Selective Inhibition of Growth of Transformed Cells by Protease Inhibitors

Abstract

Five protease inhibitors with different modes of action were found to reduce the growth of transformed mouse (Py3T3, SV3T3, and 3T12) and hamster (PyBHK) cells. Some of these inhibitors caused the transformed cells to cease growth at saturation densities characteristic for nontransformed cells.The protease inhibitors were strikingly selective with regard to the transformed cells; they had essentially no effect on the growth of the nontransformed cells. From this result, it is concluded that the inhibitors block a protease-like activity that is required for the unrestrained growth of transformed cells.The inhibitors exerted their effect directly on the cells; they did not affect growth by interacting with serum components of the medium.Normal cells in tissue culture cease to divide upon formation of a monolayer. This phenomenon, which has been termed contact inhibition (1) or density-dependent inhibition of growth (2), is observed to a much lesser degree in virally or spontaneously transformed cells.Proteolytic treatment temporarily releases normal fibroblasts from contact inhibition (3, 4), and induces the appearance of surface properties characteristic of transformed cells (5-9). This finding implies the existence of a proteolytic activity in transformed cells that might be responsible for some of the cell surface alterations associated with the release from contact inhibition of growth (5, 10). If this speculation were correct, inhibition of this proteolytic activity should lead to a decreased growth of transformed cells and, possibly, to an "induction" of contact inhibition.This report describes the strikingly selective inhibition of growth of transformed cells by five commercially available protease inhibitors, and suggests that these cells require a protease-like activity for escape from contact inhibition. Cell growth curves in the presence or absence of protease inhibitors were obtained as follows: the cells were plated in 3.5-cm petri dishes (Falcon) 48 hr before addition of the inhibitors. At the beginning of each experiment, and every 24 hr thereafter, the medium with or without inhibitors (1.5 ml per plate) was changed in each plate.Five cell counts were done on each of two petri dishes for each point. To do this, the cells were trypsinized in 1 ml of trypsin-EDTA (Gibco No. 530), and were counted in a Neubauer Hemocytometer.The following protease inhibitors were used: Tosyl-arginine methylester (TAME), Tosyl-phenylalanyl-chloromethylketone (TPCK) and Tosyl-lysyl-chloromethylketone (TLCK) all from Calbiochem; soybean trypsin inhibitor 1-S (Sigma), ovomucoid II 0 (Sigma), and Trasylol injectible (5000 units/ml) from Bayer. Trasylol was dialyzed against phosphate-buffered saline, pH 7.4 to remove a toxic bactericidal additive before use. RESULTS AND DISCUSSIONAs an example of the cell growth curves obtained in the presence or absence of protease inhibitors, the effect of TLCK on the proliferation of SV3T3 and 3T3 cells is shown in Fig. 1

Effect of a Protease Inhibitor on the Adhesion of Ehrlich Ascites Cells to Host Cells in vivo

“…We postulate, therefore, that EAT cells bind to host tissue as a result of the inhibition by TI of intrinsic proteolytic activity. This hypothesis receives support from observations which indicate that treatment of malignant cells with protease inhibitors in vitro results in an alteration of growth characteristics, which may result from modification of the cell surface (Goetz, Weinstein and Roberts, 1972;Schnebli and Burger, 1972). Our findings support the view that the abnormal growth rates and adhesive properties of tumour cells are related to modification of cell surface binding sites by the activity of intrinsic proteases.…”

Non-Selective Inhibition of Transformed Cell Growth by a Protease Inhibitor

“…The essential point is that we observed no selective effect of TPCK on the growth of the virus-transformed 3T3 cells as compared with untransformed 3T3 cells. Our results thus differ from those of Schnebli and Burger (14) who reported a selective reduction in the growth rate of SV3T3 cells treated with 10,ug/ml of TPCK. Differences in cell lines and in the manner in which TPCK-containing media were prepared might account for some of the differences in our results.…”

Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells.

“…Although the exact function and mechanism of the protease(s) with respect to specific cellular alterations are unclear, many of the effects of the proteolytic activity appear to reside at the cell surface. Thus, when exogenous proteases are added to cultures of normal cells, alterations in the growth rate, agglutinability, adhesiveness, and morphology of the cells are observed (5,9,13,29,37,53,58); conversely, when specific protease inhibitors are added to cultures of transformed cells, the opposite effects on the cells' growth rate, agglutinability, adhesiveness, and morphology are observed (21,22,52,64); and finally, specific polypeptide alterations, temporally related to the onset of malignant transformation, have been detected on the surface of transformed ceils (10,25,27,28,62,65). It has been inferred from these studies that proteolytic activity may be enhanced in cultures of transformed cells and may be involved in both the initiation and the uninterrupted maintenance of characteristic changes on the surface membrane of the malignant cell.…”