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ABSTRACT ························································································ i
TABLE OF CONTENTS ········································································ iv
LIST OF FIGURES ············································································ viii
Part 1
I. INTRODUCTION ··············································································· 1
II. MATERIALS AND METHODS ····························································· 7
1. Cell culture ······················································································· 7
2. Adenoviral transduction of HL-60 cells with TIS21 gene ································· 7
3. Differentiation analyses ········································································ 7
4. RNA extraction, semiquantitative RT-PCR and real time PCR ··························· 8
5. RNA Interference ·············································································· 10
6. Immunoblot and immunoprecipitation analyses ············································ 10
7. Statistical Analyses ············································································ 10
III. RESULTS ····················································································· 11
A. ATRA-induces granulocytic differentiation of HL-60 cells ······························ 11
B. ATRA upregulates Btg2 expression in HL-60 cells ······································· 14
C. TIS21 enhances ATRA-induced differentiation of HL-60 cells ························· 16
D. TIS21 enhances ATRA-induced differentiation via down-regulation of c-Myc in
HL-60 cell ·························································································· 19
E. TIS21 decreases stability of c-Myc protein in response to ATRA treatment ··········· 25
F. Transduction of shTIS21 abrogates TIS21 effect on c-Myc expression and HL-60
cells differentiation ··············································································· 27
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G. ATRA plus TIS21 increased Erk1/2 activity, but inhibited Akt with subsequent
GSK-3β activation ················································································ 29
H. TIS21 enhanced downregulation of c-Myc by activating GSK-3β ······················ 36
I. TIS21 enhanced ATRA-mediated c-Myc degradation in the proteosome ··············· 39
IV. DISCUSSION················································································· 42
V. CONCLUSION ················································································ 45
Part 2
I. INTRODUCTION ·············································································· 46
II. MATERIALS AND METHODS ···························································· 50
1. Cell culture ······················································································ 50
2. RNA extraction and semi quantitative RT-PCR ············································ 50
3. Immunoblot analysis ·········································································· 52
4. Cells fractionation ·············································································· 52
5. Measurement of intracellular ROS level and cell cycle analysis ························· 52
6. Chromatin immunoprecipitation (ChIP) assay ············································· 53
7. Cells Proliferation Assay ······································································ 53
8. Transfection of PKC- ········································································ 
8. Statistical analysis ············································································· 54
III. RESULTS ····················································································· 55
A. Btg2 is upregulated under serum deprivation ·············································· 55
B. Serum deprivation-induced reactive oxygen species generation upregulates
Btg2 expression ··················································································· 60
C. Serum deprivation induces NF-B activation ·············································· 63
D. NF-B regulates Btg2 expression under serum deprivation ······························ 66
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E. SP1 and DNA damage signals do not regulate Btg2 expression under serum
deprivation ························································································· 69
F. Exogenous H2O2 regulates Btg2 expression via NF-B activation in DLD-1 cells ···· 71
G. Doxorubicin induces Btg2 expression via ROS-NF-B pathway ······················· 73
H. ROS regulate NF-B activation along with Btg2 expression via PKC activity ······· 76
I. PKC- regulates NF-B activity and Btg2 expression in ROS dependent manner ···· 81
J. ROS regulate NFB-Btg2 under serum deprivation via MAPK pathway activation ·· 83
K. Btg2 reduces cells proliferation expression under serum deprivation, H2O2 and
Doxo treatment ···················································································· 86
IV. DISCUSSION················································································· 89
V. CONCLUSION ················································································ 93
Part 3
I. INTRODUCTION ·············································································· 94
II. MATERIALS AND METHODS ···························································· 97
1. Cell culture ······················································································ 97
2. Differentiation analyses ······································································· 97
3. RNA extraction, semiquantitative RT-PCR and real time PCR ·························· 97
4. Immunoblot and immunoprecipitation analyses ············································ 98
5. Cells fractionation ············································································· 98
6. Measurement of intracellular ROS level ···················································· 99
7. Statistical Analyses ············································································ 99
III. RESULTS ··················································································· 100
A. ATRA induces macrophage differentiation under reduced serum concentration ··· 100
B. ATRA enhances NF-B activation in myeloid leukemia cells ························ 104
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C. Inhibition of NF-B abrogates ATRA induced macrophage differentiation of
HL-60 cells······················································································· 107
D. Activation of NF-B induces macrophage differentiation in response to ATRA
Treatment ························································································ 109
E. ATRA enhances NF-B activation and induces macrophage differentiation via
MAPK ···························································································· 112
F. ATRA induced C/EBPα expression via MAPK is lower in cells under serum
deprivation ······················································································· 115
IV. DISCUSSION··············································································· 118
V. CONCLUSION ·············································································· 121
VI. REFERENCES ············································································· 122
국문 요약 ························································································ 148