Enhancement of transformed cell growth in agar by serine protease inhibitors

Abstract

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.

Protease Activity in Protein-Free Ns0 Myeloma Cell Cultures

“…Addition of aprotinin improved cell survival in rat renal tissue cultures by protecting membrane-bound glycoproteins from proteolytic enzymes (Davis et al, 1976). Aprotinin has also been found to increase the survival of rat hepatocytes presumably by inhibiting serine proteases located in the plasma membrane Nakamura et al, 1984) and to promote growth of transformed rat embryo fibroblasts, an effect attributed to decreased proteolysis of autocrine growth factors (Cook and Chen, 1988). In contrast, aprotinin has also been found to eliminate the growth-promoting effect of rat submaxillary gland extracts on adipose precursor cells due to inhibition of a kallikrein-like protease (Catalioto et al, 1987).…”

“…The possibility that aprotinin, because of its structural similarity to epidermal growth factor (EGF), may mimic the hormonal effect of this cytokine (Hunt et al, 1974;Cook and Chen, 1988) was rejected since supplementation of NS0 cultures with EGF at concentrations as high as 1 g/ml did not affect cell growth (unpublished data). The positive effect of aprotinin is therefore most probably caused by the inhibition of serine proteases in culture.…”

Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts.

“…Alternatively, as it is a matrix-specific inhibitor, its mecha- nism ofaction could be maintenance of ECM integrity, which is essential for supporting cell growth by binding autocrine and/or serum-derived growth factors. There is precedence for protease inhibitors to display growth-promoting activity (28,29). Experiments to distinguish between these and other possible mechanisms await acquisition of sufficient quantities of the pure native or recombinant protein.…”

Protease activity in protein-free NSO myeloma cell cultures

“…1). Aprotinin has also been found to increase the survival of rat hepatocytes presumably by inhibiting serine proteases located in the plasma membrane (Asami et al, 1984;Nakamura et al, 1984) and to promote growth of transformed tat embryo fibroblasts, an effect attributed to decreased proteolysis of autocrine growth factors (Cook and Chen, 1988). 9).…”

“…The possibility that aprotinin, because of its structural similarity to epidemml growth factor (EGF), may mimic the hormonal effect of this cytokine (Hunt et al, 1974;Cook and Chen, 1988) was rejected since supplementation of NS0 cultures with EGF at concentrations as high as 1 Ixg/ml did not affect cell growth (unpublished data). The positive effect of aprotinin is therefore most probably caused by the inhibition of serine proteases in culture.…”