臺灣博碩士論文加值系統

一第一部份：以基因微陣研究PTEN調控之基因表現型態
中文摘要
Pten 是一腫瘤抑制基因，PTEN之氨基酸序列與酪胺酸磷酸及細胞骨架蛋白質tensin 和auxilin具同質性(homology)。PTEN已被証明能抑制細胞的移動(migration)，擴散(spreading)和focal adhesions的形成，本論文證明 PTEN在肺腺癌細胞株扮演抑制癌細胞入侵(invasion)的角色，也進一步檢查其調控那些下游的基因。我們先前己經建立一系列不同入侵和轉移能力的腺癌細胞株，檢查這些腺癌細胞的pten 基因發現在高轉移能力細胞株(CL1-5)其pten在exon5有缺陷(deletion)，我們將原生型pten轉移感染(transfection)腺癌細胞株CL1-5，發現過度表現PTEN蛋白質在CL1-5細胞中會抑制其入侵的能力。我們進一步以高密度微陣列(microarray)的方法研究在過量表現PTEN的腺癌細胞株與無PTEN表現的腺癌細胞株基因表現的不同，發現 integrin 6, laminin 3, heparin-binding EGF-like growth factor, urokinase type plasminogen activator, myb protein B, Akt2 及一些EST(expressed sequence tag)等基因受pten向下調節(downregulation)。相反的，受pten向上調節(upregulation)的基因有protein phosphatase 2A1B, ubiquitin protease (unph), secreted phosphoprotein 1, leukocyte elastase inhibitor, NFKB, cAMP response element binding protein (CREB), DNA ligase 1, heat shock protein 90, 及一些EST的基因。我們更進一步的以北式墨點雜交法(northern hybridization)分析及流式細胞儀(flow cytometry)偵測PTEN 蛋白質的表現確實會抑制integrin 6 表現。我們的結果顯示過度表現的PTEN蛋白質抑制肺腺癌細胞株的入侵，可能經由調節下游的基因如integrin 6 表現。以基因微陣研究PTEN蛋白質調控之基因表現型態是很有效的工具。
第二部份：p53的339∼346氨基酸是最小的抑制功能區
中文摘要
P53是腫瘤抑制蛋白質，它可以作為某些基因轉錄的活化因子也可以是其他基因轉錄的抑制因子，目前對於抑制基因轉錄的機制並不是很清楚，為了探索它的機制，我們利用嵌合蛋白質的方法，來尋找出可抑制基因轉錄的最小胜，我們發現P53蛋白質在C-端的8個胺基酸（339-346）是最小的轉錄抑制胜，我們命名為P53MRD，這個P53 MRD正好也落在E6蛋白質結合區，然由於8個胺基酸只含有部份α-helix 故無法形成寡聚合蛋白質，進而可推知抑制基因轉錄與蛋白質聚合無關，P53MRD只抑制P53的促進基因轉錄的活性，無法抑制VP16轉錄活性，所以此抑制作用是P53 specific 。在原型P53蛋白質上笫340,341胺基酸作變異(mutant)發現其喪失對TK-CAT的抑制活性。由以上結果証明8個胺基酸是目前最小的轉錄抑制因子。

1.Profiling the Down Stream Genes of Tumor Suppressor PTEN in Lung Cancer Cells by cDNA Microarray
Abstract
PTEN is a tumor suppressor gene with sequence homology to tyrosine phosphatases and the cytoskeletal proteins, tension and auxilin. PTEN has been recently shown to inhibit cell migration, and the spreading and formation of focal adhesions. This study investigated the role of PTEN in carcinoma invasion in a lung cancer cell line and examined the down stream genes regulated by PTEN. We have previously established a cell line model in human lung adenocarcinoma with different invasive abilities and metastatic potentials. Examining the PTEN gene expression in these cell lines, we found a homozygous deletion in exon 5 is associated with high invasive ability. We then constructed stable constitutive and inducible wild-type PTEN overexpressed transfectants in highly invasive cell line CL1-5. We found that an overexpression of PTEN can inhibit invasion in lung cancer cells. To further explore the down stream genes regulated by PTEN, a high density cDNA microarray technique was used to profile gene changes after PTEN overexpression. Our results indicate that a panel of genes which can be modulated by PTEN. PTEN overexpression down regulated genes including integrin 6, laminin 3, heparin-binding EGF-like growth factor, urokinase type plasminogen activator, myb protein B, Akt2 and some EST clones. In contrast, PTEN overexpression up regulated protein phosphatase 2A1B, ubiquitin protease (unph), secreted phosphoprotein 1, leukocyte elastase inhibitor, NFKB, cAMP response element binding protein (CREB), DNA ligase 1, heat shock protein 90, and some EST genes. Northern hybridization and flow cytometry analysis also confirmed that PTEN overexpression results in the reduced expression of the integrin 6 subunit. The results of this study indicate that PTEN overexpression may inhibit lung cancer invasion by down regulation of a panel of genes including integrin 6. The cDNA microarray technique may be an effective tool to study the down stream function of a tumor suppressor gene.
2.p53 amino acids 339-346 represent the minimal p53 repression domain
Abstract
The p53 tumor suppressor protein functions as an activator and also as a repressor of gene transcription. Currently, the mechanism of transcriptional repression by p53 remains poorly understood. To help clarify this mechanism, we carried out studies designed to identify the minimal repression domain that inhibits p53 transcriptional activities. We found only eight amino acids (339-346) of the C-terminal domain (termed P53MRD) that possess activities of repression. The exact location of this minimal domain is on the E6 binding region, and it lacks the ability of tetramerization. P53MRD is able to repress the transcription of p53, while not affecting VP16. Mutants (amino acids M340P and F341D) of native p53 also lost transcriptional repression of thymidine kinase chloramphenicol acetyltransferase (TKCAT) promoter. These results suggest that this eight-amino acid element is required for the repression of p53.

目錄 (Content)
第一部份：以基因微陣研究PTEN調控之基因表現型態
中文摘要 ­­­­­­­­­­­­­­­­­­­­­­­­1
中文緒論 ­­­­­­­­­­­­­­­­­­­­­­­­2
Abstract­­­­­­­­­­­­­­­­­­­­­­­­ 3
Introduction­­­­­­­­­­­­­­­­­­­­­­­­5
Materials and Methods­­­­­­­­­­­­­­7
Results­­­­­­­­­­­­­­­­­­­­­­­­ 15
Discussion­­­­­­­­­­­­­­­­­­­­­­­­ 20
References­­­­­­­­­­­­­­­­­­­­­­­­ 27
Figure­­­­­­­­­­­­­­­­­­­­­­­­ 34
Table­­­­­­­­­­­­­­­­­­­­­­­­ 47
第二部份：p53的339∼346氨基酸是最小的抑制功能區
中文摘要 ­­­­­­­­­­­­­­­­­­­­­­­­49
中文緒論 ­­­­­­­­­­­­­­­­­­­­­­­­50
Abstract­­­­­­­­­­­­­­­­­­­­­­­­ 52
Introduction­­­­­­­­­­­­­­­­­­­­­­­­53
Materials and Methods­­­­­­­­­­­­­­55
Results­­­­­­­­­­­­­­­­­­­­­­­­ 57
Discussion­­­­­­­­­­­­­­­­­­­­­­­­61
References­­­­­­­­­­­­­­­­­­­­­­­­66
Footnotes­­­­­­­­­­­­­­­­­­­­­­­­73
Figure­­­­­­­­­­­­­­­­­­­­­­­­ 74

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