The inhibition of a tumour cell surface protease in vivo and its re-activation by oxidation

Abstract

Summary Colonic tumour cells possess a cell surface protease capable of binding 9-aminoacridine to its active centre, thus locating cells when viewed under a fluorescence microscope. In vivo and in frozen sections, the enzyme is masked by a protein inhibitor. This inhibitor can be displaced by formaldehyde fixation of the tissue and then replaced by adding a fresh extract of colon or lung tissue. The inhibitor is modified by oxidation; provided by air, oxidized glutathione or potassium permanganate, resulting in a change in conformation in the inhibitor and this then results in the enzyme binding the fluorescent probe. The effect of oxidation can be reversed by dithiothreitol. It is proposed that these changes are brought about by a disulphide exchange acting on the inhibitor which indirectly controls the activity of the cell surface enzyme in vivo.The steps described above can be conveniently followed on sections of tissue mounted on a microscope slide; this has the advantage that the same cells can be monitored during a sequence of reactions. It is believed that these techniques could well be applied to other enzyme systems than the tumour protease described in this study.

Activity of the Proteolytic Enzyme Guanidinobenzoatase in Human Basal Cell Carcinoma (With 1 colour plate)

“…This enzyme is located by the yellow, fluorescent, ring-staining of tumour cells with 9-aminoacridine. GB is uninhibited in basal cell car cinoma, unlike the inhibited GB on quies cent tumour cells [6], The pathophysiological significance of this observation seems clear. It is known that GB is capable of degrading fibronectin [ 1 ].…”

“…It can be assumed that some form of proteolysis must be involved in this process since extracellu lar fibrous proteins and probably cell mem branes of host cells must be destroyed during tumour cell migration. At least four protease systems may play a role, such as plasmino gen activator [12], the proteolytic enzyme of target cell cytolysis [13,14], GB [3,4,6] and mammalian collagénases [15].…”

“…Guanidinobenzoatase (GB) is a protease capable of degrading fibronectin and in par ticular the peptide GlyArgGlyAsp [1], This peptide is thought to link fibronectin to cell surfaces [2], Fluorescent probes which fit the active centre of GB have been designed to locate cells possessing this enzyme by means of fluorescent microscopy [3][4][5], Naturally occurring protein inhibitors of GB are present in most tissues [1,5,6] and these prevent the binding of fluorescent probes to GB on the surface of tumour cells. It has been observed that tumour cells at the ad vancing edge of a tumour mass and individ ual migrating tumour cells possess unin hibited GB activity in frozen sections [7], These cells bind the fluorescent probe for GB which enables them to be located by flu orescent microscopy [7], In the present work we are concerned with the activity of this enzyme associated with cells of basal cell car cinoma of the face.…”

Studies on the activity of a protease associated with cells at the advancing edge of human tumour masses in frozen sections

“…Guanidinobenzoatase is competitively inhibited by 9-aminoacridine (Steven et al, 1985), and this observation was used to locate tumour cells containing guanidinobenzoatase by fluorescent microscopy of formaldehyde fixed wax embedded sections. It was later established that most host tissues contained extractable proteins which were non-competitive inhibitors of guanidinobenzoatase in solution (Steven et al, 1988a). These protein inhibitors prevented the binding of 9-aminoacridine to the protease of most tumour cells in fresh frozen sections (Steven et al, 1988b), whilst some tumour cells possessed uninhibited enzyme in these frozen sections.…”