李玉梅臺灣大學：生化科學研究所周芳俞Chou, Fang-YuFang-YuChou2007-11-262018-07-062007-11-262018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/52797中文摘要 BC1是一個74KD的蛋白質，最初被發現在腦中，同時也在16個細胞株中被發現。先前的研究指出BC1能夠免除Huntingtin所誘導的聚集效應(aggregation)，同時也可以降低細胞毒性。 許多癌症的研究發現染色體20q13的位置大量增生會造成許多癌症的發生，例如：卵巢癌、乳癌、大腸癌、及胰臟癌。BC1正好座落在染色體20q13的位置。因此，我們想瞭解 (1) BC1是否具有致癌的特性？ (2) 是否抑制BC1可以降低腫瘤的形成？ 首先，我們先建立能夠大量表現BC1的細胞株 (stable cell line)，我們發現在細胞實驗過程中，BC1會促進細胞增生及誘導細胞轉型。同時，在活體老鼠實驗中，BC1會導致腫瘤的生長。 其次，我們利用BC1的反意質體(antisense plasmid)來降低細胞株中BC1的表現量。結果發現，降低BC1的表現量可以有效抑制人類乳癌細胞的增生。 綜而言之，這些結果證實BC1與致癌能力及細胞存活能力有關，同時也證實BC1具有發展抗癌藥物的潛能。We have isolated a novel gene, BC1, from the GCH (GTP cyclohydrogenase I) dominant negative cell models. BC1 is a 74KD polypeptide found in brain and also in 16 cell lines we tested. In the previous studies, BC1 could prevent Huntingtin induced aggregation as well as diminish cell toxicity. Chromosome 20q13 has been identified as gene amplification in cancers such as ovarian, breast, colon and pancreatic carcinoma. BC1 resides in this region, thus, we are interested in (1) is BC1 oncogenic? (2) Whether the inhibition of BC1 reduces tumor progression? In this study, we first established BC1 expression cell line and found that overexpression of BC1 exhibited significant advance of cell proliferation, induction transformation ability in soft agar assay, and stimulation of xenograft tumor growth in Nod-SCID mice. Second, we generate BC1-knockdown cell lines by introducing BC1 antisense plasmid. Our results reveal that down-regulation of BC1 expression can effectively inhibit the proliferation of human breast cancer cells, and BC1 may serve as a drug target in carcinogenesis. These results establish a role for BC1 in oncogenesis and cell survival and also demonstrate a potential strategy for cancer therapy.中文摘要 4 English Abstract 5 Abbreviation 6 1. Introduction 1.1 The role of oncogene and summary in cancer 7 1.2 Chromosome abnormalities in cancer 7 1.3 Growth of tumor cells in culture 8 1.3.1 Cell proliferation, loss of cell cycle control and 9 resistance to apoptosis 1.3.2 Decreased serum requirement and autocrine quality 10 1.3.3 Loss of anchorage dependence and acquisition 11 of ability to grow in soft agar 1.4 Growth of tumor cells in animals 12 1.5 Cancer cell in signal transduction pathway 13 1.6 Antisense for gene therapy 14 1.7 The role of BC1 and its known function 14 2. Material and Method 2.1 Cell Lines and Cell Culture 16 2.2 Stable Transfectants 16 2.3 Western Blot Analysis 16 2.4 WST-1 assay 17 2.5 Low-serum growth assay 17 2.6 TUNEL assay 17 2.7 Soft Agar Assay 18 2.8 Tumorigenicity in NOD-SCID Mice 18 2.9 Phospho-ERK determination 19 2.10 Wound healing assay of Scatter/Migration effect 19 3. Result PART I The Function of BC1 in tumorigenesis 3.1.1 Overexpression of BC1 in NIH3T3 cells 20 3.1.2 Effect of BC1 overexpression on cell growth in 20 NIH3T3 cells in proliferation assay 3.1.3 Overexpression of BC1 sustains cell growth 20 under low-serum conditions 3.1.4 Overexpression of BC1 shows morphological 21 transformation of NIH3T3 cells by anchorage- independent growth 3.1.5 Overexpression of BC1 displays cell motility 21 in vitro 3.1.6 BC1 induces tumor formation in NOD-SCID mice 22 3.1.7 Activation of MAPK ERK 1/2 by BC1 22 PART II The potential of antisense-BC1 for cell growth inhibition 3.2.1 Establishment of BC1 knockdown cell line in MCF7 23 3.2.2 BC1 knockdown inhibits cell growth 23 3.2.3 BC1 knockdown induces apoptosis 23 3.2.4 BC1 knockdown reverses oncogenic potential 24 in soft agar assay 3.2.5 BC1 knockdown suppresses tumor growth 24 in NOD-SCID mice 4. Discussion 4.1 Overexpression of BC1 induced cell transformation 26 in vitro and tumor progression in vivo 4.2 Multiple genes in human 20q13 chromosomal region are 26 involved in human cancer 4.3 Molecular targeting of BC1 in human cancer 27 4.4 Multiple genes in functional studies connect 27 splicing and transformation 5. Reference 29 6. Figures and Tables 41 7. Appendix 53en-US致癌基因oncogenic transformation[SDGs]SDG3other