BCAS2的雙重功能之研究：為抑癌基因p53的新穎負調控者及核接受體的共同轉錄因子

The Dual Roles of BCAS2 as a Novel Negative Regulator of the p53 Tumor Suppressor and a Transcription Cofactor of Nuclear Receptor

張紘瑋(Hung-Wei Chang)

指導教授 ： 陳小梨

國立臺灣大學/醫學院/微生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.10194

摘要

一個新穎的細胞核蛋白BCAS2（breast cancer amplified sequence 2），本實驗室利用雄性激素受體（androgen receptor, AR）當成餌，於人類HeLa MATCHMAKER cDNA基因庫中，利用yeast-two hybrid選殖系統所鑑定出來；目前的文獻顯示，BCAS2基因座落在第一號染色體1p13.3-21區域，並且在部分乳癌細胞株及檢體中具有基因增殖（gene amplification）的現象，並且BCAS2可增強雌激素受體（estrogen receptor, ER）所誘導的基因表現及可能參與在前細胞凋亡的過程中。
在本研究中，為了分析及鑑定BCAS2蛋白質的功能，使用了GST-Pull down的技術，藉由與MCF-7核萃取物共同作用，我們實驗室首次發現BCAS2與抑癌基因p53於細胞核中具有交互作用；此外，利用transiently transfection大量表現BCAS2，則會抑制p53的活性且使得p53下游基因p21表現量減少；相反的，利用RNAi技術均可knock down不論是內生或外送的BCAS2，並導致p21和BAX基因表現量上升，並更進一步減緩MCF-7及LNCaP細胞（野生型 p53）的細胞生長並增加死亡的比例，但在不具有p53的細胞株H1299中則無此效果；此外在MCF-7細胞中，處理DNA damage藥物adriamycin活化p53所引起的現象，可藉由BCAS2大量表現加以回復；因此經由以上結果推測，BCAS2可能當成p53的負調控者的角色。
此外，BCAS2曾被報導在ligand存在下，可增加雌激素受體媒介的基因轉錄作用；而在我們的研究中，BCAS2亦可與AR相互作用並增強其媒介的基因轉錄作用，且增加攝護腺癌中生化標記的PSA（prostate specific antigen）表現量，因此證明在prostate cancer細胞中，BCAS2亦可當成AR的一個轉錄輔因子，增強AR媒介的基因轉錄作用。在此研究中，我們目前已鑑定出BCAS2的雙重功能：一為核接受體的coactivator，另一個角色則為p53的負調控者。

關鍵字

p53負調控者 ； BCAS2 ； 雄激素受體 ； 細胞凋亡 ； p21

並列摘要

A nuclear protein which is encoded by a novel gene, breast cancer amplified sequence 2 (BCAS2), has been identified by applying the androgen receptor (AR) as a bait to prey a human HeLa MATCHMAKER cDNA library in a yeast-two hybrid screening system. Recent reports from the other groups show that BCAS2 is mapped on the chromosome 1p13.3-21 region, and also can enhance estrogen receptor (ER)-mediated transcription activity and regulate pro-apoptotic activity.
In this study, for characterization and identification of the functions of BCAS2, the GST pull-down assay was conducted to analyze the BCAS2 interacting proteins from nuclear extracts. Here, we first demonstrated that BCAS2 can directly interact with p53 in nucleus by in vitro GST pull-down assay, and the reciprocal IP-Western. Additionally, BCAS2 overexpression can lead to repress the activities of p53 by p21 promoter reporter assay, and can block p53-inducible p21 protein expression when treated with doxorubicin. When silencing of BCAS2 by siRNA, it can enhance the p53 transcriptional activities resulting in the increase of p21 and BAX protein expression, and also can cause the decrease of cell growth and increase cell death both in MCF-7 and LNCaP cells those containing wild type p53. It suggests that BCAS2 may play a role as a negative regulator of p53.

On the other hand, in our investigation BCAS2 also can bind to androgen receptor and can enhance AR-driven prostate specific antigen (PSA) promoter activity which is a biomarker to monitor prostate cancer. The reporter assay and RT-PCR results revealed that the decrease of endogenous BCAS2 expression caused by siBCAS2 decreased the AR-mediated PSA promoter activity and PSA mRNA expression in LNCaP cells. Additionally AR and BCAS2 can co-localize on the AR response elements within PSA promoter by the chromatin IP assay. BCAS2 is also as a transcription cofactor to enhance AR-mediated gene expression in prostate cancer cells. In this study, we demonstrate that BCAS2 has dual roles: one is a coactivator of the nuclear receptor and the other the inhibitor of p53.

並列關鍵字

p53 negative regulator ； BCAS2 ； androgen receptor ； apoptosis ； p21 ； transcription cofactor

參考文獻

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