Year

1999

Degree Name

Doctor of Philosophy

Department

Department of Biological Sciences

Abstract

Cell-surface plasminogen activation (PA) occurs by virtue of the presence of specific receptors for plasminogen (pig) and urokinase or tissue plasminogen activator (uPA and tPA respectively). Receptor binding of pig induces a conformational change in the molecule, which makes it more susceptible to activation by uPA/tPA. Plasmin (plm) generated on the cell-surface in this manner is protected from fast acting inhibitors and can subsequently participate in various extracellular proteolytic events such as cell migration (e.g angiogenesis, tumour cell invasion, inflammation, wound healing, bacterial invasion), ovulation, tissue remodelling and the activation of other protease classes and growth factors. Evidence is also emerging that PA plays as yet undefined roles in cell death either as a cytoprotective or cytotoxic agent.

125I-plg binding studies on the human monocytic leukemic cell line U937, the breast carcinoma cell lines MCF-7 and MDA-MB-231, and the colon carcinoma cell line HCT116 indicated that the presence of plg-Rs (i.e. specific pig binding) was highly variable on both U937 and MDA-MB-231 cells. This suggested that 125I-plg binding assays might not be suitable for measuring pig binding on all cell types and/or that plg-R expression is a variable phenomenon. Plg-binding proteins of 40-43kDa and 48-50kDa were consistently isolated from the plasma membranes of HCT116 cells using pigaffinity chromatography. The low concentrations of these proteins and the apparent loss of their pig-binding ability following isolation made if difficult to proceed with any further studies. Collectively these observations indicated that it was essential to develop alternative techniques for measuring plg binding.

A flow cytometric pig binding assay was developed, using fluorescein isothyocyanate-labelled plg (FITC-plg), to enable the measurement of plg-Rs (i.e specific plg binding) on various cell types (e.g adherent and nonadherent cells) and on specific subpopulations of cells (e.g viable and nonviable). A direct relationship was found to exist between cell viability [propidium iodide (PI) uptake] and the magnitude of lysine-dependent plg binding with non-viable subpopulations of cells binding up to 100-fold more plg than viable cells. This relationship was observed on numerous cell lines and indicated that the presence of non-viable cells could artifactually elevate the measurement of pig binding using non-cytometric assays, and more importantly, that plg-Rs (i.e specific pig binding) might play a role during cell death.

U937 cells that had been induced to undergo apoptosis with the protein synthesis inhibitor cycloheximide (CHX) were also shown to have highly elevated levels of plg-Rs (i.e specific plg binding) in addition to a large transient increase in cell-surface uPA. This increase in plg-Rs was a late apoptotic event, coincident with PI uptake and internucleosomal DNA fragmentation but occurring after elevations in phosphatidylserine (PS) exposure. Plg was also observed to dramatically increase the rate of CHXinduced apoptosis. These observations suggested that plg-Rs play a role during the degradative phase of apoptosis.

In order to ascertain whether apoptotic cells could activate plg, a novel flow cytometric PA assay was developed utilising fluorescein isothyocyanatelabelled aprotinin (FITC-aprotinin), to detect cell-surface plm. PA was found to be highly increased on apoptotic cells, which supports the observations that both plg-Rs and uPA are increased during apoptosis.

In summary, evidence is provided that plg-Rs (i.e-specific plg binding) are dramatically increased during cell death. The presence of both plg-Rs (i.e cell-surface plg) and uPA is critical for cell surface PA to proceed and this predominantly restricted to apoptotic U937 cells which displayed highly elevated levels of PA. These novel observations support the important role of flow cytometry in cellular plg binding and activation studies and strongly implicate the PA cascade in apoptosis, particularly on those cells that express uPA.

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