Title Page
Contents
ABBREVIATIONS 13
ABSTRACT IN KOREAN 14
Ⅰ. INTRODUCTION 16
1. Cell Biology 16
1.1. Cell Cycle 16
1.2. Mitosis 17
2. Composition of Centrosomes 20
2.1. Centrosome 20
2.2. Centrioles 21
2.3. Pericentriolar material (PCM) 23
2.4. Centrosome cycle 23
3. Cancer 26
3.1. Centrosome abnormalities 26
3.2. Chromosomal instability (CIN) 28
4. Research objectives 31
4.1. Phosphodiesterase 4D interacting protein (PDE4DIP) 31
4.2. Purpose of study 32
Ⅱ. MATERIALS AND METHOD 33
Cell lines and culture conditions 33
Transfections 33
RNA Interference 34
Immunoprecipitation 34
Immunofluorescence 36
Live-cell imaging 39
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 39
Western blot 40
Transformation of competent E. Coli DH5α bacteria 40
Isolation of plasmid DNA 41
Agarose gel electrophoresis 41
Mitotic index. 41
Statistical Analysis 42
Ⅲ. RESULTS 43
1. Depletion of PDE4DIP induces Centrosome Amplification 43
2. Depletion of PDE4DIP induces centriole over-duplication 55
3. Depletion of PDE4DIP induces multi-polar spindle. 69
4. Combination therapy 90
Ⅳ. DISCUSSION 96
Ⅴ. REFERENCES 99
ABSTRACT 106
Table 1. siRNA oligonucleotides used in this study. 35
Table 2. Antibodies were used in this study. 37
Table 3. 19 candidate genes based on transcriptome sequencing analysis results 46
Figure 1. The cell division cycle. 19
Figure 2. Schematic diagram of the centrosome cycle 25
Figure 3. Genome instability 30
Figure 4. PDE4DIP localizes at the centrosome. MKN28 cells were immunostained with PDE4DIP (green), β-tubulin... 47
Figure 5. PDE4DIP wild type, 560 mutant, and 622 mutant localize at the centrosome during mitosis.... 49
Figure 6. Depletion of PDE4DIP augments the multipolar spindle. A. PDE4DIP knockdown cells were immunostained with PDE4DIP... 52
Figure 7. Time-lapse images of mitotic cells during depletion of PDE4DIP.... 54
Figure 8. Origin of Centrosomal Defects. 58
Figure 9. Depletion of PDE4DIP mediates over-duplication of the centriole.... 61
Figure 10. PDE4DIP depletion results in a reduction of CEP350 at the centrioles.... 63
Figure 11. Depletion of PDE4DIP and CEP350 results in centriole over-duplication and PCM fragmentation.... 66
Figure 12. PDE4DIP depletion does not interfere with the centrosomal localization of Aurora A, Cep192, and PLK1.... 68
Figure 13. Phenotype-based screening by PDE4DIP depletion A-E. PDE4DIP knockdown cells were immunostained with the β-... 74
Figure 14. Depletion of PDE4DIP proteins affects the centrosomal levels of chTOG and TACC3.... 77
Figure 15. Depletion of chTOG and TACC3 associated with PCM fragmentation.... 79
Figure 16. Depletion of CEP350, chTOG, and TACC3 does not impair PDE4DIP recruitment to centrosomes.... 81
Figure 17. Immunoprecipitation of the endocytosis proteins PDE4DIP, chTOG, and TACC3... 82
Figure 18. Immunoprecipitations with overexpressed GFP-PDE4DIP WT,-560, and -622.... 84
Figure 19. Localization of chTOG by GFP-PDE4DIP WT, -560, and-622 proteins after depletion of PDE4DIP.... 86
Figure 20. Localization of chTOG by GFP-PDE4DIP WT, -560, and-622 proteins after depletion of CEP350.... 88
Figure 21. Model for the regulatory mechanism of PDE4DIP in mitosis. 89
Figure 22. Loss of Centrosome Integrity and Genome Stability 92
Figure 23. PDE4DIP regulates the colony-forming ability of gastric cancer cells.... 94
Figure 24. The target of centrosome clustering as a treatment approach. 95