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Studies on the regulation of expression of CDC20 and search for its new molecular interactors

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Title Studies on the regulation of expression of CDC20 and search for its new molecular interactors
 
Creator Das, Tania
 
Subject Molecular & Human Genetics
 
Description Advancement in the understanding of the molecular basis of cancer has led to the
emergence of tumor-specific, molecularly targeted agents that specifically kill
malignant cells more efficiently than ever. A number of cell signaling molecules, cellcycle
regulators, metabolic enzymes and cell surface macromolecules are currently
considered as potential targets for the development of novel therapeutic agents for
cancer treatment. Cdc20 a key spindle assembly checkpoint (SAC) protein, activates
APC/C during mitosis. Therefore, the major function of Cdc20 is to coordinate
mitotic progression by facilitating the orderly degradation of mitotic APC/Cb
substrates. Deregulation of Cdc20 causes erroneous mitotic division followed by
chromosomal instability (CIN). CIN is the hallmark of cancer. It is observed that the
overexpression of CDC20 occurs in several oral cell lines and primary head and neck
tumors and provide evidence that such overexpression of CDC20 is associated with
premature anaphase promotion, resulting in chromosome abnormalities in the cell
lines. More over invitro experiments have also shown that mitotic instability may be a
mechanism for developing resistance to cytotoxic drugs. Thus proper Cdc20
expression and function plays a crucial role in the context of the disease like cancer.
In this study we investigated first, the transcriptional regulation of Cdc20, second
Cdc20 mediated regulation of other cell cycle genes and finally we tried to establish
the Cdc20–Mad2 complex as a potential therapeutic target. As a result we proposed
for the first time, an oncogenic regulation of Cdc20. We showed that the protooncogene
Myb transctivates CDC20 in colorectal cancer cells and also in tumour
patient samples. We also reported that Cdc20 may negatively regulate its own
expression when the endogenous Cdc20 level goes up. We hypothesized that this
occurs probably due to maintain its own balance, since the proper level of Cdc20 is
crucial for maintaining the genomic stability of the daughter cells. To validate our
hypothesis we rescued the Myb mediated trans-activation of CDC20 from the Cdc20
auto inhibition, by lowering the endogenous Cdc20 level in the cell. More over we
showed that the Myb trans-activation response site and the Cdc20 auto-inhibition
response site resides very close to each other within 71 bp upstream of CDC20
transcription start site. All these evidences confers that the transcription of CDC20 is
a dual regulated phenomena operated by Myb and Cdc20 itself and the mode of
Summary
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operation depends upon the endogenous level of the Cdc20 protein. We also found the
involvement of p300 in Myb mediated trans-activation. As well as HDAC1 take part
in Cdc20-auto-inhibition. Additionally ubiquitination has a role in the autoregulation
of Cdc20. Unubiquitinated Cdc20 protein was found to be more prone for its own
inhibition. Finally to assess the fuctional significance of this oncogenic regulation of
Cdc20, we showed that over-expression of Myb leads to premature anaphase by over
expressing CDC20, followed by deregulation of SAC.
Cdc20 is reported to participate in the transcriptional regulation of mitotic genes by
CBP/p300 complex. Further, we have dissected the transcriptional role of Cdc20
protein in a global aspect. We performed the whole genome microarray analysis in the
altered Cdc20 expressing condition in HepG2 cells and found 252 differentially
expressed genes. The altered genes mainly belong to cell cycle and apoptotic
pathways. We found Rb, an important tumor suppressor gene is one of the major
downstream target of Cdc20. We have validated our observation in human cancer cell
line.
Finally we established Withaferin A (WA) as an inhibitor of Cdc20-Mad2 complex in
colorectal cancer cells. It causes spindle assembly checkpoint (SAC) arrest in
HCT116 and SW480 colorectal adenocarcinoma cell lines by stabilizing Cyclin B1
and its downstream molecules. Further we dissected the mechanism of action of WA
in these cell lines. We found that WA leads to proteasomal degradation of the Cdc20-
Mad2 complex that actually causes SAC arrest and apoptosis, as consequences. We
also rescued the Cyclin B1 degradation and the cell proliferation by over expression
of Mad2 in presence of WA. Together the experimental results suggests that WA
treated cells are arrested at the metaphase allowing sufficient time to activate the
apoptotic machinery leading to mitotic death of the cells.
Collectively this continuing effort is being made to identify Cdc20 as a new
oncogenic lead based on the knowledge drawn from the molecular analysis of cancer
cell lines and primary tumor that will enrich the repertoire of new molecular targets
for cancer management.
Part of this work has been published in Biochemical Pharmacology, 91 (2014) 31–39
 
Date 2015
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2108/1/Thesis_Complete__Tania_Das.pdf
Das, Tania (2015) Studies on the regulation of expression of CDC20 and search for its new molecular interactors. PhD thesis, JU.
 
Relation http://www.eprints.iicb.res.in/2108/