Download Exchange of inhibitors and proteases at tumour cell surfaces

Biochemical Society Transactions ( 1 99 1 ) 19 427s
Exchange of inhibitors and proteases at tumour cell surfaces.
FRANK S STEVEN, MARGARET M GRIFFIN, NEIL J
BULLEID and BERNARD S BROWN
Department of Biochemistry and Molecular Biology, School of
Biological Sciences, Stopford Building, Oxford Road,
Manchester MI3 9PT, U.K.
Tumour cells possess a cell-surface protease, referred to as
guanidinobenzoatase (GB), which binds the yellow-fluorescent
probe 9-aminoacridine (9-AA) at the active centre as a
competitive inhibitor [I]. It has now been shown that tissue
plasminogen activator (t-PA) cleaves guanidinobenzoates [2] in
a similar manner to GB and that GB and t-PA are closely
similar, if not identical proteases [3]. The GB of intact rat
leukaemia cells has been shown to be located at the external
surface of the cell, and this was recognised by a cytoplasmic
protein inhibitor of this cell-surface GB [4], the latter forming
an enzyme-inhibitor complex which failed to bind 9-AA.
For this study we required frozen sections of easily
recognisable tumour cells: we chose the squamous cell
carcinoma obtained from the oral cavity to illustrate our
findings. To avoid problems associated with the cytoplasmic
inhibitor of GB [4] we prepared protected sections [5] in which
the GB remained in the native state on the tumour cell surface
whilst the cytoplasmic inhibitors were extracted from thin
sections (Figure 1).
The GB on these protecteij sections was then used as a target
for potential inhibitors of GB, this interaction being followed
by use of 9-AA and fluorescent microscopy of the section.
Protected sections were used to demonstrate the transfer of
cytoplasmic inhibitors from fresh frozen sections to the
protected sections resulting in the formation of a reversible
enzyme-inhibitor complex. The GB on
sections was
inhibited by dansylglutamyl-glycylarginylchloromethyl ketone,
an inhibitor of t-PA [6] which was not displaced by 9-AA.
Y u w
Fig. 2. Fibrin removes proteases but not receptor
The GB on protected sections of tumour cells was removed
by contact with fibrin fibrils, as demonstrated by the cells'
subsequent failure to bind 9-AA (Figure 2). We assumed that
the GB on the cell surface was originally bound by a receptor
protein [7] which would remain attached to the cell surface
after fibrin treatment. If this concept was valid then these cells
might bind pure t-PA in place of GB. We treated such sections
with t-PA that had been labelled with Texas red, and
demonstrated the binding of this red-fluorescent probe to the
surface of the tumour cells. The binding of the fluorescent t-PA
was shown to be competitive with the binding of unlabelled tPA.
We suggest that p r o t e c a sections containing tumour cells
may provide a good test system for: (a) the interaction of
proteases with secreted inhibitors, (b) the exchange of proteases
which bind to the putative receptor protein on the cell surface,
and (c) studies on the receptor protein.
1.
2.
3.
4.
5.
6.
7.
GI3
Fig. 1. Preparation of protected cells
Steven, F.S., Griffin, M.M. & Al-Ahmad, R.K. (1985)
Eur. J. Biochem. 149, 35-40.
Geiger, M. & Binder, B.R. (1987) Biochim. Biophys.
Acta, 912, 34-40.
Steven, F.S., Griffin, M.M., Cederholm-Williams,
S.A., Mangel, W.F. & Maier, H. (1991) Anticancer
Res. in press.
Steven, F.S., Maier, H. & Amdt, J. (1989) J. Enz.
Inhibition, 3, 145-157.
Steven, F.S. & Griffin, M.M. (1991) J. Enz. Inhibition
in press.
Kettner, C. & Shaw, E.N. (1981) Methods in Enzym.
80, 826-842.
Stopelli, M.P., Tacchetti, C., Cubellis, M.V., Corti,
A., Heating, V.J., Cassani, G . , Appella, E. & Blasi, F.
(1984) Cell, 45, 675-684.