出版年:

研究生:

周照凱

研究生(英文姓名):

論文名稱:

壹、Ha-ras轉型之細胞細胞不著地生長及Rac訊息傳遞分析 貳、正常Neu在膀胱癌細胞株TCC-Sup中所扮演之角色

英文論文名稱:

I. Signal transduction of anchorage-independent growth and the role of Rac II.The role of normal Neu in TCC-Sup

指導教授:

劉校生

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46851136

學年度:

86

語文別:

中文

論文頁數:

0

關鍵詞:

細胞不著地生長 ; 細胞計劃性死亡 ; 膀胱癌細胞 ; 細胞訊息傳遞

英文關鍵詞:

Ras ; colony formation ; apoptosis ; Rac ;
signal transduction ; Neu(ErbB2)

被引用次數:

0

[ 摘要 ]

摘要
我們利用大腸桿菌(Escherichia coli ) 之b-galactosidase基因調節
系統在NIH/3T3細胞內建立了兩株可調控Ha-ras致癌基因之細胞株
(7-4 和2-12)，蔡(1996)於細胞不著地生長(Anchorage-independent
growth)研究中，發現2-12細胞於含10% AC-2TM(母牛初乳的粹取物，
血清之替代品)之軟洋菜膠中，需同時有Ha-ras之過量表現及Insulin
growth factor-1(IGF-1)之刺激才會形成細胞群落(colony formation)。利用此細胞群落形成之模式本研究進一步探討Ha-ras下游與細胞不著地生長相關之訊息傳遞。結果顯示在10% AC-2TM的培養液中Ha-ras的過量表現主要活化raf-1路徑，而IGF-1則是活化PI(3)k的路徑，此二路徑在0.2%血清之培養情況下亦會被活化，但不能形成細胞群落，顯示仍有未知的因子參與10% AC-2TM之培養狀況下細胞群落之形成，只是在我們的分析系統下無法偵測到。另一株可調控Ha-ras表現的7-4細胞株在含10% AC-2TM之培養液中細胞反而會apoptosis，同時在10% AC-2TM的軟洋菜膠中細胞不著地生長的能力伴隨著PI(3)k活性之降低亦顯著下降。以上結果均顯示raf-1及PI(3)k為細胞群落形成之主要路徑。
Rho家族中之Rac在Ras之訊息傳導扮演重要角色，我們把含有dominant
negative rac的質體(pZipneoN17Rac)送入7-4細胞內並建立了7-4Racd2
及7-4Racd3兩細胞株，以瞭解阻斷Rac後對細胞訊息傳遞的影響，
其中之7-4Racd2細胞週期主要分佈於G0/G1期，不同於7-4細胞的S期，
同時也改變了由Ha-ras過量表現所引起的細胞膜捲曲(membrane
ruffling)現象，有趣的是Rac下游的轉錄活化子NFkB不但沒被抑制反
而被大量活化，而且Rac下游JNK的活性及不著地生長的能力也沒有被抑制。但是7-4Racd2卻使Ha-ras過量表現所造成的細胞apoptosis減弱，總言之dominant negative rac對7-4細胞之某些生物現象之抑制並不完全，但卻可以造成NFkB之活化及抑制該細胞之apoptosis，其機制待進一步探討之。
另外我們亦分析了Receptor tyrosine kinase成員中Neu(normal Neu)
的過量表現對膀胱癌細胞TCC-Sup的影響。轉殖neu至TCC-Sup所建立的
細胞株TCC-SupN10中發現細胞生長速率減慢，並有大量細胞死亡，另外
Neu的過量表現亦使負責細胞生長之Ras及抑制腫瘤擴散的nm23H1基因表
現下降，我們的研究顯示Neu在TCC-SupN10細胞中所扮演之角色仍然十
分矛盾，不過比較傾向於細胞之分化。

[ 英文摘要 ]

Abstract
Two cell lines designed 2-12 and 7-4 cells
(derived from NIH/3T3) containing an inducible Ha-ras gene
and an E.coli lacI repressor gene have been established in our laboratory. Tsai(1996) reported that Ha-ras oncogene overexpression and Insulin growth factor-1(IGF-1) stimulation were both required for 2-12 cells to form colonies in soft agar containing 10%AC-2TM(collistrum, a substitute of serum). Using this model we analyzed colony formation related signal transduction of 2-12 cells in 10% AC-2TM containing medium. We disclosed that Ha-ras mainly activated raf-1 pathway and IGF-1 activated PI(3)k pathway. These two pathway were activated under 0.2% serum condition but cells could not form colonies, indicating the requirement of other factors for colony formation under 10%AC-2TM condition. However, the assay we used could not find them. In contrast, the Ha-ras inducible cell line 7-4 showed decreased colony formation in 10%AC-2TM containing soft agar. Further analysis demonstrated that 7-4 cells underwent apoptosis and its PI(3)kinase activity was lowered. Taken together, raf-1 and PI(3)k pathway are required for cell colony formation in soft agar.
To study the effect of Rac on Ras signaling,
we transfected dominant negative rac(pZipneoN17Rac)
into 7-4 cells to block Rac activity. Subsequently,
two cell lines 7-4Racd2 and 7-4Racd3 were established.
Among them, 7-4Racd2 showed major cell population in G0/G1
phase. 7-4Racd2 also prevented membrane ruffling which was
caused by Ha-ras overexpression. Interestingly dominant
negative rac activated NFkB, but could not down-regulate
JNK activity as well as the ability of cell anchorage-
independent growth. Moreover, dominant negative rac in
7-4Racd2 prevented cell apoptosis. In summary, dominant
negative rac in 7-4Racd2 cells is sufficient to suppress
cell apoptosis, but is not enough to block other biological
activities. Moreover, why NFkB was activated by dominant
negative rac remaining to be determined.
The role of receptor tyrosine kinase neu(normol neu)
in bladder carcinoma cell line named TCC-Sup was investigated
by introducing the neu gene into TCC-Sup. A cell line
TCC-SupN10 established by Dr. Lai*s laboratory showed
slower cell growth and is prone to cell death as compared
to TCC-Sup cells. TCC-SupN10 also showed down regulation of
Ras and nm23H1 gene expression which play critical roles in
cell growth and inhibition of tumor metastasis, respectively.
The role of Neu in TCC-Sup cell line is still contradictory
but its function is more close to cell differentiation than to
proliferation.