|
在細菌的乳糖基因調控系統中，乳糖抑制子(lac repressor, lacI) |專
一的結合於乳糖調控子序列(lacoperator, lacO)上，抑制RNA鏈結合酵
素(RNA polymerase)i行轉錄作用。利用大腸桿菌之乳糖控制子序列
(lacO) 來控制Heavy ras(Ha-ras)致癌基因於小鼠之纖維原細胞株型(
simian virus 40, SV40)的啟動子與Ha-ras基因之間而構築成
pSVlacOras質體，將pSVlacOras質體與另一個表現乳糖抑制子(lacI)之
pHblacINLSneo質體協同轉染(cotransfection)至NIH 3T3細胞,而選殖出
可調控Ha-ras基因表現之細胞株命名2-12。雖然Ha-ras基因受到誘導子
isopropyl-b-D-thiogalactoside(IPTG)的調控，然而在沒有誘導子之情
況下仍有一些抑制不夠緊密的問題，為了探究原因之所在，並謀求改進之
道，首先必須暸解Ha-ras基因插入2-12細胞中之後是否有甲基化。實驗證
實於2-12於細胞株中有多個Ha-ras基因插入染色體中，亦有基因重排現像
。而Ha-ras基因及其啟動子和lacI抑制子均發現有甲基化，但似乎不會影
響Ha-ras基因之誘導表現。而細胞生長於沒有誘導子的情況下，仍有Ha-
ras表現，可能是部分Ha-ras經重排後，失去調控性，但仍具表達力。
而2-42細胞株中僅單一Ha-ras插入染色體中，且無基因重排現像。而Ha-
ras基因及其啟動子亦發現有甲基化。實驗顯示2-12細胞中的Ha-ras基因
在IPTG誘導後，於四到八小時達到最高點，而後持續表現。若將IPTG自培
養液中去除則Ha-ras於二十四小時始下降，於七十二小時Ha-ras的表現降
至最低點而當IPTG活化Ha-ras基因表現之後，發現2-12細胞之內源性c-
jun, c-myc與nm23基因的表現降低，似乎與Ha-ras有關連性，但卻不能像
Ha-ras一樣持續表達,而在不同時間降下來，可能與細胞周期有關之因子
，對這些基因亦具調節性而nm23基因表現逐漸下降，或許是使細胞邁向轉
移期的重要因素之一，於2-12細胞中，當Ha-ras被活化，可誘導c-myc基
因之表現且細胞存活率卻下降。故Ha-ras基因是否亦可誘導計劃性細胞死
亡值得探討，然經電泳及雷射細胞流動分析，均沒有發現明顯之計劃性細
胞死亡現像。其原因可能與誘導Ha-ras表達的早期c- myc有明顯的表現，
但並不能持久有關。
|
|
An inducible system of Harvey ras(Ha-ras) gene regulation has
been established in our laboratory. It is controlled by
regula- tory elements from bacterial Escherichia coli(E. coli).
The lac operator sequence from E. coli was constructed at 3''
end of simian virus 40 (SV40) promoter region and in front of
Ha-ras oncogene The established cell line, 2-12, containing
this in- ducible Ha-ras gene can be suppressed by lac repressor(
LacI) which is in plasmid pHblacINLSneo. Furthermore, Ha-ras
oncogene can be specifically induced by a lactose analogue
IPTG. To further improve the conditions of induction and
repression, copy number, methylation and rearrangement of the
transgenes were studied. Multiple copies of Ha-ras and
lacIgenes were integrate into chromosomes, and rearrangement of
Ha-ras and lacI genes also occurred. Methylation also occurs in
all the transgenes, and it does not affect the expression of Ha-
ras gene under the admini- sion 4 to 6 hr after IPTG
induction. Expression of downstream nuclear oncogenes, c-jun
and c-myc, andmetastasis supressor gene nm23 were all actived
after Ha-ras gene activation. But, their expression is not
like Ha-ras gene at constant level but drops at various times
post-induction. It is known that decreased nm23 gene
expression correlates with metastsis of cancel cells.
Interestingly, gene expression of nm23 in 2-12 cells drops at
24 hr post-induction. It is probable that cells are further
transformed to a metastasis form after long-term over-
expression of Ha-ras gene. This study clarifies the status of
transgenes in 2-12 cells. The knowledge regarding the
relationship between Ha- ras, c-jun, and c-myc oncogene should
help us to determine the ferential screening assay, and
furthermore to identify the cofactors and secondary
messengersor correspond with Ha-ras gene expression.
|
