Abstract
在本論文中,我們開發出針對PDPK FA的高專一性抗體,並以癌細胞株作為實驗模型,分別深入探討PDPK FA在癌細胞中的訊號傳遞及功能角色.在PDPK FA訊號傳遞的研究方面,當以蛋蛋白質激脢C(PKC)抑制劑calphostinC處理人類表皮癌A431細胞時,我們發現calphostin C能透過兩個不同的途徑調控細胞中PDPK FA的活性,除了可能會透過抑制PKC(IC50~0.05mM)而引發PDPK FA酪氨酸磷酸化而活化之外,在較高濃度時則會直接抑制PDPK FA上游的酪氨激脢(IC50~1mM),而使得PDPK FA酪氨酸去磷酸化而去活化;確認酪氨酸激脢在PDPK FA 活性調控上的重要性.為了進一步證實PKC是PDPKFA上游的訊號分子,我們利用TPA、sphingosine這些高專一性PKC調節劑或抑制劑, 處理除去血清培養的人類表皮癌A431細胞,配合酪氨酸激脢抑制劑的使用,證實PKC的確透過酪氨酸激脢而調控PDPK FA的活性.免疫偵測與動態分析,更確定了 PKCe 在PDPK FA酪氨酸磷酸化過程的調控角色.在PDPK FA功能的研究方面,我們利用antisense mRNA抑制癌細胞中PDPK FA的表現量,並且因此造成細胞增殖減緩、細胞倍數期延長,甚至造成細胞程式死亡(programmed cell death, apoptosis),PDPK FA 在調控細胞增殖過程上的角色,更證明抑制癌細胞中過度表現的PDPK FA,可能是誘發程式死亡的一個重要關鍵.因此,找出適合PDPK FA抑制劑,可能對癌症治療有非常大的幫助.由於PDPKFA的調控與酪氨酸激脢息息相關,我們利用了不同的酪氨酸激脢抑制劑處理癌細胞,觀察其對PDPK FA活性的影響,結果發現如genistein、kaempferol、quercetin、apigenin這一族生物類黃酮(bioflavonoids),低濃度時(0~50mM)即可對PDPK FA造成不同程度的抑制效用.更令人訝異的是,quercetin、apigenin不但造成PDPK FA的酪氨酸去磷酸化,且也會使其絲氨酸磷酸化,將癌細胞中過度表現的PDPK FA活性控制到正常範圍.和antisense PDPK FAmRNA一樣,生物類黃酮也可以藉由抑制PDPK FA的活性而抑制癌細胞的增殖,且其抑制癌細胞增殖增殖的能力與其抑制PDPK FA活性的能力成正比,顯示利用PDPK FA的抑制劑可以達到控制癌細胞增殖的目的.另外,我們也對現有臨床上正在使用的抗癌藥物做一全面性的篩檢,試圖找出可以抑制PDPK FA的藥物. 在 十種接受測試的抗癌藥物中,我們發現沒有任何一種可以影響PDPK FA的活性及表現量,但是當以antisense mRNA或以生物類黃酮降低癌細胞中過度表現的PDPK FA時,可以增強五至三十倍抗癌藥物抑制癌細胞增殖的能力,透露出PDPK FA可能是快速抑制癌細胞增殖的關卡, 只要其訊號傳遞被抑制後就可增強抗癌藥物的療效.顯示合併PDPK FA抑制劑與抗癌藥物共同處理癌細胞的可行性,為其臨床上的應用跨出了一大步.Protein kinase FA was identified as a multisubstrate proteinkinase possibly involved in the regulation of diverse cellfunctions. Recently, kinase FA has been demonstrated as a memberof the so-called proline-directed protein kinase(PDPK) family.Based on computer-assisted sequence analysis oftranscriptionalfactors and viral oncoproteins, as well asanalysis of site-specific proteinphosphorylation both in vitroand in vivo □M7 appears that proline-directed proteinphosphorylation sites represent a unie structural motif that hasbeen conserved and canalized as a major regulatory theme. It hasbeen proposed thatoverexpression, dysregulation or viralsubversion of some certain PDPKs couldbe associated withneoplastic transformation and tumorigenesis. Initialevidencefrom clinical studies confirmed that overexpression ofPDPK FA indeed closelycorrelated with the states ofdedifferentiation/progression of human thyroidcarcinoma,cervical carcinoma, and hepatocellular carcinoma. This kinasemay,therefor represent a newly-described differentiation-blocking and/or de-differentiation-promoting agent involved inthe progression of human carcinomacells. However, little isknown about the regulation of this kinase in cells. In thisthesis the PDPK FA in the signal transductional regulation incancerbiology.