神經膠母細胞瘤是一種最具侵略性以及能夠抵抗放射線的腦部腫瘤。在先期的研究中證實，膠細胞的腫瘤在放射線治療過後會變形成為一種肉瘤的型態，而最近的文獻也指出在幹細胞及對放射線有抵抗性的細胞兩者之間的差異，另外也提到對放射線有敏感性的細胞確實能產生凋亡的情形而抑制腫瘤的生長。而那些能夠抵抗放射線的傷害而繼續生長的腫瘤細胞，除了可能具備原始腫瘤細胞型態的特性之外，也可能生長成為不同細胞形態的另一種腫瘤。我們利用神經膠母細胞瘤的細胞株經過連續性的放射線照射來檢視在放射線處理後，因為放射線而導致受損或死亡的細胞與控制組之間的特性差異。我們使用TUNEL及流式細胞技術的方法去探討在放射線處理後，對細胞凋亡及細胞週期所產生的變化；西方墨點法去檢視對放射性無敏感性的細胞中，屬於中間絲蛋白的腦幹細胞標誌及上皮-間葉過渡(epithelial-mesenchymal transition)的改變，包含它的分化以及生長情形；免疫細胞螢光染色的方法，藉由型態上去觀察在放射線處理後，特定蛋白的表現及其所在位置。這些結果顯示放射線照射過後的神經膠母細胞瘤細胞株中，Vimentin蛋白的表現與細胞的死亡率呈現顯著的負相關性，特別是在CNS-1這株神經膠母細胞瘤上可以見到Vimentin蛋白在放射線照射後會表現在細胞核當中，這與控制組有明顯的差異。另外，在CNS-1在放射線過後也會表現desmin與myogenin間葉組織的蛋白標誌以及形成三度空間的細胞聚集。放射線照射同時影響p53, Cdk-2, Cdk-4 這些調控細胞週期蛋白的表現。分析這些樣蛋白表現的相關性顯示p53對Cdk-2的調控可能是經由p21。 E-cadherin蛋白的表現在CNS-1以及13-06MG細胞株的控制組與實驗組沒有差異，但Vimentin與Fibronectin蛋白的表現卻有不同。此結果顯示有些經過放射線照射存活的細胞可能有上皮-間葉過渡 (EMT)的發生。這個結果帶給我們一個啟示，即經由研究放射線處理後所產生的放射線誘導的腫瘤，或許能幫助我們瞭解腫瘤的進展、變形以及轉移的機轉。

Glioblastoma Multiform (GBM) is one of the most devastating and radio-resistant brain cancers. The median survival in patients with glioblastoma is approximately 1 year after surgery and adjuvant chemo and/or radiotherapy. Previous studies indicated that post-irradiated gliomas can transform into certain types of sarcomas. Recent literatures had addressed the differences between stem cells and radio-resistant cells in GBMs, and others have suggested that induction of apoptosis in radio-sensitive cancer cells inhibits tumor growth. Radio-resistant cancer cells that grow to become tumors show features similar to the original tumor or present as a completely different tumor with different cell types and biological behaviors. Here, we sequentially irradiated rat and human GBM cell lines to examine cell injury and manner of cell death in pre- and post-radiated cells. We have used 1) TUNEL and flowcytometry methods to study the effects of radiation on cell apoptosis and cell cycle; 2) western blotting to exam brain stem cell marker of intermediate filament proteins and proteins involved in epithelial mesenchymal transition (EMT) in post-irradiated radio-insensitive cells, and 3) immunocytochemistry (ICC) to morphologically observe the protein expression and localization in post-irradiated GBM cells. The results showed that radiation induces apoptotic cell death, and the mortality rate on post-irradiated GBM cells and their vimentin expression (an intermediate filament stem cell protein marker) have a negative correlation. Interestingly, vimentin not only expressed in the cytoplasm(s) of post-irradiated cells of CNS-1 GBM cell line but also formed aggregates on the nuclei. Post-irradiated CNS-1 cell lines also formed three-dimensional cell clusters and expressed desmin and myogenin, which are commonly seen in cells with mesenchymal features. The administered radiation affected the expression of p53, Cdk-2, and Cdk-4 all of which are cell cycle regulation proteins expression. Correlation analysis of p53 and Cdk-2 proteins expression on day 3 after 4 Gy radiation suggested that their expression may regulate through p21 in the cell cycle. Control cells and post-irradiated cells of CNS-1 and 13-06MG cell lines do not express E-cadherin but have different protein expression patterns for fibronectin and vimentin. This indicates that EMT may occur in these post-irradiated cell lines. Given radiation-induced/associated complications in treating cancer patients, this study may help to understand the complex mechanisms of post-radiation tumor transformation, progression, and metastasis.

Agami, R. and R. Bernards (2000). Distinct initiation and maintenance mechanisms cooperate to induce G1 cell cycle arrest in response to DNA damage. Cell 102(1): 55-66.
Ailles, L. E. and I. L. Weissman (2007). Cancer stem cells in solid tumors. Current Opinion in Biotechnology 18(5): 460-466.
Attardi, L. D., A. de Vries, et al. (2004). Activation of the p53-dependent G1 checkpoint response in mouse embryo fibroblasts depends on the specific DNA damage inducer. Oncogene 23(4): 973-980.
Bølge Tysnes, B. and R. Mahesparan (2001). Biological Mechanisms of Glioma Invasion and Potential Therapeutic Targets. Journal of Neuro-Oncology 53(2): 129-147.
Bao, S., Q. Wu, et al. (2006). Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444(7120): 756-760.
Bartek, J. and J. Lukas (2001). Pathways governing G1/S transition and their response to DNA damage. Febs Letters 490(3): 117-122.
Behin, A., K. Hoang-Xuan, et al. (2003). Primary brain tumours in adults. The Lancet 361(9354): 323-331.
Bignami, A., L. F. Eng, et al. (1972). Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Research 43(2): 429-435.
Bonni, A., Y. Sun, et al. (1997). Regulation of Gliogenesis in the Central Nervous System by the JAK-STAT Signaling Pathway. Science 278(5337): 477-483.
Buscemi, G., P. Perego, et al. (2004). Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks. Oncogene 23(46): 7691-7700.
Chou, Y.-H., S. Khuon, et al. (2003). Nestin Promotes the Phosphorylation-dependent Disassembly of Vimentin Intermediate Filaments During Mitosis. Mol. Biol. Cell 14(4): 1468-1478.
Cress, A. E. (1990). The crosslinking of nuclear protein to DNA using ionizing radiation. Journal of Cancer Research and Clinical Oncology 116(4): 324.
Dahlstrand, J., L. B. Zimmerman, et al. (1992). Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments. Journal of Cell Science 103: 589-597.
Derycke, L. D. M. and M. E. Bracke (2004). N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling. International Journal of Developmental Biology 48(5-6): 463-476.
Dirks, P. B. (2008). Brain tumour stem cells: the undercurrents of human brain cancer and their relationship to neural stem cells. Philosophical Transactions of the Royal Society B-Biological Sciences 363(1489): 139-152.
Elledge, S. J. (1996). Cell cycle checkpoints: Preventing an identity crisis. Science 274(5293): 1664-1672.
Eng, L. F., J. J. Vanderhaeghen, et al. (1971). An acidic protein isolated from fibrous astrocytes. Brain Research 28(2): 351-354.
Evans, R. M. (1998). Vimentin: the conundrum of the intermediate filament gene family. Bioessays 20(1): 79-86.
Fisher, H. W. (1956). On the function of X-irradiated GBM cells in supporting growth of single mammalian cells. PNAS 42(12): 900.
Franke, W. W., C. Grund, et al. (1982). Formation of cytoskeletal elements during mouse embryogenesis. primary mesenchymal cells and the 1st appearance of Vimentin filaments. Differentiation 23(1): 43-59.
Franken, N. A. P., H. M. Rodermond, et al. (2006). Clonogenic assay of cells in vitro. Nat. Protocols 1(5): 2315-2319.
Gilbertson, R. J. and J. N. Rich (2007). Making a tumour's bed: glioblastoma stem cells and the vascular niche. Nat Rev Cancer 7(10): 733-736.
Grossman, S. A. and J. F. Batara (2004). Current management of glioblastoma multiforme. Semin Oncol 31(5): 635-44.
Gu, H., S. Wang, et al. (2002). Distribution of nestin immunoreactivity in the normal adult human forebrain. Brain Research 943(2): 174-180.
Ha, Y., J.-U. Choi, et al. (2002). Nestin and small heat shock protein expression on reactive astrocytes and endothelial cells in cerebral abscess. Neuroscience Research 44(2): 207-212.
Han, S. W. and J. Roman (2006). Fibronectin induces cell proliferation and inhibits apoptosis in human bronchial epithelial cells: pro-oncogenic effects mediated by PI3-kinase and NF-kappa B. Oncogene 25(31): 4341-4349.
Harper, J. W., G. R. Adami, et al. (1993). The p21 CDK-interacting protein CIP1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75(4): 805-816.
Jeanes, A., C. J. Gottardi, et al. Cadherins and cancer: how does cadherin dysfunction promote tumor progression. Oncogene 27(55): 6920-6929.
Jones, R. J. and W. Matsui (2007). Cancer stem cells: From bench to bedside. Biology of Blood and Marrow Transplantation 13(1): 47-52.
Kokkinos, M. I. (2007). Vimentin and Epithelial-Mesenchymal Transition in Human Breast Cancer-Observations in vitro and in vivo. Cells Tissues Organs 185(1-3): 191.
Krum, J. M. and J. M. Rosenstein (1999). Transient Coexpression of Nestin, GFAP, and Vascular Endothelial Growth Factor in Mature Reactive Astroglia Following Neural Grafting or Brain Wounds. Experimental Neurology 160(2): 348-360.
Larsson, A., U. Wilhelmsson, et al. (2004). Increased cell proliferation and neurogenesis in the hippocampal dentate gyrus of old GFAP(-/-)Vim(-/-) mice. Neurochemical Research 29(11): 2069-2073.
Lendahl, U., L. B. Zimmerman, et al. (1990). CNS stem cells express a new class of intermediate filament protein. Cell 60(4): 585-595.
M. A. Kahn, C. J. Huang, et al. (1997). Ciliary Neurotrophic Factor Activates JAK/Stat Signal Transduction Cascade and Induces Transcriptional Expression of Glial Fibrillary Acidic Protein in Glial Cells. Journal of Neurochemistry 68(4): 1413-1423.
Martin, A., H.-D. Hofmann, et al. (2003). Glial Reactivity in Ciliary Neurotrophic Factor-Deficient Mice after Optic Nerve Lesion. J. Neurosci. 23(13): 5416-5424.
Morshead, C. M., B. A. Reynolds, et al. (1994). Neural stem cells in the adult mammalian forebrain: A relatively quiescent subpopulation of subependymal cells. Neuron 13(5): 1071-1082.
O'Connor, P. M. (1997). Mammalian G1 and G2 phase checkpoints. Cancer Surveys 29: 151-82.
Oka, N., A. Soeda, et al. (2007). VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells. Biochemical and Biophysical Research Communications 360(3): 553-559.
Osoegawa, A., I. Yoshino, et al. (2008). Resection of Radiation-Induced Sarcoma of the Clavicle. Annals of Thoracic and Cardiovascular Surgery 14(3): 178-180.
Panagiotakos, G. and V. Tabar (2007). Brain tumor stem cells. Current Neurology and Neuroscience Reports 7(3): 215-220.
Piccirillo, S. G. M., B. A. Reynolds, et al. (2006). Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 444(7120): 761-765.
Poon, R. Y. C., W. Jiang, et al. (1996). Cyclin-dependent Kinases Are Inactivated by a Combination of p21 and Thr-14/Tyr-15 Phosphorylation after UV-induced DNA Damage. J. Biol. Chem. 271(22): 13283-13291.
Ransom, B., T. Behar, et al. (2003). New roles for astrocytes (stars at last). Trends in Neurosciences 26(10): 520-522.
Sancar, A., L. A. Lindsey-Boltz, et al. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry 73: 39-85.
Setsuo Hirohashi and Yae Kanai (2003). Cell adhesion system and human cancer morphogenesis. Cancer Science 94(7): 575-581.
Singh, S. K., C. Hawkins, et al. (2004). Identification of human brain tumour initiating cells. Nature 432(7015): 396-401.
Terada, Y., M. Tatsuka, et al. (1995). Requirment for tyrosine phosphorylation of CDK4 in G1 arrest induced by ultraviolet-irradiation. Nature 376(6538) : 358-362.
Thiery, J. P. and J. P. Sleeman (2006). Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7(2): 131-142.
Till, J. E. (1979). Citation classic- direct measurement of the radiation sensitivity of normal mouse bone-marrow cells.Current Contents/Life Sciences(43): L12-L12.
Uddin, S., T. Jarmi, et al. (2005). Glioblastoma multiforme. Current Opinion in Biotechnology 18(5): 460-466.
Wiese, C., A. Rolletschek, et al. (2004). Nestin expression - a property of multi-lineage progenitor cells? Cellular and Molecular Life Sciences 61(19-20): 2510-2522.