| Abstract: | MAPK路徑在細胞內扮演調控大部分的細胞反應，包括了細胞增生、分化以及來自外在刺激所造成細胞的作用機制。在本篇我們專注於一個新穎的MAPKKK分子ZAK，ZAK被歸屬於MLK family。本實驗室之前的研究證實無論是體內或體外試驗，過度表現ZAK會使肺癌細胞生長速率變慢，而在臨床病人正常肺臟組織及肺癌組織之ZAK表現也發現在正常肺臟組織中ZAK表現較強，以上訊息告訴我們ZAK在肺癌細胞株中似乎作用為一抑癌基因。接下來本研究將繼續探討ZAK所引發的抑制細胞生長反應其調控路徑為何？首先，我們發現過度表現ZAK會活化ERK及JNK分子及其下游調控之轉錄因子的活性，例如: c-Fos、c-Jun及Elk-1。接著使用抑制劑(PD98059及SP600125)作用細胞以截斷ERK及JNK路徑，發現在過度表現ZAK之肺癌細胞株其細胞生長有部份回復的現象。偵測c-Jun與c-Fos之複合體AP-1之活性，同樣的在ZAK表現之細胞株其AP-1活性增強約四倍之多。使用siRNA干擾技術阻擾c-Jun表現，其ZAL表現之細胞生長情形回復，而SEAP-AP-1活性也有被抑制的現象。另外我們又使用了siRNA-Elk-1干擾Elk-1表現也可發現在ZAK表現的細胞株其細胞生長速度有回升的情形，而SEAP-AP-1活性同樣也被抑制的現象。就以上in vitro的試驗中我們發現ZAK可藉由調控ERK及JNK路徑來控制抑制細胞生長的反應，接下來在in vivo的部份我們使用SCID mice以皮下注射ZAK過度表現之肺癌細胞，結果發現相較於控制組，ZAK表現的實驗組其腫瘤體積或重量都有被抑制的現象，其腫瘤組織蛋白也可發現c-Fos、c-Jun及Elk-1的表現較高。在細胞週期的部份我們觀察到，當ZAK被過度表現後會使細胞週期停滯於S及G2/M期，細胞週期調控分子cyclin A、E及cdc2蛋白表現下降，cyclin B1、cyclin D、CDK2、CDK4及CDK6蛋白表現增加；CDK inhibitor的部份則是p15、p16及p21蛋白表現都有增強的情形。ZZaPK為KZNF鋅指蛋白(KRAB-containing zinc finger protein)成員之一。本研究將探討ZZaPK於H460肺癌細胞株中與ZAK之間的作用關係。在細胞試驗的結果中可得知ZZaPK回復了ZAK所造成的細胞抑制作用，而在細胞週期的調控上也表現出類似H460正常肺癌細胞的反應。綜合以上結果，ZAK於體外和體內試驗中所造成的細胞及腫瘤生長抑制，可能是活化ERK及JNK路徑，間接的影響細胞週期的表現使之停滯於S及G2/M期。另外，在ZAK過度表現的H460肺癌細胞株中，導入ZZaPK可抵制ZAK之作用而使細胞生長回復正常。
The MAPK cascades regulate a wide variety of cellular responses, including the cell proliferation, differentiation and the stress responses. In this research we have identified a novel MAP kinase kinase kinase (MAPKKK) termed ZAK. ZAK is classified in MLK family. Previous studies in our laboratory demonstrated that over-expression ZAK in H460 lung cancer cells showed a decreased growth rate in in vitro and in vivo. Quantitative real time PCR results demonstrated that ZAK gene largely expressed in tumor adjacent normal part of lung tumor specimens. Here, we continued to explore the mechanisms which ZAK inhibiting lung cancer cell growth. First of all, we found ZAK will activate ERK and JNK, and the phosphorylation expression levels of down-strean transcription factor including c-Fos, c-Jun and Elk-1were increased. The effect of increased cell growth rate was significantly but incompletely reversed when ZAK overexpressing cells were treated with a specific ERK and/or JNK inhibitor. AP-1 transcription activity and the phosphorylation expression of Elk-1 were increased in ZAK overexpression cells. We transfected small interference RNA (siRNA) of Elk-1 into ZAK-overexpressing cells could restore cell growth rate. More importantly, in severe combined immunodeficient (SCID) mice, ZAK, but not the control vector, significantly suppressed tumor growth by H460 cells. Tumor tissue protein levels of c-Fos, c-Jun and Elk-1were also higher in ZAK-overexpressing group. In the cell cycle analysis, we observed that ZAK over-expression will result in cell cycle arrest in S and G2/M phase. Expressions of yclin A,cyclin E and cdc2 were down-regulated ;however, cyclin B1, cyclin D, CDK2, CDK4, and CDK6 werecup-regulated. About CDK inhibitor, p15, p16 and p21 expressions were enhanced In ZAK overexpressing H460 cells. ZZaPK belong to a known family of the KRAB-containing zinc finger protein family. In this study, we want to investigate whether ZZaPK could interact with ZAK. We found that ZZaPK counteracted ZAK function in cell counting assay, and restored ZAK-mediated cell cycle arrest in H460 cells. Based on the above results, ZAK decreased cell growth may be via activation of ERK and JNK pathways, indirectly resulted in S and G2/M phase arrest of cell cycle. ZZaPK may stimulate the ZAK-overexpressing cells re-entering the cell cycle. |
