Cancer research background
R.J.Walters 2002
The research objectives were to investigate the role of the
phosphatidylinositol 3,4,5 trisphosphate (PIP3) signalling
pathway in controlling growth and structure in the U937 cell line,
using human neutrophils as a control. U937 cells are a myeloid-derived 'immortalised'
cancer cell line, previously used as a model for signalling to neutrophils,
and, inevitably, as a model cancer cell line.
The first project involved introducing a mutant form of the p85 regulatory subunit of
phosphatidylinositol 3-kinase (PI3K) into U937 cells. The p85 mutant protein was ligated into
plasmid vectors under eukaryotic promoters using restriction endonucleases. This allowed the
laboratory to probe a wide range of PI3K functions in the U937 cell line.
A cytoskeletal assay was developed to evaluate the role of ATP and insulin in stimulating
the polymerization of the cell's actin cytoskeleton in response to changes in the activity of
phosphoinositide-3-kinase (PI3K) and phospholipase C (PLC), using a fluorescence activated cell-sorter with FITC-conjugated
phalloidin as a probe. The results showed that both ATP and Insulin stimulate actin polymerization by
activating different isoforms of PI3K. In addition, the comparatively greater action of ATP in stimulating actin polymerization from the G to F-form,
was due to a parallel activation of an increase in cell calcium, possibly via the activation of PLC. The results were the first direct
evidence that PI3K directly induces actin polymerization (increase in F-actin/G-actin content). However, the phorbol ester TPA, which stimulates actin polymerization in
neutrophils, has no effect on F-actin content in the U937 cell line.
The role of phosphatidylinositol-3-kinase (PI3K) in the regulation
of phospholipase D (PLD) in the U937 cell line was assayed by measurement of
3H-choline or 3H-phosphatidylpropanol release by ion exchange and thin
layer chromatography respectively. Using these two methods, we found that neither
ATP nor insulin exert their mitogenic (stimulation of cell growth and division) actions via the
activation of PLD, therefore excluding a role for PI3K. However, the PI3K inhibitor wortmannin did inhibit
mitogenesis in U937 cells stimulated to divide by serum growth factors. Mitogenesis in U937 cells was studied by
FACScan quantification of DNA content, made fluorescent by propidium iodide. Therefore although PI3K does exert a mitogenic
and anti-apoptotic influence upon U937 cells, there is no evidence that it does so via the activation of PLD.
The other primary interest revolves around the use of cancer cells as a means by which to study gene expression and differentiation.
The pheochromocytoma (PC12) cell line is one of the most widely studied cell lines in the world, with well over 6,000 cited publications. Derived from a rat
chromaffin cell tumor, there being no human line available, this elegant cell line has regressed down the pathway of differentiation,
from a secretory neuroendocrine cell that releases cathecholamines in response to stimulation, to a proliferating cell line with some of its mature characteristics retained.
That is to say that proliferation (active cell division, characteristically unchecked in a tumor cell) and full differentiation (expression of the mature cellular characteristics)
are, generally speaking, mutually exclusive. Thus the PC12 cell retains the potential, derived as it is from a common ancestor of nerve and neuroendocrine cells, to
differentiate back into a functional chromaffin cell under the right culture conditions, or into a nerve cell in the presence of neurotrophins such as Nerve Growth Factor.
Thus my research focused upon using the PC12 cell to measure the regulation of the expression of catecholaminergic enzymes as one of several markers of PC12 cell differentiation using a new TaqMan method which allows
both total RNA and mRNA to be measured in parallel, and to study the expression of GABAA receptors. Studies showed that PC12 cells do express GABAA receptors whose subunit composition is
differentially modulated by growth factors, and that insulin depresses the expression level of the enzymes responsible for the synthesis of adrenaline and noradrenaline.
