Complementary approaches to understanding the role of proteases and their natural inhibitors in neoplastic development: retrospect and prospect

Harry Rubin

Department of Molecular and Cell Biology, Life Sciences Addition, University of California, Berkeley, CA 94720-3200, USA

Email: hrubin{at}uclink4.berkeley.edu


    Abstract
 Top
 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
A great deal of evidence has accumulated in recent years for an important but complex role for proteases in tumor development. However, attempts to treat cancer in humans with anti-proteases have been disappointing, and it has been suggested that more basic groundwork is needed before anti-proteases can be effectively applied. Considerable basic information comes from the recognition that earlier results on transformation of chicken embryo fibroblasts (CEF) by the Bryan strain of Rous sarcoma virus (B-RSV) can be explained in terms of proteases and their inhibitors. In particular, the full but reversible normalization of discrete transformed foci by appropriate concentrations of fetal bovine or of calf serum implies a causal role for multiple proteases in transformation, and the efficacy of treatment with a physiological balance of their natural inhibitors. Addition of certain proteases to contact-inhibited normal cultures was then found to stimulate their proliferation. The toxicity of medium produced by CEF heavily transformed with B-RSV suggests that cachexia and other systemic effects of human cancer may result from vascular dissemination of peptides from pericellular proteolysis within tumors. Comprehensive studies revealed significant increases of plasminogen activator and matrix metalloproteinases (MMPs) after infection of CEF with other strains of RSV, and correlation of the proteases with aspects of transformation. A similar role for proteases is indicated in the transformation of mammalian cells by chemical and physical agents. The information gained from functional experiments on cell transformation in culture is complementary to that obtained from the molecular identification of proteases and their inhibitors in all stages of tumor development. The speed, quantification and easy manipulation of the RSV-CEF transformation assay can be combined with current methods of characterizing proteases and anti-proteases to further enrich our basic knowledge of neoplastic development in cells, and facilitate its application to the treatment of cancer.

Abbreviations: CS, calf serum; CEF, chicken embryo fibroblasts; ECM, extracellular matrix; FBS, fetal bovine serum; MMPs, matrix metalloproteinases; PA, plasminogen activator; PAHs, polycyclic aromatic hydrocarbons; RSV, Rous sarcoma virus (B-RSV, Bryan high titer strain of RSV; SR-RSV, Schmidt-Ruppin strain of RSV); TIMPs, tissue inhibitors of metalloproteinases; TPA, 12-O-tetradecanoyl phorbol-13-acetate, aka PMA (phorbol myristate acetate).


    Introduction
 Top
 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
In recent years there has been a veritable explosion of publications about the role of proteases in neoplastic development. This has come about because of the widespread belief that proteases are involved in the major features of tumor progression, namely invasiveness and metastasis. Much of the heightened research activity has been based on the recognition of a class of proteases designated matrix metalloproteinases (MMPs), which occur on the external surface of the cell and have the capacity to degrade the extracellular matrix. Methods have been developed to identify and classify these proteolytic enzymes, whose number currently stands at about 24 (1). A major part of this activity is concerned with the measurements of the MMPs in tumors and their correlation with invasiveness and metastasis. This has been accompanied by studies of inhibitors of the MMPs known as TIMPs or tissue inhibitors of metalloproteinases. The underlying assumption of this work is that tumors produce an excess of MMPs which exceed the capacity of the TIMPs to modulate their activity; or alternatively, there is a decrease in the concentration of the TIMPs which releases the activity of the MMPs to degrade extracellular matrix (ECM) (2).

Not surprisingly, as the work proceeds, increasing evidence of the diversity and complexity of the interactions becomes apparent. More and more MMPs are identified, as is the versatility of their normal functions apart from digestion of ECM (1). Furthermore, there is no simple relationship between tumor development and increase in the MMPs. In some cases, there is no increase in individual MMPs, or even a decrease, with tumor progression, nor do the TIMPs follow a predictable course. In addition, elucidation of the genome has allowed identification of more than 500 proteases, most of which are not MMPs, and many of these also play a role in neoplastic development. The result is that each type of tumor, and perhaps each individual tumor, exhibits a unique pattern of expression of proteases and TIMPs, with more patterns being almost continuously uncovered. Attempts to apply this burgeoning knowledge to the treatment of human cancer by the use of protease inhibitors has been a failure, and in some cases, actually destructive (3,4).

In the preoccupation with modern means of molecular identification of multiple proteases and their inhibitors, there has been a tendency to forget the lessons learned from a more operational approach to neoplastic behavior based on studies of relatively simple and highly efficient systems of transformation of cells in culture. These systems permit quantitative analysis of transformation over a period of days rather than the months or years required for studies in vivo. They have yielded methods of fully suppressing transformation by natural inhibitors, and of maintaining potentially transformed cells under normal regulation. They also offer models for exploitation by current methods of analysis of proteases and their inhibitors, which should add to our basic understanding of neoplastic development. It is the purpose of this article to re-evaluate the earlier studies in the light of more recent developments and show how the operational and reductionist approaches complement each other. It seems likely that the convergence of different types and levels of analysis will lead to deeper insights about the nature of the neoplastic process and improve methods for coping with it.


    Evidence for proteases and anti-proteases in transformation of chicken embryo fibroblasts (CEF) by RSV
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
The first substantive indication of a relation between excessive proteolytic activity and tumors was the observation in 1925 that cells cultured from sarcomas induced in chickens by RSV caused dissolution of plasma clots in which they were embedded when the plasma came from chickens but did not do so when it came from rabbits (5,6). Addition of a little chicken plasma allowed digestion of the clot, indicating that the sarcoma cells could only activate the extracellular digestive enzymes present in homologous serum. If the pro-enzymes of the rabbit plasma were activated by powerful shaking, and were added to the rabbit plasma clot, they lysed the clot. It was later noted that lysis of a chicken plasma clot was accompanied by rounding of the Rous sarcoma cells that otherwise have a spindle shape in the solid part of the clot (7). This showed that both the round and spindle cells seen in the sarcoma, thought previously to be different cell types, were fibroblasts.

In modern cell culture, dissociated cells are attached directly to a solid surface, usually plastic, and no plasma clots are involved. When a culture of CEF is infected with a low concentration of B-RSV and overlaid with soft agar containing 10% calf serum (CS) or less (with 1.5% chicken serum), each infected cell multiplied to form a multilayered focus of partially rounded cells (Table I) surrounded by a confluent monolayer of normal spindle-shaped cells in orderly arrangement (8). In higher concentrations of CS (15–20%), the transformed foci were reduced in size or disappeared entirely. The same suppression of focus formation occurred in 5–10% fetal bovine serum (FBS). However, the infected cells in the suppressive concentrations of serum produced transformed foci when transferred in permissive concentrations of CS, which allowed enumeration of the infected cells at any time. This ‘infective center’ assay revealed that the infected cells multiplied under the transformation-suppressive conditions as long as the uninfected cells surrounding them were multiplying, but ceased multiplication shortly after the surrounding cells became confluent and underwent contact inhibition. In contrast, exposure of CEF to high enough concentrations of B-RSV to infect more than ~10% all of the cells in the culture resulted in massive morphological transformation of the cultures (Table I), including uninfected cells, regardless of the concentration of either CS or FBS. These observations of Rous sarcoma cells raised the possibility that proteases released by these cells not only lysed plasma clots but played a major role in transforming the cells. The transformation-suppressing effect of high bovine serum concentrations in low multiplicity infections could be accounted for as the inhibition of protease activity mainly by anti-proteases in the sera, plus some released into the medium by the majority population of uninfected cells. The failure of even the highest concentration of sera to suppress transformation in concentrations of B-RSV high enough to infect a large fraction of the cells could result from the release of enough proteases to outstrip the inhibitory power of the sera, which would result in transformation of the infected cells as well as phenotypic transformation of any uninfected cells in the culture. The peptides formed by proteolytic degradation of serum and cell surface proteins may contribute to transformation. This possibility gains credence because the development of transformed foci by CEF infected with B-RSV requires the addition to the medium of tryptose phosphate broth, a tryptic digest of beef heart rich in peptides.


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Table I. Suppression of the transformed phenotype in CEF infected with low concentrations of B-RSV, as a function of serum type and concentrationa

 
The superior suppressive capacity of FBS suggested that it contained disproportionately high concentrations of one or more anti-proteases when compared with CS, especially because FBS has only ~65% the total protein concentration of CS (9). Fetuin, an {alpha}-globulin glycoprotein that constitutes ~45% of the protein in FBS (10) and has high anti-protease activity (11), suppressed focus formation in low multiplicity infection when added to the optimal transformation-permissive concentration (5%) of CS (8). Fetuin was not as complete a suppressor of focus formation as the highest concentrations of CS or FBS, suggesting that it is not the only serum protein that contributes to suppression. Other {alpha}-globulins in human plasma and presumably in bovine and other mammalian sera have been identified as their major protease inhibitors (12) and could account for the transformation-suppressive effects in CS and FBS. In human and presumably other mammalian plasmas, protease inhibitors constitute the third largest group of functional proteins after albumin and the immunoglobulins, representing nearly 10% of the total protein in plasma (13). The transformation-suppressive activity of FBS was non-dialyzable and was inactivated at 80°C for 30 min, indicating an association with proteins.

As already noted, the fact that suppression of transformation by bovine sera was effective only in cultures with a small minority of B-RSV-infected cells suggested that the majority of uninfected CEF may have released protease inhibitors that supplemented those in the sera. And indeed, several days incubation of transformation-permissive medium on CEF cultures infected with low doses of RSV and consisting predominantly of normal CEF, converted that medium into a moderate suppressor of focus formation in lightly infected cultures (8). In contrast, transformation-permissive medium conditioned by heavily transformed cultures, and freed of newly released virus, exhibited no suppression of focus formation when applied to lightly infected cultures, indicating the transformed cells either released ineffective amounts of protease inhibitors, or the latter were hydrolyzed by the protease activity of the infected cells.

A particularly interesting protease inhibitor found in human and bovine plasma is {alpha}-2-macroglobulin (14), which is also secreted by fibroblasts (15). It forms complexes with a variety of collagenases, and is a more effective inhibitor of their activity than are the TIMPs (16). It also inhibits other classes of proteases, unlike other inhibitors, which are specific for a single class of protease (13). It would therefore be of particular interest to determine the contribution of {alpha}-2-macroglobulin to suppression of B-RSV-induced foci by FBS, CS and medium conditioned by CEF.


    Relation between suppression of focus formation by normal conditioned medium and its growth-enhancing activity for low-density cultures
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
CEF multiply very poorly at very low densities in fresh medium (17), and in fact most of the cells are irreversibly damaged under such conditions (18). Medium with low concentrations of CS conditioned by large numbers of CEF becomes supportive of the survival and proliferation of very low densities of the same cells (19). It seemed plausible that there was a relationship, if not an identity, between the conditioning effect of large numbers of CEF for proliferation of small numbers of the same cells, and the transformation-suppressive effect of such media. The conditioning effect on cell proliferation was associated with large, thermolabile molecules released from the surface of CEF (19,20). Even more effective than conditioned medium in supporting proliferation of very small numbers of CEF is a feeder layer of larger numbers of X-irradiated CEF seeded on the same surface in very close proximity to the small number of living cells. This suggested that the growth-promoting factors were most effective in the immediate vicinity of the cells producing them, and this was borne out by the steep drop-off with distance from a moderate number of X-rayed cells in support of growth by very small numbers of CEF (17). Reasoning by analogy, this suggested that the modest transformation-suppressive activity of conditioned medium was a weak reflection of the suppressive effect of the majority of uninfected CEF immediately around the RSV-infected cells in lightly infected cultures. The transformation-suppressive effect of normal cells adjacent to tumor-initiated cells in vivo may be much greater than it is in vitro because the ratio of total cell volume to extracellular fluid volume is thousands of times higher in vivo than in vitro.


    Toxic activity of medium conditioned on CEF cultures heavily transformed by B-RSV
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
The analysis of transformation-suppressive activity of normal CEF by use of a surrogate growth-enhancing assay of conditioned medium was extended by examining the growth-altering activity of medium conditioned on CEF heavily infected and transformed by B-RSV (21). Such medium not only inhibited the proliferation of even moderate numbers of CEF, but caused the death of many of them. This rather surprising observation was in fact consistent with the poor growth of cultures heavily transformed by B-RSV, in sharp contrast to the robust post-confluent growth of infected cells which form discrete, multi-layered transformed foci surrounded by normal cells of lightly infected cultures in permissive medium. It also accounts for the marked improvement of transformed cell growth in medium conditioned on CEF (21). A 2–3-fold dilution of the medium conditioned by B-RSV-transformed cultures with fresh medium unmasked the growth-enhancing activity of the former medium for small numbers of CEF. This suggests that growth-enhancing conditioning factors were produced by the transformed cells but their beneficial effects were overwhelmed in undiluted medium from B-RSV-transformed cultures by an excess of toxic peptides from the partial degradation of serum proteins and conditioning factors by proteases released from the transformed cells, plus the peptides already present in the tryptose phosphate broth which was added to the medium to support transformed focus formation by B-RSV-infected CEF (22).


    Stimulation of growth in confluent CEF cultures by addition of certain proteases
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
The inference that the persistent post-confluent growth in permissive medium of B-RSV-transformed cells in isolated foci results from local release of proteases raised the question whether the addition of proteases would stimulate proliferation and other parameters of transformation in confluent cultures of uninfected CEF. CEF were grown to confluence in a medium containing only chicken serum, to avoid the anti-protease activity of bovine sera, and trypsin was added in small amounts too low to detach, or even visibly retract them (23). In the continuous presence of the trypsin there was a large increase in DNA synthesis peaking at 6–7 h, followed by a peak of mitosis at ~12 h and a doubling of cells by 20 h. Pronase and ficin also induced an increase in DNA synthesis but were much less potent than trypsin. Increased transport of glucose, as measured by 2-deoxyglucose uptake, is another indicator of RSV-transformation (24). Uptake of 2-deoxyglucose in confluent CEF cultures was increased within minutes by trypsin treatment and continued for hours, to the same extent as it did after addition of fresh chicken serum (25). The stimulation of DNA synthesis by trypsin, but not by pronase, was blocked in the presence of soybean trypsin inhibitor, which showed that the stimulation by trypsin was due to its proteolytic activity (23). Trypsin retained its growth-stimulating effect when bound on polystyrene beads, demonstrating that its action was restricted to the cell surface (26).

The stimulation of growth and associated parameters in confluent CEF by trypsin was confirmed in several laboratories (2731). Collagenase, plasmin, {alpha}-chymotrypsin and thrombin also stimulated cell division and 2-deoxyglucose uptake, but only thrombin was as effective as trypsin (29). All the stimulatory proteases except thrombin degraded the major CEF surface protein known variously as Z, fibronectin or LETS (large external transformation sensitive) (29). Thrombin also failed to remove several other surface proteins, indicating they were not essential to the regulation of DNA synthesis and cell division (30). Bromelin was later reported to fully stimulate DNA synthesis and cell proliferation in resting CEF; elastase exhibited functional activity whereas thermolysin, papain and {alpha}-protease were inactive (31). Hence, the growth-stimulating activity of the proteases for confluent CEF was fairly discriminating, but the surface target(s) of the proteases remains unknown. It is noteworthy that at least three growth factors have some proteolytic activity (32).

Although medium conditioned on cultures of B-RSV-transformed CEF was toxic to small and moderate numbers of cells, it was considered possible that it might stimulate growth in confluent, contact-inhibited CEF cultures. Medium containing small amounts of calf and chicken serum was collected daily, beginning on day 3 after infection of CEF with a high concentration of B-RSV (32). Any small toxic peptides were removed by dialysis, and B-RSV was removed by centrifugation. The medium was then diluted into fresh medium in soft agar and overlaid on confluent CEF. Although morphological transformation of the original CEF culture became apparent 2 days after infection with B-RSV, stimulation of DNA synthesis, mitosis and cell number of confluent CEF by the conditioned medium obtained from the infected cultures only became detectable in medium collected from 4 to 5 days after infection, and markedly increased with medium from 6 to 7 day cultures. No stimulation was produced by conditioned medium from cultures infected with SR-RSV. Sonically disrupted normal CEF had stimulatory activity, which rose after transformation by either B-RSV or SR-RSV (33,34). It is noteworthy that the stimulatory activity of the medium conditioned on CEF transformed by B-RSV reached its maximum several days after the cells were fully transformed, and had undergone considerable damage, presumably from accumulation of toxic materials in the medium. This raised the possibility that the stimulation was the result of proteins leaked into the medium from damaged cells. Neither collagenase nor general protease activity could be detected in the stimulatory medium as measured by standard methods (34). Those methods did not include degradation of fibrin, which underlay lysis of plasma clots containing Rous sarcoma cells (5,7). One of the substrates that the B-RSV-conditioned medium failed to digest was azacasein, although casein in overlays of CEF transformed by either B-RSV or SR-RSV was digested (35). Casein digestion was most effective in the presence of chicken serum, and was inhibited by CS and FBS. The presence of CS in the medium used in the growth stimulation by conditioned medium may have inhibited the digestion of azacasein, especially since fibrinolytic activity of Rous sarcoma cells depends on the conversion of serum plasminogen to plasmin, which has only a limited specificity for casein (35).

One of the more interesting proteases that have mitogenic activity when added to CEF is thrombin (29,30,36). It is also mitogenic for mouse embryo fibroblasts and human diploid foreskin fibroblasts (37). Thrombin is derived by cleavage from plasma prothrombin through the combined action of other plasma proteins and thromboplastin activity supplied by cells. The magnitude of the mitogenic response of CEF to thrombin in serum-free medium was very similar to that produced by adding serum (36). Prothrombin exhibited very little mitogenic activity, as did factors purified from serum that are required in converting prothrombin to thrombin. However, the combination of prothrombin with these factors, plus brain thromboplastin, converts the prothrombin to thrombin and induces cell proliferation. These experiments illustrate that prothrombin, although not a mitogen itself, can be a source of mitogenic activity for CEF. As much as 50% of the mitogenic activity of fibrinogen-free plasma is inhibited by a potent protease inhibitor, and it was estimated that the conversion of prothrombin to thrombin accounted for most of the lost activity.

Absorption of the proteins of oxalated plasma and elution from BaSO4 yields two fractions (36). The unabsorbed supernatant had ~68% of the recovered mitogenic activity and most of the protein of the plasma. The eluted fraction had very little mitogenic activity unless thromboplastin was added, when it exhibited ~30% of the total recovered activity. The specific mitogenic activity of the second fraction was 125 times greater than that of CS. The actual contribution of prothrombin as a source of mitogenic activity under physiological conditions is difficult to estimate since thrombin is readily neutralized by the anti-thrombin of plasma and serum. In the microenvironment of a wound clotting area, it was conjectured that the activated proteases, including thrombin, might initiate the process of wound healing before the thrombin is neutralized or sequestered (36).

The mitogenic activity of thrombin raised the question of what surface proteins of CEF are targets for its mitogenic actions. Thrombin exhibits a high specificity in the cleavage of peptide bonds limited to a few proteins like fibrinogen and actin (30). Several proteases that stimulate DNA synthesis in CEF were examined for their capacity to hydrolyze a particularly sensitive protein of the cell surface of CEF of 250 kDa molecular weight that is not present in most transformed cells. One category of proteases, including trypsin, pronase and bromelin, removed the 250 kDa protein in 10 min, but its removal did not increase DNA synthesis measured 12 h after removal of the enzymes. Thrombin did not catalyze the cleavage of the 250 kDa protein even in high concentration of the enzyme acting for an extended period of 30 min. However, thrombin was at least as effective in stimulating DNA synthesis as 2% CS when either was added for 12 h. Two other transformation-sensitive surface proteins were not hydrolyzed by thrombin. Chymotrypsin, which did not stimulate DNA synthesis in CEF in these experiments, did remove the 250 kDa protein, indicating that its removal was not a factor in mitogenesis. Thrombin does bind to and cleave a 43 kDa cell surface component that is responsive to its mitogenic action (38). It must cleave the cell surface receptor to stimulate cell division.


    Characterization of the factors involved in fibrinolysis by transformed cells
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
A new era in neoplastic transformation opened with a series of investigations by Reich and colleagues originally motivated by the observations of Fischer (5,6) on the lysis of plasma clots by RSV-transformed CEF. Instead of using plasma clots for the detection of the proteolytic activity of transformed cells, they employed a film of radioactively labeled fibrin spread on the floor of the culture dishes, to which they attached cells, and measured the release of label from the fibrin into the medium (39). They began with CEF infected with SR-RSV grown in medium supplemented with 10% FBS, but soon extended their studies to other strains of RSV, and the effects of other sera, cell types and tumor viruses on fibrinolysis. Transformed cultures degraded ~84% of the fibrin to peptide fragments of heterogeneous size. A major fraction of the peptide fragments were large enough to be acid insoluble, which implied that the enzyme(s) responsible for fibrinolysis had restricted specificity (39). CEF transformed by all tested strains of RSV exhibited substantial fibrinolytic activity, but uninfected CEF and those infected with non-transforming avian leukosis viruses or cytocidal viruses produced little or no fibrinolytic activity. CEF were infected with a temperature-sensitive mutant of SR-RSV that did not transform at high temperatures but did so at low temperatures. There was no significant fibrinolytic activity at a non-permissive high temperature, but it began to appear 8–10 h after a downshift to a permissive temperature. The fibrinolytic activity appeared several hours before morphological transformation became evident, and slowly disappeared when the temperature was raised to the non-permissive level. These findings were consistent with, but did not prove, a causal relationship between fibrinolytic activity and morphological transformation.

Attempts were made, without success, to demonstrate fibrinolytic activity in medium containing 10% FBS, which had been conditioned on CEF transformed by SR-RSV. That failure suggested that the fibrinolysin was largely bound to the cell surface. This suggestion was borne out by attaching transformed cells to only one side of uncoated coverslips, and placing them either facing down and in contact with labeled fibrin coating the bottom of the culture dish, or facing up and in contact with the medium but away from the fibrin. Only the coverslip cells in direct contact with the fibrin film exhibited fibrinolytic activity. The restriction of proteolytic activity to cell contact with the substratum appeared to hold only under certain conditions and was dependent on the nature of the serum present in the medium.

CEF transformed by SR-RSV were seeded on fibrin, and the initial medium was replaced by media with different types of sera. Chicken and monkey sera allowed much more lysis of fibrin than FBS or CS (39). Mixing FBS with chicken serum decreased the fibrinolytic activity of the medium, indicating there were inhibitors of fibrinolysis in FBS. The suppression of focus formation by 10% FBS in cultures lightly infected with B-RSV (8) suggests the possibility of a relation between fibrinolytic activity and transformation.

Medium containing no serum was collected after incubation with SR-RSV-transformed cells, and added to fibrin-coated dishes with or without the addition of various sera. The serum-free medium displayed no fibrinolytic activity unless serum was added. The addition of chicken serum gave the highest lysis of fibrin, and FBS gave much less. The results indicated that fibrinolysis depended on an interaction between a factor secreted by SR-RSV-transformed cells and a factor in chicken serum, and was inhibited by factors in FBS. Small, synthetic and large, biosynthetic molecules known to inhibit trypsin inhibited the fibrinolytic activity of the medium from transformed cells, indicating that some step in the lysis had trypsin-like specificity. Sera from chickens bearing tumors initiated by SR-RSV were inhibitory to fibrinolysin, suggesting that the inhibitor is formed by the host in response to the tumor and might play an important role in the frequent regression of such tumors. This intriguing observation obviously merits further study on a more quantitative level accompanied by attempts to isolate and test the inhibitory material(s) for anti-protease activity.

To evaluate the generality of the relationship between transformation and fibrinolysis found in RSV-transformed CEF, it was studied in mammalian cells transformed by other RNA viruses and by DNA viruses (40). Hamster embryo fibroblasts transformed by SV40, a DNA virus, had high fibrinolytic activity when grown in hamster, dog or monkey serum, but were negative in chicken serum, FBS or CS. The outstanding difference of the SV40-transformed hamster cells from SR-RSV-transformed CEF is that chicken serum had the highest fibrinolytic activity, and hamster serum had no activity when added to serum-free medium obtained from the transformed CEF (39). FBS and soybean trypsin inhibitor inhibited fibrinolysis by medium from either transformed CEF or hamster embryo fibroblasts. Morphological transformation of the infected hamster cells was correlated with fibrinolysis.

A similar set of experiments was done with mouse embryo fibroblasts transformed by mouse sarcoma virus, like RSV an RNA virus. Hamster serum had little fibrinolytic activity in medium from the non-transformed mouse cells, unlike its high activity in medium from transformed hamster cells. Both chicken serum and FBS inhibited fibrinolysis by medium from the mouse cells. A line of SV40 transformed 3T3 cells exhibited a similar serum specificity for fibrinolysis as those transformed by mouse sarcoma virus. Rat cells and CEF transformed by B77 RSV exhibited different specificities for fibrinolysis. The cumulative results showed the serum specificity for fibrinolysis depended on the cell rather than the virus. None of the uninfected, normal-appearing cells produced fibrinolytic activity in any serum. The similarity of the response to diverse DNA and RNA viruses of any particular cell type suggested that the cellular response might involve a very small number or even a single enzymatic pathway as an obligatory common event. This event may also occur in chemically transformed rat mammary carcinomas, mouse sarcomas and human tumor lines since they all produced a cell factor that activates fibrinolysis (40).

The relation between fibrinolysin and morphological transformation was further studied in a series of experiments with SV40 transformed hamster embryo fibroblasts (41). Most of the experiments were done in dog serum because of its ready availability and its activation for fibrinolysis by transformed hamster cells. The rates of change of both fibrinolysin production and morphological transformation proceeded more rapidly with increases in dog serum concentration, although rapid fibrinolysis production continued for many hours before the full extent of morphological change appeared. When the protein that promotes fibrinolysis was precipitated from the dog serum, the ability to induce morphological transformation was also lost, and both properties were restored when the precipitate was added back to the serum. Once the serum factor was clearly identified as plasminogen (42), both fibrinolysis and morphological change were blocked by removal of plasminogen, and were restored by addition of the purified protein to depleted sera (but see below for contravening results). Co-cultivation of normal and transformed hamster cells sharing the same dog serum medium but separated by a barrier resulted in morphological transformation of the normal cells (41). There was, however, no morphological transformation of the normal cells in FBS or chicken serum in which the specific plasminogen could not be activated by the factor from transformed hamster cells. These experiments suggested that the activation of serum plasminogen by the cell factor was necessary for both fibrinolysis and morphological transformation.

Another experiment, however, showed than an additional factor was needed for morphological transformation (41). The material in FBS that inhibited the activation of plasminogen was removed by acid treatment, which allowed fibrinolysis by the depleted FBS in the presence of transformed hamster cells. The acid-treated FBS did not however support morphological transformation of the hamster cells. Therefore, an inhibitor remained in the FBS that blocked factors necessary for morphological transformation but not for fibrinolysis. The implication of this finding is that either at least one other factor was produced by SV40 transformed hamster cells in addition to the plasminogen activator (PA) that was necessary for morphological transformation, or that PA induced transformation by an activity other than the activation of plasminogen to plasmin.

The serum factor that participates in fibrinolytic activity associated with tumor and transformed cells was purified and definitively identified as plasminogen (42). Chicken plasminogen is activated only by the factor released from cultures of CEF transformed by RSV but not by those released from transformed mammalian cells. The mammalian plasminogens, including that in FBS, are activated to varying extents by a factor released from all the transformed cells tested, including SR-RSV-transformed CEF. The cell factor or PA that plays a role in tumor-associated fibrinolysis was partially purified from the serum-free culture medium of CEF transformed by SR-RSV (43). It is an arginine-specific serine protease of molecular weight ~39 kDa that converts chicken plasminogen to plasmin. It also occurs in cell-associated form in the transformed but not normal cells that were tested. The cell factor acts preferentially on one of two electrophoretic classes of plasminogen. Elevated levels of PA are found in chicken, hamster, mouse and rat embryo fibroblast cultures transformed by DNA and RNA viruses in comparison with the normal primary or secondary cultures from which they are derived. Primary cultures prepared from chemically induced rat mammary carcinomas, mouse skin carcinomas, virus-induced mouse mammary carcinomas and avian viral sarcomas all show increases in cell factor production when compared with their normal tissue counterparts. Hence, there appears to be an association between high levels of cell factor production and transformation both in culture or in animals. However, the production of PA is not restricted to neoplastic cells since high levels of activators have been found in, or released by, granulocytes, activated macrophages, sperm, seminal plasma and endometrial fluid (43). Nevertheless, there is a significant and consistent difference in the amount of activator between the transformed and tumor cells listed above and their normal counterparts when primary cultures of both are compared. Some established cell lines that are non-tumorigenic do release PA, and some that are tumorigenic do not, so the comparisons are valid only for primary or secondary cultures of neoplastic cells and comparable normal cells. Even the restriction to primary cultures of correlation of PA with transformation has to be qualified because primary cells isolated from human lung and prostate, as well as from calf bladder, display high levels of fibrinolytic activity (44).

The intracellular distribution of PA was examined in SR-RSV-transformed CEF (45). The results suggested that a transformation-dependent, neutral serine protease is firmly associated with specific cell membranes, with no significant association with the nucleus, mitochondria, endoplasmic reticulum or lysosomes. The SR-RSV CEF produce 50-fold more of the protease PA than do normal CEF (46). Treatment of the transformed cells with the tumor promoter TPA (12- O-tetradecanoyl phorbol-13-acetate), also known as PMA, further enhances the level of PA ~10-fold, and requires protein synthesis. The transformed cells treated with TPA underwent further morphological changes, including cell elongation followed by retraction and formation of large clusters of rounded, refractile cells, which eventually detach from the surface of the dish. The cultures were initially grown in FBS depleted of plasminogen, with or without TPA. They were then washed and the rest of the experiment, with or without TPA, including the observation of morphological changes, was done in serum-free medium. Therefore, the morphological changes appeared not to result from the conversion of plasminogen to plasmin but are correlated with the increase in PA protease induced by TPA (46).

The additional morphological alterations of CEF transformed by SR-RSV, that were induced by TPA in serum-free medium, were inhibited by several protease inhibitors, including leupeptin, benzamidine and DFP, a specific inhibitor of serine proteases (47). A number of protease inhibitors are ineffective in preventing the TPA-induced morphological changes, including inhibitors of trypsin, chymotrypsin elastase, thrombin and, most importantly, plasmin. The results indicate that the PA can catalytically alter cell morphology and do so independently of plasminogen, which was previously its only known natural substrate.


    Cell surface and extracellular protein targets of proteases
 Top
 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
The cell surface protein commonly known as fibronectin is also called LETS, CSP and Z protein. It is the major glycoprotein on the surface of fibroblasts and, as previously noted, is usually, but not always, decreased in transformed and tumor cells (48). Its removal does not stimulate DNA synthesis or mitosis (30). However, its absorption to the surface of SR-RSV-transformed CEF, which are characteristically shortened, rounded and unaligned, results in their flattening, elongation and a normal pattern of alignment (49). Fibronectin has similar effects on SV40 transformed mouse cells and a variety of other transformed cell lines. The cells assume a more fibroblastic shape, and there is less nuclear overlap between cells. However the addition of fibronectin does not restore growth control in the form of contact inhibition at high density (49), nor does it lower the heightened rate of uptake of 2-deoxyglucose (50). CEF or Balb/c 3T3 fibroblasts transformed by SR-RSV degrade fluorescence-labeled fibronectin covalently linked to the surface of a fixed gelatin film (51). The degradation of fibronectin in the presence of 10% FBS occurs at sites of cell contact with the film, and along their migratory paths. The released peptides are not precipitated by 10% TCA, consistent with a molecular weight of <30 kDa. The release of peptides was partially inhibited by protease inhibitors, but that inhibition did not visibly affect the disappearance of the fluorescence-labeled fibronectin. However, a compound which forms complexes with metal ions inhibited the release of peptides and the visible degradation of the fibronectin, suggesting the involvement of MMPs. The localized degradation of fibronectin at contact sites could break attachments of the cell membrane to the substratum and play a role in the rounding up of transformed cells, but the partial detachment apparently does not account for the loss of growth regulation (36).

ECM is a complex network of proteins and carbohydrate polymers that underlies epithelial cells and surrounds connective tissue cells such as fibroblasts (52). CEF ECM contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant components. Radiolabelled ECM is deposited on the culture dish by CEF, and the cells are removed by EDTA followed by NH4OH. The cells are seeded on the radiolabelled ECM film. Fibronectin constitutes >75% of the total ECM and is also a major component of the surface of fibroblasts (30,49,50). Trypsin treatment removes most of the fibronectin and some of the procollagen-collagen bands from the deposited ECM. The prior removal of fibronectin and other trypsin-sensitive glycoproteins allows the release of collagen components by collagenase treatment. After the seeding of SR-RSV-transformed CEF on ECM in serum-free medium, the cells appear at first more elongated and less refractile than they are on plastic. The clustering of the cells induced by the addition of the promoter TPA is at first completely prevented on ECM; the cells remain elongated and completely aligned. With additional incubation of the TPA-treated cultures for 25 h the ECM appears to be lost, and the cells become disorganized and eventually cluster. The later changes in the TPA-treated cultures correlated with the especially high plasminogen activity of these cultures.

The degradation of ECM by SR-RSV-transformed CEF in TPA is greatly accelerated by the addition of purified plasminogen to the medium (52). The rate of degradation is returned to its value before the addition of plasminogen by including trasylol, an inhibitor of plasmin, which shows that the conversion of plasminogen to plasmin is responsible for the acceleration. Little or no intact fibronectin is present in the medium, but most of it remains acid precipitable, which indicates the fibronectin is only partially degraded. Use of different types of protease inhibitors alone or in combination (Table II) indicates that serine proteases, cysteine proteases, MMPs, arginine proteases and arginine-preferring serine and thiol proteases are all involved in the degradation of ECM by the TPA-treated CEF transformed by SR-RSV in serum-free medium.


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Table II. Degradation of [35S]methionine-labeled ECM by SR-RSV- transformed CEF, with TPA in serum-free medium (no added plasminogen)a

 
Addition of FBS-containing plasminogen to the serum-free medium increases the degradation of ECM by the TPA-treated transformed CEF, but not as much as the addition of purified plasminogen (52). Removal of most of the protease-inhibitory activity from the FBS enhanced the degradation of ECM to the level produced by the addition of purified plasminogen. Removal of plasminogen from FBS reduces the degradation of ECM, indicating that increased degradation in FBS is dependent on its plasminogen content. It should be kept in mind that FBS suppresses transformation of B-RSV-infected CEF, but only when they are a small fraction of the total population, which consists mainly of normal, uninfected CEF. Suppression does not occur when the number of B-RSV-infected CEF is high, presumably because they then degrade the inhibitory components of the FBS. In contrast, when normal CEF constitute a large majority, they add to the transformation-suppressing components of the serum (8).

ECM degradation by transformed cells in direct contact with the ECM film was compared with degradation by cells separated from the ECM (52). The effects of adding plasminogen and {alpha}-2-macroglobulin, a natural protease inhibitor, were studied in both situations. Serum-free medium with TPA was used throughout. Cells in direct contact with ECM degraded the matrix at a relatively slow but progressive rate. When the cells were separated from the ECM, there was a substantial reduction in ECM degradation. This suggests that cell-mediated degradation of ECM is brought about mainly by cell surface proteases. Addition of plasminogen to the medium leads to rapid degradation of ECM whether or not the cells are attached to the ECM, although it is a little slower when the cells are not attached. The large increase in ECM degradation in the presence of plasminogen is consistent with its conversion to plasmin by cellular activator: the plasmin being soluble diffuses rapidly into the medium where it can degrade the ECM, which has no attached cells. The broad-spectrum protease inhibitor {alpha}-2-macroglobulin inhibits ECM degradation by the soluble, diffusable enzymes. The inhibitor has no effect on degradation with cells that are in contact with the matrix in the absence of plasminogen, and little effect in the presence of plasminogen. Each representative of three classes of protease inhibitor induced a partial inhibition of ECM degradation, and the combination of all three inhibitors had an additive effect, while combining two of the same class had no additive effect (Table II). The overall results therefore indicate ECM is degraded by a variety of protease types bound to the surface of and released from transformed cells. These proteases could play a role in the invasiveness of the transformed cells and in determining their morphological appearance although not in their unregulated growth per se according to the evidence presented so far (30,49,50).

A specific monoclonal antibody to avian PA was used to examine the catalytic role of the activator in ECM degradation and morphological alterations induced by SR-RSV-transformed CEF (53). The antibody fully inhibits the conversion of plasminogen to plasmin mediated by pure PA, and inhibits by 60–65% the degradation of ECM by plasminogen-free conditioned medium from the transformed cells, which indicates a direct catalytic role of PA in the process. The anti-PA antibody also inhibits the morphological changes and, to some extent, the overgrowth associated with transformation in the complete absence of plasminogen, showing direct catalytic involvement of PA in these processes as well. This still leaves open the question whether PA can fully account for the transformed phenotype in light of the partial effects of protease inhibitors on ECM degradation (52) and on other critical observations made soon after the report of increased PA in transformed cultures (39), which will now be considered.


    Critical analysis of the relation between PA and key properties of neoplastic transformation
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
The biochemical identification of PA as a serine protease in RSV-transformed CEF that is responsible for fibrinolysis raised the question of its relation to key properties of neoplastic transformation such as growth in suspension, increased saturation density and increased uptake of hexoses. The first indication of a relation between PA level and neoplastic behavior was that soybean trypsin inhibitor reduced colony formation of SV40-transformed hamster embryo fibroblasts in agar by a factor of four (40). Given the broad range of substrate specificity of the trypsin inhibitor, the reduction in colony formation could be attributed to inhibition of proteases other than the one produced by PA cleavage of plasminogen into plasmin. It was then reported that the depletion of plasminogen from FBS lowered the colony count of SV-transformed hamster embryo fibroblasts in agar by a factor of three, and reconstituting the plasminogen partially restored the count (54). This evidence was later contravened by the finding that the procedure that had been used for depleting plasminogen from FBS also removed factors required for conventional growth of cells attached to plastic, and this growth could not be restored with a pure sample of plasminogen (55). There was no correlation between PA production by rat embryo fibroblasts transformed by polyoma virus and their capacity to produce colonies in agar (56). Although PA activity is high in most sarcomas, it was absent in most carcinomas (44,57). It was also present in some normal tissues and in non-tumorigenic cell lines (44). There was no correlation between fibrinolytic activity and transformed growth characteristics in 10 different types of serially cultured mammalian cells (58). Suppression of fibrinolysin activity in a transformed line of 3T3 mouse cells failed to restore normal density growth regulation (59).

The evidence from mammalian cells therefore argues against a general role of PA and fibrinolysis in neoplastic growth. There remains the question of their role in morphological transformation of mammalian cells. There was an excellent correlation between fibrinolysis and development of characteristic morphological changes in SV40-transformed hamster embryo fibroblast cultures (41). Co-cultivation of normal and transformed cells leads to morphological changes in the normal cells similar to those in the transformed cells. However, fibrinolysis and morphological change could be dissociated from one another in FBS. Destruction of the macromolecular inhibitors of fibrinolysis in FBS by acid treatment does not affect the inhibition of morphological change by the FBS. It was therefore concluded that an active fibrinolytic system is a necessary but not sufficient requirement for the morphological changes that accompany transformation of hamster fibroblasts (41). However, there is no convincing evidence of a correlation between fibrinolysis and neoplastic growth in mammalian cells in light of the evidence presented earlier.

The original evidence, however, for an association between PA or fibrinolytic activity and neoplastic growth behavior came from studies of RSV-induced transformation of CEF (39). Evidence against such an association in CEF came from the isolation of colonies from agar of CEF transformed by either of two strains of RSV (60). There was a wide range of variation in PA production among the colonies during their growth on plastic, but no correlation with either transformed morphology, uptake of 2-deoxyglucose, or efficiency of colony formation when resuspended in agar. The lack of correlation does not necessarily rule out some relation between PA and those properties because there is so much heterogeneity in the ordering of these properties between the variant clones and among subclones that a consistent quantitative correlation would be difficult, if not impossible, to establish.

A further attempt to examine correlations was made in CEF with a series of partial transformation mutants of SR-RSV. These mutants varied in transformed morphology, colony size in agar, hexose transport and surface fibronectin content (61). There was a dissociation in degree of expression of these properties among the mutants. CEF infected with the mutants were tested for tumorigenicity in chickens and nude mice to determine its relation to the previously studied properties and to PA (62). All the mutants were weaker tumor producers than wild type SR-RSV (62), and the levels of the transforming gene product pp60src were also lower in cells infected with the mutants (61). Another complication was that some of the mutant tumors regressed after initial growth while others grew progressively. Because the different parameters of transformation are dissociated from one another in the mutant-infected cells, the data were interpreted as supporting a model in which the transforming protein kinase phosphorylates multiple primary targets in generating the transformed phenotype (61). It was concluded that colony formation in agar and PA production were highly correlated with tumor-forming ability of the CEF infected with the mutants. Because of the complexity of the results with PA, the difficulty in quantifying some parameters, and the lack of a statistical evaluation of the correlations, the significance of the asserted correlation is not convincing, especially in light of conflicting evidence (60). The question became moot with the direct demonstration of increases of other proteases in transformed cells.


    Multiplicity of proteases and their inhibitors involved in transformation
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
The focus to this point has been restricted to the possible role of PA in the process of neoplastic transformation. It has been noted that PA can act directly on CEF infected with SR-RSV, as well as by activating plasminogen (47), and that inhibitor studies suggest indirectly that several different types of protease are involved in hydrolyzing ECM by these cells (52). It is also relevant in this regard that the only treatment of RSV-infected CEF that has fully normalized their morphology and growth behavior is the addition of moderate concentrations of FBS or high concentrations of CS (8), both of which undoubtedly contain multiple proteins, especially {alpha}-globulins, that inhibit the whole range of proteases (12,13). In more recent years a new class of proteases known as MMPs, which are localized at the cell surface and digest ECM, has grown to about 24 members (1). DNA sequence analysis indicates 2–3% of the mammalian genome codes for more than 500 proteases (63). Not surprisingly, this proliferation of proteases has led to the identification of more proteases in RSV-transformed CEF, as well as in transformed mammalian cells and in tumors. CEF transformed by SR-RSV secrete a 70 kDa metallo-gelatinase at higher levels than normal CEF (64). It is categorized as an MMP-2, which degrades denatured collagens but not native collagens. It is co-isolated in association with a 22 kDa protein which is a tissue inhibitor of MMPs (TIMP). Two other MMPs were later isolated from SR-RSV-transformed CEF, suggesting that oncogenes stimulate the secretion of MMPs that may play an important role in tumor invasion and metastasis (65). Several different oncogene products from different strains of RSV stimulate the secretion of MMPs through activation of a protein kinase pathway (66). Thus, the cell seems to channel different pathways to produce the same end result of secreting several MMPs.

In addition to the involvement of several MMPs in transformation, there are several TIMPs that modulate the activity of the MMPs (67). One such TIMP in SR-RSV-transformed CEF has already been mentioned in connection with the 70 kDa MMP-2 derived from these cells (64). Another was found in association with the substratum of CEF infected with a temperature-sensitive mutant of the Prague A strain of RSV (68). It is a 21 kDa protein that is stimulated within a few hours of transfer of infected cells to a permissive temperature, and is not present in uninfected CEF or those fully transformed by the wild-type Prague A virus. Biochemical characterization indicates its relation to TIMP-1 and TIMP-2, and it was classified as TIMP-3 (69). To illustrate the complexity of these characterizations, this asserted MMP inhibitor actually accelerates the morphological changes of cell transformation, and stimulates the proliferation of growth-retarded, non-transformed cells maintained in low serum concentrations. The relation of TIMPs to invasiveness of tumors was studied in a line of fibrosarcomas derived from cultured mouse embryo fibroblasts by spontaneous transformation and serial transplantation in mice (2). There was no correlation of invasiveness with collagenase secretion or growth of the cells in agar. There was, however, a striking correlation with TIMP secretion, which was 10–20-fold lower in cells of high invasive potential than in the normal parental fibroblasts or tumor cells with low invasive potential. In CEF transformed by SR-RSV, the activity of TIMP-2 is reduced through a non-covalent complex with an undefined molecule (70). Transformed cells can therefore alter the protease–anti-protease balance by transcriptional or post-transcriptional modification yielding inhibitor-free protease.

PA can be modulated within wide limits by a variety of stimuli, including hormones, inflammatory agents and retinoids as well as by tumor viruses (71). Antibodies against cell surface constituents of two mammalian cell lines caused cell rounding within 2 h of treatment followed by a progressive rise in PA production for 24–36 h. Both effects were reversible and depended on the continued presence of the antibody for their maintenance (71). The increase in PA during RSV-infection of CEF can be accounted for by an increase in mRNA for the specific urokinase PA produced during transformation from less than 1 molecule to 25–60 molecules per cell (72). TPA treatment of normal CEF and of SR-RSV-transformed CEF raised the levels of PA mRNA to 20 and 260 molecules per cell respectively. It is apparent that the virus-induced PA is a cellular, not a viral, gene product yet it is only one of about 1000 different RNA transcripts that accumulate after RSV transformation (73). These observations point to a role for at least three other gene products in modulation of PA expression in CEF. This does not exclude the involvement of other nuclear gene products and transcription factors in response to the single transforming pp60src gene of RSV, and is another example of the complexity of the cellular response.


    The role of proteases in transformation of mammalian fibroblasts by chemical and physical carcinogens
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
Major emphasis to this point has been on the effects of proteases and protease inhibitors on cells transformed by viruses. Evidence suggesting that they also mediate the transforming effects of chemical and physical carcinogens has come from experiments with the C3H 10T1/2 line of mouse embryo fibroblasts. In one set of experiments 10T1/2 cells exposed to carcinogenic polycyclic aromatic hydrocarbons (PAHs) or to X-rays were incubated in a series of increasing concentrations of FBS (74) that paralleled in several respects similar treatment of CEF infected with B-SRV (8). High concentrations of FBS markedly inhibited development of transformed foci in 10T1/2 cells that had been exposed to carcinogenic PAHs or X-rays. The high concentrations of FBS also inhibited focus formation by 10T1/2 cells that had already been transformed by PAHs as long as they were added to a confluent layer of non-transformed 10T1/2 cells. The inhibition was attributed to the increased saturation density and crowding induced by raising the concentrations of FBS, with the assumption that this would cause greater membrane interaction, and allow the preponderance of non-transformed cells to modify the growth of adjacent transformed cells (74). Close analysis of the data effectively rules out this explanation. One of the FBS samples (‘serum A’) supported the same saturation density at concentrations of 5 and 10% (higher concentrations of serum A were not tested for saturation density), but induced 40% reduction in transformed foci between 5 and 10% FBS, and a >24-fold reduction with 15% FBS (Table III). The other FBS sample (‘serum B’) supported only a 17% increase in saturation density between 10 and 20% FBS but reduced total focus formation ~50-fold in that interval (chart 4 of ref. 74). The results are therefore quite similar to the precipitous reduction of foci in CEF infected with low doses of B-RSV when the concentration of FBS was raised from 2.5 to 5%, or CS was raised from 10 to 15% (8). In these concentration ranges there is little effect on saturation density of CEF. The suppression of focus formation is therefore more plausibly accounted for by the direct anti-protease activity of the increased serum concentrations than by non-existent or marginal increases in saturation density.


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Table III. Transformed focus formation by DMBA-treated C3H/10 T1/2 mouse fibroblasts as a function of FBS concentration and saturation densitya

 
Interpretation of later experiments with the 10T1/2 and other mammalian cells in a single concentration (10%) of FBS concluded that the inhibition of focus formation was the result of junctional communication with non-transformed cells through cell-to-cell contacts (75). This was thought to allow the transmission to the transformed cells of agents from the non-transformed cells that increased cyclic AMP phosphorylation and partially inhibited the growth of the transformed cells, although it apparently did not change their morphology. In another set of experiments with rat cells transformed by five different oncogenes, total inhibition of junctional communication did not prevent growth inhibition by surrounding non-transformed rat cells (76). Furthermore, no relation was found between ionic coupling and tumor production in seven fibroblastoid and seven epithelioid mammalian cell lines (77). High population density of normal cells probably contributes to local inhibition of macromolecular proteases by release of anti-proteases with a sharp gradient of concentration from the cell surface outward (17), which would limit the suppression of transformation to a short distance from the normal cells. The immediate contact of the normalized transformed cells with a confluent, contact-inhibited sheet of normal cells would then result in regulated growth of the former. The combination of local effects might seem at first glance to support the idea that junctional communication is needed to inhibit the growth of transformed cells, when a more plausible explanation is that pericellular anti-proteases are effective only at short distances from the cell surface in suppressing transformation. There is, however, a possible alternative explanation, which involves cyclic AMP, junctional communication and proteases. Cyclic AMP strongly inhibits the increase in PA induced by SR-RSV infection or TPA treatment of CEF (78). If this were also true in mammalian cells, junctional communication between non-transformed mammalian cells, with a relatively high concentration of cyclic AMP, and their transformed counterparts would raise the cyclic AMP levels in the latter and inhibit the increased PA and perhaps other proteases that characterize transformed cells. Their altered growth pattern would then directly reflect the reduction in protease activity.

Additional support for a role of proteases in transformation of 10T1/2 cells by chemical and physical carcinogens comes from experiments in which protease inhibitors were added to cultures after exposure to the carcinogens. The 10T1/2 cells in 10% FBS were exposed to PAH for 24 h and then treated separately with each of five protease inhibitors for the entire 6 week period of transformed focus formation (79). Foci were suppressed by four of the five protease inhibitors, but the foci did appear upon removal of the inhibitors. If the anti-protease treatment was restricted to the first 48 h after exposure to MCA, there was no suppression of subsequent focus formation. Treatment of five chemically transformed lines and one tumor cell line with the protease inhibitors revealed marked heterogeneity in reduction of saturation density among the lines, which suggested that more than one protease was involved in transformation (79).

In another set of experiments exposure of the 10T1/2 cells to X-irradiation was followed by separate treatment with each of three protease inhibitors for the 6 week duration of the transforming assay (80). The degree of suppression of focus formation varied between the three protease inhibitors, again suggesting that more than one protease is involved in transformation. Evidence was also presented that antipain, the most effective of the protease inhibitors, could suppress transformation when present for only one day in the first 10 days after irradiation, suggesting an effect on a DNA repair process (80,81). However, this result conflicts with both the failure of suppression by anti-proteases in similarly brief, early treatment of chemically induced transformation of 10T1/2 cells, and the reversibility of long term treatment (79). It also conflicts with the full reversibility of suppression by FBS of B-RSV-induced-transformation of CEF (8), and of chemical transformation of 10T1/2 cells (74). There were fewer than 0.5 foci per culture in control cells that were X-rayed but untreated with anti-proteases in the experiments that reported suppression of transformation by brief, early treatment with anti-proteases (80,81). In these experiments, only a few hundred colony-forming cells per culture survived exposure to the X-rays, but statistical analysis indicates that carcinogen-induced transformation cannot be distinguished from spontaneous transformation of the 10T1/2 line when such small numbers of cells are used (82). The evidence for irreversible suppression of carcinogen-induced transformation by brief, early treatment with anti-proteases (80,81) therefore needs independent confirmation using much larger cell numbers. In contrast, the evidence for reversible suppression during long-term treatment of viral- and carcinogen-induced transformation with anti-proteases was obtained using large numbers of cells, and is therefore on solid ground (8,79). The suppression by specific anti-proteases (79,80) is only partial and therefore adds to the evidence that several proteases are involved in causing the transformed phenotype.


    In vivo evidence for a causal role of proteases in tumor development
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
An early study that drew attention to the relation between proteases and tumors was chiefly concerned with a variety of transplantable carcinomas and sarcomas of mice, but included a few spontaneous and induced tumors (83). The general conclusion of this work was that the small-sized, multiplying cells in the periphery of solid tumor transplants are characterized by high protease activities usually exceeding that of their normal multiplying counterparts. High protease levels were later found in the interstitial fluid of the tumors (84). More recently, there has been a greatly expanded interest in the relationship of proteases to tumors, which has focused on the effect of MMPs on ECM in facilitating invasion and metastasis (85). The MMPs are associated with a number of normal and pathological functions, such as inflammation, cell migration during wound healing, embryo implantation and ovulation (86). They occur mainly as pro-enzymes, which require activation in tumors to acquire the invasive phenotype. The balance of active enzymes and their inhibitors determine whether ECM degradation occurs. It has become apparent, however, that the MMPs have functions in tumors other than facilitating invasion, and have substrates other than ECM. The level of complexity has also increased because proteases in some situations actually inhibit rather than promote tumor development, presumably through their apoptotic effect, and anti-proteases sometimes promote tumor development by activating proteases. To add to the complexity, much of the MMP production in human tumors comes from reactive stromal cells (4). Proteases also appear to function in tumor development at earlier stages than invasion (1). For example, overexpression of a tissue inhibitor of MMPs in mice blocks the development of liver cancers through inhibition of events associated with tumor initiation (87). Non-invasive human basal cell epitheliomas exhibited four to 70 times greater collagenolytic activity than normal skin on a reconstituted collagen substrate (88). Strong inhibition of the enzyme activity by dilutions of human serum indicated the collagenase was in the tumor cells rather than in granulocytes, of which, in any case, there were very few in the tumors. Short-term treatment of mouse skin with a carcinogenic PAH increased collagenase activity, and very high levels of collagenase were detected in papillomas and carcinomas that followed long-term treatment (89).

Transgenic mice have provided more recent examples of MMP influence in pre-invasion stages of tumor development. Overexpression of MMP-7 induced a 13 week reduction in the time to development of primary mammary tumors (90). Overexpression of MMP-13 caused epidermal hyperplasia and a marked increase in tumor incidence when dimethylbenzanthracene (DMBA) and TPA were applied to skin in an initiation and promotion sequence (91). And knockout of MMP-7 in a mouse model of intestinal cancer reduced the incidence of these tumors by 60% (92). The accumulated evidence therefore shows that MMPs play a significant role in the earliest stages of tumor development in a variety of organ systems.

Various methods of inhibiting MMP activity have been tested for the treatment of cancer in Phase II and Phase III trials (1,4). Although some trials have been successful in experimental animals, the results in treating human cancer have been disappointing, and in some cases even harmful (3,93). Some hope has been held out for yet to be tested treatment of early stages of cancer. One of the difficulties is undoubtedly that more than one protease is likely to be activated in any tumor. Another is that inhibition of normal functions of the proteases are damaging to the host. The view has been expressed that much basic groundwork remains to be done before anti-proteases can be effectively applied as therapeutic agents in cancer (4). Much of the basic groundwork can be most efficiently done in relatively simple systems in cell culture, particularly in CEF transformed by RSV, which has already yielded much information. Even there the level of complexity of proteases and their inhibitors is bound to be high, but certainly less so than in the variety of tumors in the organism.


    Complementarity of approaches
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 
Although a relation between proteases and tumors was implied in 1925 by the capacity of a chicken sarcoma induced by RSV to lyse a plasma clot (5), a more general interest in this relation awaited identification of the proteases involved in that lysis (39,40,42,54). CEF infected with SR-RSV displayed a large increase in PA, a serine protease that was correlated with transformation of the cells, and caused lysis of a labeled fibrin film to which the cells were attached. The lysis resulted from activation of plasminogen to plasmin, also a serine protease, which digested the fibrin. Inhibition of PA in SV40-transformed hamster fibroblasts by soybean trypsin inhibitor (54) or by removal of plasminogen from the serum (40) decreased the capacity of the transformed hamster cells to form colonies when suspended in agar. Since proliferation in agar is considered a key characteristic of the neoplastic state, it indicated that the increase of PA was an essential feature of transformation. However, that conclusion had to be qualified because soybean trypsin inhibitor blocks the action of many serine proteases in addition to PA and plasmin, and the method for removing plasminogen also reduced the proliferative capacity of cells attached to a solid surface (55). Furthermore, no correlation was found in rat cells between PA activity and capacity for proliferation in agar (56). Additional doubt is cast on the relation between fibrinolytic activity and transformation by failure to find a correlation between these properties among transformed colonies of SR-RSV-transformed CEF, which had been removed from agar (60). Fibrinolytic activity tended to be absent in carcinomas as opposed to its presence in sarcomas (44,57) and suppression of its activity did not restore contact inhibition in transformed 3T3 cells (59). These and other related findings (e.g. ref. 58) tended to dampen interest in the relation between increased PA activity as expressed in fibrinolysis, and neoplastic transformation.

The problem took another turn when it was found that PA had a direct effect on cells independent of the activation of plasminogen (47), and that other proteases increase in the transformation of CEF by SR-RSV infection (52,6466). Monoclonal antibody to PA inhibited the degradation of ECM, and the morphological changes of the SR-RSV-infected CEF and it limited their overgrowth, indicating a direct role of PA in transformation (53). The inhibition of ECM degradation by antibody was incomplete, in accord with the operation of multienzyme systems, including the MMPs, in the matrix degradation.

The rapid expansion of the protease field, which included the involvement of many MMPs and their inhibitors in tumor development, called my attention to much earlier observations that high concentrations of bovine serum suppressed the development of transformed foci in low dose infection of CEF by B-RSV (8), and that overgrowth of confluent normal CEF was stimulated by low concentrations of trypsin and other proteases (23,26,29,31). FBS was two to three times more effective than CS in suppressing the transformed foci. The suppression did not decrease the number of CEF that produced virus, nor the proliferation rate of the infected CEF at subconfluent densities, but it did maintain the sensitivity of the infected cells to contact inhibition. This normalization of the morphology and growth behavior of B-RSV-CEF is most plausibly attributed to the presence of multiple protease inhibitors in mammalian sera (12,13), which inactivated the multiple proteases later found to increase during transformation of CEF by RSV (39,65,66). One caveat to this conclusion is that most of the experiments demonstrating proteases in the CEF employed strains of RSV other than the B-RSV strain used in the serum-suppression experiments (39,6466). Increased PA has, however, been demonstrated in B-RSV infection of CEF (94), and it is likely that increases in other proteases will be found when examined there.

Some important operational details have come out of the B-RSV transformation studies that may help to explain the experimental failure of protease inhibitors to normalize the full range of neoplastic behavior in transformed cells. Suppression by the bovine sera was effective only at low multiplicities of B-RSV infection, which would result in discrete multilayered transformed foci surrounded by monolayers of normal cells. At concentrations of B-RSV that infect a large number of CEF there was no suppression of transformation by bovine sera even with the highest concentration used. Surprisingly, there was no increase in saturation density of CEF cultures heavily transformed by B-RSV, apparently because transformation was accompanied by the accumulation of toxic material in the medium. This may be explained by the need to add tryptose phosphate broth (TPB) to the medium in order to obtain transformation by B-RSV (22). TPB is a tryptic digest of beef heart and already rich in peptides. The further digestion of these peptides and of proteins in the medium by the proteases of heavily infected cultures might act as toxohormones, which are low molecular weight peptides that have been isolated from tumors and their cultures (9597). Toxohormones induce systemic effects upon inoculation into animals such as cachexia, anemia and immunosuppression which are similar to those found in cancer patients, but the mechanism of their origin has hitherto been unknown. The demonstrated importance of proteases in tumor development and cell transformation suggests that the toxic peptides arise from proteolytic digestion of pericellular proteins in the tumors. The transformation of CEF by B-RSV infection under conditions, which release cytotoxic products could be a model system to investigate the cause of the systemic effects of cancer which are a major cause of morbidity.

The low dose B-RSV-CEF system could be used to identify the transformation-suppressing proteins in serum, and determine whether they are indeed protease inhibitors. Identifying the inhibitors would in turn help to establish how many proteases contribute to the transformed phenotype. A particularly interesting question is the relation of age and tumor-suppressing activity of serum. The results to date indicate that fetal serum is 2–3 times more suppressive of transformation than serum from 4 to 8 month old calves, but no tests were made with adult serum (8). Could the loss of capacity to suppress transformation with age be related to the increase of cancer with age? Adult chicken serum does not inhibit fibrinolysis, but serum from chicken embryos and young chicks has not been tested. It is noteworthy that RSV infection of fibroblasts in connective tissue of chick embryos does not produce sarcomas within the embryo (98100). The appearance of an inhibitor of fibrinolysis in the serum of chickens bearing sarcomas induced by SR-RSV (39) suggests that the host mounts a physiological reaction to inhibit tumor proteases and thereby may account for the frequent regression of avian sarcoma (54). It would be of great interest to determine whether the tumor-bearing chickens also develop serum inhibitors of the MMPs associated with RSV transformation and sarcoma development.

There is already evidence that serum (or plasma) contains several inhibitors of transformation-related proteases. FBS inhibited one or more of the elements involved in fibrinolysis, which includes direct and indirect effects of PA (39,47). Removal of the inhibitors of fibrinolysis from FBS does not remove the inhibitors of morphological transformation (41). The collagenase of human basal cell epitheliomas is inhibited by large dilutions of human serum (88) and it seems likely that bovine serum contains inhibitors of the several MMPs in RSV-transformed CEF (6466). Lest it be thought that FBS only inhibits RSV-transformation of CEF, high concentrations of FBS reverse transformation of mouse fibroblasts induced by chemical and physical carcinogens (74). There has been little or no success in treating human cancer with individual synthetic anti-proteases or with TIMPs and they produce significant side effects (1,3,4). It is therefore worth considering trials with combinations of natural anti-proteases in the balanced concentrations found in serum, which produced complete normalization of B-RSV-transformed CEF and of 10T1/2 cells with no evidence of cell damage.

The failure of anti-proteases in therapy of advanced human cancer, which was originally based on evidence for the role of proteases in the invasion of connective tissue and metastasis, has resulted in calls for more groundwork with particular attention to the role of proteases in early stages of carcinogenesis (1,4). The transformation of CEF by RSV, including the suppression of transformed foci by high concentrations of bovine serum, and the production of cytotoxins in heavily infected cultures, is an ideal system for studying basic aspects of the transformation. A variety of temperature-sensitive mutants of RSV can be enlisted in these studies. The role of proteases, anti-proteases and potential systemic toxohormones can be investigated where precise, quantitative results can be obtained within a week. All the functional molecular components should be identified by up-to-date molecular and biochemical methods. In addition, the simple biological techniques that yielded so much quantitative information with minimum effort should be extended to all the strains and mutants of RSV to obtain information that is complementary to that obtained by molecular analysis. It is possible, and perhaps even probable, that complex combinations of multiple proteases, anti-proteases and toxic peptides in a given context are required to reproduce their true pathogenic effects, and these conditions may not be easily reproduced by reconstituting the separate elements. In that sense, the integrated, holistic and the analytical approaches may be truly complementary to one another in a biological sense, as proposed in Elsasser's theory of organisms (101).


    Acknowledgments
 
Special thanks are due to Dorothy Rubin for editing the manuscript throughout its various drafts. Support for the present work came from the National Institutes of Health grant # G13 LM07483.


    References
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 Abstract
 Introduction
 Evidence for proteases and...
 Relation between suppression of...
 Toxic activity of medium...
 Stimulation of growth in...
 Characterization of the factors...
 Cell surface and extracellular...
 Critical analysis of the...
 Multiplicity of proteases and...
 The role of proteases...
 In vivo evidence for...
 Complementarity of approaches
 References
 

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Received January 2, 2003; accepted February 6, 2003.





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