神經膠質瘤是一種非常高度惡性的中樞神經腫瘤，而且放射治療與化學治療都能有效的治療。近幾年的證據指出，phosphoinositide 3-kinase/Akt的訊息傳遞路徑在對抗傳統治療上扮演了一個很重要的因素。在此我們測量利用藥物或者基因調控來抑制PI3K/Akt訊息傳遞，造成了神經膠質瘤細胞的放射敏感性增強。我們發現實驗室的12株神經膠質瘤細胞株，有6株的Akt有過度活化的現象，其中大部分是因為PTEN的去活化(可能是失去作用的突變造成)而不是PIK3CA的增強活性的突變造成。U87和U373兩株神經膠質瘤細胞株都有PTEN的突變伴隨著Akt在ser473上的高度磷酸化，我們利用LY294002來抑制PI3K並且將細胞照射0、2.5、5、7.5四個劑量的放射線來測量放射敏感性。結果顯示LY294002可以抑制神經膠質瘤細胞的Akt活性，細胞的存活率也隨著放射線劑量而有更明顯的降低。我們又利用帶有dominant-negative Akt或PTEN 基因的腺病毒來感染D54MG、U87和U373等神經膠質瘤細胞發現他也會有類似LY294002的結果。接著我們使用PDK1跟mTOR的抑制劑來試驗放射敏感性測試，但結果發現兩種抑制劑都沒有辦法造成神經膠質瘤細胞的放射敏感性增強。
神經膠質瘤的轉移與惡性程度有著極大的關聯。 最近第一型的類胰島素生長因子被發現會經由PI3K/Akt的訊息傳遞路徑調控造成細胞的入侵能力變強。因此我們使用LY294002來看看神經膠質瘤細胞的轉移能力的影響。細胞轉移分成入侵跟平移，我是用Boyden chamber來偵測細胞的入侵能力，利用wound colonization來估計細胞的平移能力。而結果是顯示利用LY294002抑制Akt活化可以造成大部分神經膠質瘤的入侵能力下降。我們也又利用帶有PTEN或dnAkt的腺病毒來證明一次這個結果。其中PTEN腺病毒抑制細胞入侵的效果非常好。後來我們假設神經膠質細胞瘤的轉移與放射敏感性主要是經由PI3K-PDK1-Akt-mTOR這條訊息傳遞路徑造成的，所以我們再來就用PDK1和mTOR的抑制劑來試試看能不能抑制神經膠質瘤細胞的轉移現象。不幸地，這兩種抑制劑抑制腦瘤細胞入侵的效果很有限。總而言之，我們的結果顯示使用基因或藥物來抑制PI3K/Akt都是非常適合來造成腦瘤細胞的放射敏感性增強及減少腦瘤細胞的入侵能力，雖然腦瘤的放射敏感性與入侵能力的調控也許還有其他的訊息調控機制來影響它。

Gliomblastoma is a highly malignant tumor of the central nervous system that is resistant to radiation and chemotherapy. Evidences accumulated over recent years have indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. In this study we determined whether inhibition of PI3K/Akt signal pathway through pharmacological and/or genetic manipulation could enhance radiation sensitivity in glioma cells. Our results showed 6 of 12 glioma cell lines with activated Akt mostly due to reciprocal down-regulation of PTEN activity (loss-of-function mutations) but not by PIK3CA gain-of-function mutations. U87 and U373 glioma cell lines with PTEN mutation showing strong Akt Ser473 phopshorylation were treated with PI3K inhibitor LY294002 and irradiated with 0, 2.5, 5 and 7.5 Gy of radiation dosages. The results showed LY294002 inhibited Akt actvation in the glioma cells and decreased clonogenic survival in a radiation dose-dependent manner. Expression of dominant-negative Akt and PTEN through adenovirus mediated gene delivery in U87 and U373 glioma cells sensitized tumor cells to radiation treatment. Furthermore, PDK1 and mTOR inhibitors were also used on radiation sensitivity test. But both inhibitors had no radiosensitization in glioma cells.
Glioma invasion was linked to advanced tumor stages. Recently, Type 1 insulin-like growth factor regulates tumor invasion have been showed to be mediated through the PI3K/Akt signaling pathway. In this study, we treated glioma cells with LY294002 to analyze its effects on invasion and migration potentials of the tumor cells. The results showed LY294002 inhibited both abilities in most glioma cell lines in vitro. In addition we used adv-PTEN and adv-dnAkt to confirm these results. Adv-PTEN performed dramatic decrease in glioma cell invasion potentials. Furthermore, we investigated whehter PI3K downstream PDK1, and mTOR involved in tumor cell invasion. We used PDK1 and mTOR inhibitors in glioma and determined their effects on invasion by Boyden chamber assay. Unfortunately, both of inhibitors had only limited inhibition on glioma invasion. Take together, our results indicate the feasibility of using PI3K/Akt inhibiting genetic and pharmacological agents to induce glioma cells to become more sensitive to radiation treatment and reduced invasion potentials. However, glioma radiosensitization and invasion may also be regulated by other signaling pathway.

Contents 1
Abbreviations 2
中文摘要 3
Abstract 5
General Introduction
1.1 Backgrounds and Significances 6
1.2 Specific Aims 22
Materials and Methods 24
Results 35
Figures 48
Discussion 86
Future Perspectives 94
References 97
Appendix 117

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