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研究生：	李宜蓁 Li, I-Chen
論文名稱：	開發降低氧化傷害誘導的DNA微衛星不穩定之化合物 Discovery of compounds that reduce microsatellite instability induced by oxidative stress
指導教授：	張玲 Chang, Christina Ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	41
中文關鍵詞：	直腸結腸癌、氧化傷害、DNA錯誤配對修復機制、DNA微衛星序列不穩定
外文關鍵詞：	Colorectal Cancer, Oxidative stress, DNA mismatch repair system, DNA microsatellite instability
相關次數：	點閱：133 下載：0
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在台灣及西方國家，大腸直腸癌發生率及死亡率為居癌症第三。過去研究顯示，遺傳性非瘜肉症大腸直腸癌（Hereditary Non-Polyposis Colon Cancer），或慢性潰瘍性大腸炎（Chronic Ulcerative Colitis）的病人是產生大腸癌的高危險群。然而潰瘍性結腸炎（Ulcerative Colitis）以及90% 的遺傳性和15% 的偶發性大腸癌均發現有DNA微衛星序列不穩定（即Microsatellite Instability, MSI）的現象。此外在癌症化療之後所復發的非轉移癌組織中也發現微衛星序列不穩定的現象。因此本研究假設發炎反應及化療誘導的MSI可能與細胞受到氧化傷害（Oxidative stress）有關，而因氧化傷害造成的MSI是可以被預防的。藉由實驗室先前建立的雙螢光MSI reporter system，本研究以帶有13個胞嘧啶及腺嘌呤（cytosine: adenine, (CA) ）重複序列MSI reporter 之大腸癌細胞（HCT116-(CA)13）做為研究對象，以過氧化氫（H2O2）或已知會產生氧化壓力的化療藥物Methotrexate（MTX）做為氧化因子誘導細胞產生MSI，並尋找抑制氧化傷害引起MSI的化合物。流式細胞儀分析顯示，H2O2濃度與MSI程度呈劑量依存性。此外在經過重複MTX處理的HCT116-(CA)13細胞中，由DNA定序發現到在MSI reporter上的減少了一組CA鹼基對，並且在美國癌症協會提供的MSI marker分析上，也發現五個marker中有其中一個減少六個鹼基對。此外H2O2也造成hMSH6、CHK1與TGFβRII在編碼區產生序列移位，證明了氧化壓力會造成MSI。經由初步篩選五種可能抑制MSI的化合物，我發現兩種化合物能夠在不影響細胞存活率的情況降低H2O2及MTX引起的MSI現象。而這些發現也指出，在我們細胞實驗下，氧化壓力引起的MSI是可以被這兩種化合物抑制，因此找尋更多能夠降低MSI的化合物，有助於控制MSI發生率，並減緩因MSI誘導直結腸癌發生。

Colorectal cancer (CRC) ranks the third most common cancer in Taiwan and in other developed countries. CRC comprises hereditary non-polyposis colorectal cancer (HNPCC) families and sporadic CRC. In addition, patients with chronic ulcerative colitis (UC) are at the high risk of CRC. Microsatellite instability (MSI) occurs in ~10% patients with ulcerative colitis, as well as in ~90% HNPCC and ~15% of sporadic CRC. Moreover, chemotherapy-related secondary malignancies display a high frequency of MSI. These clinical observations strongly link MSI with oxidative stress, during chronic and cancer-associated inflammation or from ROS-generating chemotherapeutic agents. However, it is largely unclear whether the MSI phenotype induced by oxidative stress is preventable. In this study, I used a newly developed dual-fluorescent reporter system harboring an artificial (CA)13 microsatellite in the colorectal cancer HCT116 cell model to examine compounds that induce or suppress frameshift mutations. Flow cytometric analysis shows that oxidative stress generated by H2O2 increased the frameshift frequency of the (CA)13 microsatellite in a dose-dependent manner. Methotrexate (MTX), an ROS-generating anti-cancer drug, induced a frameshift mutation by deleting one repeat unit of the artificial (CA)13 microsatellite and which restored the in-frame reading of RFP. Based on an internationally accepted MSI assay, H2O2 altered the D17S250 dinucleotide-repeat microsatellite and not four other markers recommended by National Cancer Institute in the US. Furthermore, H2O2 caused frameshift mutations in the coding microsatellite of the hMSH6, CHK1 and, to a lesser extent, TGFβRII genes. After examining five different compounds, I discovered that Compounds x and y, without affecting cell viability, decreased H2O2-induced frameshift mutations in a dose-dependent manner. These findings indicate that the MSI phenotype, including frameshift mutations, induced by oxidative stress can be prevented by Compounds x and y at least in our cell model. Identification of additional MSI-reducing compounds will allow better control of MSI development hence the incidence and progression of CRC.

TABLE OF CONTENTS
中文摘要	I
ABSTRACT	II
誌謝	III
1. INTRODUCTION	1
1.1 Colorectal Cancer (CRC)	1
1.2 Microsatellite instability (MSI)	1
1.3 DNA Mismatch repair (MMR) system and MSI	2
1.4 MSI occurs in patients with ulcerative colitis	2
1.5 MSI occurs in cancer patients	3
1.6 Chemotherapy related MSI	3
1.7 Preventive measures to reduce CRC	4
1.8 Hypothesis	5
Figure 1. Proposed development and prevention of MSI in CRC	5
1.9 Specific Aims	5
2. MATERIALS AND METHODS	6
2.1 Cell lines and cell culture	6
2.2 Dual fluorescence MSI reporter	6
2.3 Treatment of H2O2, anti-oxidants and/or methotrexate	6
2.4 Cell viability assay	7
2.5 Analysis of frameshift mutations by flow cytometry	7
Figure 2. (A) Insertion of the (CA)13 microsatellite shifts RFP encoded by DsRed out of its reading frame. Re-appearance of red fluorescence in p(CA)13-RFP-IRES-GFP-Hyg-transfected cells would indicate frameshift mutations. (B) Flow Cytometric analysis of HCT116-(CA)13 cells	8
2.6 PCR-based MSI analysis	8
Fig. 3 Analysis of PCR-based MSI	9
2.7 Detection of Reactive Oxygen Species (ROS)	9
Fig. 4 Flow Cytometric analysis of ROS	10
2.8 Western blot analysis	10
2.9 Statistical analysis	10
3. RESULTS	11
3.1 A non-lethal dose range of H2O2 in HCT116 cells and derivatives	11
3.2 H2O2 increases the MSI frequency in a dose-dependent manner	11
3.3 Confirmation of H2O2 increased frameshift mutations by a high-content fluorescent microscopy	12
3.4 H2O2 alters endogenous microsatellite loci in HCT116-(CA)13 cells	12
3.5 Frameshift mutations in coding microsatellites result in decreased protein levels	13
3.6 Methotrexate generates ROS and induces MSI	14
3.7 MTX increases the frameshift mutations	14
3.8 Determination of non-cytotoxic doses of selected compounds in HCT116 cells and the derivatives	15
3.9 Effects of selected compounds on the frameshift frequency induced by H2O2	16
5. DISCUSSIONS	17
6. REFERENCES	22
7. TABLES	25
Table 1. Microsatellites markers recommended by the US National Cancer Institute (NCI) for the PCR-based MSI assay	25
Table 2. Coding microsatellites for the PCR-based MSI assay	26
8. FIGURES	28
Figure 5. H2O2 affects the viability of HCT116-derived stable transfectants.	28
Figure 6. H2O2 increases the frameshift frequency in a dose-dependent manner.	29
Figure 7. Confirmation of H2O2-increased frameshift mutations by high content fluorescent microscopy.	31
Figure 8. H2O2 induces instability of endogenous microsatellites in HCT116-(CA)13 cells.	32
Figure 9. H2O2 decreased the steady-state levels of proteins involved in MMR and DNA damage response in HCT116-(CA)13 cells.	33
Figure 10. H2O2 decreased the steady-state levels of proteins involved in MMR and DNA damage response in HCT116 cells.	34
Figure 11. Methotrexate generates ROS in HCT116 cells.	35
Figure 12. A long-term treatment of methotrexate results in the MSI phenotype in HCT116-(CA)13 derivatives.	37
Figure 13. Determination of non-cytotoxic doses of selected compounds in HCT116-(CA)13 cells.	39
Figure 14. Effects of selected compounds on the MSI frequency induced by H2O2 in HCT116-(CA)13 cells.	41


                                    

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