Driver, Glenn Alan2008-05-192008-05-192008-05-19http://hdl.handle.net/10539/4842Abstract Integrin-linked Kinase (ILK) is an integrin-associated protein kinase, which regulates growth factor-signalling pathways and cell-ECM adhesion events. Abrogated ILK expression or activity has been implicated in contributing to oncogenic transformation. We examined the role played by ILK in growth factor-stimulated and integrin signalling events in five human oesophageal squamous cell carcinoma cell lines (HOSCCs), known to overexpress the EGF receptor. Western blot analysis revealed the presence of ILK (59kDa) in all the moderately differentiated HOSCC lines. ILK1 was confirmed as being the predominant isoform. Densitometrically analysed Western blots showed that, per unit of protein, ILK is expressed uniformly across the cell lines under standard culture conditions. Following EGF (10 ng/ml) and TGFβ1 (1 ng/ml) treatment, ILK expression increased in all five HOSCCs. Indirect immunofluorescence microscopy showed the majority of ILK to localise at a cytoplasmic/nuclear level, with a proportion of ILK localising at the membrane, which resembled the distribution pattern of the β3 integrin subunit. This membranal distribution most likely follows that of the adhesion plaques although lesser, and variable, amounts were also identified throughout the cytoplasm. The functionality of the ILK1 kinase domain was demonstrated using myelin basic protein (MBP)-based kinase assays. EGF and TGFβ1 treatment produced an increase in ILK activity in the WHCO3 cell line of 3.5 fold, but a decrease in activity in the other cell lines, which are suggested to involve the actions of PTEN. The identification of nuclear ILK was surprising, and the mechanism for nuclear ILK localisation was suggested to involve a caveolae-associated protein, caveolin-1. Cell adhesion assays revealed that KP-392-mediated inhibition of ILK resulted in a nonsignificant reduction in cell adhesion to collagen and fibronectin. These data provide distinctive evidence for the influence of growth factors on ILK expression, but a duality in the effect on ILK activity. This apparent dichotomy is noteworthy and may be of particular relevance in HOSCC. It is further suggested that KP-392-induced ILK inhibition destabilises the αβ integrin heterodimer and that PI3K acts upstream of ILK-mediated cell adhesion events in HOSCCs. This suggests that ILK mediates integrin associated processes in human oesophageal SCC cell lines.8946187 bytesapplication/pdfenIntegrin-linked Kinaseoesophageal carcinomaEGFTGFβ1Thesis