Functional Study of the Regulation of DNA Topoisomerase II and its Cellular Role
in Drug Responsiveness
BY
Cheng-Fen (Terri) Chen
B.S., Taipei Medical University, 2004
THESIS
Submitted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Biopharmaceutical Sciences
in the Graduate College of the
University of Illinois at Chicago, 2011
Chicago, Illinois
Defense Committee:
William T. Beck, Chair and Advisor
Debra Tonetti
John L. Nitiss
Zaijie Jim Wang
Xiaolong He
Yin-Yuan Mo, Southern Illinois University
ACKNOWLEDGEMENTS
First of all, I would like to express my heartfelt thanks to my advisor, Dr. William T. Beck, for
his encouragement, for treating me with respect and for his guidance that helped this project
to be accomplished.
I would like to also thank my thesis committee: Drs. Xiaolong He, Yin-Yuan Mo, John L. Nitiss,
Debra A. Tonetti, and Zaijie Jim Wang, for their precious input and valuable advice.
Furthermore, I thank all the Beck lab members who supported me and shared their invaluable
expertise with me. I would like to extend my special thanks to Martina Vaskova for her being
kind, patient and making the lab such a great place to work in.
I would like to extend deep thanks to my friends in Chicago for always being there for me.
Last but not least, I thank my family for their support and for believing that I can successfully
complete this training.
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TABLE OF CONTENTS
CHAPTER
PAGE
Chapter 1
1.1. Summary…………………………………………………………………………………………..1
1.2. Introduction………………………………………………………………………………………..5
1.2.1. Overview: Drug Resistance in Cancer……………………………………………………….5
1.2.2. Overview: DNA Topoisomerase ……………………………………………………………..7
1.2.2.1. DNA Topoisomerase Cellular Function……………………………………………8
1.2.2.2. DNA Topoisomerase Family………………………………………………………..9
1.2.2.3. DNA Topoisomerase II (Top2) Biochemistry ……………………………………11
1.2.2.4. DNA Topoisomerase II (Top2) Isoforms…………………………………………11
1.2.2.5. Expression of DNA Topoisomerase II (Top2)…………………………………...12
1.2.2.6. DNA Topoisomerase II (Top2) and Anti-cancer Agents……………….............13
1.2.2.7. Resistance to Anti-Top2 Agents………………................................................14
1.2.2.8. Regulation of DNA Topoisomerase II (Top2)……………………………………17
1.3. Transcription Factor, NF-Y (Nuclear Factor-Y)………………………………………………20
1.4. MicroRNA and its Involvement in Chemoresistance………………………………………..22
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CHAPTER
PAGE
Chapter 2
2.1. Hypothesis and Specific Aims…………………………………………………………………26
2.2. Significance……………………………………………………………………………………...28
Chapter 3
Material and Methods
3.1. Cell Lines and Culture Conditions…………………………………………………………….29
3.2. Antibodies………………………………………………………………………………………..30
3.3. Preparation of Protein Extracts………………………………………………………………..30
3.4. Western Blot Analysis…………………………………………………………………………..32
3.5. Isolation of Total RNA and Reverse Transcription (RT)-PCR…………………………...…33
3.6. Expression Vectors……………………………………………………………………………..34
3.7. DNA Sequencing………………………………………………………………………………..35
3.8. Preparation of Virus Particles………………………………………………………………….35
3.9. Establishment of Stable Cell Lines……………………………………………………………36
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3.10. Transient Transfection………………………………………………………………………..37
3.11. Cell Proliferation Assay and Doubling Time………………………………………………..37
3.12. Cytotoxicity Assay…………………….……………………………………………………….38
3.13. Cell Cycle Analysis…………………………………………………………………………….39
3.14. Flow Cytometry for Cell Phenotype………………………………………………………….40
3.15. Apoptosis Assay……………………………………………………………………………….40
3.16. Luciferase Assay………………………………………………………………………………41
3.17. Statistical Analysis…………………………………………………………………………….42
Chapter 4
Determine the Role of Top2 in Mediating Drug Sensitivity
4.1. Background………………………………………………………………………………………43
4.2. Results……………………………………………………………………………………………44
4.2.1. Expression of Top2 is decreased in anti-Top2 agent-resistant Cancer Cell
Lines…………………………………………………………………………………………...44
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4.2.2. Suppression of Top2 by RNAi……………………………………………………..46
4.2.3. Suppression of Top2 Expression Results in Resistance to Top2 Poisons……49
4.2.4. Cancer Cells are Able to Proliferate with Relatively Low Level of Top251
4.3. Discussion……………………………………………………………………………………….53
Chapter 5
Examine the Role of NF-YB in Regulating Top2
5.1. Background………………………………………………………………………………………55
5.2. Results……………………………………………………………………………………………56
5.2.1. Expression of Top2 and NF-YB is Inversely Correlated………………………..56
5.2.2. NF-YB Mediated Top2 Expression Indirectly…………………………………….59
5.3. Discussion……………………………………………………………………………………….60
Chapter 6
Examine the Role of microRNAs in Top2-Mediated Drug Resistance
6.1. Background………………………………………………………………………………………64
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CHAPTER
PAGE
6.2. Results……………………………………………………………………………………………65
6.2.1. MicroRNAs are Differentially Expressed in Drug-Sensitive CEM and DrugResistant CEM/VM-1-5 Cells………………..………………………………………………65
6.2.2. NF-YB is a Direct Target of miR-485-3p……………………………………………67
6.2.3. MiR-485-3p Modulates the Sensitivity of CEM/VM-1-5 Cells to Top2 Poisons..69
6.3. Discussion……………………………………………………………………………………….71
Chapter 7
Examine the Cytotoxic Effect Mediated by the Antibiotic, Doxycycline (Doxy) and its
Role in DNA Top2 and Top2 Expression
7.1. Background………………………………………………………………………………………75
7.2. Results……………………………………………………………………………………………76
7.2.1. Knockdown of Top2 expression by tetracycline inducible system……………..76
7.2.2. Doxycycline Causes Growth Inhibition and Cytotoxicity in Cancer Cells……….78
7.2.3. Doxycycline Induces Apoptosis in Cancer Cell Lines…………………………….80
7.2.4. Doxycycline Causes Dysregulation Top2 and Top2 Expression in Cancer Cell
Lines…………………………………………………………………………………...81
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7.2.5. Differentiating Agents Mediate Top2 and Top2 Expression in Cancer Cells
Lines…………………………………………………………………………………....83
7.3. Discussion……………………………………………………………………………………….86
Chapter 8
8.1. Conclusion and Future Directions……………………………………………………………..89
8.2. References………………………………………………………………………………………94
8.3. VITA……………………………………………………………………………………………..110
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LIST OF TABLES
TABLE
PAGE
Table 1. DNA Topoisomerase classifications....…………………………………….……….9
Table 2. Anti-Topoisomerase II Agents……………….…. ………………………………....13
Table 3. Cancer cell lines resistant to Anti-Topoisomerase II agents…………………….16
Table 4. miRNAs Involvement in Cancer Chemoresistance ………………………………25
Table 5. Cell Lines and Culture Conditions……………………….…………………………30
Table 6. NF-YB and Top2 mRNA expression levels in the NCI-60 panel of cancer cell
lines.………………………………..………………………………….………………63
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LIST OF FIGURES
FIGURE
PAGE
Figure 1. A schematic representation of molecular mechanisms of
chemoresistance in a cancer cell………………………………………….………6
Figure 2. DNA topology……………………………………. …………………………………8
Figure 3. Reactions of DNA Topoisomerases………. ……………………………………10
Figure 4. Relative position of cis-acting elements in the human Top2a promoter and
associated transcription factors………. ………………………………………….19
Figure 5. CEM/VM-15 cells express reduced Top2a protein and mRNA compared to
CEM cells………………………………. ………………………………………….45
Figure 6. Knockdown Top2 expression by vector-based Top2shRNA…..………….48
Figure 7. Suppression of Top2 expression causes resistance to Top2 poison……….50
Figure 8. Cell growth curve, doubling time and cell cycle distribution…………………..52
Figure 9. Inverse correlation between the protein levels of Top2 and NF-YB in CEM
and CEM/VM-1-5 cells and Rh30 and Rh30/v1 cells…………………………..57
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Figure 10. Similar mRNA level of Top2 and NF-YB in drug-sensitive and –resistant
cell lines……………………………………………………………….……………58
Figure 11. Knock down of NF-YB does not affect Top2a protein levels.….…….……....59
Figure 12. NF-YB is a putative target for hsa-miR-485-3p………………….…………….66
Figure 13. MiR-485-3p targets NF-YB 3′-UTR…………………….………….……………68
Figure 14. MiR-485-3p inhibited NF-YB expression and mediated drug
responsiveness………………………………………….………….……………..70
Figure 15. Knockdown of Top2a expression by tetracycline inducible system…………77
Figure 16. Growth Inhibition of cancer cell lines after Doxycycline-treatment.…………79
Figure 17. Cell apoptosis after Doxy-treatment………………………………....…………80
Figure 18. Top2 and Top2 protein expression in Doxy-treated cells…………………82
Figure 19. Effects of Doxy, DMSO and TPA on HL-60 and CEM morphology …………84
Figure 20. Effects of Doxy, DMSO and TPA on Top2 and Top2 expression….……..85
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LIST OF ABBREVIATIONS
3’/5’-UTR
3’/5’- Untranslated region
Abs.
Absorbance
Ct
Cycle threshold
DMSO
Dimethyl sulfoxide
Doxy
Doxycycline
GFP
Green fluorescent protein
ICEs
Inverted CCAAT Elements
KD
Knock down
MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide )
MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
NF-YB
Nuclear factor-YB
PCNA
Proliferating cell nuclear antigen
PI
Propidium iodide
PTEN
Phosphatase and tensin homolog
Sp
Specificity protein
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Top2
DNA topoisomerase II
TPA
12-O-tetradecanoylphorbol-13-acetate
RA
Retinoic acid
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