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Role of surface alterations in cell transformation: the importance of proteases and surface proteins
Cell, Vol. 1, No. 4, April
1974,
Copyright
Q 1974 by MIT
Role of Surface Alterations in Cell Transformation: the Importance of Proteases and Surface Proteins Richard 0. Hynes Imperial Cancer Research Fund Lincoln’s Inn Fields London WC2A 3PX, England
The purpose of this review is to discuss recent results in three related areas of research concerning the nature of the changes which occur when normal fibroblasts are transformed. These topics comprise the agglutination of transformed cells by lectins, the production of proteases by transformed cells, and changes in cell surface proteins. The idea that proteases are involved in transformation is discussed in light of the possible nature of the surface changes occurring on transformation and after proteolytic treatment. Testable models for the nature of this change in surface architecture are presented and discuss their implications for the biological performance of the cell are discussed. In particular, it is suggested that loss or alteration of one or a few proteins could have pleiotropic effects on the behaviour of many surface proteins by influencing their freedom of mobility and thus their interactions with each other and with the interior of the cell. (Choice of subject matter has been selective and other aspects of transformation have been omitted because of lack of space. Rather than attempt to give a complete bibliography, I have quoted recent papers, where references to earlier work may be found.) Surface Changes Detected by Lactlns Lectins are proteins with affinity for specific carbohydrate groups. Several show the property of agglutinating transformed cells, whereas similar concentrations do not agglutinate normal cells (for review see Burger, 1973). This observation indicates that an alteration in surface architecture is associated with transformation. The question this poses is: what is the nature of the change? A simple explanation would be a difference in number of lectin binding sites, but apparently this is not the case. While some authors detect increased binding by transformed cells (Noonan, Levine, and Burger, 1973), others do not (Cline and Livingston, 1971; Ozanne and Sambrook, 1971; Arndt-Jovin and Berg, 1971; Sela et al., 1971). Even where differences have been detected, they were much smaller in magnitude than the differences in agglutinability (Noonan, Levine, and Burger, 1973). Since there is no a priori reason to assume that lectins bind only to one class of glycoprotein, or that all the binding sites are involved in agglutination, however, discovery of differences or similarities in overall binding does not actually answer the question. Moreover, normal cells can be rendered
Review
agglutinable by treatment with proteolytic enzymes (Burger, 1969; lnbar and Sachs, 1969; Sela et al., 1971; Nicolson and Blaustein, 1972) which proves that the relevant binding sites are present in normal cells, but in some way are rendered nonfunctional for agglutination. To summarize, differences in numbers of binding sites are inadequate to explain the differences in agglutinability. An alternative explanation would be that there is a difference in spatial arrangement of binding sites. Such a difference has been detected by labeling membranes with lectin-ferritin conjugates (Nicholson 1971, 1972). Normal fibroblasts show a random, dispersed distribution of binding sites, while transformed and trypsinised cells show clumps of sites. However, membrane proteins are free to diffuse in the plane of the membrane (Frye and Edidin, 1970; Singer and Nicolson, 1972) and binding of multivalent ligands (such as lectins) can cause them to clump together (Taylor et al., 1971). This “patching” phenomenon is inhibited by prefixation of the membrane or by low temperatures. When normal, transformed, and trypsinised fibroblasts were examined, either after prefixation or at O-4%, all showed a random, dispersed pattern of lectin binding sites (Rosenblith et al., 1973; lnbar and Sachs, 1973; de Petris, Raff, and Mallucci, 1973; Nicolson, 1973). Fixation or low temperatures also blocked agglutination, although lectin binding itself was not affected (de Petri% Raff, and Mallucci, 1973; Noonan and Burger, 1973; lnbar et al., 1973a). When cells were labeled with marked lectins at 4°C and then warmed to 25-37”C, or when cells were treated throughout at the higher temperature, both patching of sites and agglutination of transformed and trypsinised cells returned. Both patching and agglutinability thus require movement of sites in response to binding of the lectins. In the contact regions between agglutinated cells, there are always concentrations of lectin binding sites (Nicolson, 1972; de Petris, Raff, and Mallucci, 1973), suggesting that the sequence of events in transformed or trypsinised fibroblasts is: (1) binding of lectins; (2) clumping of binding sites into patches; and (3) formation of multivalent bridges between patches on different cells, leading to agglutination. According to this argument, the reason that normal fibroblasts do not agglutinate would be that their binding sites are not so free to move and therefore do not patch. Exactly this result has been obtained by most authors (Rosenblith et al., 1973: lnbar and Sachs, 1973; Nicolson, 1973); but in another study de Petris, Raff and Mallucci (1973) found patching in all three cell types, although the normal fibroblasts did not agglutinate. The reason for the discrepancy is unclear: there is a direct conflict of results. There may be technical reasons for
Cell 148
the different results. Nicolson (1973) showed that, while normal fibroblasts do not generally show patching, under certain experimental conditions patching can be observed on these cells. It is therefore possible to conclude that agglutination requires movement of lectin binding sites in the plane of the membrane, and that the lectin binding sites on normal fibroblasts are in some way restricted in their lateral movement, since with most techniques used they remain dispersed while those of transformed or trypsinised cells form patches. This restriction of motility is removed by transformation and by proteolysis, and may perhaps be overridden by certain experimental manipulations.
Proteolytic Enzymes Produced by Transformed Cells The fact that superficially similar alterations can be produced in the surfaces of fibroblasts by transformation and by proteolysis raises the question of whether the transformation change is due to proteolysis. That is, do transformed cells produce proteases; and do these proteases act upon the surfaces of cells? The answer to the first question is positive. A wide variety of fibroblasts from different species, transformed in several different ways, show proteolytic activities greatly in excess of those shown by their normal counterparts (Bosmann, 1972; Schnebli, 1972; Unkeless et al., 1973; Ossowski et al., 1973a). Whether these proteases produced by transformed cells actually have any role in transformation is a more complicated question, but several facts support this idea. First, factors released by transformed cells have been shown to affect the properties of normal cells in the direction of transformation, leading to stimulation of overgrowth (Rubin, 1970; B&k, 1973) or migration (Btirk, 1973) under conditions in which these would not normally occur. It is not actually known if these factors are proteases. However, known proteases can stimulate growth of normal cells (Burger, 1970; Sefton and Rubin, 1970). A recent report showed that a pronase treatment sufficient to render normal cells agglutinable did not cause them to divide (Glynn, Thrash, and Cunningham, 1973). This is in apparent disagreement with the earlier results but, if true, might suggest that the surface change detected by lectins is not sufficient to signal growth even if it is involved. A more direct involvement of proteolytic activities generated by transformed cells has been demonstrated. Transformed cells release a factor which activates plasminogen to plasmin (Ossowski et al., 1973b). This is potentially an amplification step since plasminogen is a component of serum. Using serum depleted of plasminogen by affinity chromatography (Deutsch and Mertz, 1970) Ossowski et
al. (1973b) showed that plasmin activity is required for growth in agar, migration into a wound in the absence of serum, and a characteristic series of morphological changes. All these are characteristics of transformed cells, which therefore appear to depend on proteolytic activity on the part of these cells.
Changes In External Proteins In considering the surface changes detected by lectins and the question of whether exogenous proteases produce the same effects as transformation (whether or not transformation itself involves proteases), it is important to know more about the details of these changes. Investigations of molecular events at the cell surface have revealed alterations that take place in its proteins upon transformation. Procedures have been developed in studies on erythrocytes for labeling specifically those proteins that extend wholly or partially outside the cell membrane. The techniques use probes which will not enter the cell either because of their size (Phillips and Morrison, 1971; Hubbard and Cohn, 1972) or their charge (Bender, Gavan, and Berg, 1971; Bretscher, 1971). When the method of iodination catalyzed by lactoperoxidase is applied to fibroblasts, a limited number of proteins is labeled. This analysis shows that normal fibroblasts possess a major external protein which is absent or much reduced in virus-transformed derivatives (figures 1 a, 1 b; Hynes, 1973; Wickus, Branton, and Robbins, 1974; Hogg, 1974). No other changes in external proteins have been detected after transformation. (Poduslo, Greenberg, and Glick (1972) found no differences, but were concerned solely with trypsinised cells.) This transformation-sensitive protein is very sensitive to proteolysis (figures lc, Id) and is removed from the cells by treatments similar to those used to render cells agglutinable by lectins (Hynes, 1973). The protein has a nominal molecular weight of 250,000 and appears to be a glycoprotein (Hynes and Humphryes, 1974). The presence or absence of the 250K protein is temperature sensitive in chicken embryo fibroblasts transformed by ts mutants of Rous sarcoma virus (Wickus, Branton, and Robbins, 1974; Hynes and Wyke, unpublished), and the glycoprotein detected by metabolic labeling is apparently absent from transformed or trypsinised cells, not merely masked (Hynes and Humphryes, 1974) although this is not conclusively proven. If absence of the 250K protein from transformed cells is due to its removal by proteolysis, then cocultivation of transformed cells with normal cells should remove the protein from the normal cells.
Surface 149
Alterations
in Transformation
in the absence of this protein: the evidence is consistent with their being involved, but it is not proven that they are. It is equally possible, given the present data, that transformed cells do not synthesize the 250K protein at all. It is conceivable, though unlikely, that it is modified by transformation so that it is not detected either by iodination or by labeling with sugar precursors. However, it will be assumed below that it is absent on transformed cells. Despite these reservations, we can conclude that a definable alteration in the array of proteins at the cell surface occurs on transformation, and this alteration can be mimicked by proteolytic digestion. This leaves open the question of whether this event is biologically relevant, or whether it is a secondary result of transformation.
Similar Alterations Figure 1. Autoradiographs blast Proteins, Labeled dase; Composite Figure
of SDS-Polyacrylamide by lodination, Catalysed
Gels of Fibroby Lactoperoxl-
(a, b) Comparison of normal hamster fibroblasts NIL6 (a), with virus transformed derivative NIL&HSV6 (b). (c, d) Trypsin treatment after iodination of NIL8 : 3 pg/ml trypsin for 5 min (c); 1 pg/ml trypsin for 10 min (d). (e, f) NIL6 cells were iodinated and then overlaid for 24 hr with either NIL8 (e) or NIL&HSV (f). (g-i) NIL8 cells labeled at different stages in their growth: exponential (g); confluent but still dividing (h); confluent arrested culture (i). The samples represent equal amounts of cell protein. Electrophoresis species travel
was from further.
top to bottom:
lower
molecular
weight
This is, in fact, what happens (figures le, If; Hynes and Pearlstein, unpublished). This observation, while consistent with proteolysis, does not prove it. The labeled proteins on normal cells turn over in any case (unpublished data), and an increase in this turnover for any reason would produce the same result, although the increase would need to be specific for this protein. Several qualifications should be noted in interpreting this change in surface molecules. The first concerns the labeling procedure, which is specific for exposed tyrosine residues (Phillips and Morrison, 1971; Hubbard and Cohn, 1972) so only surface proteins having such residues become labeled. There may well be other proteins, some of which may change on transformation and/or be sensitive to proteolysis. It would be desirable to obtain results using probes specific for different exposed groups. Second, although the 250K protein is defined as external, it is not clear whether It is a true membrane protein or an extracellular differentiated product. It does purify with membrane fractions, and it is not identical with either mucopolysaccharides or collagen, two possible differentiated products (Hynes and Humphryes, 1974). Finally, concerning the possible role of proteases
In Normal Ceils
One question that arises concerning any difference between normal and transformed cells is whether the normal cells demonstrate the same change at any point in their growth. Mitotic cells do exhibit surface changes, some of which are similar to those described above for transformed and trypsinised cells. Normal mitotic cells thus bind lectins more readily than do interphase cells of the same type (Shoham and Sachs, 1972; Noonan, Levine, and Burger, 1973) and are agglutinable (Shoham and Sachs, 1972; Burger, 1973). There are also increases in the expression of some antigens at mitosis and in early Gl (Kuhns and Bramson, 1966; Cikes and Friberg, 1971; Thomas, 1971). Similarly, certain antigens absent from normal cells are expressed in transformed and trypsinised cells (Hayry and Defendi, 1970; Burger, 1971 b). Pardee and Rozengurt (1974) have reviewed the evidence for changes in cell surface properties occurring at or soon after mitosis. Many of these changes are also observed in transformed cells. The changes include an increased rate of transport and biochemical changes as well as the antigenic changes discussed above. Mitotic cells also have lowered cyclic AMP levels (Burger, et al., 1972; Sheppard and Prescott, 1972) and transformed cells and rapidly growing normal cells show reductions in cyclic AMP levels compared with arrested normal cells (for references, see Pardee and Rozengurt, 1974). The labeling of the 250K external protein on the surfaces of normal fibroblasts also shows variations with growth state and stage in the cell cycle. Rapidly growing normal cells show lower levels of iodination of this protein than confluent arrested cells (figures lg-li), although it is never completely absent as in most transformed cells (figure 1 b). This difference may be due to the particular stage of the cell cycle at which confluent cells arrest. There is
Cell 150
a marked reduction in iodination of 250K protein on cells blocked at mitosis, and an increase in cells arrested in early Gl by low serum. Cells arrested in hydroxyurea (late Gl) do not show such an increase in labeling of this protein (Hynes, manuscript in preparation). From these results, it appears that the difference between normal and transformed cells is not merely one of growth rate, but that normal fibroblasts at certain times show a transient change in surface architecture similar to that observed in transformed cells. Table 1 summarises, in highly simplified form, the data discussed in the previous sections, making clear the parallels between transformed cells, protease-treated normal cells, and mitotic cells.
in agglutinability by lectins and an apparent absence of a particular surface protein, although these are not necessarily related. Of course, other antigenic and biochemical changes also occur (and are discussed here only briefly). 2. Similar, but not necessarily identical, changes can be produced by proteolytic treatment of normal cells. 3. Transformed cells produce proteolytic enzymes. 4. The increased agglutinability of transformed and trypsinised cells depends on some freedom of mobility, in the plane of the membrane, of the sites which bind lectins. 5. Normal cells also show fluctuations in some of these properties, especially at mitosis.
Construction of Models for Surface Changes in Transformation In conclusion, I shall attempt to interrelate the results discussed above, and present models arising from them. Some of the ideas have been expressed elsewhere, and many of them are speculative, but my intention in setting them out here is to present working hypotheses and to point out areas where further investigation is necessary. In particular, here I shall discuss hypotheses concerning, (a) the role of proteases in transformation, and (b) the nature and results of the surface changes associated with transformation. The basic relevant facts may be summarized (see Table 1) as: 1. Changes in surface architecture occur in transformed cells. Specifically, these involve an increase
Do Proteases Play a Role In Transformation? One obvious model is that transformed cells release proteolytic enzymes which act upon their surfaces, producing a series of changes which are responsible for the transformed phenotype (Burger, 1973; Pardee, Jimenez de Asua, and Rozengurt, 1974). It should therefore be possible to mimic this phenotype by addition of proteases and to block its expression by inhibiting proteolytic activity. While this model is an attractive one, it remains unproven. Several of the steps in the argument are only weakly supported, some are disputed, and the details of many of them are unclear. It is possible to assemble a list of phenotypic characteristics of transformed cells (by no means complete) and to ask which of these can be produced by proteases in general, and of these, which
Table
1. Properties
of Different
Types
of Cell
Parameter
“Normal”
Density dependent inhibition of growth
+
Migration
Growth
Cells
into wound
Proteolytic AMP
Nutrient
Low plasminogen dependent
enzymes
Low
levels
High
transport
Agglutinability Patching
by lectins
of lectin
Transformed Cells
-a
-
NT
High plasminogen dependent
NT
+ Plasminogen dependent
sites
lodination of 250K external orotein
at confluence
Low
Other
references
in text
NT
Low at all densities
LOW
High
High at all densities
Low
High
High
High
-b
+
+
NT
+
opinion, opinion,
High
Low at confluence
NT Not Tested a For dissenting b For dissenting
Mitotic Cells By definition
in agar
Cyclic
Protease-Treated “Normal” Cells
see Glynn, Thrash, and Cunningham (1973). see de Pet&. Raff, and Malluccl (1973).
Surface 151
Alterations
in Transformation
can be produced by specific proteolytic activities produced by transformed cells, or which can be blocked in the transformed cell by inhibition of proteolysis. Agglutination by lectins can be induced in normal fibroblasts by proteases (see above). Increased agglutination induced by transformed cell proteases has not been described. It would be interesting to know whether plasmin, a proteolytic enzyme generated indirectly by transformed cells (Ossowski et al., 1973b), or the cell factor, a plasminogen activator and therefore a protease (described by Unkeless et al., 1973) can render normal cells agglutinable. Evidence for inhibition of agglutinability in transformed cells by protease inhibitors has not been reported, and it is not known whether growth in plasminogen-depleted serum will render transformed cells nonagglutinable. Loss of the 250K external glycoprotein can be produced by trypsin and also, less effectively, by plasmin (unpublished results). Attempts to repeat this with medium conditioned by transformed cells have so far been negative; but in the experiment described earlier, transformed cells accelerated loss of this protein from normal cells. Transport of 2-deoxyglucose, which is higher in transformed cells than in arrested normal cells, can also be elevated in normal cells by treatment with proteases (Sefton and Rubin, 1971). Transformed cells exhibit reduced levels of cyclic AMP (Otter-r, Johnson, and Pastan, 1971; Sheppard, 1972). This too can be mimicked in normal cells by treatment with proteases (Otten, Johnson, and Pastan, 1972; Sheppard, 1972; Burger et al., 1972). Increased
Table
2. Effects
of Proteolytic
Enzymes Produced
in Normal
Added proteases
Transformed characteristic
rates of transport and reductions in cyclic AMP have been produced by addition of a partially purified factor from transformed cell conditioned medium (Biirk, 1973; Rozengurt and Jimenez de Asua, personal communication). Transformed cells continue to grow under conditions where normal cells stop. Overgrowth of normal cells can be stimulated by defined proteases (Burger, 1970; Sefton and Rubin, 1970) or by factors released by transformed cells (Rubin, 1970; Biirk, 1973). The latter are not known to be proteases. Growth stimulation by pronase has recently been disputed by Glynn, Thrash, and Cunningham, (1973). A speedy resolution of this conflict of data is to be hoped for. Burger (1971a) reported that addition of leukemic cells to normal fibroblasts stimulated growth of the latter, as did isolated membranes from the leukemic cells. This effect was inhibited by protease inhibitors. Such inhibitors also reduced the culture density to which transformed cells grow (Schnebli and Burger, 1972) although it is not clear that these cells were blocked in Gl, as are arrested normal cells. Migration into a wound in the absence of serum and growth in agar are also characteristic of transformed cells. Both these properties depend on plasminogen (Ossowski et al., 1973b), and growth in agar can be prevented by protease inhibitors (Ossowski et al., 1973a). It is not known whether exogenous proteases will give normal cells these properties, but factors produced by transformed cells will stimulate migration of normal ones (Biirk, 1973). These data are summarized in Table 2. It can be seen that many of the required observations are
Cells
by
Transformed cell factors proteases
In Transformed or
Transformed cells
Plasminogen dependent
Cells Blocked protease inhibitors
Surface Agglutinability
+
Absence protein
of 250K +
Increase transport
in
+b
+
i-b
+
+P
+a
+b +b
Internal Reduction
in CAMP
Biological Overgrowth Migration Growth in agar
~This observation was not confirmed by Glynn, b No evidence that this is due to proteolysis. Other
references
in text.
Thrash,
and Cunningham
+
+ + +
(1973).
+
by
Cell 152
lacking. It should also be noted that, where effects have been produced on normal cells by transformed cells or by factors derived from them, this has not usually been proved to be via proteolytic activity. The gaps in Table 2 indicate areas for fu: ture work. It is also necessary to determine which, if any, of these observations are related. For example, are surface changes instrumental in producing the change in cyclic AMP, and are either of these involved in the change in growth properties? Several reports in which various of these changes have been dissociated from one another were mentioned earlier. The surface change detected by lectins can be observed without changes in cyclic AMP (Burger et al., 1972) or growth stimulation (Glynn, Thrash, and Cunningham, !973), and serum or insulin stimulate growth but not agglutinability. Hsie, Jones, and Puck (1971) and Sheppard (1971) reported that addition of dibutyryl cyclic AMP to transformed cells reduced their agglutinability. This suggests that either this surface change is secondary to the cyclic AMP change (which would not be consistent with the results of Burger et al.); or that some feedback exists between the two changes; or that the surface changes produced by transformation, proteolysis, and low cyclic AMP, although superficially similar, are in fact distinct. This latter explanation is perhaps rendered less likely by the fact that the molecular changes detected at the cell surface in transformed and trypsinised cells are the same (Hynes, 1973) given the proviso that additional, undetected differences may also exist. Furthermore, as normal cells stop growing, their cyclic AMP levels rise (Otten, Johnson, and Pastan, 1971, 1972; Seifert and Paul, 1972) as does labeling of 250K external protein. Mitotic cells show low levels of both, and treatment with proteases lowers both (see above). I shall not consider here the sequence of changes occurring inside the cell, for example those involving cyclic AMP, which might be concerned in growth control (Biirk, 1968) since these have been reviewed elsewhere (Pardee and Rozengurt, 1974; Pardee, Jimenez de Asua, and Rozengurt, 1974). These authors suggested a reciprocal feedback relationship between the metabolism of cyclic AMP inside the cell and the state of the surface membrane. It is clearly important to investigate whether such a relationship exists, and what might be its role in growth control. Studies are required in which the various parameters of transformation are ordered and their interdependence during transitions in growth or transformation are determined. The time courses of shifts of several of these parameters have been studied, but unfortunately, not all in the same system. In general, alterations in cyclic AMP
level occur early after a stimulus (Otten, Johnson, and Pastan, 1972; Sheppard, 1972; Rozengurt and Jimenez de Asua, 1973; Jimenez de Asua, Rozengurt, and Dulbecco, 1974) whereas alterations in agglutinability occur quite late (Benjamin and Burger, 1970; Ben-Bassat, Inbar, and Sachs, 1970; Eckhart, Dulbecco, and Burger, 1971). However, in the case of proteolysis, surface changes are detected very early (Burger, 1971a). It is of course possible, given a reciprocal relationship as proposed by Pardee, Jimenez de Asua, and Rozengurt (1974) to accommodate different sequences of events, depending on the nature of the stimulus, the same end result being achieved via different routes. Pardee, Jimenez de Asua, and Rozengurt (1974) have suggested that (at least) two states of the cell surface exist: one (Q) characteristic of quiescent cells, and the other (P) characteristic of proliferating cells (for example, transformed cells). They suggested that this second state is involved in stimulating activity on the part of the cells, and that changes from the Q to the P state are produced by a variety of stimuli, such as serum, proteases, and transformation, thus triggering growth.
What Is the Nature of the Surface Change? Agglutination of cells by lectins requires binding of the lectins to sites on the cell surface, followed by movement of the bound sites to collect in patches. Inhibition of the gathering into patches blocks agglutination (Inbar et al., 1973a; de Petris, Raff, and Mallucci, 1973; Noonan and Burger, 1973). In most cases, precise correlation between ability to patch and to agglutinate has been observed, and normal fibroblasts were found to do neither; but one report showed patching in these cells, without agglutination (de Petris, Raff, and Mallucci, 1973). This suggests that, while patching alone may be insufficient for agglutination, under most conditions the lectinbinding sites on normal cells are less mobile than those on transformed cells. This will be assumed here despite some conflicting results. What, then, is the nature of this restriction? It could lie in differences in lipid composition, although the fact that mobility of surface proteins can be produced by proteolysis both in fibroblasts (see above) and in other cell types (Tillack, Scott, and Marchesi, 1972; lnbar and Sachs, 1973; lnbar et al., 1973b; Nicholson, 1972) suggests that proteins are involved, and once this restriction is removed, the lipid nature of the membranes does not block movement. Microfilaments, which are affected by cytochalasin B, are not involved either in patching or in agglutination; and energy, or even live cells, are not necessary either for agglutination or for its absence
Surface 153
Alterations
in Transformation
in normal cells (de Petris, Raff, and Mallucci, 1973; Noonan and Burger, 1973). A passive inhibition of mobility therefore exists. Edelman, Yahara, and Wang (1973) have developed the idea that surface receptor sites of lymphocytes are attached to structures inside the cell, and that this interaction may be involved in transducing stimuli from the surface to the interior. If this were the situation in fibroblasts, one could postulate that transformation or proteolysis releases the lectin binding sites from this attachment, thus making them freer to move in response to lectin binding. I should like to propose as an additional hypothesis that the lectin binding sites might be attached, either permanently or under the influence of lectins, to a protein on or in the outer surface of the membrane. This might restrict their motion, and its loss or alteration would then release the restriction. An obvious candidate is the 250K protein discussed above. For this model, it does not matter whether the absence of this protein on transformed cells is due to proteolysis or not. The general models could equally well apply to other, as yet undetected, surface proteins, which could fulfill the same role as that suggested here for the 250K protein. The reason for describing models specifically in terms of the latter protein is that they are testable. Several alternative models for the situation in normal cells can therefore be proposed. 1. Surface proteins are attached individually to structures inside the cell; specifically, assemblies of colchicine-binding proteins (Edelman, Yahara, and Wang, 1973). These internal structures control the mobility of the surface molecules, and interactions between different surface molecules occur via the internal structures. 2. Restriction of the mobility of surface proteins is due to the effects of other membrane proteins. Two distinct versions of this model can be considered. a) The restriction on mobility is direct and constitutive; that is, the surface proteins are ordered into small groups by noncovalent bonding. b) The restriction only arises after interaction with multivalent ligands such as lectins; for example, by cross-linking into small groups, which prevents lateral diffusion previously allowed. In its extreme form, this model suggests that the restriction of mobility involves only proteins in or on the membrane. 3. A model which is a hybrid of the previous two, in that only some surface molecules are linked directly to internal structures (for example, microtubules), while the rest are free except in so far as they are bound to the first set. Either constitutive or induced binding could be envisaged as in the second model proposed above. This sort of model could also be used to explain the concanavalin
A-mediated restriction of surface immunoglobulin movement observed in lymphocytes (see Edelman, Yahara, and Wang, 1973). It is then necessary to ask how each of these models fits the experimental results concerning transformation, and what would be their predictions. Attachment to Internal Structures According to the first model, freedom of movement of surface molecules would be produced by alterations in the colchicine-binding protein assembly inside the cell. On this model, loss of a major surface protein, for example the 250K protein, might derange this assembly so that all the other receptors are released from their association with it, thus becoming free to move. Alternatively, the primary alteration might be in the colchicine-binding protein assembly, leading to the release of the surface receptors and allowing them to move freely. Two observations of interest in this context are that plasma membranes of transformed cells have reduced quantities of actin and of another protein of similar size (Wickus and Robbins, 1973) and that the 250K protein is reduced on the surfaces of cells blocked in mitosis by colchicine (Hynes, unpublished) or vinblastine (E. Pearlstein, personal communication). Whether this is due to the inhibitors or the state of mitosis is not clear; although cells that were not rounded up in inhibitors still had the 250K protein. Investigation of mitotic cells not treated with inhibitors is indicated. If colchicine-binding proteins were concerned in anchoring membrane proteins, then treatment with colchicine might be expected to produce increased mobility. This appears to be the case in lymphocytes (Edelman, Yahara, and Wang, 1973). Further, if freedom of mobility is a requirement for lectinmediated agglutination, as it seems to be, colchitine might increase it. In fact, Yin, Ukena, and Berlin (1972) showed that colchicine and similar agents actually inhibit concanavalin-mediated binding of erythrocytes to transformed fibroblasts. It is not clear if this is an assay for agglutination as generally understood. A more direct determination of the effect of colchicine on the mobility of surface molecules is required. Restrlction of Mobility by Membrane Proteins In considering the second model, the restriction of mobility by membrane proteins, it is obvious that an alteration of one or a few of these might remove the restriction. It is more difficult to see how the restriction is imposed in the first place. Considering this model specifically in terms of the 250K protein, the other surface glycoproteins would either be associated with it permanently, or become associated
Cell 154
via the binding of lectins. The latter would require that the 250K protein binds lectins, and this appears to be the case (unpublished). The question then arises as to how binding of the 250K protein (or any other) could prevent movement. One might suppose that, being a large molecule, it would diffuse more slowly, especially when associated with other proteins. As Edidin (1972) points out, the dependence of diffusion rate on size is linear, so that the restriction produced in this way would probably be too small. Alternatively, an extended network of 250K protein might exist, forming a sort of exoskeleton. Finally, as suggested above, presence of the 250K protein might affect membrane fluidity by interacting with the lipids, although the mechanism by which this might occur is obscure.
Internal Linkage of the 250K Protein The third possibility, that the 250K protein (but not the other proteins, or at least not all of them) is directly anchored by linkage to internal structures, combines features of both the previous models. In this case, removal of this protein or its linkage to the internal structures would release the restriction on mobility of the other surface molecules, and also remove their communication with the interior. The linkage might be direct or indirect (e.g., via intramembranous particles). It would be possible to test whether the 250K protein extends through the membrane as does the major glycoprotein of erythrocyte membranes (Bretscher, 1971). Both these latter two models would predict that mobility of some surface proteins would be affected by the presence or absence of others, and by the degree of cross-bridging which existed between them. For example, the use of non cross-linking probes could test whether the lectin binding sites on fibroblasts are freely diffusing or not; and whether presence or absence of the 250K protein has any effect on this mobility, either intrinsically, or after cross-linking by multivalent ligands. Similar tests could be applied to surface antigens and surface enzymes, given development of the relevant probes (see Edelman, Yahara, and Wang, 1973). Nearest neighbour analysis, using chemical crosslinks followed by investigation by biochemical techniques, could also allow investigation of the associations existing prior to, and after, perturbation of the surface. A similar approach has been applied to erythrocytes (Steck, 1972); but given recent knowledge about the readiness of cell surface molecules to redistribute, these studies need repeating under conditions in which care is taken to avoid perturbation by the probes themselves. Another prediction is that growing normal cells might be more readily agglutinable and show greater free-
dom of movement of surface molecules than would confluent arrested cells, since the latter apparently have more of the 250K protein and show changes in other membrane properties (Pardee and Rozengurt, 1974). It is necessary also to consider the possible biological relevance of such surface changes, although such discussion must at present be speculative. If the change detected by lectins involves a release of many surface molecules into a condition of greater freedom of movement than normal, then pleiotropic changes in surface properties would be expected. Presumably, many surface molecules function as enzymes, receptors for biplogical signals, or in transport. Disruption of the normal interactions between surface molecules and their contact with the inside of the cell should interfere with their functions. lnbar et al. (19736) have shown a correlation between mobility of surface sites and ability to respond to external signals. And Edelman, Yahara, and Wang (1973) found that, if sufficient concanavalin A was added to lymphocytes to restrict mobility, the mitogenic activity of the concanavalin A was blocked, suggesting that some surface movement is required for mitogenesis. At present, the meaning of these observations is not clear; but they may indicate that mobility of surface sites is important in their function, so that disturbance of the normal controls on this mobility is likely to have biological effects. One of the models considered earlier involved surface molecules permanently arranged in groups. Obviously, disruption of such an assemblage by loss of one of its members would affect the activity of the others-especially if, at the same time, they lost their interaction with the interior of the cell. There exist many observations of changes in antigenie and biochemical characteristics at the surfaces of transformed cells. Some of these are also produced by proteolysis (e.g. Hdyry and Defendi, 1970; Burger, 1971 b). Disruption of preexisting relationships among surface proteins in the way I have proposed could well lead to such changes: for example, exposure of previously buried antigens (see Collins and Black, 1974) altered interrelationships between surface enzymes and their substrates, either allowing interactions previously blocked (Roth and White, 1972), or preventing the normal interactions. Similarly, alterations in the control of nutrient transport and in the metabolism of cyclic AMP by membrane bound enzymes could be produced by such a membrane alteration (see Pardee, Jimenez de Asua, and Rozengurt, 1974). Reciprocally, cyclic AMP could affect the membrane via protein kinase activity (Johnson et al., 1972; Rubin and Rosen, 1973).
Surface 155
Alterations
in Transformation
Mobility of Surface Molecules in Normal Cells Finally, it is of interest to consider the fact that several of the changes observed in transformed and protease-treated cells are also shown by mitotic cells (see above). It has been suggested before (Burger, 1971 b, 1973; Pardee, Jiminez de Asua, and Rozengurt, 1974) that the transient expression of a surface change triggers the next cycle of replication in normal cells. Normally this alteration disappears as cells enter Gl . This is true for lectin binding (Noonan, Levine, and Burger, 1973), and for the 250K surface protein (Hynes, unpublished), as well as for cyclic AMP levels (Burger, et al., 1972). It would be of great interest to know whether there is a transient release of proteases at mitosis, triggering these mitotic changes. A unifying hypothesis would then be that transformed cells become locked in this state. This is, of course, far from proven; and in any case, there is no reason why similar changes cannot be produced by a variety of mechanisms. As discussed above and elsewhere (Pardee and Rozengurt, 1974), several of the parameters considered in this review are modulated in normal cells. The suggestion implicit in the models presented earlier is that this modulation could be effected by controlling the freedom of mobility of surface molecules, and that the control mechanisms are aborted by proteolysis or transformation. Thus, growth stimulation by serum or insulin would be envisaged as leading to greater freedom of mobility, but not to the same degree as complete removal of the controls. According to the nomenclature of Pardee, Jimenez de Asua, and Rozengurt (1974), the surface states of quiescent cells, stimulated normal cells, and transformed cells could be labeled Q, Pa, and Pb, respectively, and in order of increasing mobility of surface molecules. The aim of this review has been to collect some of the various facts related to these hypotheses, and to analyse how far they support the ideas presented above. These are intended to form working hypotheses, which derive from many previous suggestions. I have tried to indicate the many points on which further data are needed, in the hope that some or all of the hypotheses can be eliminated. In two recent papers, Gahmberg and Hakomori (1973, Proc. Nat. Acad. Sci. USA 70, 3329), using external labeling by galactose oxidase plus sodium borotritiide, detected a large external glycoprotein absent on transformed cells, and Berlin et al. (1974, Nature 247, 45) presented evidence consistent with a model in which surface proteins are attached to microtubule proteins.
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Q 1974 by MIT
Role of Surface Alterations in Cell Transformation: the Importance of Proteases and Surface Proteins Richard 0. Hynes Imperial Cancer Research Fund Lincoln’s Inn Fields London WC2A 3PX, England
The purpose of this review is to discuss recent results in three related areas of research concerning the nature of the changes which occur when normal fibroblasts are transformed. These topics comprise the agglutination of transformed cells by lectins, the production of proteases by transformed cells, and changes in cell surface proteins. The idea that proteases are involved in transformation is discussed in light of the possible nature of the surface changes occurring on transformation and after proteolytic treatment. Testable models for the nature of this change in surface architecture are presented and discuss their implications for the biological performance of the cell are discussed. In particular, it is suggested that loss or alteration of one or a few proteins could have pleiotropic effects on the behaviour of many surface proteins by influencing their freedom of mobility and thus their interactions with each other and with the interior of the cell. (Choice of subject matter has been selective and other aspects of transformation have been omitted because of lack of space. Rather than attempt to give a complete bibliography, I have quoted recent papers, where references to earlier work may be found.) Surface Changes Detected by Lactlns Lectins are proteins with affinity for specific carbohydrate groups. Several show the property of agglutinating transformed cells, whereas similar concentrations do not agglutinate normal cells (for review see Burger, 1973). This observation indicates that an alteration in surface architecture is associated with transformation. The question this poses is: what is the nature of the change? A simple explanation would be a difference in number of lectin binding sites, but apparently this is not the case. While some authors detect increased binding by transformed cells (Noonan, Levine, and Burger, 1973), others do not (Cline and Livingston, 1971; Ozanne and Sambrook, 1971; Arndt-Jovin and Berg, 1971; Sela et al., 1971). Even where differences have been detected, they were much smaller in magnitude than the differences in agglutinability (Noonan, Levine, and Burger, 1973). Since there is no a priori reason to assume that lectins bind only to one class of glycoprotein, or that all the binding sites are involved in agglutination, however, discovery of differences or similarities in overall binding does not actually answer the question. Moreover, normal cells can be rendered
Review
agglutinable by treatment with proteolytic enzymes (Burger, 1969; lnbar and Sachs, 1969; Sela et al., 1971; Nicolson and Blaustein, 1972) which proves that the relevant binding sites are present in normal cells, but in some way are rendered nonfunctional for agglutination. To summarize, differences in numbers of binding sites are inadequate to explain the differences in agglutinability. An alternative explanation would be that there is a difference in spatial arrangement of binding sites. Such a difference has been detected by labeling membranes with lectin-ferritin conjugates (Nicholson 1971, 1972). Normal fibroblasts show a random, dispersed distribution of binding sites, while transformed and trypsinised cells show clumps of sites. However, membrane proteins are free to diffuse in the plane of the membrane (Frye and Edidin, 1970; Singer and Nicolson, 1972) and binding of multivalent ligands (such as lectins) can cause them to clump together (Taylor et al., 1971). This “patching” phenomenon is inhibited by prefixation of the membrane or by low temperatures. When normal, transformed, and trypsinised fibroblasts were examined, either after prefixation or at O-4%, all showed a random, dispersed pattern of lectin binding sites (Rosenblith et al., 1973; lnbar and Sachs, 1973; de Petris, Raff, and Mallucci, 1973; Nicolson, 1973). Fixation or low temperatures also blocked agglutination, although lectin binding itself was not affected (de Petri% Raff, and Mallucci, 1973; Noonan and Burger, 1973; lnbar et al., 1973a). When cells were labeled with marked lectins at 4°C and then warmed to 25-37”C, or when cells were treated throughout at the higher temperature, both patching of sites and agglutination of transformed and trypsinised cells returned. Both patching and agglutinability thus require movement of sites in response to binding of the lectins. In the contact regions between agglutinated cells, there are always concentrations of lectin binding sites (Nicolson, 1972; de Petris, Raff, and Mallucci, 1973), suggesting that the sequence of events in transformed or trypsinised fibroblasts is: (1) binding of lectins; (2) clumping of binding sites into patches; and (3) formation of multivalent bridges between patches on different cells, leading to agglutination. According to this argument, the reason that normal fibroblasts do not agglutinate would be that their binding sites are not so free to move and therefore do not patch. Exactly this result has been obtained by most authors (Rosenblith et al., 1973: lnbar and Sachs, 1973; Nicolson, 1973); but in another study de Petris, Raff and Mallucci (1973) found patching in all three cell types, although the normal fibroblasts did not agglutinate. The reason for the discrepancy is unclear: there is a direct conflict of results. There may be technical reasons for
Cell 148
the different results. Nicolson (1973) showed that, while normal fibroblasts do not generally show patching, under certain experimental conditions patching can be observed on these cells. It is therefore possible to conclude that agglutination requires movement of lectin binding sites in the plane of the membrane, and that the lectin binding sites on normal fibroblasts are in some way restricted in their lateral movement, since with most techniques used they remain dispersed while those of transformed or trypsinised cells form patches. This restriction of motility is removed by transformation and by proteolysis, and may perhaps be overridden by certain experimental manipulations.
Proteolytic Enzymes Produced by Transformed Cells The fact that superficially similar alterations can be produced in the surfaces of fibroblasts by transformation and by proteolysis raises the question of whether the transformation change is due to proteolysis. That is, do transformed cells produce proteases; and do these proteases act upon the surfaces of cells? The answer to the first question is positive. A wide variety of fibroblasts from different species, transformed in several different ways, show proteolytic activities greatly in excess of those shown by their normal counterparts (Bosmann, 1972; Schnebli, 1972; Unkeless et al., 1973; Ossowski et al., 1973a). Whether these proteases produced by transformed cells actually have any role in transformation is a more complicated question, but several facts support this idea. First, factors released by transformed cells have been shown to affect the properties of normal cells in the direction of transformation, leading to stimulation of overgrowth (Rubin, 1970; B&k, 1973) or migration (Btirk, 1973) under conditions in which these would not normally occur. It is not actually known if these factors are proteases. However, known proteases can stimulate growth of normal cells (Burger, 1970; Sefton and Rubin, 1970). A recent report showed that a pronase treatment sufficient to render normal cells agglutinable did not cause them to divide (Glynn, Thrash, and Cunningham, 1973). This is in apparent disagreement with the earlier results but, if true, might suggest that the surface change detected by lectins is not sufficient to signal growth even if it is involved. A more direct involvement of proteolytic activities generated by transformed cells has been demonstrated. Transformed cells release a factor which activates plasminogen to plasmin (Ossowski et al., 1973b). This is potentially an amplification step since plasminogen is a component of serum. Using serum depleted of plasminogen by affinity chromatography (Deutsch and Mertz, 1970) Ossowski et
al. (1973b) showed that plasmin activity is required for growth in agar, migration into a wound in the absence of serum, and a characteristic series of morphological changes. All these are characteristics of transformed cells, which therefore appear to depend on proteolytic activity on the part of these cells.
Changes In External Proteins In considering the surface changes detected by lectins and the question of whether exogenous proteases produce the same effects as transformation (whether or not transformation itself involves proteases), it is important to know more about the details of these changes. Investigations of molecular events at the cell surface have revealed alterations that take place in its proteins upon transformation. Procedures have been developed in studies on erythrocytes for labeling specifically those proteins that extend wholly or partially outside the cell membrane. The techniques use probes which will not enter the cell either because of their size (Phillips and Morrison, 1971; Hubbard and Cohn, 1972) or their charge (Bender, Gavan, and Berg, 1971; Bretscher, 1971). When the method of iodination catalyzed by lactoperoxidase is applied to fibroblasts, a limited number of proteins is labeled. This analysis shows that normal fibroblasts possess a major external protein which is absent or much reduced in virus-transformed derivatives (figures 1 a, 1 b; Hynes, 1973; Wickus, Branton, and Robbins, 1974; Hogg, 1974). No other changes in external proteins have been detected after transformation. (Poduslo, Greenberg, and Glick (1972) found no differences, but were concerned solely with trypsinised cells.) This transformation-sensitive protein is very sensitive to proteolysis (figures lc, Id) and is removed from the cells by treatments similar to those used to render cells agglutinable by lectins (Hynes, 1973). The protein has a nominal molecular weight of 250,000 and appears to be a glycoprotein (Hynes and Humphryes, 1974). The presence or absence of the 250K protein is temperature sensitive in chicken embryo fibroblasts transformed by ts mutants of Rous sarcoma virus (Wickus, Branton, and Robbins, 1974; Hynes and Wyke, unpublished), and the glycoprotein detected by metabolic labeling is apparently absent from transformed or trypsinised cells, not merely masked (Hynes and Humphryes, 1974) although this is not conclusively proven. If absence of the 250K protein from transformed cells is due to its removal by proteolysis, then cocultivation of transformed cells with normal cells should remove the protein from the normal cells.
Surface 149
Alterations
in Transformation
in the absence of this protein: the evidence is consistent with their being involved, but it is not proven that they are. It is equally possible, given the present data, that transformed cells do not synthesize the 250K protein at all. It is conceivable, though unlikely, that it is modified by transformation so that it is not detected either by iodination or by labeling with sugar precursors. However, it will be assumed below that it is absent on transformed cells. Despite these reservations, we can conclude that a definable alteration in the array of proteins at the cell surface occurs on transformation, and this alteration can be mimicked by proteolytic digestion. This leaves open the question of whether this event is biologically relevant, or whether it is a secondary result of transformation.
Similar Alterations Figure 1. Autoradiographs blast Proteins, Labeled dase; Composite Figure
of SDS-Polyacrylamide by lodination, Catalysed
Gels of Fibroby Lactoperoxl-
(a, b) Comparison of normal hamster fibroblasts NIL6 (a), with virus transformed derivative NIL&HSV6 (b). (c, d) Trypsin treatment after iodination of NIL8 : 3 pg/ml trypsin for 5 min (c); 1 pg/ml trypsin for 10 min (d). (e, f) NIL6 cells were iodinated and then overlaid for 24 hr with either NIL8 (e) or NIL&HSV (f). (g-i) NIL8 cells labeled at different stages in their growth: exponential (g); confluent but still dividing (h); confluent arrested culture (i). The samples represent equal amounts of cell protein. Electrophoresis species travel
was from further.
top to bottom:
lower
molecular
weight
This is, in fact, what happens (figures le, If; Hynes and Pearlstein, unpublished). This observation, while consistent with proteolysis, does not prove it. The labeled proteins on normal cells turn over in any case (unpublished data), and an increase in this turnover for any reason would produce the same result, although the increase would need to be specific for this protein. Several qualifications should be noted in interpreting this change in surface molecules. The first concerns the labeling procedure, which is specific for exposed tyrosine residues (Phillips and Morrison, 1971; Hubbard and Cohn, 1972) so only surface proteins having such residues become labeled. There may well be other proteins, some of which may change on transformation and/or be sensitive to proteolysis. It would be desirable to obtain results using probes specific for different exposed groups. Second, although the 250K protein is defined as external, it is not clear whether It is a true membrane protein or an extracellular differentiated product. It does purify with membrane fractions, and it is not identical with either mucopolysaccharides or collagen, two possible differentiated products (Hynes and Humphryes, 1974). Finally, concerning the possible role of proteases
In Normal Ceils
One question that arises concerning any difference between normal and transformed cells is whether the normal cells demonstrate the same change at any point in their growth. Mitotic cells do exhibit surface changes, some of which are similar to those described above for transformed and trypsinised cells. Normal mitotic cells thus bind lectins more readily than do interphase cells of the same type (Shoham and Sachs, 1972; Noonan, Levine, and Burger, 1973) and are agglutinable (Shoham and Sachs, 1972; Burger, 1973). There are also increases in the expression of some antigens at mitosis and in early Gl (Kuhns and Bramson, 1966; Cikes and Friberg, 1971; Thomas, 1971). Similarly, certain antigens absent from normal cells are expressed in transformed and trypsinised cells (Hayry and Defendi, 1970; Burger, 1971 b). Pardee and Rozengurt (1974) have reviewed the evidence for changes in cell surface properties occurring at or soon after mitosis. Many of these changes are also observed in transformed cells. The changes include an increased rate of transport and biochemical changes as well as the antigenic changes discussed above. Mitotic cells also have lowered cyclic AMP levels (Burger, et al., 1972; Sheppard and Prescott, 1972) and transformed cells and rapidly growing normal cells show reductions in cyclic AMP levels compared with arrested normal cells (for references, see Pardee and Rozengurt, 1974). The labeling of the 250K external protein on the surfaces of normal fibroblasts also shows variations with growth state and stage in the cell cycle. Rapidly growing normal cells show lower levels of iodination of this protein than confluent arrested cells (figures lg-li), although it is never completely absent as in most transformed cells (figure 1 b). This difference may be due to the particular stage of the cell cycle at which confluent cells arrest. There is
Cell 150
a marked reduction in iodination of 250K protein on cells blocked at mitosis, and an increase in cells arrested in early Gl by low serum. Cells arrested in hydroxyurea (late Gl) do not show such an increase in labeling of this protein (Hynes, manuscript in preparation). From these results, it appears that the difference between normal and transformed cells is not merely one of growth rate, but that normal fibroblasts at certain times show a transient change in surface architecture similar to that observed in transformed cells. Table 1 summarises, in highly simplified form, the data discussed in the previous sections, making clear the parallels between transformed cells, protease-treated normal cells, and mitotic cells.
in agglutinability by lectins and an apparent absence of a particular surface protein, although these are not necessarily related. Of course, other antigenic and biochemical changes also occur (and are discussed here only briefly). 2. Similar, but not necessarily identical, changes can be produced by proteolytic treatment of normal cells. 3. Transformed cells produce proteolytic enzymes. 4. The increased agglutinability of transformed and trypsinised cells depends on some freedom of mobility, in the plane of the membrane, of the sites which bind lectins. 5. Normal cells also show fluctuations in some of these properties, especially at mitosis.
Construction of Models for Surface Changes in Transformation In conclusion, I shall attempt to interrelate the results discussed above, and present models arising from them. Some of the ideas have been expressed elsewhere, and many of them are speculative, but my intention in setting them out here is to present working hypotheses and to point out areas where further investigation is necessary. In particular, here I shall discuss hypotheses concerning, (a) the role of proteases in transformation, and (b) the nature and results of the surface changes associated with transformation. The basic relevant facts may be summarized (see Table 1) as: 1. Changes in surface architecture occur in transformed cells. Specifically, these involve an increase
Do Proteases Play a Role In Transformation? One obvious model is that transformed cells release proteolytic enzymes which act upon their surfaces, producing a series of changes which are responsible for the transformed phenotype (Burger, 1973; Pardee, Jimenez de Asua, and Rozengurt, 1974). It should therefore be possible to mimic this phenotype by addition of proteases and to block its expression by inhibiting proteolytic activity. While this model is an attractive one, it remains unproven. Several of the steps in the argument are only weakly supported, some are disputed, and the details of many of them are unclear. It is possible to assemble a list of phenotypic characteristics of transformed cells (by no means complete) and to ask which of these can be produced by proteases in general, and of these, which
Table
1. Properties
of Different
Types
of Cell
Parameter
“Normal”
Density dependent inhibition of growth
+
Migration
Growth
Cells
into wound
Proteolytic AMP
Nutrient
Low plasminogen dependent
enzymes
Low
levels
High
transport
Agglutinability Patching
by lectins
of lectin
Transformed Cells
-a
-
NT
High plasminogen dependent
NT
+ Plasminogen dependent
sites
lodination of 250K external orotein
at confluence
Low
Other
references
in text
NT
Low at all densities
LOW
High
High at all densities
Low
High
High
High
-b
+
+
NT
+
opinion, opinion,
High
Low at confluence
NT Not Tested a For dissenting b For dissenting
Mitotic Cells By definition
in agar
Cyclic
Protease-Treated “Normal” Cells
see Glynn, Thrash, and Cunningham (1973). see de Pet&. Raff, and Malluccl (1973).
Surface 151
Alterations
in Transformation
can be produced by specific proteolytic activities produced by transformed cells, or which can be blocked in the transformed cell by inhibition of proteolysis. Agglutination by lectins can be induced in normal fibroblasts by proteases (see above). Increased agglutination induced by transformed cell proteases has not been described. It would be interesting to know whether plasmin, a proteolytic enzyme generated indirectly by transformed cells (Ossowski et al., 1973b), or the cell factor, a plasminogen activator and therefore a protease (described by Unkeless et al., 1973) can render normal cells agglutinable. Evidence for inhibition of agglutinability in transformed cells by protease inhibitors has not been reported, and it is not known whether growth in plasminogen-depleted serum will render transformed cells nonagglutinable. Loss of the 250K external glycoprotein can be produced by trypsin and also, less effectively, by plasmin (unpublished results). Attempts to repeat this with medium conditioned by transformed cells have so far been negative; but in the experiment described earlier, transformed cells accelerated loss of this protein from normal cells. Transport of 2-deoxyglucose, which is higher in transformed cells than in arrested normal cells, can also be elevated in normal cells by treatment with proteases (Sefton and Rubin, 1971). Transformed cells exhibit reduced levels of cyclic AMP (Otter-r, Johnson, and Pastan, 1971; Sheppard, 1972). This too can be mimicked in normal cells by treatment with proteases (Otten, Johnson, and Pastan, 1972; Sheppard, 1972; Burger et al., 1972). Increased
Table
2. Effects
of Proteolytic
Enzymes Produced
in Normal
Added proteases
Transformed characteristic
rates of transport and reductions in cyclic AMP have been produced by addition of a partially purified factor from transformed cell conditioned medium (Biirk, 1973; Rozengurt and Jimenez de Asua, personal communication). Transformed cells continue to grow under conditions where normal cells stop. Overgrowth of normal cells can be stimulated by defined proteases (Burger, 1970; Sefton and Rubin, 1970) or by factors released by transformed cells (Rubin, 1970; Biirk, 1973). The latter are not known to be proteases. Growth stimulation by pronase has recently been disputed by Glynn, Thrash, and Cunningham, (1973). A speedy resolution of this conflict of data is to be hoped for. Burger (1971a) reported that addition of leukemic cells to normal fibroblasts stimulated growth of the latter, as did isolated membranes from the leukemic cells. This effect was inhibited by protease inhibitors. Such inhibitors also reduced the culture density to which transformed cells grow (Schnebli and Burger, 1972) although it is not clear that these cells were blocked in Gl, as are arrested normal cells. Migration into a wound in the absence of serum and growth in agar are also characteristic of transformed cells. Both these properties depend on plasminogen (Ossowski et al., 1973b), and growth in agar can be prevented by protease inhibitors (Ossowski et al., 1973a). It is not known whether exogenous proteases will give normal cells these properties, but factors produced by transformed cells will stimulate migration of normal ones (Biirk, 1973). These data are summarized in Table 2. It can be seen that many of the required observations are
Cells
by
Transformed cell factors proteases
In Transformed or
Transformed cells
Plasminogen dependent
Cells Blocked protease inhibitors
Surface Agglutinability
+
Absence protein
of 250K +
Increase transport
in
+b
+
i-b
+
+P
+a
+b +b
Internal Reduction
in CAMP
Biological Overgrowth Migration Growth in agar
~This observation was not confirmed by Glynn, b No evidence that this is due to proteolysis. Other
references
in text.
Thrash,
and Cunningham
+
+ + +
(1973).
+
by
Cell 152
lacking. It should also be noted that, where effects have been produced on normal cells by transformed cells or by factors derived from them, this has not usually been proved to be via proteolytic activity. The gaps in Table 2 indicate areas for fu: ture work. It is also necessary to determine which, if any, of these observations are related. For example, are surface changes instrumental in producing the change in cyclic AMP, and are either of these involved in the change in growth properties? Several reports in which various of these changes have been dissociated from one another were mentioned earlier. The surface change detected by lectins can be observed without changes in cyclic AMP (Burger et al., 1972) or growth stimulation (Glynn, Thrash, and Cunningham, !973), and serum or insulin stimulate growth but not agglutinability. Hsie, Jones, and Puck (1971) and Sheppard (1971) reported that addition of dibutyryl cyclic AMP to transformed cells reduced their agglutinability. This suggests that either this surface change is secondary to the cyclic AMP change (which would not be consistent with the results of Burger et al.); or that some feedback exists between the two changes; or that the surface changes produced by transformation, proteolysis, and low cyclic AMP, although superficially similar, are in fact distinct. This latter explanation is perhaps rendered less likely by the fact that the molecular changes detected at the cell surface in transformed and trypsinised cells are the same (Hynes, 1973) given the proviso that additional, undetected differences may also exist. Furthermore, as normal cells stop growing, their cyclic AMP levels rise (Otten, Johnson, and Pastan, 1971, 1972; Seifert and Paul, 1972) as does labeling of 250K external protein. Mitotic cells show low levels of both, and treatment with proteases lowers both (see above). I shall not consider here the sequence of changes occurring inside the cell, for example those involving cyclic AMP, which might be concerned in growth control (Biirk, 1968) since these have been reviewed elsewhere (Pardee and Rozengurt, 1974; Pardee, Jimenez de Asua, and Rozengurt, 1974). These authors suggested a reciprocal feedback relationship between the metabolism of cyclic AMP inside the cell and the state of the surface membrane. It is clearly important to investigate whether such a relationship exists, and what might be its role in growth control. Studies are required in which the various parameters of transformation are ordered and their interdependence during transitions in growth or transformation are determined. The time courses of shifts of several of these parameters have been studied, but unfortunately, not all in the same system. In general, alterations in cyclic AMP
level occur early after a stimulus (Otten, Johnson, and Pastan, 1972; Sheppard, 1972; Rozengurt and Jimenez de Asua, 1973; Jimenez de Asua, Rozengurt, and Dulbecco, 1974) whereas alterations in agglutinability occur quite late (Benjamin and Burger, 1970; Ben-Bassat, Inbar, and Sachs, 1970; Eckhart, Dulbecco, and Burger, 1971). However, in the case of proteolysis, surface changes are detected very early (Burger, 1971a). It is of course possible, given a reciprocal relationship as proposed by Pardee, Jimenez de Asua, and Rozengurt (1974) to accommodate different sequences of events, depending on the nature of the stimulus, the same end result being achieved via different routes. Pardee, Jimenez de Asua, and Rozengurt (1974) have suggested that (at least) two states of the cell surface exist: one (Q) characteristic of quiescent cells, and the other (P) characteristic of proliferating cells (for example, transformed cells). They suggested that this second state is involved in stimulating activity on the part of the cells, and that changes from the Q to the P state are produced by a variety of stimuli, such as serum, proteases, and transformation, thus triggering growth.
What Is the Nature of the Surface Change? Agglutination of cells by lectins requires binding of the lectins to sites on the cell surface, followed by movement of the bound sites to collect in patches. Inhibition of the gathering into patches blocks agglutination (Inbar et al., 1973a; de Petris, Raff, and Mallucci, 1973; Noonan and Burger, 1973). In most cases, precise correlation between ability to patch and to agglutinate has been observed, and normal fibroblasts were found to do neither; but one report showed patching in these cells, without agglutination (de Petris, Raff, and Mallucci, 1973). This suggests that, while patching alone may be insufficient for agglutination, under most conditions the lectinbinding sites on normal cells are less mobile than those on transformed cells. This will be assumed here despite some conflicting results. What, then, is the nature of this restriction? It could lie in differences in lipid composition, although the fact that mobility of surface proteins can be produced by proteolysis both in fibroblasts (see above) and in other cell types (Tillack, Scott, and Marchesi, 1972; lnbar and Sachs, 1973; lnbar et al., 1973b; Nicholson, 1972) suggests that proteins are involved, and once this restriction is removed, the lipid nature of the membranes does not block movement. Microfilaments, which are affected by cytochalasin B, are not involved either in patching or in agglutination; and energy, or even live cells, are not necessary either for agglutination or for its absence
Surface 153
Alterations
in Transformation
in normal cells (de Petris, Raff, and Mallucci, 1973; Noonan and Burger, 1973). A passive inhibition of mobility therefore exists. Edelman, Yahara, and Wang (1973) have developed the idea that surface receptor sites of lymphocytes are attached to structures inside the cell, and that this interaction may be involved in transducing stimuli from the surface to the interior. If this were the situation in fibroblasts, one could postulate that transformation or proteolysis releases the lectin binding sites from this attachment, thus making them freer to move in response to lectin binding. I should like to propose as an additional hypothesis that the lectin binding sites might be attached, either permanently or under the influence of lectins, to a protein on or in the outer surface of the membrane. This might restrict their motion, and its loss or alteration would then release the restriction. An obvious candidate is the 250K protein discussed above. For this model, it does not matter whether the absence of this protein on transformed cells is due to proteolysis or not. The general models could equally well apply to other, as yet undetected, surface proteins, which could fulfill the same role as that suggested here for the 250K protein. The reason for describing models specifically in terms of the latter protein is that they are testable. Several alternative models for the situation in normal cells can therefore be proposed. 1. Surface proteins are attached individually to structures inside the cell; specifically, assemblies of colchicine-binding proteins (Edelman, Yahara, and Wang, 1973). These internal structures control the mobility of the surface molecules, and interactions between different surface molecules occur via the internal structures. 2. Restriction of the mobility of surface proteins is due to the effects of other membrane proteins. Two distinct versions of this model can be considered. a) The restriction on mobility is direct and constitutive; that is, the surface proteins are ordered into small groups by noncovalent bonding. b) The restriction only arises after interaction with multivalent ligands such as lectins; for example, by cross-linking into small groups, which prevents lateral diffusion previously allowed. In its extreme form, this model suggests that the restriction of mobility involves only proteins in or on the membrane. 3. A model which is a hybrid of the previous two, in that only some surface molecules are linked directly to internal structures (for example, microtubules), while the rest are free except in so far as they are bound to the first set. Either constitutive or induced binding could be envisaged as in the second model proposed above. This sort of model could also be used to explain the concanavalin
A-mediated restriction of surface immunoglobulin movement observed in lymphocytes (see Edelman, Yahara, and Wang, 1973). It is then necessary to ask how each of these models fits the experimental results concerning transformation, and what would be their predictions. Attachment to Internal Structures According to the first model, freedom of movement of surface molecules would be produced by alterations in the colchicine-binding protein assembly inside the cell. On this model, loss of a major surface protein, for example the 250K protein, might derange this assembly so that all the other receptors are released from their association with it, thus becoming free to move. Alternatively, the primary alteration might be in the colchicine-binding protein assembly, leading to the release of the surface receptors and allowing them to move freely. Two observations of interest in this context are that plasma membranes of transformed cells have reduced quantities of actin and of another protein of similar size (Wickus and Robbins, 1973) and that the 250K protein is reduced on the surfaces of cells blocked in mitosis by colchicine (Hynes, unpublished) or vinblastine (E. Pearlstein, personal communication). Whether this is due to the inhibitors or the state of mitosis is not clear; although cells that were not rounded up in inhibitors still had the 250K protein. Investigation of mitotic cells not treated with inhibitors is indicated. If colchicine-binding proteins were concerned in anchoring membrane proteins, then treatment with colchicine might be expected to produce increased mobility. This appears to be the case in lymphocytes (Edelman, Yahara, and Wang, 1973). Further, if freedom of mobility is a requirement for lectinmediated agglutination, as it seems to be, colchitine might increase it. In fact, Yin, Ukena, and Berlin (1972) showed that colchicine and similar agents actually inhibit concanavalin-mediated binding of erythrocytes to transformed fibroblasts. It is not clear if this is an assay for agglutination as generally understood. A more direct determination of the effect of colchicine on the mobility of surface molecules is required. Restrlction of Mobility by Membrane Proteins In considering the second model, the restriction of mobility by membrane proteins, it is obvious that an alteration of one or a few of these might remove the restriction. It is more difficult to see how the restriction is imposed in the first place. Considering this model specifically in terms of the 250K protein, the other surface glycoproteins would either be associated with it permanently, or become associated
Cell 154
via the binding of lectins. The latter would require that the 250K protein binds lectins, and this appears to be the case (unpublished). The question then arises as to how binding of the 250K protein (or any other) could prevent movement. One might suppose that, being a large molecule, it would diffuse more slowly, especially when associated with other proteins. As Edidin (1972) points out, the dependence of diffusion rate on size is linear, so that the restriction produced in this way would probably be too small. Alternatively, an extended network of 250K protein might exist, forming a sort of exoskeleton. Finally, as suggested above, presence of the 250K protein might affect membrane fluidity by interacting with the lipids, although the mechanism by which this might occur is obscure.
Internal Linkage of the 250K Protein The third possibility, that the 250K protein (but not the other proteins, or at least not all of them) is directly anchored by linkage to internal structures, combines features of both the previous models. In this case, removal of this protein or its linkage to the internal structures would release the restriction on mobility of the other surface molecules, and also remove their communication with the interior. The linkage might be direct or indirect (e.g., via intramembranous particles). It would be possible to test whether the 250K protein extends through the membrane as does the major glycoprotein of erythrocyte membranes (Bretscher, 1971). Both these latter two models would predict that mobility of some surface proteins would be affected by the presence or absence of others, and by the degree of cross-bridging which existed between them. For example, the use of non cross-linking probes could test whether the lectin binding sites on fibroblasts are freely diffusing or not; and whether presence or absence of the 250K protein has any effect on this mobility, either intrinsically, or after cross-linking by multivalent ligands. Similar tests could be applied to surface antigens and surface enzymes, given development of the relevant probes (see Edelman, Yahara, and Wang, 1973). Nearest neighbour analysis, using chemical crosslinks followed by investigation by biochemical techniques, could also allow investigation of the associations existing prior to, and after, perturbation of the surface. A similar approach has been applied to erythrocytes (Steck, 1972); but given recent knowledge about the readiness of cell surface molecules to redistribute, these studies need repeating under conditions in which care is taken to avoid perturbation by the probes themselves. Another prediction is that growing normal cells might be more readily agglutinable and show greater free-
dom of movement of surface molecules than would confluent arrested cells, since the latter apparently have more of the 250K protein and show changes in other membrane properties (Pardee and Rozengurt, 1974). It is necessary also to consider the possible biological relevance of such surface changes, although such discussion must at present be speculative. If the change detected by lectins involves a release of many surface molecules into a condition of greater freedom of movement than normal, then pleiotropic changes in surface properties would be expected. Presumably, many surface molecules function as enzymes, receptors for biplogical signals, or in transport. Disruption of the normal interactions between surface molecules and their contact with the inside of the cell should interfere with their functions. lnbar et al. (19736) have shown a correlation between mobility of surface sites and ability to respond to external signals. And Edelman, Yahara, and Wang (1973) found that, if sufficient concanavalin A was added to lymphocytes to restrict mobility, the mitogenic activity of the concanavalin A was blocked, suggesting that some surface movement is required for mitogenesis. At present, the meaning of these observations is not clear; but they may indicate that mobility of surface sites is important in their function, so that disturbance of the normal controls on this mobility is likely to have biological effects. One of the models considered earlier involved surface molecules permanently arranged in groups. Obviously, disruption of such an assemblage by loss of one of its members would affect the activity of the others-especially if, at the same time, they lost their interaction with the interior of the cell. There exist many observations of changes in antigenie and biochemical characteristics at the surfaces of transformed cells. Some of these are also produced by proteolysis (e.g. Hdyry and Defendi, 1970; Burger, 1971 b). Disruption of preexisting relationships among surface proteins in the way I have proposed could well lead to such changes: for example, exposure of previously buried antigens (see Collins and Black, 1974) altered interrelationships between surface enzymes and their substrates, either allowing interactions previously blocked (Roth and White, 1972), or preventing the normal interactions. Similarly, alterations in the control of nutrient transport and in the metabolism of cyclic AMP by membrane bound enzymes could be produced by such a membrane alteration (see Pardee, Jimenez de Asua, and Rozengurt, 1974). Reciprocally, cyclic AMP could affect the membrane via protein kinase activity (Johnson et al., 1972; Rubin and Rosen, 1973).
Surface 155
Alterations
in Transformation
Mobility of Surface Molecules in Normal Cells Finally, it is of interest to consider the fact that several of the changes observed in transformed and protease-treated cells are also shown by mitotic cells (see above). It has been suggested before (Burger, 1971 b, 1973; Pardee, Jiminez de Asua, and Rozengurt, 1974) that the transient expression of a surface change triggers the next cycle of replication in normal cells. Normally this alteration disappears as cells enter Gl . This is true for lectin binding (Noonan, Levine, and Burger, 1973), and for the 250K surface protein (Hynes, unpublished), as well as for cyclic AMP levels (Burger, et al., 1972). It would be of great interest to know whether there is a transient release of proteases at mitosis, triggering these mitotic changes. A unifying hypothesis would then be that transformed cells become locked in this state. This is, of course, far from proven; and in any case, there is no reason why similar changes cannot be produced by a variety of mechanisms. As discussed above and elsewhere (Pardee and Rozengurt, 1974), several of the parameters considered in this review are modulated in normal cells. The suggestion implicit in the models presented earlier is that this modulation could be effected by controlling the freedom of mobility of surface molecules, and that the control mechanisms are aborted by proteolysis or transformation. Thus, growth stimulation by serum or insulin would be envisaged as leading to greater freedom of mobility, but not to the same degree as complete removal of the controls. According to the nomenclature of Pardee, Jimenez de Asua, and Rozengurt (1974), the surface states of quiescent cells, stimulated normal cells, and transformed cells could be labeled Q, Pa, and Pb, respectively, and in order of increasing mobility of surface molecules. The aim of this review has been to collect some of the various facts related to these hypotheses, and to analyse how far they support the ideas presented above. These are intended to form working hypotheses, which derive from many previous suggestions. I have tried to indicate the many points on which further data are needed, in the hope that some or all of the hypotheses can be eliminated. In two recent papers, Gahmberg and Hakomori (1973, Proc. Nat. Acad. Sci. USA 70, 3329), using external labeling by galactose oxidase plus sodium borotritiide, detected a large external glycoprotein absent on transformed cells, and Berlin et al. (1974, Nature 247, 45) presented evidence consistent with a model in which surface proteins are attached to microtubule proteins.
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