||To address the role of ubiquitin-specific peptidase 24 in DNA damage
||Department of Pharmacology
在一般的狀況下，細胞可能會遭遇到許多來自環境的壓力，包含放射線或是氧化壓力，導致DNA產生受損。DNA的受損可能導致細胞凋亡或是癌化的產生，因此細胞內部有許多種類的DNA修復蛋白以及訊息傳遞路徑在進行DNA的修復。然而，許多重要的DNA修復蛋白，例如RAD51也被發現與癌細胞的抗藥性產生有著重要的關聯性。所以研究細胞如何調控DNA修復蛋白的基因表達對於維持基因體穩定性以及避免癌症抗藥性的產生是一個重要的議題。我們實驗室先前的研究發現在肺腺癌細胞株A549當中抑制泛素特異性胜肽酶USP24的基因表達會顯著增加RAD51這個在同源重組修復(homologous recombination repair; HR)當中扮演重要角色的蛋白質的表現。因此本篇研究想要釐清USP24是否可以藉由影響RAD51基因表達進而影響同源重組修復。我們發現在A549細胞當中抑制USP24的表達會促進RAD51的基因表達，然而在H1299細胞當中則無。經過文獻搜尋這兩細胞株的基因背景差異，我們懷疑抑癌基因p53有可能參與在USP24調控RAD51的訊息傳遞路徑當中。但是實驗結果顯示p53並沒有參與在USP24調控RAD51的訊息傳遞路徑當中。所以我們使用不同片段的RAD51啟動子透過報導基因測試(reporter assay)想要藉此找出USP24所影響的轉錄因子(transcription factor)可能結合在RAD51的啟動子上的位點。我們發現RAD51的啟動子上有一E2F轉錄因子家族結合的位點對於USP24調控RAD51基因表達扮演重要的角色。接著我們探討USP24是否可以透過調控RAD51的基因表達進而影響同源重組修復的能力與影響癌細胞抗藥性的產生。我們發現USP24會降低RAD51在DNA斷裂處形成foci的能力。同源重組修復試驗也發現USP24可以透過影響RAD51的表達進而降低同源重組修復的能力。最終，我們探討USP24是否可以藉由影響RAD51基因表達而影響癌細胞的抗藥性產生。透過細胞週期及細胞凋亡比例的分析與群落形成能力試驗(colony formation assay)，我們證實USP24可以藉由調控RAD51基因而增加癌細胞的抗藥性。總結我們的實驗結果發現，USP24可以藉由E2F家族的轉錄因子去調控RAD51的基因表達，更進一步影響同源重組修復的能力以及癌細胞抗藥性的產生，希望這個新發現能對於癌症治療產生新的策略。
Recent studies have found many deubiquitinating enzymes play an important role in
cancer progression. Previously, we found mRNA expression of RAD51, an important
homologous recombination factor, is dramatically increased in ubiquitin-specific peptidase
24 (USP24) knockdown A549 cells. DNA damage may cause nucleotide exchange and
result in genetic mutation or genomic instability. In addition, DNA repair ability is also
important for cancer cell drug resistance; therefore, understanding the molecular
mechanism by which DNA repair is regulated may provide us an insightful view of cellular
response to DNA damage. We found RAD51 gene expression is upregulated in USP24
knockdown A549 cells but not H1299 cells. Previous study demonstrated that p53 acts as a
repressor of RAD51 gene; interestingly, genotype of p53 in A549 and H1299 are wild-type
and null, respectively. Thus we confirmed the role of p53 in USP24 and RAD51 pathway
using overexpression and knockdown strategy in H1299 and A549 cells. However, the
result indicated p53 is not involved in the regulation of RAD51 by USP24. Next, we used
different truncated RAD51 promoter to confirm the transcription factors which are
regulated by USP24. Our data suggested E2F4 may be a critical regulator of
USP24-RAD51 pathway. We also examined the homologous recombination repair activity
and cell survival in USP24 knockdown cells treated with DNA damage drug camptothecin
(CPT). Our data indicated homologous recombination repair activity and drug resistance
are both increased in USP24 knockdown cells. In conclusion, we found USP24 inhibits
RAD51 gene expression via E2F4 to affect the homologous recombination repair activity
and cancer cell drug resistance.
When cells suffered from environmental stresses, such as UV irradiation and some chemical reagents, or from internal stress such as faults in DNA replication, DNA damage occurs. DNA damage may cause nucleotide exchange and result in genetic mutation or genomic instability. DNA damage may lead to apoptosis or tumorigenesis; therefore, understanding the underlying mechanism by which DNA repair is regulated may provide us an insightful view of cellular response to DNA damage.
Ubiquitin-Specific Peptidase 24 (USP24), a novel and less studied ubiquitin specific peptidase, can regulate protein stability by modifying protein ubiquitination. Recently, we found that RAD51 mRNA and protein level were elevated in USP24 knockdown lung adenocarcinoma A549 cell line.
RAD51 is an important protein for homologous recombination that accumulates at damage site of DNA and helps strand invasion during DNA repair. However, RAD51 also plays an important role in the development of drug resistance in a number of cancer types like colorectal cancer and non-small cell lung cancer. As described above, how RAD51 gene expression is controlled appropriately is an important issue in cancer biology.
In this study, we found RAD51 gene expression is upregulated in USP24 knockdown A549 cells but not H1299 cells. Previous study demonstrated that p53 acts as a repressor of RAD51 gene; interestingly, genotype of p53 in A549 and H1299 are wild-type and null, respectively. Thus, we investigated the role of p53 in this process. We found ubiqutinated p53 protein is increased in USP24 knockdown A549 cells and RAD51 protein level is similar in both USP24 and p53 knockdown cells. Also, we confirmed p53 overexpression could not rescue RAD51 protein level in USP24 knockdown H1299 cells. These data indicated that p53 is not involved in USP24-regulated RAD51 gene expression. Next, we used different truncated RAD51 promoter to search the transcription factors regulated by USP24. Our data suggested that E2F4 may be a critical regulator in the USP24-RAD51 pathway. We also examined the homologous recombination repair activity and cell survival in USP24 knockdown cells treated with DNA damage drug camptothecin (CPT). These data indicated homologous recombination repair activity and drug resistance are both increased in USP24 knockdown cells. In conclusion, we suggested that USP24 inhibits RAD51 gene expression via E2F4 to affect the homologous recombination repair activity and cancer cell drug resistance.
MATERIALS AND METHODS
All experiments about USP24 knockdown cells were done by infection of cells by lentivirus containing USP24 shRNA plasmid for 24 h and the medium were changed to fresh medium, and cells were harvested after 72 h. RAD51 gene expression was analyzed by reporter assay, RT-PCR, and Western blot analysis. RAD51 foci formation was stained by immunofluorescent staining and pictured by fluorescence microscopy. Cell cycle and annexin V- propidium iodide staining were analyzed by flow cytometry. Cell survival-related functional assays were carried out after treatment with CPT for 24 h.
RESULTS AND DISCUSSION
Rad51 gene expression was increased in USP24 knockdown A549 cells. Previous study revealed that p53 represses Rad51 gene expression in breast cancer, so p53 may play a role in USP24-mediated down-regulation of RAD51 expression. However, our results revealed that overexpression GFP-p53 in H1299 cells did not change the USP24-regulated RAD51 gene expression. Also, p53 protein level was not changed in USP24-knockdown A549 cells. In addition, we found that knockdown of p53 can reduce Rad51 gene expression, but cannot abolish the up-regulation of Rad51 by USP24 knockdown, suggesting that p53 positively regulates Rad51 expression, but is not involved in USP24-regulated Rad51 expression. Next, we used different truncated RAD51 promoter to search the transcription factors regulated by USP24. Our data indicated E2F may be a critical regulator in USP24-RAD51 pathway. Rad51 foci formation was increased in USP24 knockdown U2OS cells under CPT treatment when compared to that of control cells. The effect of USP24 in homologous recombination (HR) activity was studied by HR assay and our result indicated that USP24 knockdown can increase DNA damage repair activity through increasing Rad51 expression. We also checked apoptotic cell death in control and USP24 knockdown cells treated with CPT. We were found USP24 knockdown cells were more resistant to DNA damage drug. Finally, cell survival was increased in USP24 knockdown U2OS cells treated with CPT. These data indicated that USP24 regulates HR activity through modulating RAD51 gene expression. We also interested in how USP24 regulates RAD51 gene expression. So, we investigated Rad51 promoter activity by using different truncated forms of RAD51 promoter and identified several potential transcription factor binding sites within human RAD51 promoter. The involvement of USP24-regulated transcription factors such as E2F4 will be clarified by gene knockdown via shRNA.
In conclusion, in this study we found USP24 regulates RAD51 gene expression by affecting E2F, and this signaling pathway is important for homologous recombination activity and cancer cell drug resistance. This study uncovers the role of USP24 in DNA damage repair activity via modulation of RAD51 expression, which might be important for the regulation of genomic stability and tumorigenesis.
簡寫表......... . .Xl
第一節 泛素修飾. . ..1
第二節 去泛素酶...... . . . 2
第五節 研究動機....... .. . . . . . . . . . . 8
第一節 在肺癌細胞株A549 細胞中抑制USP24 會促進RAD51 基因
表達，然而在H1299 細胞當中則無作用.. . . . . . . . . . . . . .27
第二節 抑癌基因p53 並沒有參與在USP24 調控RAD51 基因表達的
訊息傳遞路徑. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 8
第三節 利用RAD51 啟動子來尋找USP24 所調控之轉錄因子結合位進而找出USP24 可能調控之轉錄因子...30
第四節USP24 藉由影響RAD51 基因表達進而影響同源重組修復........ . . . . 3 1
第一節USP24 對於抑癌基因p53 在肺癌當中的影響 ...34
第二節 USP24 可能藉由影響其他轉錄因子進而影響同源重組修復中的重要蛋白或是直接與同源重組修復中的重要蛋白有交互作用而影響同源重組修復之能力. . . . 3 5
第三節USP24 可能藉由影響E2F4 進而影響RAD51 基因表達與肺癌發展...... .36
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