Bortezomib誘導人類Burkitt淋巴癌細胞異質性核蛋白K 之SUMOylation分子機制探討

Bortezomib是一種蛋白酶體 (proteasome) 抑制劑，其對於許多腫瘤均具有毒殺能力；在2003年5月美國食品暨藥物管理局核淮此藥用於復發性的多發性骨髓瘤的治療。本實驗以具有c-Myc 基因高度表現的 Burkitt’s lymphoma細胞株－Daudi做為研究模型。異質性核蛋白 (heterogeneous nuclear ribonucleoprotein, hnRNP) 是與核醣核酸相結合的蛋白質，而hnRNP K為hnRNP 家族的成員，參與許多生物功能，包括調控轉錄以及轉譯。hnRNP K 是 ubiquitin E3 連接酶－HDM2/MDM2的一個目標蛋白，可被proteasome分解。先前本實驗室以蛋白質體學分析，Bortezomib作用在Daudi細胞會降低分子量約為 65 kDa的hnRNP K表現量，卻增加50 kDa的表現，而未經轉譯後修飾的 hnRNP K，預估分子量為50 kDa。研究顯示 hnRNP K 可進行多種轉譯後修飾，例如SUMOylation。本實驗探討 Bortezomib對 hnRNP K SUMOylation以及c-Myc 表現的調控，進而影響腫瘤細胞生長的機制。結果發現，Bortezomib可抑制Daudi細胞的增生。從西方墨點法可觀察到此藥物降低hnRNP K在65 kDa的表現量，而50 kDa的表現則明顯增加；Bortezomib也同時發現可以降低 pan-SUMO於65 kDa的表現量，兩者兼具時間及藥物濃度的相依性。透過免液沉澱法以及暫時性基因轉殖技術發現hnRNP K於65 kDa的表現是發生SUMOylation，由此推測Bortezomib會導致hnRNP K SUMOylation下降。在營養缺乏的環境下，我們發現 hnRNP K SUMOylation 以及c-Myc蛋白質的表現量均降低，因此推測SUMOylated-hnRNP K會藉由調控c-Myc影響細胞增生。Bortezomib會降低c-Myc蛋白質，卻不影響mRNA。此外，hnRNP K可藉由c-Myc mRNA 5’端上IRES結構結合，進而正向調控c-Myc的轉譯；從轉殖 pGL3-myc-5’UTR 帶有IRES序列的冷光分析中，除了發現Bortezomib可降低冷光值之外，在共同轉殖hnRNP K和SUMO 1時，冷光值也明顯上升。我們還發現hnRNP K上Lys422可能為SUMO的主要修飾位置。由此得知，在Burkkit’s lymphoma細胞株-Daudi中，Bortezomib藉由降低hnRNP K SUMOylation，進而以轉錄後階層減弱c-Myc的轉譯，抑制細胞的生長。

關鍵字

Bortezomib ；異質性核蛋白K ； SUMOylation ； c-Myc

並列摘要

Bortezomib is a proteasome inhibitor. The U.S. FDA recently approved the first proteasome inhibitor Bortezomib for the treatment of refractory multiple myeloma. In this study, we use Burkitt’s lymphoma cell line, Daudi, as a model, characterized by deregulation of c-Myc gene. Heterogeneous nuclear ribonucleoprotein K (hnRNPK), a member of the hnRNP family, exhibits a high-affinity, sequence-specific interaction with RNA. hnRNP K has been linked to a variety of cellular processes, including the regulation of transcription and translation. Induction of hnRNP K ensues through the inhibition of its ubiquitin-dependent proteasomal degradation mediated by the ubiquitin E3 ligase HDM2/MDM2. Our recent studies have shown that Bortezomib downregulated the expression of hnRNP K in 65 kDa, but increased in 50 KDa by proteomic techniques. The molecular weight of non-modification hnRNP K might be 50 kDa. Previous reports have shown that hnRNP K has various post-translational modifications, including SUMOylation. In this study, we investigate the molecular mechanism of Bortezomib on the regulation of hnRNP K SUMOylation. Here we report Bortezomib inhibited cell proliferastion by MTT assay. Western blot analysis revealed that Bortezomib decreased hnRNP K protein in 65 kDa, but elevated in 50 kDa. Bortezomib also declined pan-SUMO protein in 65 kDa. Furthermore, not only SUMO 1, but also hnRNP K was increased on the band of 65 kDa in SUMO 1 overexpression cells, suggested that hnRNP K occurs SUMOylation in Daudi cells. Immunoprecipitation and transfection experiments established that Bortezomib declined SUMOylated hnRNP K. The level of SUMOylated hnRNP K and c-Myc was lower in starvation Daudi cells, suggested this modification has an effect on cell proliferation. Additionally, Bortezomib treatment and luciferase assay revealed that lower SUMOylated hnRNP K reduced c-Myc on post-transcriptional level through c-Myc mRNA-5’UTR-IRES. We also found Lys422 may be the sumoylation site of hnRNP K. These data support the downregulation of SUMOylated hnRNP K by Bortezomib may play a role of cell growth through reducing c-Myc in Burkitt’s lymphoma.

並列關鍵字

Bortezomib ； hnRNP K ； SUMOylation ； c-Myc

參考文獻

1. Bellan C, Stefano L, Giulia DF, Rogena EA, Lorenzo L: Burkitt lymphoma versus diffuse large B-cell lymphoma: a practical approach. Hematol Oncol 2009.

2. Thomas RK, Re D, Wolf J, Diehl V: Part I: Hodgkin's lymphoma--molecular biology of Hodgkin and Reed-Sternberg cells. Lancet Oncol 2004, 5:11-18.

3. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML, Delsol G, De Wolf-Peeters C, Falini B, Gatter KC, et al.: A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 1994, 84:1361-1392.

4. Cheson BD: What is new in lymphoma? CA Cancer J Clin 2004, 54:260-272.

5. Burkitt D: A sarcoma involving the jaws in African children. Br J Surg 1958, 46:218-223.

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Bortezomib誘導人類Burkitt淋巴癌細胞異質性核蛋白K 之SUMOylation分子機制探討

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