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논문명/저자명
Cloning and functional characterization of a novel gene, Romo1 = 새로운 유전자 Romo1의 클로닝 및 기능 규명 / 정영민 인기도
발행사항
서울 : 고려대학교 대학원, 2005.2
청구기호
TD 616.994 ㅈ272c
형태사항
viii, 104 p. ; 26 cm
자료실
전자자료
제어번호
KDMT1200536507
주기사항
학위논문(박사) -- 고려대학교 대학원, 의생명과학, 2005.2
원문

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Title Page

Abstract

Contents

1. Introduction 12

1.1. Drug resistance 12

1.1.1. Overview 12

1.1.2. Multidrug resistance (MDR) 12

1.1.3. MDR proteins: ATP-binding cassette (ABC) transporters 15

1.1.4. ABC transporters in human cancers 16

1.2. Mechanisms of cancer drug resistance in cellular pathways. 20

1.2.1. p53 20

1.2.2. Bcl-2 21

1.2.3. Nuclear factor κB 22

1.2.4. Other resistance mechanisms 23

1.3. Mechanisms of increased ROS stress in animal cells 25

1.3.1. ROS and cellular generation 25

1.3.2. Effects of ROS on DNA damage and regulators of cell cycle progression 26

1.3.3. ROS in cellular senescence 30

1.3.4. ROS in oncogenic transformation 31

1.3.5. Persistent oxidative stress and drug resistance 33

1.4. Purpose of this study 38

2. Materals and methods 39

2.1. Cell culture condition and reagents 39

2.2. RNA preparation 39

2.3. PCR by reverse transcriptase 40

2.4. Differential display PCR 40

2.5. Northern blot analysis 41

2.6. Construction of Romo1 expressing vector 42

2.7. Transient transfection 42

2.8. Stable transfection 43

2.9. Cell cycle analysis 43

2.10. BrdU uptake analysis 44

2.11. Cytotoxicity assay 44

2.12. Mitochondrial fractionation 45

2.13. Fluorescence microscopy 45

2.14. Measurement of intracellular ROS 46

2.15. Anti-sense oligonucleotide treatment 47

2.16. Western blot analysis 47

2.17. Statistical analysis 48

3. Results 49

3.1. Differential gene expression carried out between anti-cancer drug sensitive and resistant tissue. 49

3.2. Cloning of Romo1 51

3.2.1. Genomic sequence and expression of Romo1 51

3.2.2. Construction of pcDNA3/Romo1 sense and anti-sense expressing vector and establishment of Romo1 stable expression cells. 53

3.3. Romo1 induces drug resistance against anti-tumor agents 58

3.3.1. Romo1 induces drug resistance against anti-tumor agents in SNU 638 cells 58

3.3.2. Romo1 induces drug resistance against anti-tumor agents in H1299 cells 58

3.4. Romo1 induces cell cycle arrest at G1 phase 63

3.4.1. Romo1 induces cell cycle arrest at G1 phase in SUN 638 63

3.4.2. Romo1 induces cell cycle arrest at the G1 phase in H1299 cells 68

3.5. Romo1 is localized in the mitochondria. 76

3.6. Romo1 enhances ROS in the mitochondria 79

3.6.1. Romo1 enhances ROS generation in the mitochondria of SNU 638 and H1299 cells 79

3.6.2. Anti-sense ODN suppressed ROS increases in the Romo1 over-expressing cells 84

3.6.3. Decreased proliferation in the Romo1-overexpressing cells was reversed by anti-sense ODN 87

4. Discussion 89

5. Conclusions 93

6. References 94

국문초록 113

Figure 1. This cartoon summarizes many of the ways in which cultured cancer cells have been shown to become resistant to cytotoxic anticancer drugs. 14

Figure 2. A hypothetical 2-D model of human P-glycoprotein, based on hydropathy analysis of the amino acid sequence and its functional domains. 18

Figure 3. ABC transporters with known drug substrates. 19

Figure 4. A model for oxidative stress in tumor formation. 35

Figure 5. Scheme, representing the multitude of effects that ROS can have on signal transduction and cell cycle progression. 36

Figure 6. Differential display PCR of mRNA in anti-cancer drug sensitive and resistant tumor tissues. 50

Figure 7. Nucleotide and amino acid sequences of human Romo1 cDNA 52

Figure 8. Northen blot of Romo1 in cancer cell lines. 55

Figure 9. Construction of pcDNA3/Romo1 sense/anti-sense 56

Figure 10. Northern blot of Romo1 in candidate Romo1 overexpressing cell lines (SNU 638). 57

Figure 11. Photography after 5-FU and paclitaxel treatment for 3 days. 60

Figure 12. Cytotoxicity assay by cell counting. 61

Figure 13. Cytotoxicity assay by cell counting in H1299 cells. 62

Figure 14. Growth curves of pcDNA3 or pcDNA3/Romo1 stably transfected cell lines. 64

Figure 15. Cell cycle analysis of Romo1 stable expressing cells. 65

Figure 16. Western blot analysis of p53, phosphorylated Rb, Cyclin E, cdk2 and β-actin in 638/C2 and 638/S6 cells. 67

Figure 17. Construction of pcDNA3/Romo1-myc-his expression vector. 69

Figure 18. Cell cycle analysis by flow cytometry after transient transfection. 71

Figure 19. Growth curves of vector or pcDNA3.1/Romo1-myc stably transfected cell lines. 72

Figure 20. Cell cycle analysis using BrdU uptake. 73

Figure 21. Western blot analysis of Romo1-myc-his, p21, p27, bcl-2 and β-actin after tranfection of Romo1-myc-his in course of time. 75

Figure 22. Romo1-GFP localized in mitochondria. 77

Figure 23. Romo1-myc-his is localized in mitochondria. 78

Figure 24. The effect of Romo1 transfection on ROS modulation in H1299 cells. 80

Figure 25. Effects of Romo1 transfection on the generation of ROS in SNU 638 cells. 81

Figure 26. Effects of Romo1 transfection on the generation of ROS in H1299 cells. 82

Figure 27. Generation of intracellular ROS in H1299/Romo1-S7 cells 83

Figure 28. Inhibitory effects of ROS generation by AS-ODN in H1299/Romo1-S7 cells 85

Figure 29. AS-ODN of Romo1 decrease the ROS level in H1299/C2 cells and H1299/Romo1-S7 cells 86

Figure 30. AS-ODN of Romo1 induces increased proliferation in H1299/Romo1-S7 cells 88

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