Comp. Biochem. Physiol. Vol. 103B,No. 4, pp. 785-793, 1992

0305-0491/92 $5.00+ 0.00 Pergamon Press Ltd

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MINI REVIEW PROTEINASES A N D PROTEINASE INHIBITORS AS MODULATORS OF A N I M A L CELL GROWTH (3. KENNETHSCOTT Department of Biochemistry, University of Auckland, Auckland, New Zealand (Fax 649 373-7452) (Received 11 May 1992)

Atetract--1. Three distinct lines of evidence indicate that proteinases are involved in the growth of cultured animal cells. 2. Endogenous growth-related proteinases have been identified, and exogenous proteinases can also stimulate cell proliferation, probably by different mechanisms. In some cases, higher concentrations of proteinases are cytotoxic. 3. Proteinase inhibitors, not surprisingly, inhibit cell growth, but can also be mitogenic at sub-inhibitory concentrations. 4. There must, therefore, be at least three major cellular processes in which proteinases or proteinase inhibitors can operate to exert a direct effect on cell proliferation. 5. Details of one action of an exogenous proteinase, typified by thrombin and the thrombin receptor, are becoming clear at the molecular level, but thrombin probably activates at least two intracellular signalling systems, as well as acting as a growth inhibitor in some situations. 6. Much remains to be investigated in other examples.

INTRODUCTION Proteinases are growth stimulators for many types of animal cells in culture. This statement is supported by three main areas of experimental evidence which have been accumulated over nearly a quarter of a century. Two of these areas, relating to endogenous growthrelated proteinases, directly identified as such, and to growth inhibition by proteinase inhibitors, support the concept of a proteolytic step or steps as an integral part of cellular growth-regulatory processes. The third, involving the identification of exogenous proteinases as mitogens, need not necessarily operate through the same mechanism. In the last few years it has been discovered that proteinase inhibitors may also be mitogenic. Although this discovery is superficially paradoxical when considered alongside the earlier observations, these phenomena may represent different aspects of a more complex growth-stimulatory mechanism or, as the following review will indicate, may be only indirectly related, if at all. In writing a short review, it is inevitable that citation has been representative rather than comprehensive and, at various points in the text, reviews have been cited where appropriate. As a general introduction to the field, Vasiliev and C-elfand (1981), though obviously lacking many recent references, have considered growth-related proteolysis within the wider context of cellular growth control. EXOGENOUS PROTEINASES AS MrrOGENS

The evidence for proteinases as mitogens has been previously reviewed in more detail (Scott, 1987). The earliest work in this field concerned the addition of

exogenous proteinases to tissue explants and established cell cultures. Growth stimulation was observed in both situations (Simms and Stillman, 1937). A mitogenic effect of proteinases on human lympbocytes was first reported by Mazzei et al. (1966). Trypsin and other proteinases were shown to stimulate DNA synthesis and cell division in quiescent, confluent mouse fibroblasts (Burger, 1970; Sefton and Rubin, 1970). Though of intrinsic interest, it has already been stated, and should be emphasized, that the mitogenic action of exogenous proteinases may not be related to any endogenous mechanisms of growth control or stimulation. However, the possible physiological relevance of mitogenesis by exogenous proteinases is reinforced by the observation that proteinases secreted by the rat submaxillary gland are mitogenic/n vitro (Catalioto et al., 1987). Thrombin has been shown to be an effective mitogenic proteinase (Chert and Buchanan, 1975), and it has been suggested that this enzyme may have a physiological role in wound-healing (Gospodarowicz et al., 1978). Thrombin has been the focus of much of the subsequent work in this area, and it is known that the enzyme must be catalytically active to stimulate fibroblast growth (Glenn et al., 1980), although a peptide sequence distinct from the active site is mitogenic for macrophages (Bar-shavit et a/., 1986). Insolubilized thrombin is mitogenic, indicating that it acts at the cell surface (Carney and Cunningham, 1978). Thrombin substrates, receptors and inhibitors have been identified (Glenn and Cunningham, 1979; Baker et al., 1982; Moss and Cunningham, 1981). Thrombin stimulates the hydrolysis of phosphatidylinositol-4,5-bisphosphate to generate the intracellular second-messengers inositol trisphosphate and diacyl-

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glycerol, which are known to have a growthpromoting role (Stiernberg et al., 1983; Pouyssegur et al., 1985). The mechanism of activation of the phospholipase by thrombin is unknown, but can perhaps be inferred from recent experiments on the platelet thrombin receptor, for which the gene has been cloned, sequenced and used to express a recombinant receptor (Vu et al., 1991). This transmembrane protein is not a receptor in the traditional sense; proteolytic cleavage at a unique thrombin site near the N-terminus results in a conformational change, allowing the receptor to mediate intracellular events. The new N-terminal region generated by thrombin acts, in effect, as a "tethered ligand" for the receptor. A similar mechanism for thrombin receptor activation has recently been demonstrated in hamster lung fibroblasts (Hung et al., 1992). Some reports on the mechanism of action of thrombin have identified catalytic activity and "receptor occupancy" as distinct components of the process. Alpha-thrombin inactivated with diisopropylphosphorofluoridate, which will bind to cellsurface thrombin receptors, is not itself mitogenic for hamster embryonic fibroblasts, but does stimulate D N A synthesis in conjunction with either 7thrombin, which is catalytically active but does not bind to cells, or phorbol myristate acetate (Gordon and Carney, 1986). This latter reagent stimulates protein kinase C, and it has been suggested that this stimulation could also follow from the proteolytic action of thrombin; this would be consistent with the known second-messenger function of diacylglycerol, generated by the thrombin-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate. Several species of cell-surface thrombin-binding proteins have been identified (Frost et al., 1987), in addition to the well-characterized platelet receptor, and it is possible that one or more of these proteins acts as a transducer for a different intracellular signal in response to thrombin binding. Separation of the two thrombin signals (receptor occupancy and proteolytic action) does not result in a mitogenic response in human epithelial cells (He et al., 1992). Another aspect of thrombin-mediated mitogenesis is the activation of the S I S proto-oncogene in human endothelial cells (Daniel et al., 1986); this response is also elicited by catalytically-inactivated thrombin and so may be due to the mitogenic peptide sequence already mentioned. A cyclic AMP-dependent mechanism may be involved in this, or related, responses (Kavanaugh et al., 1988). The S I S gene product is a form of platelet-derived growth factor which is known to act as an autocrine growth stimulator; transforming growth factor fl can also act as an indirect mitogen by inducing S I S gene expression (Moses et al., 1990). Thrombin-induced protein phosphorylation may also be involved in the proliferation of human endothelial cells (Dupuy et al., 1989), implying the activation of a protein kinase, but these authors note that not all such cells respond to thrombin. Cytotoxic effects of exogenous proteinases on blood cells have been observed, usually at higher concentrations than those which cause mitogenic effects (Kaplan and Bona, 1974; Hatcher et al., 1978; Fraser and Scott, 1984). Similar experiments on

monolayer cell cultures are complicated by proteolytic detachment of cells from the substratum. This phenomenon may explain the differential effects of thrombin on human fibroblasts reported by Hall and Ganguly (1980), but Vittet et al. (1992) have demonstrated a cyclic AMP-mediated antiproliferative effect of thrombin on human megakaryoblastic cells growing in suspension culture. This effect is dependent on the proteolytic activity of thrombin, as it is blocked by simultaneous administration of hirudin, but is only observed in cells growing in low-serum growth medium. Inositol trisphosphate production is also stimulated but there is no evidence for any growth stimulation by thrombin throughout the concentration range tested. It seems that thrombin may activate intracellular messengers which have both proliferative and antiproliferative consequences; it is not yet clear how these antagonistic effects are summated in the cell. ENDOGENOUS GROWTH-RELATED PROTEINASES

One rationalization of the mitogenic effects of exogenous proteinases could be that they mimic endogenous proteinases which have a role in cell growth or division. A positive correlation between growth rate and cell-surface neutral proteinase activity was shown for several human cell cultures (Hatcher et al., 1976), and the activity was also shown to increase prior to mitosis in synchronized cultures. More recently, it has been shown that antibodies to a human cell-surface proteinase will inhibit DNA synthesis and cell proliferation in fibroblasts, leukocytes and some human tumour cell cultures (Pitts and Scott, 1983; Fraser and Scott, 1984; Scott and Scow, 1985). Fibroblast growth arrest resulting from inhibition of this enzyme, the so-called growth-related proteinase (GRP), can be reversed by thrombin (Allen et al., 1981), but inhibition of the GRP does not affect phosphoinositide metabolism, suggesting different mechanisms of action for thrombin and the GRP (Scott et al., 1989). This latter report also indicates that inhibition of the GRP blocks the mitogenic stimulus exerts by several different peptide growth factors, but not by exogenous calcium. A recently-discovered transmembrane serine proteinase has been implicated in cell proliferation. Hepsin was first identified as the product of a cloned human liver cDNA, and has been shown to present its catalytic domain outside the cell and, though ubiquitous, to be more highly expressed in activelydividing cells (Tsuji et al., 1991). In addition to the evidence implicating cell-surface proteinases in cell proliferation, there have been several reports of mitogens which stimulate the release of proteinases from leukocytes or lymphocytes (Weissman et al., 1972; Hatcher and Norin, 1982; Ku et al., 1983). In the last case, the enzymes responsible are similar to the leukocyte form of the G R P (Fraser and Scott, 1984). There is also evidence that secreted proteinases may be cytotoxic, though most of the work in this area relates to enzymes from cytotoxic lymphocytes acting on the other blood cell types, and may thus represent a special case. The enzymes responsible are serine proteinases, and are secreted from granules in cytotoxic lymphocytes (Pasternack

Proteinases and proteinase inhibitors and Eisen, 1985; Masson and Tschopp, 1987). It is possible that such enzymes exert a cytotoxic effect by way of a mechanism which is mitogenic at a lower degree of activation. This would imply that different leukocytes have different sensitivities to growthpromoting and growth-inhibiting proteinases. The plasminogen activators (urokinase and tissue plasminogen activator) are the most extensively studied of the endogenous cell-surface and extracellular proteinases (Laiho and Keski-Oja, 1989). Secreted urokinase can bind to cell-surface receptors in an active form (Vassalli et al., 1985). The earlier investigations indicated that these enzymes were not implicated directly in cell growth, but were indirectly involved in vivo through a role in metastasis and tissue invasion (Adelman et al., 1982; Ossowski and Reich, 1983; Scott, 1988). In this role, these enzymes may form part of a proteolytic cascade which results in degradation of extracellular matrices (He et al., 1989). More recently, urokinase has been shown to stimulate the proliferation of a human epidermal tumour cell culure (Kirchheimer et aL, 1987). The enzyme must be catalytically active, and only highmolecular-weight urokinase, containing a binding site for the cell-surface receptor, is effective. Further recent evidence suggests that urokinase may act as an autocrine growth stimulator for human renal glomerular epithelial cells (He et al., 1991). These cells secrete urokinase and possess urokinase receptors, and exogenous urokinase stimulates DNA synthesis and cell division. However, this phenomenon also has some similarity to thrombin-mediated mitogenesis as the effects of the two proteinases (urokinase and thrombin) are not additive. PROTEINASE INHIBITORS AS GROWTH INHIBITORS

The third line of evidence implicating proteinases as growth regulators concerns the addition of exogenous proteinase inhibitors to cell cultures. Although this has been the most popular experimental approach, there are some ambiguities in the interpretation of experiments with synthetic or microbial inhibitors (Scott, 1987). In part, these relate to intracellular effects following from the rapid cellular uptake of small or lipophilic inhibitors. Inhibition of intracellular proteolysis may indirectly influence proliferation by virtue of inhibition of protein turnover, or of protein processing. However, apparently specific growth-related intracellular proteinases have been identified (Billings et al., 1987; Wong et al., 1987; Bories et al., 1989), and the distinction between this experimental approach and that described in the previous section has become arbitrary. The general conclusion of earlier experiments, namely that inhibition of pericellular proteinases correlates with inhibition of cell proliferation, has been borne out by parallel studies with highmolecular-weight protein proteinase inhibitors (Schnebli, 1975). Over the time scale of cell culture experiments, it is possible that even macromolecular proteinase inhibitors have been taken up by endocytosis, and exerted their effect by virtue of inhibition of intracellular proteinases. It has been shown that soybean trypsin inhibitor (SBTI) and human ~t-lantitrypsin (~tlAT) inhibited the GRP and also

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inhibited cellular growth. In the case of • IAT, this inhibition occurred even when the inhibitor was coupled to polystyrene beads, indicating that the proteinase--inhibitor interaction occurred at the cell surface (Scott and Seow, 1985). From the outset, there has been interest in exploiting this research in the inhibition of tumour growth (Troll et aL, 1970), and some success has been claimed in animal systems for the Bowman-Birk soybean proteinase inhibitor as an inhibitor of chemicallyinduced tumour proliferation (Weed et aL, 1985). The target enzyme, isolated from transformed mouse fibroblasts, was similar in some respects to the GRP but was mainly located in the cytoplasm (Billings et al., 1987; Scott, 1987). Recent cell-fractionation studies showed that the equivalent human enzyme was also predominantly cytoplasmic (Billings et al., 1991), though some of the activity was in an uncharacterized particulate fraction. Yavelow et al. (1987a,b) have suggested that similar enzymes act as extracellular membrane receptors for the binding and internalization of proteinase inhibitors; thus an extracellular proteinase which exerted an intracellular effect following internalization would be readily inhibited by extracellular proteinase inhibitors. This mechanism has features in common with the autocrine action of some growth factors but, as the proteinase is not secreted in a soluble form, the word "pericrine" could be coined to signify the pericellular localization of the effector enzyme. In vivo, a growthstimulatory pathway of this type would be amenable to modulation by extracellular elements, in this case by circulating or tissue fluid proteinase inhibitors. If this hypothesis is correct, then it will be worthwhile to search for intracellular proteinase substrates which have a signalling function. Intracellular events following from proteinase activation have already been identified. The Bowman-Birk inhibitor has recently been shown to inhibit expression of the F O S proto-oncogene in transformed mouse fibroblasts. This is a specific effect, rather than one of generalized inhibition of transcription, and indicates that proteinases, and their inhibitors, can affect genes involved in the regulation of cell proliferation (Caggana and Kennedy, 1989). Earlier research by the same group has shown that transcription of the myc protooncogene in mouse fibroblasts was inhibited by antipain, although there was no effect on cell growth (Chang et al., 1985) and, more recently, the inhibition of v - H a - r a s gene expression in transformed NIH 3T3 cells has also been ascribed to antipain (Cox et al., 1991). Discussion of the effects of growth-inhibitory proteinase inhibitors on proto-oncogene expression leads naturally back to the consideration of their effects on tumour cell proliferation. Some such experiments have already been discussed in the present review, as has some earlier work in the field (Scott, 1987). A report that anti-GRP antibodies distinguished between groups of tumour or transformed cells that were either dependent upon the GRP, or independent of it (i.e. their growth was not inhibited by the antibodies; Pitts and Scott, 1983), has been supported by the finding that ~IAT also would inhibit the growth of some human tumour cell lines, but not others (Scott, 1988; Scott and Tse, 1992). In all cases,

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a cell-surface proteinase activity corresponding to the G R P was detected and inhibited. It is possible to infer from these experiments that the expression of some oncogenes may override the effect of GRP inhibition, whereas, for example, the U 2 0 S osteosarcoma cell line, which expresses v - s i s (Weich et al., 1986), is still sensitive to GRP inhibition (Scott and Tse, 1992). This last result is consistent with our observation that G R P inhibition blocks the mitogenic action of platelet-derived growth factor (Scott et al., 1989). The identity of the oncogenes expressed in most of the tumour cells used in these studies is not known, and further work in this connection is clearly warranted. There appears, also, to be some specificity with respect to the actual proteinase inhibitors which inhibit tumour cell growth. Recent experiments with human urinary trypsin inhibitor (UTI), a component of inter-~-trypsin inhibitor (Gebhard et al., 1990), demonstrated the inhibition of growth of a human lymphoma cell line (Chawla et al., 1990), in which role ~ IAT, SBTI and other macromolecular proteinase inhibitors were ineffective.

POSSIBLE MECHANISMS OF GROWTH-RELATED PROTEOLYSIS

Apart from the effects on proto-oncogenes by a putative intracellular enzyme, and the previouslydiscussed stimulations of phosphoinositide and cyclic AMP metabolism by thrombin, there are few clues to the mechanism whereby proteinases could stimulate cell growth (Scott, 1987). Inhibition of the GRP blocks the mitogenic action of epidermal growth factor (EGF), but binding and internalization of EGF are not affected (Scott and Seow, 1985, 1986). Proteolytic activation of protein kinase C has been demonstrated, but depends upon cytoplasmic or cytoskeletal proteinases (Tapley and Murray, 1984; Pontremoli et al., 1990). This requirement could be met by the re-internalization of a "pericrine" proteinase as previously discussed, but inhibition of the GRP has no effect on protein kinase C activity in human fibroblasts (Scott, G. K. and Tse, C. A., unpublished results). The mitogenic action of urokinase on human renal cells may involve protein kinase C, as downregulation or inhibition of this enzyme inhibits the mitogenic effect (He et al., 1991). Proteolytic release or activation of an autocrine growth factor (Lieberman, 1983) has been ruled out as the mode of action of the GRP (Scott et al., 1989). An intriguing possibility, which has not yet been experimentally tested, is that an autocrine growth inhibitor could be continually degraded by a growth-related extracellular proteinase, but would accumulate if the enzyme were inhibited. The galactose-binding protein, identified as a labile, negative growth regulator in mouse fibroblasts (Wells and Malluchi, 1991), is also known to exist in humans (Couraud et al., 1989). It may be significant in this regard that GRP levels decline in confluent fibroblasts (Harper et al., 1984); if the galactose-binding protein is a substrate for this enzyme, then it could perhaps accumulate in confluent cultures. A variety of other negative growth regulators (chalones; Mizayaki and Horio, 1989) could act in a similar way, and

there is evidence that SBTI may substitute for one such protein (Strobel-Stevens and Lacey, 1981). A situation which bears superficial comparison with the regulation of cellular proliferation is the modulation of neurite outgrowth from neuroblastoma cells by thrombin and protease-nexin 1. Thrombin causes neurite retraction, and this is inhibited by the nexin, which thus stimulates neurite outgrowth (Monard, 1988; Gerwurz and Cunningham, 1990), but this interplay can be explained in terms of cell-substratum interactions. A similar explanation for cell proliferation is inadequate; numerous experiments with suspension culture cells and, in particular, the parallel effects of GRP inhibition in monolayer and suspension cultures of mouse fibroblasts argue against it (Scott and Seow, 1985). The evidence that a proteinase-induced increase in membrane fluidity is a very early event, prior to the generation of intracellular signals, when cytotoxic lymphocytes interact with target cells may also have some relevance to the problem of growth control (Utsunomiya and Nakaniski, 1986). Older concepts of the mode of action of growthrelated proteolysis may not yet have been fully exploited. Exogenous trypsin stimulates DNA synthesis in isolated nuclei from Chinese hamster fibroblasts, a process that could mimic an intracellular proteinase (Brown et al., 1977), and effects of exogenous proteinases on membrane transport systems have been previously reviewed (Scott, 1987). A model for cellular growth control by a pericellular inhibitor of nutrient uptake, which is also susceptible to proteolytic degradation, has been proposed (Bhargava and Chandani, 1988). MACROMOLECULAR PROTEINASE INHIBITORS AS MITOGENS

A totally new dimension to these studies was introduced by reports that pancreatic secretory trypsin inhibitors (PSTIs) could act as mitogens. In two cases, the human protein was studied (Ogawa et al., 1985; McKeehan et al., 1986). In the latter example, a protein produced by a human hepatoma cell line was identified as a growth factor and then shown to be identical with human PSTI (McKeehan and McKeehan, 1987). Fukuoka et al. (1986) have identified a mitogen in rat pancreatic secretions which they suggest is the rat equivalent of PSTI. McKeehan et al. (1986) have shown that another hepatoma growth factor is a proteinase inhibitor related to UTI and inter-~-trypsin inhibitor, and that other proteinase inhibitors are also mitogenic, though much less effective on a molar basis than human PSTI. It should be noted that McKeehan's group has demonstrated mitogenicity towards human endothelial cells, and has failed to demonstrate activity with fibroblasts, whereas the other reports relate to human or mouse fibroblasts. Cook and Chen (1988) have reported that a range of proteinase inhibitors, including some macromolecular inhibitors, can enhance the growth of transformed rodent fibroblasts. In view of our earlier results in this area, we have reinvestigated the action of various protein proteinase inhibitors on human fibroblasts. We were unable to demonstrate stimulation of cell proliferation with

Proteinases and proteinase inhibitors bovine pancreatic trypsin inhibitor, or with human ~t1AT, but with human and ovine PSTI some stimulation was seen. All of these reagents showed a stimulation of DNA synthesis. For the PSTIs, these effects were seen at concentrations of 10-Ll0-2/~mol.1-1. At higher concentrations, cell proliferation and D N A synthesis were both inhibited. This includes the concentration range used in earlier studies reporting inhibition of proliferation (Scott and Tse, 1988; Hamilton et al., 1990). A biphasic effect on human fibroblast growth by UTI, with a stimulatory effect at low concentrations and inhibition at higher concentrations, has also been observed, though the absolute concentrations required for mitogenesis were much higher than for PSTI. Stimulation of DNA synthesis by 102 nmol.1-1 UTI was inhibited by 2 # m o l . l -j slAT. Some similar effects on bovine endothelial cells were also observed (J. K. Perry and G. K. Scott, unpublished results). The relationship between the mitogenic and the antimitogenic actions of proteinase inhibitors is of considerable interest, but there is, as yet, little experimental evidence which bears on the molecular mechanisms of these processes. It seems unlikely that the two effects represent simply a biphasic process analogous to growth factor-receptor interaction and its down-regulation, though it is possible that downregulation of a mitogenic receptor could contribute to the inhibitory effect seen at higher concentrations of proteinase inhibitors. The antimitogenic action of an individual inhibitor is maximal at approximately the same concentration at which GRP inhibition is maximal. In the case of PSTI, the mitogenic effect is seen at much lower concentrations, at which inhibition of the GRP is not detectable, but with other proteinase inhibitors the two effects occur in a narrower concentration range. This circumstantial evidence suggests that, when proteinase inhibitors act as mitogens, they bind to receptor(s), which is, or are, distinct from the growth-related proteinases so far identified. Nevertheless, given that mitogenic proteinase inhibitors vary considerably with respect to structure, the common feature which we might expect in a "universal" receptor is that it will be a proteinase, or related protein. This hypothesis does not accommodate evidence that thiol proteinase inhibitors have growth-modulatory properties (Sun, 1989); until now only serine proteinases and their inhibitors have been considered in this review. It should also be realized that thiol proteinases can be mitogenic (Suhar et al., 1986; Troen et al., 1988). In addition, one of the tissue inhibitors of metalloproteinases can inhibit endothelial cell growth and has recently been shown to have growth factor activity for human erythroid cells (Stetler-Stevenson et al., 1992). Many cell types in culture produce endogenous proteinase inhibitors (Long and Williamson, 1983; Scott, 1987). Interest has focused on protease-nexins, of which several types have been identified, inhibiting thrombin, plasminogen activators and other proteinases (Knauer et al., 1983). Although the protease-nexins are secreted by cells into the culture medium, the complexes that they form with proteinases become bound to the cell surface and internalized. There is no evidence, as yet, for a direct

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growth-regulatory role for protease-nexins, other than the neurite-outgrowth model already discussed, but their ability to inactivate thrombin and urokinase could imply at least an indirect role (Low et al., 1982). Specific inhibitors for plasminogen activators have also been discovered and, in view of a possible growth-related role for these enzymes, should also be considered in the present context (Laiho and Keski-Oja, 1989). Transforming growth factor ~ is another agent which induces first stimulatory and then inhibitory growth responses in some cells as its concentration increases (Myoken et al., 1990). In the latter case, separate receptors were identified, and there is some evidence for separate mechanisms of action for the two types of response (Moses et al., 1990). These authors have suggested a model for the role of TGF// in wound-healing. TGFp, diffusing from platelets at the site of injury, would have a mitogenic effect on cells at the periphery of the zone of diffusion, but would inhibit cell division and promote differentiation of cells migrating into the immediate area of the injury. The known chemotactic property of TGF~, and its ability to promote the synthesis of components of the extracellular matrix, are consistent with this model. It is possible that circulating proteinase inhibitors could reinforce this effect via the "pericrine" system previously discussed. The loss of sensitivity to growth inhibition by proteinase inhibitors in many tumour cells has already been discussed. In these circumstances, proteinase inhibitors produced and secreted by tumour cells may act as autocrine growth stimulators, or may serve to protect the cells from secreted proteinases involved in invasion and metastasis. The detection of proteinase-inhibitor production in surveys of tumours has usually been qualitative, and there is little evidence as to the growth-promoting effect of these inhibitors on tumour cells. A survey of human colon carcinomas indicated that virtually all of them expressed the mRNA for PSTI, whereas other tumour types did not (Tomita et al., 1990). There was no apparent correlation between the extent of expression and tumour size, degree of differentiation, progression and metastasis. Similar surveys indicate that 1AT is frequently produced by colorectal tumours, though there is, as yet, no consensus view of the significance of this finding, with claims that it has both a positive and a negative correlation with patient survival (Wittekind et al., 1986; Karashima et al., 1990). Of some interest is the fact that PSTI shares some sequence homology with EGF (Hunt et al., 1974). However, there is no evidence for an interaction between PSTI and the EGF receptor (Niinobu et al., 1986; Hamilton et al., 1990). The mitogenic action of proteinase inhibitors, which are structurally unrelated to PSTI or EGF, also argues against a mechanism involving the EGF receptor. EGF has some activity as a proteinase inhibitor, and the EGF-binding protein is an esterase/proteinase which enhances the mitogenic activity of EGF (Lembach, 1976). There is, as yet, no evidence that the EGF-associated enzyme has a specific role in the mitogenic mechanism of the growth factor; it may simply act as an exogenous proteinase, with a synergistic effect on cell

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growth, as is seen with thrombin and E G F (Zetter et al., 1977). Associations between other growth factors and proteinases have been reviewed (Scott, 1982), though there has been no subsequent evidence for the proposed hypothesis linking their actions. Two recent reports indicate that hepatocyte growth factor (HGF) has extensive structural homology with plasmin, though H G F is not itself proteolytically active, and that H G F inhibits the growth of some tumour cell lines (Tashiro et al., 1990; Tajima et aL, 1991). Taken in conjunction with the accumulated evidence already discussed, these reports suggest a hypothetical, evolving "superfamily" of growthmodulatory proteins and receptors, in which the earliest ancestral component was a serine proteinase/proteinase inhibitor complex, exploiting the specific interaction of these two components to give specific growth control. In this hypothesis, evolutionary divergence has resulted in some present-day family members which retain proteinase or inhibitor functions, and others which have lost these functions as a consequence of selection for greater specificity in growth modulation, but which retain structural homology with serine proteinases or inhibitors. The existence of thiol and metalloproteinases and inhibitors, which also affect cell growth, implies convergent evolution and the continued existence of growthrelated reactions which depend upon functional proteinases or inhibitors. Though attractive as a unifying hypothesis, this model is extremely speculative, and does not account for all the known experimental evidence.

SUMMARY AND CONCLUSIONS In reviewing and categorizing the foregoing information, it appears that there are at least three separate major phenomena which have been observed in the field of proteolytic processes involved in cell proliferation. The mitogenic action of exogenous proteinases, typified by experiments with thrombin, is rapidly becoming explicable at the molecular level, though complicated by the existence of multiple effector pathways, by mitogenic effects not caused by proteolytic action, and by antiproliferative mechanisms in some cells. It is almost certainly distinct from the growth-stimulatory activities of endogenous proteinases, in which intracellular and pericellular processes may be distinct, or may form two phases of a single process. Proteinase inhibitors may have a modulatory role by virtue of the inhibition of these enzymes, but also appear to be growth factors in their own right. When we have a better understanding of these discrete processes, we may be in a better position to appreciate the significance of cell-surface, secreted and circulating proteinases and proteinase inhibitors to cell growth in vivo. Acknowledgements--The experiments carried out in the

author's laboratory were made possible with the support of the Auckland Medical Research Foundation, the Auckland University Research Grants Committee, the Cancer Society of New Zealand and the Medical Research Council (now the Health Research Council) of New Zealand. The author thanks Mrs S. Buglass for help in the preparation of the manuscript.

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