- 學位論文
PMA 引發 HL-60 細胞分化早期事件之研究
Early Events Associated with Phorbol Myristate Acetate- Stimulated HL-60 Cells Differentiation
摘要
中文摘要 人類前骨髓性白血病 ( human promyelocytic leukemia ) HL-60細胞, 能被 Phorbol Myristate Acatate (PMA) 誘發, 而分化成為巨噬細胞 ( Macrophage )。 但是, PMA 誘發 HL-60 細胞分化的早期控制機轉, 知道 的並不多。在我們的實驗報告中發現, HL-60 細胞經 PMA ( 10 nM ) 處 理後, 結果造成細胞G1期停滯 (G1 arrest)。蛋白質激酵素C ( protein kinase C ; PKC ) α 同功酵素也因 PMA 的刺激, 而在 5 分鐘時開始轉 位 ( translocation ) 至細胞膜, 而最大值則在 30 分鐘被看見, 之後 PKCα 慢慢地消失, 而在 24 小時後 down regulation。隨著 PKCα 的 轉位,同時也發現Rb proteins 出現underphosphorylation form , Rb underphosphorylation form 在 PMA 刺激後 5 分鐘出現, 這樣情形也隨 著時間消失在 24 小時, 與 PKCα 轉位結果, 非常相符 。Rb proteins 的磷酸化受 D type cyclins,CDK4 和 CDK4 抑制者 ( P21cip1 ，P16 INK4a)的調節 。Cyclin D3在PMA處理後10小時被表現出來.雖然我們無法 證實 CDK4 和 p21cip1,p16INK4a, 受 PKCα 的調節, 但若跟據Zang et al(1995) 的發現, 很可能 PMA 誘導 HL-60細胞分化為Macrophage, 是經 PKC α活性, CDK4 inhibitor p21 cip1 的誘導,繼而造成Rb dephosphorylation, 最後造成細胞生長週期的 G1 arrest及細胞分化。
並列摘要
Abstract The human promyelocytic leukemia HL-60 cells can be induced to differentiation toward macrophage by treatment with PMA. However, very little is known regarding the early events that control differentiation of HL-60 by PMA. In this report, we demonstrated that PMA (10nM) treatment results in cell cycle arrest in G1 phase. Addition of PMA stimulated PKCα translocation in 5 minutes andthe maximum response was seen 30 minutes after PMA was added. Upon activation,PKCα is gradually degraded. The PKCα down regulation was observed within 24hours after PMA treatment. The PKCα translocation is associated withRb underphosphorylation. Rb underphosphorylation was apparent at 5 minutes and gradually disappeared within 24 hours, consistent with the time course of PKCα activation. Because Rb phosphorylation can be regulated by D type cyclins andCDK4, we also examinedthe expression of D type cyclins, CDK4 and CDK4 inhibitors(p21cip1, p16INK4a) in HL-60 cells after PMA treatment. Cyclin D3 but not cyclinD1 or Cyclin D2 was expressed 10hrs after PMA addition. However, expression of both CDK4 and CDK4 inhibitors(p21cip1, p16INK4a) were not affected by PMAtreatment. Although we can not directly prove that CDK4, p21 cip1 and p16INK4a are regulated by PKCα. According to what Zang et al. (1995)found, PMA inducingHL-60 differentiateinto macrophage, was probably due to PKCα activation, and the induction of CDK4 inhibitor p21cip1, followed by Rb dephosphorylation, and eventually thecell cycle arrested at G1 and committed cell differentiation.