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摘要

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.

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摘要

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  1. 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 • 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 ( proteinkinase 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 , Rbunderphosphorylation 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 ，P16INK4a)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 etal(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Rbdephosphorylation, 最後造成細胞生長週期的 G1 arrest及細胞分化。

  2. The human promyelocytic leukemia HL-60 cells can be induced todifferentiation toward macrophage by treatment with PMA.However, very little is known regarding the early events thatcontrol differentiation of HL-60 by PMA. In this report, wedemonstrated that PMA (10nM) treatment results in cell cyclearrest in G1 phase. Addition of PMA stimulated PKCαtranslocation in 5 minutes andthe maximum response was seen 30minutes after PMA was added. Upon activation,PKCα is graduallydegraded. The PKCα down regulation was observed within 24hoursafter PMA treatment. The PKCα translocation is associatedwithRb underphosphorylation. Rb underphosphorylation wasapparent at 5 minutes and gradually disappeared within 24 hours,consistent with the time course of PKCα activation. Because Rbphosphorylation can be regulated by D type cyclins andCDK4, wealso examinedthe expression of D type cyclins, CDK4 and CDK4inhibitors(p21cip1, p16INK4a) in HL-60 cells after PMAtreatment. Cyclin D3 but not cyclinD1 or Cyclin D2 was expressed10hrs after PMA addition. However, expression of both CDK4 andCDK4 inhibitors(p21cip1, p16INK4a) were not affected byPMAtreatment. Although we can not directly prove that CDK4, p21cip1 and p16INK4a are regulated by PKCα. According to what Zanget al. (1995)found, PMA inducingHL-60 differentiateintomacrophage, was probably due to PKCα activation, and theinduction of CDK4 inhibitor p21cip1, followed by Rbdephosphorylation, and eventually thecell cycle arrested at G1and committed cell differentiation.

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