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我們用利用大腸桿菌 (Escherichia coli) 之乳糖調節組在 NIH3T3 細胞
內建立了可誘導 Ha-ras 致癌基因表達之 212 細胞株，並以軟洋菜膠分
析法 (soft agar assay) 研究 Ha-ras 致癌基因與相關因子對細胞不著
地生長(anchorage-independent growth) 的影響。當細胞培養在含 10%
牛血清的軟洋菜膠中，並以誘導子 Isopropyl-b-D-thiogalatoside
(IPTG) 誘導 Ha-ras 致癌基因過度表達，可刺激 212 細胞形成群落，然
而若是培養在含 0.2% 牛血清的軟洋菜膠中，雖活化 Ha-ras 致癌基因卻
無法促使細胞形成群落，顯示血清中的某些因子和活化之 Ha-ras是細胞
不著地生長所必需的。與不著地生長有關之生長因子 (growth factor)
已知的有epidermal growth factor (EGF)，platelet -derived growth
factor(PDGF)，basic fibroblast growth factor (bFGF)，胰島素 及
Insulin-like growthfactor-1 (IGF-1) 等，若將其直接加入含 0.2% 牛
血清培養液中，依然無法刺激細胞形成群落。以牛初乳萃取物(bovine
colostrum, AC-2, Valio, Finland) 取代牛血清以提供細胞所需的基本
養分，於活化 Ha-ras 情況下仍然無法刺激細胞形成群落，顯示 AC-2 培
養液中或許含有抑制子或缺乏不著地生長所需的因子或量不足。實驗結果
沒有發現任何抑制子存在，因此將上述生長因子分別加入含 AC-2 的培養
液中，並以 IPTG 活化 Ha-ras 致癌基因，發現除了 transforming
growth factor-b1 (TGF-b1) 外皆可促使細胞形成不同程度之群落。而以
膠體分離 (gel filtration) 將牛血清依分子量大小分為四個部份，將之
分別加入含 AC-2 的培養液中，並活化 Ha-ras 致癌基因，發現分子量
含小於 14 Kd 的第四部份可刺激細胞群落形成。以上結果顯示至少兩個
路徑是細胞不著地生長所必需的。當我們加入抑制 Ras 癌蛋白轉置到細
胞膜的藥物： lovastatin 及pravastatin 時造成細胞群落之下降，若加
入抗 IGF-1接受體 (receptor) 的抗體，阻斷訊息經由 IGF-1 接受體傳
遞，亦明顯的抑制細胞群落之形成。這些結果進一步顯示細胞不著地生長
需活化之 Ha-ras 外之其它途徑共同作用，而 IGF-1 可能為其中之一。
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The effects of Ha-ras oncogene and related factors on anchorage-
independent growth of a212 cell line derived from NIH3T3 cells
in soft agar was investigated. The 212 cell contains an
inducible Ha-ras oncogene which is under the control of
Escherichia coli lactose regulatory elements. When cells were
maintained in soft agar containing 10% calf serum,
overexpression of Ha-ras oncogene stimulated colony formation.
When cells were cultured in soft agar containing only 0.2% calf
serum, no colony was detected even with IPTG induction,
indicating that besides activated Ha-ras oncogene , some factor(
s) in serum are also required for anchorage- indenpendent
growth. However, simply adding anchorage- independent growth
related growth factors (EGF, PDGF, bFGF, Insulin or IGF-1) into
soft agar containing 0.2% calf serum could not induce colony
formation. While 0.2% calf serum was replaced with bovine
colostrum (AC-2 , Valio, Finland) to compensate nutrient
inadequacy, the characteristic of unable to form colony
remained, indicating that AC-2 either contains inhibitors or
lacks the factor(s) for anchorage- independent growth. So far
no inhibitor was detected in AC-2, and diverse levels of colony
formation were stimulated by growth factors except TGF-b1 in
AC-2 containing medium with IPTG induction. Calf serum was
separated by gel filtration into 4 fractions, and each fraction
was added into AC-2 containing medium with IPTG induction. The
4th fraction could stimulate cell colony formation, and its
molecular weight is smaller than 14 Kd. When we added the
inhibitors (lovastatin and pravastatin) of Ras oncoprotein,
colony formation efficiency declined. When anti-IGF-1 receptor
antibody was added (which block the signal pathway of IGF-1 ),
colony formation was also inhibited. Again these data suggest
that at least two pathways are required for anchorage-
independent growth : one is ras pathway, and IGF-1 is one of
the other pathways.
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