週期蛋白激酶(Cdk2)與腫瘤抑制基因(p53)在紫外線照射中國倉鼠卵巢細胞所誘發細胞凋亡之角色

Roles of Cdk2 and p53 in UV-induced apoptosis of Chinese hamster ovary cells

廖助彬(Chu-Bin Liao)

指導教授 ： 劉銀樟

國立清華大學/生命科學院/生命科學系/博士(2006年)

摘要

當細胞遭受基因傷害時，會啟動檢查控制機制，此機制可透過ATR/ATM- p53-p21(Waf1/Cip1) 訊息傳遞路徑來達成，進而保護基因的完整性。而中國倉鼠卵巢細胞中因缺乏p21蛋白質的表現，故當細胞遭受紫外線傷害時，無法將細胞停滯於G1期中。進一步的研究中發現，紫外線照射過的細胞於24小時後會出現大量的細胞凋亡，且其週期蛋白激酶(Cdk2)在紫外線照射後，活性明顯的上升，而且此活性的增加與細胞凋亡的情形是具有相關性的。此外，過度活化的Cdk2會造成Rb蛋白質的過度磷酸化，進而活化E2F蛋白。而紫外線造成的細胞凋亡也會被Cdk2的抑制劑－roscovitine所抑制。另一方面，在進行紫外線照射實驗時給予不同的抗氧化物，發現抗氧化物並無法減低紫外線造成的細胞凋亡。因此我們推論活性氧分子似乎並不參與紫外線造成的細胞凋亡。但是在進行pyrrolidine dithiocarbamate(PDTC)這類的抗氧化物質處理中，我卻發現到PDTC可以透過抑制Cdk2活性，來抑制細胞的生長與減少細胞的凋亡。因此我假設不正常活化的Cdk2會使細胞藉由Rb/E2F活化路徑，走向細胞凋亡。實驗結果中我發現，p53雖然因為紫外線照射而大量產生，但是其下游因子－Bax蛋白卻沒有增加。緊接著利用siRNA減少內源性 p53 的表現，其結果並無法阻止紫外線所造成的細胞凋亡。而實際上我卻發現到p53於中國倉鼠卵巢細胞的核酸修復中，扮演著一個相當重要的角色。總而言之，於此研究的結果中我推論出，不正常活化的Cdk2會使得細胞藉由Rb/E2F路徑，但非p53，以促使細胞進入紫外線所造成的細胞凋亡。另一方面，我更證明出p53直接參與核酸的修補。

關鍵字

中國倉鼠卵巢細胞 ； 紫外線 ； 細胞凋亡 ； 週期蛋白激酶 ； 腫瘤抑制基因

並列摘要

DNA damage checkpoint control which involves the pathway of ATR/ATM- p53-p21(Waf1/Cip1) provides a mechanism to protect the genomic integrity. Chinese hamster ovary cells (CHO-K1) which cannot express p21 fail to arrest at G1 phase following UVC irradiation. The UV-irradiated CHO-K1 cells exhibit substantial apoptosis at 24 h after irradiation. Activities of cyclin-dependent kinases especially, Cdk2 are found elevated following the irradiation. The elevation of Cdk2 activity correlates with the increase of UV-induced apoptosis. Further, the increase in Cdk2 activity causes hyperphosphorylation of retinoblastoma (Rb) protein, which consequently activates E2F protein. Moreover, the UV-induced apoptosis was suppressed by roscovitine, a small-molecule Cdk2 inhibitor. On the other hand, the reactive oxygen species (ROS) seems not to be involved in UVC-induced apoptosis, which was proved by pretreatment with different kinds of antioxidant except pyrrolidine dithiocarbamate (PDTC). The PDTC reduces cell proliferation, and its inhibition of UV-induced apoptosis is likely to due to the inhibition of Cdk2 activity. Thus, we hypothesize that deregulation of Cdk2 may lead to apoptosis via the Rb/E2F-mediated pathway. Besides, p53 is induced in UV-irradiated CHO-K1 cells, but the expression of pro-apoptotic Bax protein, a downstream effector of p53, is not elevated. Further study indicates decrease of endogenous p53 by p53 specific siRNA cannot suppress UV-induced apoptosis. In fact, p53 plays an important role in excision of UV-induced DNA adducts. Taken together, my study demonstrates that deregulation of Cdk2 via Rb/E2F and p53-independent pathway induces apoptosis in UV-irradiated cells. Furthermore, p53 is involved in early stage of nucleotide excision repair.

並列關鍵字

CHO-K1 cells ； ultraviolet ； apoptosis ； Cdk2 ； p53

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