https://scholars.lib.ntu.edu.tw/handle/123456789/163795
標題: | 行政院國家科學委員會專題研究計畫成果報告:細胞凋亡調控基因(sFRP2)在犬乳腺腫瘤之細胞凋亡調控角色及相關調節因子的研究(3/3) | 作者: | 林中天 | 關鍵字: | 分泌性細胞凋亡基因;分泌性 frizzled 蛋白基因;細胞凋亡;訊息傳遞;乳 腺腫瘤;secreted apoptosis related protein secreted frizzled related protein;apoptosis;signal transduction;gene transfection;mammary neoplasia | 公開日期: | 2005 | 出版社: | 臺北市:國立臺灣大學獸醫學系暨研究所 | 摘要: | 乳腺腫瘤對動物與人類皆是重要且常見的 腫瘤,其產生的病因是複雜為多因子牽涉的結 果。Secreted frizzled related proteins (sFRPs)是近年的報告發現與Wnt-Frizzled 訊 息傳遞傳導路徑的調節和細胞凋亡(apoptosis) 的調節中扮演著雙重角色的蛋白質。我們實驗 室最近發現sFRP2 基因在人類與犬隻乳腺腫瘤 中有大量的表現及活化,但是在正常犬乳腺組 織中則沒有表現(前NSC project, 已發表於期 刊Breast Cancer Research and Treatment)。 我們為了有系統地進一步研究犬隻乳腺腫瘤中 的sFRP2 在功能上的角色與腫瘤分子生物學上 的機制,擬定了下列的幾項研究策略。 這個計畫包括了六個主要的部分,需要3 年的研究時間:第一年首先主要的工作在於建 立並分析多種新犬隻乳腺腫瘤組織的初代培養 (primary culture)細胞株並且純化、分析乳腺 上皮細胞。在第一年我們已成功地建立並分析 數個本地病例之犬乳腺腫瘤細胞株。這些細胞 利用下列技術分析其特性,包括增殖速度(by MTT assay)、反轉錄鏈聚合脢反應(RT-PCR)、 原位雜交法( in situhybridization)與免疫化 學染色(immunohistochemistry)及西方墨點法 偵測sFRP2 的表現。結果發現sFRP2 基因之 mRNA 及蛋白質在犬乳腺腫瘤細胞株有大量之 表現,然而在犬正常乳腺細胞及其他非MGT 細 胞株則無表現,第一年的成果已發表刊登於期 刊In vitro cellular and developmental biology-Animal(2003)。在第二階段,犬sFRP2 被轉殖入含有GFP 基因與CMV 啟動子的哺乳類 細胞表現載體, 藉由lipofection 方式將 GFP-sFRP2 穩定地轉染入(transfect)犬隻乳 腺腫瘤初代培養與商品化乳腺腫瘤的細胞株, 在本年度之計畫中進行更進一步的sFRP2 調控 細胞凋亡的功能分析。 在第二年度之研究方面,我們已非常辛苦 地分析確認sFRP2 基因確具有抗細胞凋亡之功 能,且分析複雜的調控細胞凋亡之相關訊息傳 遞途徑為fibronectin-integrin signal transduction pathway,此重要發現為此新基因族之首篇調控 細胞凋亡功能訊息傳遞途徑之新發現,研究成 果也已發表。 在第三年度之研究方面,研究sFRP2 之抗 細胞凋亡與其他訊息因子或轉錄因子之關聯 性。實驗結果發現,表現sFRP2 基因之細胞受 到紫外光誘發細胞凋亡後,FAK 之tyrosine 磷 酸化程度會增加,且有NF-κB 之活性增加及 JNK 之活性抑制的情況。 本研究之結果,預期提供重要之學術資 訊,以了解sFRP 基因族在犬乳腺腫瘤細胞之 細胞凋亡調控之訊息傳遞情形。此外,此計劃 也為未來進一步研究sFRP 基因族不同成員之 各種功能,及了解乳腺腫瘤複雜之病因,提供 進一步研究分析之基礎。 Mammary neoplasms are important and common tumors in both animals and humans and the etiology is complex. The secreted frizzled related proteins (sFRPs) are newly identified proteins and implicated to have dual roles of modulation of Wnt-Frizzled signal transduction pathway and regulation of apoptosis. We have recently found that sFRP2 was expressed abundantly in human and canine mammary gland tumors (MGT) tissues but was undetectable in normal canine mammary gland. To systematically investigate the functional roles and molecular mechanisms of sFRP2 in canine MGT, several strategies are to be carried out as described below. The project is comprised of six major parts for a period of 3 years: In the 1st year, new primary cell cultures from native canine MGT tissues has been established and purified for mammary epithelial cells. We have successfully established and analyzed more native primary MGT cell lines from surgically excised MGT specimens. The cells are characterized for their cell origins, proliferation rate (by MTT assay), expression of sFRP2 by RT-PCR, in situ hybridization, and immunohistochemistry, and Western blotting. Expression experiments revealed the sFRP2 was abundantly expressed in canine MGT cell lines, but not expressed in normal canine MG cells nor other commercial non-MGT cell lines (previous NSC project, published in the Breast Cancer Research and Treatment). Canine sFRP2 is cloned into a mammalian expression vector with GFP reporter gene and CMV promoter. The GFP-sFRP2 is stably transfected into primary canine MGT and commercial MGT cell lines by lipofection for further analysis from the next stage of the project. In the 2nd year, apoptosis regulation mediated by SFRP2 was investigated by overexpression of SFRP2 in MGT and MCF7 cells. DNA fragmentation and caspase 3 activity analyses showed that the susceptibility of the cells to UV-induced apoptosis decreased in the context of SFRP2 overexpression. To analyze the pathways through which SFRP2 transduces anti-apoptosis signals, co-immunoprecipitation and cell adhesion assays were carried out. SFRP2 was found secreted from cells and associated with the fibronectin-integrin protein complex and could promote cell adhesion. Moreover, by using heparin to block the SFRP2-fibronectin interaction or anti-integrin 51 antibody to interrupt the fibronectin-integrin connection, the anti-apoptosis activity of SFRP2 was decreased. Taken together, these results suggest that SFRP2 exert its anti-apoptotic function in mammary cancer cells through association with the fibronectin-integrin signal transduction pathway, not the Wnt signaling as previous thought. The important data has been published. In the 3rd year, analysis of the relation of sFRP2 transduced anti-apoptosis with other signaling and transcription factors, multiple-color immunofluorescence staining, immunoprecipitation, and immunoblotting were carried out. SFRP2 was found co-localized in the extracellular matrix of MGTs and the tyrosine phosphorylation of FAK was enhanced. Moreover, JNK was suppressed and NF-kB was activated in the cells expressing SFRP2 after UV-induced apoptosis analyzed by immunoblotting and electrophoretic mobility shift assay (EMSA). Taken together, these results suggest that SFRP2 exerts its anti-apoptotic function in mammary cancer cells with NF-κB activation or JNK suppression. The results of this project should offer important scientific basis and information to understand the roles of sFRP-mediated signaling in canine mammary tumor cells. It also provides a basis for further analysis of functions of different members of the sFRP gene family and elucidation of the complex etiology and signaling pathways of mammary tumors. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/28735 | 其他識別: | 932313B002022 | Rights: | 國立臺灣大學獸醫學系暨研究所 |
顯示於: | 獸醫學系 |
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932313B002022.pdf | 1.77 MB | Adobe PDF | 檢視/開啟 |
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