바이오와 나노기술을 이용한 세포사멸 기전과 구조에 대한 연구
Functional and Structural Analysis of Cell Death using Bio/Nano-technologies
- 주제(키워드) Cell death , Biotechnology , Nanotechnology
- 발행기관 아주대학교
- 지도교수 최상돈
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000018791
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Even though various chemotherapeutic drugs are being used for clinical trials, until now, the field of scientific research keeps on carrying several studies aiming the development of relatively safe and stable anti-cancer drugs. Unfortunately, the continued use of the chemotherapeutic drug has the major limitation of severe side-effects in various organs. In particular, in case of p53, which is a key transcription factor known for its important role in the modulation of multiple cellular signaling pathways. The mutation occurring in p53 gene has been reported to be the cause of 75% of cancer’s cases suggesting that the loss of p53 function might lead to tumorigenesis. However, the exact mechanism by which the p53 causes cell death by DNA damage through apoptosis or necrosis, is not yet fully understood. Moreover, a clear understanding of the various modifications occurring in both cell adhesion and nuclear envelope features related to cell death remains largely unknown. To address this subject, our present study investigated the observed modifications in cell adhesion ability and nuclear envelope changes as well as DNA damage caused by p53 regulated apoptosis and necrosis, by using the Topoisomerase II targeting chemotherapeutic drugs such as etoposide and/or doxorubicin. For this, a variety of bio/nano-technological methods were used during. The first results of this study showed that the treatment of the human renal proximal tubule derived cells (HK-2) by etoposide induced DNA damage, p53 activation, cell cycle arrest and triggered the generation of reactive oxygen species (ROS) as well the mitochondrial biogenesis. Moreover, cell morphological changes such as cell swelling and plasma membrane rupture were also confirmed by carbon nanotube atomic force microscopy (CNT/AFM) analysis. In the other hand, the treatment of the p53-defective cells by etoposide limited cell cycle arrest and the mitochondrial biogenesis while it induced the increase of DNA damage, ROS mitochondrial generation and nitric oxide (NO) synthesis. Moreover, mitochondria outer membrane protein degradation as well as caspase 3 activation also increased. The morphological changes such cell shrinkage and the formation of apoptotic body were confirmed by the use of the CNT/AFM analysis. These findings show that the p53 activation, through the DNA damage signals, is responsible for the inhibition of cell death by apoptosis and the activation of necrosis. The second result of this study showed that though the treatment of the HK-2 cell by etoposide and doxorubicin induced the change of morphological features into those noticed in necrosis, in the case of cells treated by etoposide both apoptosis and necrosis features were observed while in the case of doxorubicin only necrosis morphological characteristic were reported. Based on the above experimental conditions, the morphological changes of cell during the mechanism of cell death by apoptosis were measured in real time by the xCELLigene: cells size, cell adhesion area and cell adhesion speed were formulated based on that adhesive properties of the cells were confirmed. The results showed that in apoptosis the cells presented a decrease in cell adhesion area as well as a decrease in their speed of adhesion. In the other hand in case of necrosis the speed of cell adhesion didn't present any significant change while an increase in the cell adhesion area was reported. In addition, in both cases of apoptosis and necrosis the changes of the nuclear envelope were investigated using CNT/AFM analysis and the result of the investigation showed that in necrotic cells the nuclear envelope did not present any damage. The nuclear pore complex as well as the general form of the nuclear envelope was confirmed. However, in case of apoptosis the rupture of the nuclear envelope as well as the nuclear pore destruction due to the activated caspase were also showed. Moreover, in the present work, DNA fragmentation protein translocation in nucleus through the rupture of the nuclear envelope caused by caspase, were also detected. In the present study, even though apoptosis and necrosis features such as changes in cell adhesion properties and changes in the nuclear envelope features were characterized, new methods of measurement using the nano-technology will be recommended in order to overcome the limitations of conventional measurement methods. In conclusion, through the fusion of various bio- and nano-chemical technologies we studied cell death, used a variety of methods that helped to understand cell death mechanisms.
more목차
Contents
ACKNOWLEDGMENTS
ABSTRACT
Chapter 1. Etoposide Induces Necrosis through P53-mediated Antiapoptosis 1
1.1 Abstract 1
1.2 Introduction 2
1.3 Results. 4
ETO Induced DNA Damage Response (DDR) Proteins, Formation of γ-H2AX Foci, and a Cell Cycle Arrest. 4
ETO Induced Oxidative Stress and Mitochondrial Biogenesis. 5
Inhibition of P53 Abrogated a Cell Cycle Arrest and Induction of DDR Proteins; These Events Enhanced ETO-induced DNA Damage and Cytotoxicity. 6
Inhibition of P53 Affected Mitochondrial Biogenesis, Oxidative Stress and Caspase 3 Activation. 6
Inhibition of P53 Induced Cell Adhesion Disruption and Apoptosis. 7
Inhibition of P53 Induced a Morphological Transition from Necrosis to Apoptosis. 8
A P53 Knockdown Reduced the cROS Generation and Induced Caspase 3 and Apoptosis. 9
1.4 Discussion 10
1.5 Methods 14
1.6 Figure and Figure legends 20
1.7 Supplements Figure and Supplements Figure legends. 33
1.8 References 41
Chapter 2. New Label-free Measurement Methods for The Analysis of Nuclear Envelope Topography and Cell Adhesion in Necrosis and Apoptosis 46
2.1 Abstract 46
2.2 Introduction 47
2.3 Results. 49
DOX Completely Induced Necrosis, while ETO Induced Necrosis in Combination with Apoptosis 49
Necrosis and Nepoptosis Generated Cell Swelling, but Yield Different Effects on Cell Adhesion 49
Preservation of Necrotic Morphological Changes during Necrosis and Nepoptosis 50
Necrosis and Nepoptosis Generated Nucleus Swelling and Altered Nucleus Morphology 51
Nepoptosis Induced NE Rupturing and DNA Leakage from The Nuclei 52
Cell Adhesion Disruption and NE Rupturing Are Induced by Caspase Activation during Nepoptosis. 52
ENDOG Is Translocated in The Nucleus through Caspase-induced NE Rupturing during Nepoptosis. 53
2.4 Discussion 54
2.5 Methods 57
2.6 Figure and Figure legends 63
2.7 Supplements Figure and Supplements Figure legends 71
2.8 References 78
Chapter 3. APPENDIX 81
1. Paper 1: ATF3 Plays a Key Role in Kdo2-Lipid A-Induced TLR4-Dependent Gene Expression via NF-kB Activation 81
2. Paper 2: Capric Acid Inhibits NO Production and STAT3 Activation during LPS-Induced Osteoclastogenesis 91
3. Paper 3: Doxorubicin Induces Cytotoxicity through Upregulation of pERKDependent ATF3 100
4. Paper 4: Discovery of an Integrative Network of MicroRNAs and Transcriptomics Changes for Acute Kidney Injury 111
Chapter 4. 국문요약 123
