Doctor of Philosophy
School of Biological Sciences - Faculty of Science
Al-Ejeh, Fares, Binding and fate of plasminogen activator inhibitor type-2 in breast carcinoma cells: rationale for development as a delivery vehicle of cytotoxins for anti-urokinase therapeutic strategies, PhD thesis, School of Biological Sciences, University of Wollongong, 2004. http://ro.uow.edu.au/theses/383
Plasminogen activator inhibitor type 2 (PAI-2) is one of the inhibitors of the plasminogen activation cascade mediated by urokinase (uPA) which is overexpressed on the surface of metastatic cancer cells. The plasminogen activation cascade is the generator of plasmin proteolytic activity on the cell surface which is required under normal physiological conditions and is largely exploited during cancer invasion and metastasis. The overall aim of this thesis was mainly to characterise the inhibition of cell-surface uPA by exogenously added PAI-2 and the elucidation of the fate of uPA:PAI-2 complexes once formed on the cell surface. PAI-2 was directly confirmed as a potent and rapid inhibitor of cell-surface uPA and specifically targets this antigen. PAI-2 internalised rapidly in a uPA-dependent manner after complex formation with cell-surface receptor (uPAR)-bound uPA. Internalised PAI-2 localised in endosomes/ lysosomes where it was presumably degraded. While the majority of uPAR/uPA:PAI-2 complexes formed on the cell surface were internalised, cellsurface uPA appeared to be replenished and uPAR was recycled to the cell surface. These findings were significant as they provide new information regarding cell-surface regulation of the plasminogen activation system as well as a biological rationale for the use of PAI-2 as a delivery vehicle of cytotoxins for anti-uPA therapeutic strategies. To extend the analyses of PAI-2 binding to cells and its subsequent fate, breast cancer cells were genetically modified to provide material for this study. Modulation of uPA-receptor (uPAR) expression was found to induce changes in components of the plasminogen activation cascade on the cell-surface. Upregulation of cell-surface uPAR on non-invasive, low uPAR/uPA-expressing T-47D breast cancer cells caused a concomitant increase in cell-surface uPA. This caused an increase in PAI-2 binding capacity to cells, thus directly confirming uPA-specificity of PAI-2. In addition, uPAR upregulation induced an increase in the lysine-dependent binding of plasminogen to the surface of these cells. Although cell-surface uPA was not confidently excluded as receptor for plasminogen binding on uPAR-modulated cells, the increase in plasminogen binding on these cells is ultimately due to increase in cell-surface plasminogen receptors. Finally, upregulation of uPAR also affected cell morphology and all together the data confirmed that uPAR/uPA-upregulation is associated with adoption of a metastatic genotype/phenotype. These results support other interests in our laboratory directed towards concluding whether uPA may act as a plasminogen receptor via an active-site independent epitope and elucidating plasminogen receptors which may be modulated due to modulation of uPAR/uPA expression. Finally, this thesis aimed at optimising the preparation of PAI-2 as a uPA-targeting vehicle for cancer radiotherapy and radioimaging applications. PAI-2 was modified by the addition of the metal chelator cDTPA; the product was characterised and radiolabelling with a radioisotope suitable for radiotherapy was optimised. These results allowed the development of standard operating procedures for use in pre-clinical and potentially clinical studies.