Cellular and molecular effects of CDC6-induced senescence in human bronchial epithelial cells

Postgraduate Thesis uoadl:2780791 443 Read counter

Unit:
Specialty Molecular Biomedicine Mechanisms of Disease, Molecular and Cellular Therapies, and Bioinnovation
Library of the School of Health Sciences
Deposit date:
2018-09-11
Year:
2018
Author:
Karousi Foteini
Supervisors info:
Γοργούλης Βασίλειος, Καθηγητής, Ιατρικής, ΕΚΠΑ
Κοτσίνας Αθανάσιος, Επίκουρος Καθηγητής, Ιατρικής, ΕΚΠΑ
Χαβάκη Σοφία, Επίκουρη Καθηγήτρια, Ιατρικής, ΕΚΠΑ
Original Title:
Cellular and molecular effects of CDC6-induced senescence in human bronchial epithelial cells
Languages:
English
Translated title:
Cellular and molecular effects of CDC6-induced senescence in human bronchial epithelial cells
Summary:
The human body is a complex and wondrous living machine. Like any machine, our bodies are composed by smaller parts that work together – the organs. Organs consist of million cells that cooperate with each other to perform a specialized task. Cancer is the result of a long time process that typically happens when the cells of the human body present ‘an antisocial behavior’ and cannot work with other cells in harmony. Recently, it has been revealed that a type of cellular senescence, the Oncogene-Induced Senescence (OIS), may act as an anti-tumor barrier.
Cell division cycle 6 (CDC6) is an essential protein for cell’s fate as it acts as replication licensing factor and prevents the cell from re-replication and genomic instability. Its over-expression has been associated with aberrant DNA replication and its deregulation has been linked with several types of cancer. Recent data have revealed a new oncogenic role of CDC6 and its newly identified participation in transcription regulation.
In this thesis, an epithelial cellular model in which over-expression of CDC6 was achieved in an inducible way through a doxycycline-inducible promoter is studied. Immortalized human bronchial epithelial cells (HBECs) (hTERT/CDK4) are used for that purpose. Most of the cancers are of epithelial origin and the above cell system simulates the whole spectrum of epithelial cancer development from the non-malignant stage to complete transformation of the normal cells into a mesenchymal - cancerous state. Importantly, in this thesis the part of carcinogenesis that we focus on is the cellular and molecular mechanisms controlling anti-tumor barrier-senescence.
The aim of this project was to study the role of CDC6 in cancer initiation and development. To facilitate our study, we examined how HBEC cells can be synchronized in the cell cycle. We confirmed by phase contrast microscopy and Flow Cytometry that simple deprivation of supplements was sufficient to reversibly block a large majority of the treated cells in G0/G1 phase. These experimental conditions thus enabled us to define a framework for addressing the second aim of this study: to monitor accurately the changes driven by CDC6 induction and the consequences on cell cycle dynamics and gene expression changes. We discovered a premature and accelerated entry of the cells in S phase. In addition, cells overexpressing CDC6 showed difficulties to carry on normal S phase and we suggest that replication may be blocked before the completion of the S phase. A third aim consisted in identifying the functional consequences of CDC6 induction at the chromatin structure and gene expression levels. In particular, we performed Chip-seq experiments to gain insights in the changes observed in the genome-wide binding of CTCF between HBEC OFF-control and HBEC cells that expressed CDC6 for 3 days (HBEC 3d Tet-ON). In HBEC 3d Tet-ON cells, we discover that the binding of CTCF on TSS is decreased at a subset of Transcription start sites (TSS). We propose that such structural changes may affect the gene expression program of the CDC6 overexpressing cells. To validate this hypothesis, we analyzed ATAC-seq data that were generated in the lab in order to understand changes in chromatin accessibility upon CDC6 induction and we monitored the number and localization of Differentially Accessible Regions (DAR) as well as their correlated effect on gene expression.
Main subject category:
Health Sciences
Keywords:
Cancer, Cellular senescence, CDC6
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
206
Number of pages:
59
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