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

Abstract

Guanidinobenzoatase is a trypsin-like protease associated with tumour cells (Steven et al., 1985) which may be assayed in solution by the cleavage of methylumbelliferone from the fluorogenic substrate 4-methylumbelliferyl-p-guanidinobenzoate (Steven & Al-Ahmad, 1983). This protease has been shown to cleave the tetrapeptide GlyArgGlyAsp which is thought to link cell surfaces to fibronectin (Pierschbacher & Ruoslahti, 1984). 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. In the present paper we have studied those tumour cells in frozen sections which possess uninhibited guanidinobenzoatase. When treated with 9-aminoacridine and examined by fluorescent microscopy these uninhibited cells exhibit cytoplasmic and cell surface yellow fluorescence; whilst other morphologically similar tumour cells with fully inhibited guanidinobenzoatase appear to be non-fluorescent with blue-green cytoplasm and cell surfaces. Those tumour cells which possess uninhibited guanidinobenzoatase were located at the advancing edge of the tumour mass or were detected as small groups of individual tumour cells outside the main tumour mass. This paper is concerned with the inhibition of the guanidinobenzoatase on these cells at the advancing edge of the tumour mass, since these can be more easily located than single cells in sequential sections. The role of host tissue protein inhibitors in the control of cell migration will be discussed.BZAR [bis-(N-benzyloxycarbonyl-L-argininamido)-Rhodamine] was first described by Leytus et al. (1983). BZAR inhibits guanidinobenzoatase in solution and on the surface of tumour cells (Steven et al., 1988), but is cleaved by other trypsin-like enzymes (Leytus et al., 1983). As both BZAR and 9-aminoacridine bind to the active centre of guanidinobenzoatase we used these inhibitors, one after the other, on Materials and methods 9-Aminoacridine was purchased from Sigma Chemical Company, St Louis, MO, USA. A stock aqueous solution, 10-3 M, of 9-aminoacridine was used for fluorescent staining. We employed 1O6 M BZAR dissolved in isotonic saline for the inhibition of cell bound guanidinobenzoatase. Frozen sections of human tumours taken from the head and neck region were kindly provided by the Pathology Department of the Justus-Liebig University, Giessen, West Germany. In all, over 500 frozen sections were provided from 55 subjects; these sections contained normal tissue as well as tumour cells, and the fluorescent examinat...

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

“…The lack of an inhibitor in the skin extract, capable of inhibiting GB on basal cell carci noma cells, may help to explain the local invasive character of these tumour cells. It is therefore of interest that a synthetic inhibitor of GB is available in the form of B7.AR; this reagent inhibits GB [7], but does not inhibit a number of other trypsin-like enzymes [11],…”

“…Other frozen sections were treated with BZAR (10~6 M) in isotonic saline for 2 h, prior to 9-aminoacridine stain ing. BZAR [11], acronym for bis-(N-benzyloxycarbonyl-Z.-Argininamido)-Rhodamine, is an inhibitor of GB [7] and also a substrate for trypsin-like enzymes [11], BZAR was kindly provided by Dr. W.F. Mangel, Brookhaven National Laboratory, Assoc.…”

“…The tumour tissue was embedded in paraplast and corresponding sections, stained with haematoxylin-eosin, were subjected to conventional pathohistological analysis. 9-aminoacridine (Sigma Chemicals) was used for the location of cells possessing GB [3][4][5][6][7], The frozen sections were placed in 200 ml isotonic saline containing 10~3 A/ 9-aminoacridine for 2 min, the excess reagent was washed off and the slides were placed in 200 ml iso tonic saline for I min prior to fluorescent microscopy. Using this technique with the Leitz Diaplan fluores cent microscope and filter block G (excitation 350-460 nm), a yellow fluorescence indicated the presence of active GB.…”

“…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.…”