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Contents

Preface 11

I/II. Chapter 1. Phospholipase D as a survival factor during anti-cancer drug induced cell death 12

1. Abstract 1 13

2. Introduction 15

3. Materials and Methods 18

Materials 18

Cell culture 18

PLDs retroviral vector construction and virus production 19

Stable transfection of PLDs 19

Reverse transcriptase polymerase chain reaction 20

PLD activity assay 21

Western blot analysis 22

Measurement of phosphatidylserine (PS) exposure. 23

Cell viability 23

Small Interference RNA (siRNA) synthesis and transfection 23

Statistical analysis 24

4. Results 25

Apoptosis of stomach cancer cells (SNU 484 cells) induced by taxotere. 25

Effect of taxotere on Bcl-2 and PLD expression level in stomach cells. 25

Effect of PLD on taxotere-induced cell death in stomach cells. 26

Overexpression of either PLD1 or PLD2 inhibits taxotere induced apoptosis in cultured SNU 484 cells. 28

Expression of PLD suppresses taxotere-induced cell death through Bcl-2 regulation. 29

Bcl-2 expression was inhibited by down regulation of PLD1 or PLD2 using small interfering PLD1 or PLD2 RNA. 29

PA is involved in upregulation of Bcl-2 expression. 30

5. Discussion 32

II/III. Chapter 2. Molecular biological study on AP-180 as a phospholipase D inhibitor 51

1. Abstract 2 52

2. Introduction 54

3. Materials and methods 57

Materials 57

Cell culture 58

PLD activity assay 58

Reverse transcriptase polymerase chain reaction 59

Western blot analysis 60

Cloning of hAP180 from human cDNA library 61

Cell viability 62

Transient transfection 62

Immunocytochemistry 63

Construction of human AP180 deletion mutants 64

Immunoprecipitation 65

In vitro synthesis of amino acids and transient transfection. 66

Site-directed mutagenesis 66

Nucleotide Sequencing analysis 67

Statistical analysis 67

4. Results 69

PLD is activated by PMA, and hAP180 inhibits PLD activity stimulated by PMA in variable cell lines 69

hAP180 inhibits PLD1-but not PLD2-induced Bcl-2 expression 70

The hAP180 deletion mutants, in which amino acids from Thr310 to Lys320 are deleted, lost inhibition ability upon PLD activation 71

The amino acids from Thr310 to Lys320 are responsible for binding with PLD and the three amino acids, Thr312, Ser313, and Pro314 are important for inhibition of PLD activity 73

5. Discussion 75

III/IV. References 100

IV/V. 국문요지 110

V/VI. 감사의 글 135

List of figures

Chapter 1 8

Figure 1-1. PLD signaling and PLD-catalyzed hydrolysis and transphosphatidylation reactions 37

Figure 1-2. Apoptosis induced by taxotere in SNU484 stomach cancer cells. 38

Figure 1-3. Expression patterns of molecules involved in taxotere signaling. 39

Figure 1-4. Effect of PLD on taxotere-induced cell death in stomach cells. 40

Figure 1-5. Overexpression of either PLD1 or PLD2 in SNU 484 cells increased Bcl-2 expression resulting in significant inhibition of apoptosis induced by taxotere. 41

Figure 1-6. Expression of PLD suppresses taxotere-induced cell death through Bcl-2 regulation. 42

Figure 1-7. Effects of PLD1 or PLD2 inhibition using siRNA on expression of Bcl-2 in SNU484 cells. 43

Figure 1-8. Effects of PA on expression of Bcl-2 in SNU484 cells 44

Figure 1-9. Schematic drawing of survival signaling pathway generated by PLDs during taxotere-induced cell death in stomach cancer cells 45

Chapter 2 9

Figure 2-1. Establishment of model cell lines ; screening of high PLD activity using PMA 80

Figure 2-2. PMA induced PLD activity inhibited by hAP180 wild type in KATOIII and SiHa cells 81

Figure 2-3. PA, product of PLD, increases Bcl-2 expression, but AP180 inhibits Bcl-2 expression induced by PLD1 82

Figure 2-4. Human AP180 inhibits PLD1 induced increment of survival rate and exacerbates cell death during anticancer agents induced apoptosis. 83

Figure 2-5. Immunostaining after hAP180 and PLD1 cotransfection into SiHa cells 84

Figure 2-6. Features of hAP180 homologs, c-Myc-hAP180 constructs, and effects of C-terminal deletion mutants on inhibition of PLD activity. 85

Figure 2-7. C-terminal truncated mutants, △314 and △320 can not inhibit PLD activity 86

Figure 2-8. N-terminal truncated mutants, △314 and △320 can not inhibit PLD1 induced increment of survival rate during anticancer agents induced apoptosis. 87

Figure 2-9. Synthetic amino acids (specific residues responsible for inhibition of PMA induced PLD activity ; 310-TVTSPNSTPAK-320, 310-TVTSP-314, and 315-NSTPAK-320) inhibits PLD activity. 88

Figure 2-10. Interaction between PLD1 or PLD2 and hAP180 89

Figure 2-11. Site-directed mutagenesis, T312A, S313A, and T314A can not inhibit PMA-induced PLD activity and S313 is critical motif for binding with PLD1 90

Figure 2-12. Schematic drawing ; hAP180 inhibits survival signaling pathway generated by PLD1 91

Figure 2-13. Suitable target for development of anticancer drug ; inhibitor for PLD acting as survival factor during differentiation, chemoresistance, and apoptosis of the cancer cells. 92

Supplement Figures

Supplement Figures 1-1. Retrovirus constructions for PLDs overexpression. 113

Supplement Figures 1-2. PA treatment did not change the expression of the anti-or pro-apoptotic Bcl-2 protein family and propranolol did not affect cell viability. 114

Supplement Figures 2-1. Sequencing results of cloned full coding sequences of hAP180 into pCMV-Myc mammalian expression vector. 115

Supplement Figures 2-2. Sequencing results of cloned C-terminal deletion series and N-terminal truncated mutants of hAP180 into pCMV-Myc mammalian expression vector. 118

Supplement Figures 2-3. Sequencing results of site-directed mutagenesis point mutants of hAP180 into pCMV-Myc mammalian expression vector. 127

초록보기

인지질 분해 효소 D(phospholipase D, PLD)는 세포내·외부의 호르몬, 싸이토카인, 성장인자 등에 의해 유발되는 항세포사, 세포 내 물질이동, 세포분열 등에 밀접하게 관여되어 있다. PLD는 phosphatidyl choline을 phosphatidic acid (PA)와 choline으로 가수분해 하는 효소이다. PLD의 산물인 PA는 내세포작용에서 clathrin-coated vesicles에서 clathrin의 조립에 꼭 필요한 물질이며, amphiphysins와 synaptojanin과 AP 180과 같은 단백질들도 vesicle의 구조를 형성하는데 조력하는 단백질이다. 기존연구에서 확인된 결과를 바탕으로 볼 때 주목할 것은 amphiphysins, synaptojanin, AP 180 단백질이 PLD의 활성 억제에도 관여한다는 것이다. 본 논문에서는 인간 AP180(hAP180)을 대상으로 PLD 활성 억제에 중요한 구체적 motif를 찾아냈으며 hAP180에 의한 PLD 활성 억제는 암세포의 항암제 민감성을 유도하였다. 이는 hAP180에 의한 PLD 활성 억제가 암세포의 항암제민감도를 높여 항암제 효력 증강을 시사한다. 이러한 가능성을 확인하기 위해 첫 번째로 hAP180 유전자를 인간 cDNA pool로부터 클로닝하였다. 클로닝된 hAP180을 암세포주에 발현시킨 결과 phobol-myristate-acetate(PMA)에 의해 증가된 PLD 활성을 억제하였고 Thr-310에서 Lys-320까지의 11개 아미노산(TVTSPNSTPAK)이 PLD 활성 억제에 중요함을 밝혔다. 이를 아미노말단 부분 5개(310-TVTSP-314)와 카르복실말단 6개(315-NSTPAK-320)로 나누어 처리한 결과 아미노말단 부분의 5개 아미노산이 카르복실말단 6개 아미노산 대비 PLD 활성 억제가 매우 유의하였다. 이 결과를 토대로 볼 때 hAP180을 구성하는 아미노산 중 310-TVTSP-314 부위가 PLD 활성 억제에 중요한 motif임을 시사한다. 또한, AP180의 C-terminal deletion series와 N-terminal truncated mutant series와 이용한 실험 결과에서도 310-TVTSP-314가 PLD 활성 억제에 직접 관여하였다. hAP180의 PLD 활성 억제는 두 단백질의 결합에 의해 일어남을 시사하는 결과로 PMA에 의한 PLD 활성 증가에 따라 세포 안에서 PLD1과 hAP180의 colocalization이 하였다.

PLD 활성 억제에 중요한 5개(310-TVTSP-314) 아미노산을 alanine(Ala)으로 각각 단일 아미노산 치환 돌연변이 연구 결과 Thr312, Ser313, Pro314 아미노산이 PLD 활성 억제에 있어 중요한 역할을 가짐이 확인되었다. 면역침강법을 이용하여 단백질 수준에서의 상호작용을 확인한 결과에서 Ser313이 Ala으로 치환된 돌연변이의 경우 PLD1과의 결합강도가 매우 낮았다. 이는 Ser313이 PLD1과의 결합에서 매우 중요한 motif임을 시사한다. 현재까지의 연구를 바탕으로, PLD1의 아미노산 중 hAP180(특히 310-TVTSP-314 부위)과 결합하는 부위를 탐색할 계획에 있으며, PLD 활성억제제 개발을 위해 3개 아미노산(Thr312, Ser313, Pro314)을 표적으로 한 peptido mimetic drug 제작에 착수할 것이다. 만약 PLD 활성 억제제의 개발이 성공적으로 마무리될 경우 PLD 매개 항세포사를 조절하여 항암치료 시 암세포의 약제내성 감소, 세포사 증진, 항암제의 효능 및 민감도 증가, 항암제 dose-limit toxicity 감소 등의 효과를 가져 올 수 있을 것으로 사료된다.

참고문헌 (81건) : 자료제공( 네이버학술정보 )

참고문헌 목록에 대한 테이블로 번호, 참고문헌, 국회도서관 소장유무로 구성되어 있습니다.
번호 참고문헌 국회도서관 소장유무
1 Estimating the world cancer burden: Globocan 2000. 네이버 미소장
2 Docetaxel in the treatment of gastric cancer. 네이버 미소장
3 Establishment and characterization of human gastric carcinoma cell lines 네이버 미소장
4 Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. 네이버 미소장
5 Inactivation of Bcl-2 by Phosphorylation 네이버 미소장
6 Phosphatidylcholine-derived phosphatidic acid and diacylglycerol are involved in the signaling pathways activated by docetaxel 네이버 미소장
7 Role of phospholipase D2 in anti‐apoptotic signaling through increased expressions of Bcl‐2 and Bcl‐xL 네이버 미소장
8 Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family. 네이버 미소장
9 Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization 네이버 미소장
10 Phospholipid signalling through phospholipase D and phosphatidic acid 네이버 미소장
11 Phospholipase D: Enzymology, Mechanisms of Regulation, and Function 네이버 미소장
12 Regulation of phospholipase D 네이버 미소장
13 Phospholipase D and membrane traffic - Potential roles in regulated exocytosis, membrane delivery and vesicle budding 네이버 미소장
14 Possible role of phospholipase D in cellular differentiation and apoptosis 네이버 미소장
15 Overexpression of phospholipase D1 in human breast cancer tissues 네이버 미소장
16 Pharmacological importance of phospholipase D and phosphatidic acid in the regulation of the mitogen-activated protein kinase cascade 네이버 미소장
17 Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid 네이버 미소장
18 Alternative phospholipase D/mTOR survival signal in human breast cancer cells. 네이버 미소장
19 Anti-apoptotic role of phospholipase D isozymes in the glutamate-induced cell death 소장
20 Roles of phospholipase D in apoptosis and pro-survival. 네이버 미소장
21 Anti-apoptotic role of phospholipase D in spontaneous and delayed apoptosis of human neutrophils 네이버 미소장
22 Phospholipase D Activity in Human Gastric Carcinoma 네이버 미소장
23 A Stable Human-Derived Packaging Cell Line for Production of High Titer Retrovirus/Vesicular Stomatitis Virus G Pseudotypes 네이버 미소장
24 Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: involvement of protein kinase C-alpha. 소장
25 A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION 네이버 미소장
26 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding 네이버 미소장
27 Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays 네이버 미소장
28 Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7 네이버 미소장
29 A Crucial Role of Caspase 3 and Caspase 8 in Paclitaxel-Induced Apoptosis 네이버 미소장
30 Survival Signals Generated by Estrogen and Phospholipase D in MCF-7 Breast Cancer Cells Are Dependent on Myc 네이버 미소장
31 (1998). Kinetic analysis in mixed micelles of partially purified rat brain phospholipase D activity and its activation by phosphatidylinositol 4,5-bisphosphate. Neurochemical research 23, 589-599. 미소장
32 Taxol-induced Apoptosis and Phosphorylation of Bcl-2 Protein Involves c-Raf-1 and Represents a Novel c-Raf-1 Signal Transduction Pathway 네이버 미소장
33 Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. 네이버 미소장
34 Involvement of Microtubules in the Regulation of Bcl2 Phosphorylation and Apoptosis through Cyclic AMP-Dependent Protein Kinase 네이버 미소장
35 The effect of antimicrotubule agents on signal transduction pathways of apoptosis 네이버 미소장
36 Phorbol-12-myristate-13-acetate activation of phospholipase D in human neutrophils leads to the production of phosphatides and diglycerides 네이버 미소장
37 Standard cisplatin/infusional 5-fluorouracil (PF) vs docetaxel (T) plus PF (TPF) as neoadjuvant chemotherapy for nonresectable locally advanced squamous cell carcinoma of the head andneck (LA-SCCHN): a phase III trial of the EORTC Head and Neck Cancer Group (EORTC #24971) 네이버 미소장
38 Docetaxel plus Prednisone or Mitoxantrone plus Prednisone for Advanced Prostate Cancer 네이버 미소장
39 Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. 네이버 미소장
40 (2003). Randomized, multinational, phase III study of docetaxel plus platinum combinations versus 미소장
41 Phase III Randomized Trial of Docetaxel Plus Cisplatin Versus Vindesine Plus Cisplatin in Patients With Stage IV Non-Small-Cell Lung Cancer: The Japanese Taxotere Lung Cancer Study Group 네이버 미소장
42 Prospective Randomized Trial of Docetaxel Versus Best Supportive Care in Patients With Non-Small-Cell Lung Cancer Previously Treated With Platinum-Based Chemotherapy 네이버 미소장
43 Randomized Phase III Trial of Docetaxel Versus Vinorelbine or Ifosfamide in Patients With Advanced Non-Small-Cell Lung Cancer Previously Treated With Platinum-Containing Chemotherapy Regimens 네이버 미소장
44 Bcl2 Is the Guardian of Microtubule Integrity 네이버 미소장
45 Synergistic Effects of 8-Chlorocyclic-AMP and Retinoic Acid on Induction of Apoptosis in Ewing's Sarcoma CHP-100 Cells 네이버 미소장
46 The Bcl-2 Protein Family: Arbiters of Cell Survival 네이버 미소장
47 Inhibition of axotomy-induced neuronal apoptosis by extracellular delivery of a Bcl-XL fusion protein 네이버 미소장
48 Bcl-2 and the Outer Mitochondrial Membrane in the Inactivation of Cytochrome c during Fas-mediated Apoptosis 네이버 미소장
49 Cooperative Interception of Neuronal Apoptosis by BCL‐2 and BAG‐1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species 네이버 미소장
50 Prevention of Apoptosis by Bcl-2: Release of Cytochrome c from Mitochondria Blocked 네이버 미소장
51 Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells. 네이버 미소장
52 Decreased Phospholipase D (PLD) Activity in Ceramide- Induced Apoptosis of Human Keratinocyte Cell Line HaCaT 네이버 미소장
53 Changes of phospholipase D activity in TNF-alpha and anti-Fas/Apo1 monoclonal0aantibody induced apoptosis in HL-60 and A20 cells 네이버 미소장
54 Activation of Phospholipase D Participates in Signal Transduction Pathways Responsive to gamma-Radiation 네이버 미소장
55 Role of mitochondria in apoptosis 네이버 미소장
56 Tyrosine kinases as targets for cancer therapy. 네이버 미소장
57 Genes: an Open Access Journal 네이버 미소장
58 Prognostic markers in triple‐negative breast cancer 네이버 미소장
59 EGFR, HER2 and VEGF Pathways: Validated Targets for Cancer Treatment 네이버 미소장
60 Tumor Selective G2/M Cell Cycle Arrest and Apoptosis of Epithelial and Hematological Malignancies by BBL22, a Benzazepine 네이버 미소장
61 Methods in molecular biology. Preface. 네이버 미소장
62 DNA repair deficiency as a therapeutic target in cancer 네이버 미소장
63 Phospholipase D provides a survival signal in human cancer cells with activated H-Ras or K-Ras 네이버 미소장
64 Phospholipase D Prevents Etoposide-Induced Apoptosis by Inhibiting the Expression of Early Growth Response-1 and Phosphatase and Tensin Homologue Deleted on Chromosome 10 네이버 미소장
65 Mutant p53 in MDA-MB-231 breast cancer cells is stabilized by elevated phospholipase D activity and contributes to survival signals generated by phospholipase D. 네이버 미소장
66 Overexpression of phospholipase D suppresses taxotere-induced cell death in stomach cancer cells 네이버 미소장
67 Synaptojanin Inhibition of Phospholipase D Activity by Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate 네이버 미소장
68 Inhibition of phospholipase D by amphiphysins 네이버 미소장
69 AP-1 and Oct-1 transcription factors down-regulate the expression of the human PIT1/GHF1 gene. 네이버 미소장
70 Simultaneous Binding of PtdIns(4,5)P2and Clathrin by AP180 in the Nucleation of Clathrin Lattices on Membranes 네이버 미소장
71 LIM Kinase 1 Activates cAMP-responsive Element-binding Protein during the Neuronal Differentiation of Immortalized Hippocampal Progenitor Cells 네이버 미소장
72 (1991). Phospholipid metabolism in bradykinin-stimulated human fibroblasts. II. Phosphatidylcholine breakdown by phospholipases C and D; involvement of protein 미소장
73 Role of phospholipase D2 in anti-apoptotic signaling through increased expressions of Bcl-2 and Bcl-xL 네이버 미소장
74 Phosphoproteomic Analysis of the Developing Mouse Brain 네이버 미소장
75 Curvature of clathrin-coated pits driven by epsin 네이버 미소장
76 The ENTH domain 네이버 미소장
77 Promotion of microtubule assembly in vitro by taxol. 네이버 미소장
78 Etoposide: discovery and medicinal chemistry. 네이버 미소장
79 The potential for phospholipase D as a new therapeutic target 네이버 미소장
80 Mammalian phospholipase D structure and regulation 네이버 미소장
81 The Phosphorylation of Coated Membrane Proteins in Intact Neurons 네이버 미소장

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