不同細胞株在氧化壓力下啟動細胞週期停滯之機轉探討

Mechanisms associated with oxidative stress in triggering cell cycle arrest in various cell lines

林宗德(Tzung-De Lin)

指導教授 ： 鄭鈞文

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00067

摘要

反應性氧化分子 (reactive oxygen species, ROS) 為不穩定分子，具有攻擊其他分子之特性，當細胞處於高氧化壓力的環境時，易導致細胞DNA結構受到破壞，而促使細胞發生變異、老化甚至是細胞的癌化。當細胞曝露於高氧化性物質環境時，導致DNA受到損壞後，會透過腫瘤抑制蛋白的啟動，有效地對細胞週期進行調控，以進行DNA修復等作用。本篇研究目的即是要藉由短暫性活性氧化物質H2O2作用下，觀察不同細胞株在DNA受氧化性傷害後，其調控DNA修復之機轉。我們以HEK 293、MCF-7、HCC1937及HCC1937（BRCA1）轉殖株等四株細胞株，以不同濃度的H2O2對各細胞株作短暫性的處理。藉由Comet assay、Western blotting、Flow cytometry、DNA fragmentation及Enzyme activity assay等實驗方法，加以分析各細胞株受H2O2作用後之細胞生理生化改變。結果發現：腎細胞株HEK 293對於DNA氧化傷害最為敏感；其次為乳癌細胞株MCF-7、HCC1937（BRCA1）最後是HCC1937。從細胞週期分佈和細胞分裂素的表現，得知在氧化物質曝露的環境下，HEK 293細胞株會透過Chk 1、p53和磷酸化p53蛋白的活化，調控Cyclin A及Cyclin B1的蛋白表現，使細胞週期停滯於G2/M時期。相較於HEK 293細胞株，不論p53蛋白表現與否，在乳癌細胞株中（MCF-7、HCC1937和HCC1937（BRCA1）），p21為反應DNA受氧化性傷害之主要活化的腫瘤抑制蛋白，並受H2O2濃度影響而呈現劑量依存（dose-dependant）關係。此外，在細胞週期分析中SubG1也有些微增加之現象，這也意味誘導著細胞凋亡的進行。再者，在細胞生理代謝方面，曝露於氧化性傷害的環境中，皆會加快誘導catalase的活化，這將有利於細胞將氧化性分子加以解毒代謝。本篇論文以H2O2對組織來源及基因型差異之細胞株進行短暫性的氧化性傷害處理，嘗試模擬生物體中，真正受到外生性及內生性之生化代謝過程中，所可能產生ROS對細胞所產生的傷害。說明細胞株之間由於抗氧化物質排除能力之高低，因此，對於氧化性物質傷害的敏感度而有所不同之外；氧化性傷害後啟動腫瘤抑制蛋白活化路徑亦有所差異。本篇研究結果對不同組織受到氧化性傷害與細胞癌化特異性之間提供一重要關鍵的訊息。

關鍵字

反應性氧化分子 ； DNA ； 氧化性傷害

並列摘要

Oxidative damage to DNA via reactive oxygen species（ROS）is considered to play a crucial role in cell aging, tumorigenesis and cancer. Under exposure to environment of high oxidative stress, cells are predisposed to DNA structure damage, cell senescence and tumorigenesis. To prevent oxidative damage, cells therefore, evolve mechanisms involving in the activation of tumor suppress proteins, cell cycle control and DNA damage repair for the maintenance of DNA integrity. However, there are few data discussed about the cellular response under exposure of transient oxidative stress. The aim of this study was to clarify the possible mechanisms against oxidative injuring in different cell lines. Various cell lines, including a kidney cell line HEK 293 and breast cancer cell lines MCF-7, HCC1937 and HCC1937 (BRCA1), were enrolled, and all of them were treated with different concentrations of H2O2. Several techniques, including Comet assay, Flow cytometry, DNA fragmentation, Western blotting and Enzyme activity assay were used to analysis the physiological alternations of these cell lines. After the H2O2 treatment, we found that HEK 293 cells is the most sensitive to oxidative stress, showing of the largest moment tail among these four cell lines. In addition, up-regulated expression of Chk 1, p53 and phosphorylated-p53 proteins will correlated with down-regulation of A- and B1- cyclins in HEK 293 cells, resulting in the cell cycle arrested in G2/M stage.
On the other hand, either p53 positive or p53 negative, p21 were apparently expressed in MCF-7, HCC1937 and HCC1937 (BRCA1) cells. Interestingly, these breast tumor cell lines showed an increasing trend of accumulation of cells in SubG1 phase after to high dosage of H2O2 treatment. Catalase activity will be forced to enhance after exposure to environment of high oxidative stress among these cell lines.
In this study, we established a cell model with the temporal oxidative stress for the mimic of real oxidative stress surrounding that cells met with. We were able to define a real physiological status of cells under exposure to oxidative substance. The susceptibilities to oxidative injury were also ascribed to different genetic profiles of tumor suppressor gene.
Therefore, the molecular mechanisms of cell cycle arrest for the potential benefit in triggering oxidative DNA damage repair were dependent upon the tissue- and genetic specificities. In conclusion, these findings may reassure the mechanisms of tumor suppressor gene against oxidative damage but also provide the functional role of the scavengers and cell cycle regulation in the response of oxidative stress.

並列關鍵字

ROS ； Oxidative damage ； DNA

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