGTP的含量會隨刺激時間的增加而上升。同時由於活化態的RasGTP，會活化訊息傳遞途徑下游的蛋白,由西方墨點分析磷酸化c-Raf-1及MAPK的實驗中，證實了c-Raf-1及MAPK的磷酸化也隨著RasGTP的增加而明顯的跟著上升。另外，由MEK 磷酸化活性測定實驗中，證實MEK磷酸化活性亦隨著RasGTP的量而增加。同時經由PD098059(MEK磷酸化活性專一性的抑制劑)的處理，証實可有效抑制突變K-ras促使MEK活性上升的現象。由此可證明經IPTG誘導產生的活化態突變Ras蛋白確實是經由Raf-MEK-MAPK的訊息途徑傳遞。而抑制劑PD098059亦可用來驗證Raf-MEK-MAPK訊息傳遞與cortisol的生合成及分泌的關連性。因此，吾等對pK568MRSV穩定轉染的人類腎上腺皮質細胞分析其P450scc, P450c17及3βHSD的mRNA及cortisol分泌量，證實皆會隨著IPTG刺激時間延長而增加，同時亦可被PD098059抑制。綜合上述的結果,可證實突變的K-ras致癌基因不只是促進細胞增生，同時還調控類固醇cortisol的生合成,並且是經由Ras-RAF-MEK-MAPK的訊息傳遞途徑。
進一步吾等希望能利用cDNA微矩陣排列(microarray)對突變K-ras穩定轉染的人腎上腺皮質細胞進行分析，期待更進一步了解Ras-to-MAPK訊息途徑與類固醇荷爾蒙生合成之間相互調控的分子機制。研究結果，可得知有些基因有up regulation的現象,其中較顯著的有human zinc finger protein 22上升28.5倍,Osteopontin上升5.8倍,LIM domain Kinase 2 (LIMK2)上升3.3倍,Homo sapiens dual-Specificity tyrosine-(Y)-Phosphorylation regulated Kinase 2 (DYRK2)上升2.2倍及human syntaxin 3上升2.0倍。另外,有較顯著down regulation的基因有Retinoblastoma binding protein 1 (RBBP1)、Homo sapiens craniofacial development protein 1(CFDP1), DAP Kinase-related-apoptosis-inducing protein Kinase 1 (DRAK1), SKI-interacting protein (SKIP)及human poly(A)-Binding protein (PABP)等基因，表現量分別下降4.0、2.4、2.3、2.2及2.1倍。這些有差異性表現的基因與突變K-ras致癌基因穩定轉染人類腎上腺皮質細胞株的外部型態改變，細胞增生率增加，類固醇生合成及分泌能力增加之關連性，尚待進一步研究確認。
吾等希望能利用深入了解，突變的K-ras致癌基因如何參與腎上腺皮質細胞腫瘤的形成及促進類固醇賀爾蒙分泌過程的致病機轉。期望可協助臨床醫師針對K-ras致癌基因突變的腎上腺皮質腫瘤，做為發展新的治療理論的基礎研究。

關鍵字

突變致癌基因 K-ras ； 功能性腎上腺腫瘤 ； 人類腎上腺皮質細胞 ； 類固醇賀爾蒙生合成 ； RAF ； MEK

並列摘要

Abstract
In our previous study on the tumorigenesis of human functional adrenal tumors, we observed high frequency of K-ras gene point mutations in clinical adrenal tumors. Furthermore, we cloned the mutated K-ras gene from the tumors and inserted it into vectors to transfect normal bovine adrenocortical cells purchased from ATCC (American Tissue Culture Center) to express mutated K-ras gene. The results showed that the mRNA level of steroidogenic enzyme, such as Cholesterol side-chain cleavage enzyme (P450SCC), 17a-Hydroxylase/17, 20-lyase (P450c17) and 3b- Hydroxysteroid dehydrogenase (3bHSD) in the mutant K-ras stable transfected cells was elevated. To increase the credibility of our experiments, we had cultured the human adrenaocortical cell from the human adrenal cortex, the cortical tissues were separated from the medulla after the adrenal tissue was dissected from patients. The cultured human adrenocortical cells synthesize and secrete steroid hormones after stimulated with ACTH and Angiotensin II. Then, we transfected the cells with mutant K-ras expression plasmids constructed from human functional adrenal tumors. The results showed that cells after stable transfection grew faster than normal cells. Additionally, cell morphological change was observed in mutant K-ras transfected cells. Moreover, when analyzed the synthesis pathway of hormones, the mRNA of P450SCC, P450C17 and 3βHSD was found to have increased and the level of cortisol increased 16 to 22 times higher than control cells. Additionally, the increased steroid hormone production in mutant K-ras transfected cells was reversed by lovastatin, a pharmacological inhibitor of p21ras function. These results combined with our previous reports of steroidogensis in bovine adrenocortical cells suggest that the ability of K-ras oncogene indeed involved in steroidogenesis in human adrenocortical cells.
Then we had transfected the pK568MRSV , an IPTG inducible mutated K-ras expression plasmid, into the cultured human adrenaocortical cell. Then, the stable transfectants were selected with G418 (400 ug/ml). Additionally, the results have shown that the increase of RasGTP levels in the pK568MRSV transfected cells was time-dependent manner after IPTG induction. Western blot analysis results revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase (MAPK). The increase of mRNA amount in P450scc、P450c17 and 3βHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal -regulated kinase inhibitor. In our report, we proved that lovastatin, a pharmacological inhibitor of p21ras function, also reversed the increase of cortisol level in mutant K-ras stable transfectants. Taken together, these findings proved that the active mutant Ras enhanced not only cell proliferation but also steroidogenesis in steroidogenic phenotype cells by activating Raf-MEK-MAPK related signal transduction pathway. Therefore, we believe that K-ras mutants influence regulation of steroidogenesis in adrenocortical cells through RAF-MEK—MAPK pathway
The ever-increasing rate at which genomes are being sequenced is attracting attention to functional genomics ─ an area of genome research that is concerned with assigning biological function to DNA sequences. Therefore, we use the cDNA microarray, genes involved in cell cycles, signal transduction, apoptosis, tumorigenesis and some expressed sequence tags (ESTs), totally over 8000 unique human genes arrayed on glass slides, to examine the difference between the control cell and the K-ras transfected cell to understand the cross talks between the Ras-to-MAPK pathway and the regulation of the steroidogenesis process. For example, we interesting, human zinc finger protein 22, osteopontin, LIM domain kinase 2 (LIMK2), Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) and human syntaxin 3 were shown to be 28.5, 5.8, 3.3, 2.21 and 2.0- fold up-regulated, respectively, by microarray. Similarly, Retinoblastoma binding protein 1 (RBP1), Homo sapiens craniofacial development protein 1(CFDP1), DAP kinase-related-apoptosis-inducing protein kinase1 (DRAK1), SKI-interacting protein (SKIP) and human poly(A)-binding protein (PABP) gene were shown to be 4.0, 2.4, 2.3, 2.2 and 2.1-fold down-regulated, respectively. The roles of those targeted genes involve in cell proliferation, cell morphologic change, tumorigenesis and steroidogenesis still need to be confirmed and further investigation.

並列關鍵字

K-ras oncogene mutants ； functional adrenal tumour ； normal human adrenocortical cells ； steroidogenesis ； RAF ； MEK

參考文獻

第六章參考文獻

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