以過氧化氫誘發細胞老化之分子機制

Abstract

根據細胞所處環境之刺激，可能會有下列三種特殊的樣態呈現在細胞週期進展裡面：細胞生長、細胞死亡以及細胞老化。細胞老化為不可逆且永不再進行細胞複製之現象，因此可與細胞死亡一起作為抑制癌症生長之機制。就如同可以從 p53 dependent 以及 p53 independent 的路徑來誘發細胞死亡，雖可由不同的路徑去誘發細胞老化，但細胞以一整體來反應環境的刺激，因此這些路徑彼此互為交通，最後這些發自細胞質的訊息於核內被整合，而細胞老化亦於該處被執行。 本實驗以 hydrogen peroxide 誘發 WI38 細胞老化，發現以 250μM 的 hydrogen peroxide 會獲得較好的結果，其老化比例為33.9% ；在相同的處理條件下，缺乏 pl6 及 ARF 的 U20S 細胞並無明顯的老化現象。另外，調節細胞週期進展之分子變化方面，p53的量沒有明顯增多，但由 p21 量累積的現象得知 P53 的活性增加； p16 亦逐漸累積，而 pRb 大多是處於低磷酸化的狀態；此外，我們還發現一分子量為 140kDa 的 HDM2 。在 WI38 中，其蛋白質含量隨著誘發後之觀察天數增加而逐漸減少；在 U20S 細胞則無此趨勢，不過與 WI38 相比較，其蛋白質含量卻特別多。以λ－phosphatase 進一步檢視的結果發現該分子為被磷酸化、以sumo－1進行免疫沉澱反應發現其被sumoy－1修飾以及分別以辨識 N 端及 C 端的抗體推測其為 N－terminal truncate 的 HDM2 。此外 ， p140－ HDM2 與pRb有實質交互作用。 細胞老化需要停滯細胞週期進展以及抑制細胞死亡的產生，並且需要 SA heterochromatin foci 的建立，而這些事件都與 pRb 有密切之關聯，既然 P140－HDM2蛋白質含量與老化趨勢相關，且其具有與 pRb 交互作用之能力，因此，或許可以籍此分子一究更深層的細胞老化機制。

Based on the different stimuli, cells could have three major responses. They are cell cycle progression, cell death, and cellular senescence. Cellular senescence is an irreversible and permanent phenomenon of cell cycle arrest. Consequently, cellular senescence as well as apoptosis is recognized as tumor suppressive mechanism. Just like apoptosis induced by p53 dependent and independent pathways, cellular senescence could be elicited from different routes. However, cells react to stimuli coordinately and systematically. Senescent signals from cytosol are integrated in the nucleus and senescence mechanism is executed there. We attempt to induce cellular senescence in WI38 by hydrogen peroxide and find the dose of 250μM to be most effective (33.9%). In contrast, the same treatment could not induce cellular senescence in U20S cells, which lacks p16 and ARF. In addition to morphological phenotype, the amount of p53 is increased slightly. But the increasing activity of p53 is indicated by the accumulation of p21. Besides, the amount of p16 is also accumulated and pRb is hypophosphorylated. Other than 90kDa, we find HDM2 antibody recognized a band of l40kDa size in SDS-PAGE. The p140-HDM2 in WI38 cells decreased day by day after exposure to hydrogen peroxide. Compared with WI38 cells, the protein content of p140-HDM2 was much more plentiful in U20S cells. Revise by λ-Phosphatase, immunoprecipitation with sumo-1 antibody, and immunoblot with anti-N terminal HDM2 antibody suggesting that p140-HDM2 was phophoraylated, sumoylated and N-terminal truncated, respectively. Moreover, it physically interacts with pRb. Cell cycle arrest, inhibition of apoptosis, and construction of SA heterochromatin foci are all required for execution of cellular senescence. Given that p140-HDM2 correlates cellular senescence and could interact with pRb, delving the further mechanism of cellular senescence could be set about from it.