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Synergistic revitalization of senescent cells via sequential activation of FOXM1 and E2F1

Title
Synergistic revitalization of senescent cells via sequential activation of FOXM1 and E2F1
Translated Title
FOXM1 및 E2F1 활성화를 통한 세포 노화 회복 효과
Authors
Eun Jae Yang
DGIST Authors
Eun Jae Yang; Young-Sam Lee; Daehee Hwang
Advisor(s)
이영삼
Co-Advisor(s)
Daehee Hwang
Issue Date
2022
Available Date
2022-03-08
Degree Date
2022/02
Type
Thesis
Keywords
Aging, Cellular senescence, Synergism, senomorphic effect, FOXM1, E2F1.
Description
Aging, Cellular senescence, Synergism, senomorphic effect, FOXM1, E2F1.
Abstract
Age-associated cellular senescence is characterized by cessation of cell division. However, the molecular pathways underlying cell cycle reactivation in senescent cells remain elusive, hampering the development of senomorphic compounds to rescue ceased cell division. The multifaceted nature of cel-lular senescence further necessitates targeting multiple senomorphic factors. We report synergistic senomorphic effects, including reversal of cell cycle arrest and mitochondrial or lysosomal impairment, of KU-60019 and Y-27632 in human diploid fibroblasts undergoing replicative senescence. Time-course transcriptomic analysis identified FOXM1 and E2F1 as crucial cell cycle reactivation regulators. The synergistic senomorphism arose from activation of cell cycle-dependent kinases via co-inhibition of the ATM and ROCK kinases by FOXM1, promoting the G2/M transition, with subsequent E2F1-induced activation of the G1/S transition. Our results demonstrate a cooperative senomorphic mecha-nism involving sequential activation of FOXM1 and E2F1 for coordinated escape of senescent cells from cell cycle arrest, whose anti-aging effects can be tested in vivo.|본 논문은 노화된 세포에 ROCK 억제제인Y27632와 ATM 억제제인 KU60019를 처리하였을 때, 전사인자인 FOXM1과 E2F1의 활성화로 인해 세포 노화 회복 효과가 나타난다는 내용을 담고 있다. 특히, 두 화학물을 같이 처리하였을 경우, 세포 분열 증가, 베타갈락토즈 염색 감소, 리포퓨신 발현량 감소, ROS 생성량 감소, DNA 손상 회복 등 여러 세포 노화의 생물지표들이 정상화되고, 전반적으로 노화된 세포가 노화를 극복하는 것을 관찰하였다. 전사체 분석을 통해 약물 처리 후, FOXM1과 E2F1이 활성화된다는 사실을 추론하였고, 실제로 이 두 전사인자들이 노화 회복 효과를 유도한다는 것을 알 수 있었다. FOXM1 및 E2F1 활성화 기제를 조사해보니, FOXM1은 ATM-PLK1-CDC25C-CDK1 인산화 과정과 ROCK-AKT-PLK1-CDC25C-CDK1 인산화 과정의 합류에 의해 약물 동시 처리 시 더욱 활성화가 되고, E2F1은 ATM-CHK2-CDC25A-CDK2-Rb 인산화 과정과 ROCK-AKT-CDK2-Rb 인산화과정의 합류로 인해 약물 동시 처리 시 더욱 활성화된다는 사실을 밝혔다. 이와 같은 사실들을 통해 본 논문은 그 동안 잘 알려지지 않았던, 세포 노화 회복 효과 방법과 기제를 명확히 한 연구결과라 할 수 있다.
Table Of Contents
I. Introduction 1 II. Experimental Method & Materials 4 2.1. Cell culture 4 2.2. Cell proliferation assay 4 2.3. SA-β-gal staining 5 2.4. Quantification of lipofuscin 6 2.5. Immunocytochemistry 6 2.6. Subcellular fractionation 7 2.7. Western blot analysis 7 2.8. Measurement of mitochondrial functions 9 2.9. Transcriptome analysis 10 2.10. qRT-PCR analysis 11 2.11. Identification of differentially expressed genes (DEGs) 11 2.12. Functional enrichment analysis and TF enrichment analysis 12 2.13. Transcriptome analysis in other models 12 2.14. Chromatin immunoprecipitation (ChIP) 13 2.15. Cell cycle analysis 13 2.16. coefficient of drug interaction (CDI) calculation 14 III. Results 15 3.1. KU and Y cooperatively restore the senescence phenotypes of HDFs 15 3.2. Transcriptome analysis reveals key candidate regulators of the synergistic senomorphism of KU and Y 17 3.3. FOXM1 acts as a triggering regulator of the synergistic senomorphism of KU and Y 20 3.4. E2F1, as a direct target of FOXM1, also plays a role in senomorphism 21 3.5. KU+Y treatment activates CDK-cyclin pathways to release FOXM1-dependent G2 arrest and subsequently release E2F1-dependent G1 arrest 23 IV. Discussion 26 V. Conclusion 30 VI. Figures 31 VII. References 62
URI
http://dgist.dcollection.net/common/orgView/200000597358
http://hdl.handle.net/20.500.11750/16331
DOI
10.22677/thesis.200000597358
Degree
Doctor
Department
New Biology
University
DGIST
Related Researcher
  • Author Lee, Young-Sam Senescence-Associated Mechanism Lab
  • Research Interests DNA replication and repair; Restoration of cellular senescence; Structural and functional relationship of proteins
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Collection:
Department of New BiologyThesesPh.D.


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