在腫瘤形成初期，大多會臨近於層狀上皮細胞旁形成細胞團塊。為何生長中的轉型細胞傾向於形成凸起的細胞團塊而不是鑲嵌於上皮細胞層中目前仍不清楚。我們建立了綠螢光蛋白標定及可受到IPTG所誘導致癌基因Ha-RasV12表現的MK4細胞。當將細胞養滿時，不論是與正常的MDCK細胞共同培養或是單獨培養，只有Ha-RasV12表現的MK4細胞(MK4H-Ras)可形成向上突起的細胞團塊。這樣所觀察的結果與MEK/ERK路徑相關但與基質順應性及細胞增殖無關。此外MK4H-Ras細胞呈現底部stress fiber減少與junctional actin增加。在內層的細胞團塊中的microtubules似乎被下降調控了。而在細胞團塊中，仍然可看到E-cadherin和p120 catenin出現在紊亂的細胞層間。我們猜測當擾亂細胞黏附因子的極性分佈時可促進細胞團塊的產生。因此我們將帶有綠螢光標定的細胞種在不同的單層細胞上一小時接著量化細胞的黏附能力。結果顯示只有MK4H-Ras單層細胞表面具有黏附細胞的能力。此外綠螢光標定的MK4H-Ras細胞也表現出較大的黏附能力去黏在MK4H-Ras單層細胞上。我們接著使用不含鈣離子的培養液來評估鈣離子在細胞黏結作用中所扮演的角色。結果顯示當細胞培養在不含鈣離子的培養液時，綠螢光標定的MK4H-Ras細胞黏著在MK4H-Ras單層細胞上的數目就會減少。總結，我們的數據推測過度表現Ha-RasV12所促進MK4細胞團塊的產生可能是由於破壞了膜蛋白的極性分佈或透過鈣離子依賴性的細胞黏附因子之表現來增強細胞黏結作用。

In the initiation of tumorigenesis, cell mass always forms adjacent to the sheet-like epithelia. Why the growing transformed cells tend to form extruded cell mass rather than incorporating into the epithelial layer remains unknown. We established GFP-tagged and IPTG-inducible oncogene Ha-RasV12 expressed-MK4 cell. When cultured cells at confluence, only Ha-RasV12 expressed MK4 cells (MK4H-Ras) can form extruded cell aggregates (foci) no matter in co-cultured with normal MDCK cells or cultured alone. Such observation is dependent of MEK/ERK pathway, but independent of the compliance of culture substrate and cell proliferation. Besides, MK4H-Ras cells exhibit loss of basal stress fibers while displaying intensified junctional actin. Microtubules seemed to be downregulated in the inner cell mass. In the foci, both E-cadherin and p120 catenin were found between the disorganized cells layer. We proposed that the disturbance of polarized distribution in cell adhesion molecules promote the formation of cell aggregate. We then plated GFP-tagged cells on different cell monolayer for one hour and then evaluated the cell adhesion ability. The result revealed that only MK4H-Ras monolayer surface was susceptible to cell adhesion. In addition, GFP-tagged MK4H-Ras cells also exhibited the highest capacity to adhere to the MK4H-Ras monolayer. We then used calcium free medium to evaluate role of calcium in cell adhesion. The result showed that the number of GFP-tagged MK4H-Ras cells adhered to the MK4H-Ras monolayer was diminished in the calcium free medium. Taken together, our data suggests that Ha-RasV12 overexpression facilitate foci formation in MK4 cell might be due to the disruption of the polarized distribution of membrane proteins or the augmentation of the cell adhesion via calcium–dependent adhesion molecules.

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