Title Page
Abstract
Contents
I. Introduction 9
II. Materials and Methods 11
1. Cell lines and cell culture 11
2. Cell viability assay 11
3. Clonogenic assay 12
4. Cell migration assay 12
5. Cell cycle analysis 13
6. Hoechst staining 13
7. RNA isolation and quantitative Real Time PCR(q-RT PCR) 14
8. Immuno-precipitation (IP) assay 14
9. Western blot analysis 15
10. Chromatin Immuno-precipitation (ChIP) assay 15
III. Results 20
1. BET protein inhibitors PFI-1 and JQ1 lead to growth retardation in a p53-independent manner. 20
2. BET inhibitors repress cancer properties. 22
3. Apoptosis was not induced by BET inhibitors. 25
4. BET inhibitors block cell cycle progression from G1 to S phase. 27
5. The expression levels of p21 were elevated by BET inhibitors in a p53-independent manner. 29
6. Disruption of activity of BET proteins leads to Rb dephosphorylation and E2F1 inactivation. 31
7. Treatment of BET inhibitors results in down-regulation of E2F1 target genes. 34
8. Recruitment of BRD2 was disrupted by BET inhibitors on the regulatory region of C-MYC. 36
9. BET inhibitors attenuate growth ability by regulating histone methylation at the E2F1 binding site of C-MYC promoter. 40
IV. Discussion 45
V. References 48
국문초록 54
Figure 1. The effect of BET inhibitors on cell growth. 21
Figure 2. Inhibitory effects of BET inhibitors on cell proliferation and migration. 23
Figure 3. No effect on apoptosis in BET inhibitors-treated cells. 26
Figure 4. Cell cycle analysis in growth-arrested cells. 28
Figure 5. Increased expression of p21 as a result of growth retardation. 30
Figure 6. Reduced level of phosphorylation of retinoblastoma protein (Rb) by... 32
Figure 7. Interaction between E2F1 and BRD2. 33
Figure 8. Decreased expression of E2F1 target genes by BET inhibitors. 35
Figure 9. ChIP assay showing recruitment of E2F1-BRD2 complex on promoter... 39
Figure 10. Effect of BET inhibitors on epigenetic histone modifications. 43
Figure 11. The mechanism of BET inhibitors on the regulation of C-MYC... 44