轉錄作用調控DNA 損壞訊息路徑活化之探討

Abstract

第一型去氧核糖核酸拓蹼異構酶(DNA topoisomerase I)目標藥物，喜樹鹼(camptothecin)，為一種針對複製期細胞特性的抗癌藥物。目前對細胞經抗癌藥物camptothecin 處理所引起之細胞反應仍未明瞭。近來研究陸續顯示，在高濃度camptothecin 處理下，對非複製期與不具週期性的細胞造成毒殺作用，而camptothecin 亦能引起與複製無關的細胞反應，例如: 轉錄作用(transcription)引發之topoisomerase I-DNA 可切性複合體(cleavable complex)遭蛋白解體(proteasome)程序降解(degradation)。本篇論文指出，由camptothecin 對細胞所誘發的DNA 損壞訊息(DNA damage signals)，例如: ATM 蛋白的自體磷酸化(autophosphorylation)，細胞週期有關的Chk2蛋白與腫瘤抑制蛋白p53 的磷酸化現象，在處以藥物抑制細胞轉錄作用後(阻礙topoisomerase I-DNA 可切性複合體降解)，能降低蛋白磷酸化程度。此外，利用蛋白解體抑制劑亦能減少由camptothecin 引起DNA 損壞訊息活化程度，達到與細胞轉錄作用抑制劑雷同的效果。綜合上述實驗結果，我們推論: 轉錄作用引發之topoisomerase I-DNA 可切性複合體遭蛋白解體程序降解能有效暴露DNA 斷點(DNA breaks)。本篇論文顯示，RNA 聚合酶(RNA polymerase)與topoisomerase I-DNA 可切性複合物之間的撞擊，與伴隨而來的可切性複合體遭蛋白解體降解，在camptothecin所引起非複製期細胞之細胞反應與毒害效果上，扮演不可或缺的決定性角色。

Camptothecin, a DNA topoisomerase I-targeting drug, has been suggested to be a S-phase specific anticancer drug. However, the cellular responses to camptothecin remain largely unclear. Recent studies indicated that the high concentration of camptothecin could kill the non-S phase and non-cycling cells. In addition, camptothecin also induces the replication-independent cellular responses, such as the transcription- and proteasome-dependent degradation of topoisomerase I-DNA cleavable complexes. In this thesis, we showed that inhibition of RNA transcription, thereby blocking topoisomerase I-DNA cleavable complexes degradation, prevented various camptothecin-induced DNA damage signals, e.g. ATM autophosphorylation, phosphorylation of Chk2 and p53 activation. Proteasome inhibitors also prevented the activation of camptothecin-induced signaling pathways like transcription inhibitors did. Taken together, our results indicate that the transcription- dependent and proteasome-mediated degradation of topoisomerase I-DNA cleavable complexes removes topoisomerase I away from DNA damage and thereby exposes DNA breaks. Our results suggest a novel pathway in which the collision between RNA polymerase and cleavable complexes coupled with subsequent proteasomal degradation of topoisomerase-DNA cleavable complexes play essential roles in determining cellular responses and cytotoxicity of camptothecin in the non-S phase cells.