Title Page
Contents
Abstract 9
I. INTRODUCTION 11
II. MATERIALS AND METHODS 14
2.1. Drugs 14
2.2. Cell culture and resistant cell line establishment 14
2.3. Cell viability assay 15
2.4. Western blot and co-immunoprecipitation 15
2.5. Quantitative real-time PCR (qRT-PCR) 17
2.6. siRNA transfection 18
2.7. Gene expression microarray analysis 19
2.8. Cancer Cell Line Encyclopedia (CCLE) analysis 20
2.9. Fluorescence-activated cell sorting analysis (FACS) for cell cycle 20
2.10. Immunohistochemistry of cell blocks 20
2.11. Senescence associated (SA)-β-galactosidase staining 21
2.12. Animal studies 22
2.13. Clinical samples 23
2.14. Statistical analysis 24
III. RESULTS 25
3.1. Generation and confirmation of palbociclib resistant cell lines 25
3.2. RB loss and cyclin E overexpression are found in CDK4/6 inhibitor resistant cells 28
3.3. CDK2 inhibitor synergizes with palbociclib to inhibit cell proliferation 30
3.4. Inhibition of CDK2 increases senescence by inhibiting phospho-C-MYC, which is responsible for acquired resistance to palbociclib 32
3.5. Combined inhibition of CDK2 and CDK4/6 overcomes resistance to palbociclib in palbociclib-resistant xenograft model 39
3.6. Patients' pleural effusion data support the correlation of cyclin E overexpression and palbociclib resistance 42
IV. DISCUSSION 46
V. REFERENCES 50
ABSTRACT IN KOREAN 54
Table 1. Primary antibodies used for western blot or immunohistochemistry 17
Table 2. Primers used for qRT-PCR 18
Figure 1. Derivation and confirmation of CDK4/6 inhibitor resistance using HR-... 27
Figure 2. Alteration of cell cycle related genes and protein expression in palbociclib-... 29
Figure 3. CDK2 inhibitor synergizes with palbociclib to inhibit cell proliferation. (A-B) 32
Figure 4. Inhibition of CDK2 increases senescence by inhibiting phospho-C-MYC,... 37
Figure 5. CDK2 siRNA plus palbociclib regresses palbociclib-resistant breast cancer... 41
Figure 6. Pleural effusion analysis from HR-positive breast cancer patients support... 44