Patel, Sabina
(2008)
The Development of Tetracycline Dependent Pancreatic Cancer Cells and the Evaluation of CapG and Gelsolin Expression on Pancreatic Cancer Cell Motility In Vitro.
PhD thesis, University of Liverpool.
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Abstract
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<O.OOO 1). High nuclear gelsolin was associated with reduced patient survival (P=O.OI). RNA interference (RNAi) mediated depletion of gelsolin in Panc-I, Suit-2 and MiaPaCa-2 cells led to significantly impaired motility, as assessed by either Boyden chambers or wound healing assays. To summarise, the generation of tetracycline-inducible pancreatic cancer cell lines provided a platform for functional analysis of CapG. These cell lines will also prove useful in the future, for the study ofa wide variety of proteins. The analyses of CapG and gelsolin showed that they can modulate the motility of pancreatic and hepatoma cancer cells in vitro. This suggests a potentially important role for these proteins in cancer cell dissemination. Supplied by The British Library - 'The world's knowledge'
| Item Type: | Thesis (PhD) |
|---|---|
| Depositing User: | Symplectic Admin |
| Date Deposited: | 20 Oct 2023 09:25 |
| Last Modified: | 20 Oct 2023 09:28 |
| DOI: | 10.17638/03174590 |
| Copyright Statement: | Copyright © and Moral Rights for this thesis and any accompanying data (where applicable) are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3174590 |
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