Title:
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The Development of Tetracycline Dependent Pancreatic Cancer Cells and the Evaluation of CapG and Gelsolin Expression on Pancreatic Cancer Cell Motility In Vitro
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Precise control of the level of protein expression in cells can facilitate functional
studies providing information on the role of given proteins. In this thesis, I describe
the generation of tetracycline-inducible pancreatic cancer cells and the subsequent
use of these in the functional characterisation of an actin capping protein, CapG.
Such cells were obtained in three pancreatic cancer cell lines, Panc-I, Suit-2 and
MiaPaCa-2 cells through consecutive transfections with two plasmid constructs. The
first of these harboured a second-generation reverse tetracycline-controlled
transactivator protein (rtTA) whilst the second, contained the gene of interest (CapG
or luciferase) under the control of a tetracycline response promoter element (pTRE).
Suit-2 derived tetracycline inducible clones, along with stable doxycycline-inducible
hepatoma cell lines, were used to study the effect of modulating CapG expression on
cell motility. Here I report that stable introduction of a pTRE2hygCapG construct
into two doxycycline-inducible clones derived from Suit-2 cells, Suit-2 ptet1I and
Suit-2 ptet29 clones resulted in a dose and time-dependent increase of CapG
expression in response to doxycycline. Moreover, doxycycline-mediated upregulation
of CapG expression led to a significant increase in the wound healing
capacity of Suit-2 ptet29 cells.
The expression of a related actin binding and cell motility protein, gelsolin was also
determined. Immunostaining of benign (n=24 patients) and malignant (n=68 patients)
pancreatic ductal cells revealed higher gelsolin expression in the malignant state
(P
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