Cell, Vol. 6, 137-147,

October

1975,

Copyright

‘c 1975

by MIT

Effect of Proteases on Activation of Resting Chick Embryo Fibroblasts and on Cell Surface Proteins Peter M. Blumberg’ and Phillips W. Robbins Center for Cancer Research and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary The relationship between activation of resting chick embryo fibroblasts by proteases and proteolytic alteration of the cell surface has been investigated. Five different proteases were examined: trypsin, collagenase, plasmin, a-chymotrypsin, and thrombin. All of these proteases, when added to the culture medium at concentrations of 0.08-2.2 pg/ml, stimulated deoxyglucose uptake and induced cell division. The absolute levels of stimulation depended on the specific protease. Activation ranged from a doubling in cell number In 24 hr for trypsin and thrombin down to a 47% increase in cell number for a-chymotrypsln. Except in the case of thrombin, the stimulatory effects of these proteases correlated wlth breakdown of 2, a protein which is the major chick surface proteln as revealed by lactoperoxldase-catalyzed lodination and which disappears upon transformation. In the case of thrombin, stlmulatory concentrations brought about no detectable loss of surface components. Thus loss of Z Is not a necessary condition for activation of chick fibroblasts; it may be a sufficient condition for activation of part of the cell population. Introduction Considerable interest has focused upon the changes in cell surface structure accompanying transformation. In the case of chick embryo fibroblasts, transformation causes the decrease or disappearance of three proteins-A, 52, and 2 (Wickus and Robbins, 1973; Wickus, Branton, and Robbins, 1974; Robbins et al., 1975). Z (230,000 daltons), also called LETS or SF antigen, has attracted particular attention (Hynes, 1973; Hynes, 1974; Hynes and Humphryes, 1974; Hogg, 1974; Ruoslahti et al., 1973; Ruoslahti and Vaheri, 1974; Gahmberg, Kiehn, and Hakomori, 1974). It is the major surface protein in chick embryo fibroblasts as revealed by lactoperoxidase-catalyzed iodination. It is found on the surface of chick embryo myoblasts as well as fibroblasts and is also present on the surface of cultured cells from many species other than chicken. *Present School,

address: Department Boston, Massachusetts

of Pharmacology, 02115.

Harvard

Medical

Studies in a number of laboratories have shown that Z disappears upon transformation. Unlike A and g, Z has been reported to be unusually sensitive to proteases such as trypsin and collagenase (Wickus et al., 1974; Hynes, 1973; Hynes and Humphryes, 1974). Likewise, trypsin has been reported to release resting cells from densitydependent inhibition (Burger, 1970; Sefton and Rubin, 1970). While some workers have been unable to repeat these findings for 3T3 cells (Glynn, Thrash, and Cunningham, 1973), the phenomenon of protease stimulation has been independently confirmed for the case of chick embryo fibroblasts (Vaheri, Ruoslahti, and Hovi, 1974; Blumberg and Robbins, 1975). The relation between activation of the chick embryo fibroblasts by proteases and proteolytic cleavage of Z is therefore of interest. Activation of cells by proteases has possible physiological significance. Lack of growth control is a characteristic of transformed cells. In addition, most transformed cells produce elevated levels of a protease, plasminogen activator, which in turn activates the serum pro-enzyme plasminogen to the serine protease plasmin. Plasmin, in turn, may be responsible for some but not all of the altered properties of the transformed cell (Goldberg, 1974; Unkeless et al., 1973, 1974a; Unkeless, Gordon, and Reich, 1974b; Ossowski et al., 1973a, 1973b; Ossowski, Quigley, and Reich, 1974; Quigley, Ossowski, and Reich, 1974; Reich, 1974). Results Cleavage of Z For the studies on the effects of proteases on the cell surface and on activation, enzyme was added to the medium of cultures of resting fibroblasts, after which incubation was continued in the presence of the enzyme until the end of the experiment. It should be noted that chicken serum has much lower levels of trypsin inhibitors than does calf serum. Under such conditions, low concentrations of trypsin appeared to cleave a single protein on the fibroblast cell surface. This effect could most readily be visualized by surface labeling of the cells with lactoperoxidase-catalyzed iodination (Figure 1). This cleaved protein was that previously identified as Z or LETS. As discussed above, this protein is the major surface protein on chick fibroblasts as revealed by lactoperoxidase-catalyzed iodination and disappears or decreases upon transformation. Weak bands, not readily visualized on the above gel, might have been altered by the trypsin in addition to Z. However, prolonged exposure of the autoradiograms failed to reveal such cleavage by these low concentrations of trypsin. In contrast, as

Cell 138

would have been expected from the broad specificity of trypsin, high concentrations of the enzyme led to extensive degradation of the surface polypeptides. The degradation of 2 by trypsin appeared to proceed through several intermediate fragments. As is apparent in Figure 1, the most noticeable was a fragment of molecular weight about 14,000 daltons less than Z. At higher trypsin concentrations, a lower molecular weight fragment can be seen at approximately 72,000 daltons. Similar effects on Z to those caused by trypsin can be seen with other proteases. The effect of increasing concentrations of collagenase is illustrat-

ed in Figure 2. Z, once again, was the only visible protein cleaved. In an effort to reveal proteolytic changes not apparent with iodinated cells, cultures were labeled with I%-proline or WS-methionine before protease treatment. In the case of I%-prolinelabeled whole cells, the only apparent band cleaved by either trypsin or collagenase was that identified as Z (Figure 3). Similarly, no alteration other than loss of Z could be seen when W-methioninelabeled cells were treated with protease; for these experiments either extracts of whole cells or membranes prepared according to the method of Brunette and Till (1971) were examined.

Effect of trypsin on iodinated chick fibroblasts origin

z-

4

marker dye

Concentration of trypsin /4/m~ Figure

1, Effect

of Trypsin

on the Cell Surface

Trypsin at the indicated final concentrations for 4’/2 hr. the cells were iodinated, dissolved Procedures.

of Chick

Embryo

Fibroblasts

was added to the medium of cultures of secondary in SDS, and subjected to SDS slab gel electrophoresis

chick embryo fibroblasts. on 7% gels as described

After incubation in Experimental

Activation 139

by Proteases

Correlation 01 Activation and Cleavage How do the protease concentrations which cleaved 2 correspond to the concentrations required to activate resting fibroblasts? Increase upon activation in three parameters-deoxyglucose transport, cell number, and thymidine incorporation-were reported by Sefton and Rubin (1971). For the experiments reported here, activation was routinely measured as stimulation of deoxyglucose transport. This measure was technically most convenient and provided a large response over background. In addition, since increase in cell number represents perhaps the most fundamental measure of cell activation, this was also monitored. As shown below, the concentrations of protease required for activation as measured by either of these methods, deoxyglucase uptake or increase in cell number, were comparable. Stimulation of 3H-thymidine incorporation, which was not usually measured, gave similar results.

Effect of collagenose on iodinated chick fibroblasts

Cleavage of Z by trypsin gave excellent correlation with activation of the resting chick fibroblasts (Figure 4A). The concentrations for half-maximal cleavage of Z and half-maximal stimulation of deoxyglucose transport were within 10% of each other. Trypsin is a relatively nonspecific enzyme. It was therefore significant that cleavage of Z by collagenase and cellular activation gave an excellent correlation (Figure 4B). An important question was whether the cleavage and stimulation arose from the collagenolytic activity of the enzyme or from a contaminating protease. Two assays were used to detect the presence of such a protease in the collagenase preparations (see Experimental Procedures). As measured by caseinolytic activity, two separate batches of Worthington collagenase had the equivalent of only 0.32% and 0.25% trypsin contamination. Since a protease might have little activi-

Effect of Proteases on 14C proline labeled chick fibroblasts

[ 1

a .x-- origin z-m

-’

origin

marker dye marker dye

c

Figure 3. Effect of Trypsin Chick Embryo Fibroblasts

Figure 2. Effect Fibroblasts

of Collagenase

on the Cell Surface

of Chick

Embryo

Cells were treated as described in the legend to Figure 1, except that collagenase was added at the indicated concentrations,

and Gollagenase

on “C-Proline-Labeled

Secondary chick embryo fibroblasts were grown in medium supplemented with 14C-proline (2 pCi/ml). After 2 days, trypsin and collagenase at the final concentrations indicated above were added to the medium on the plates. The plates ware washed extensively; the cells were then dissolved in SDS and subjected to SDS slab gel electrophoresis on 7% gels.

Cell

140

LO-

e.o-

c

-35 El-

I 4

2 5

- 3.0 9: 8 1 g : 16$ -2.5 g .c 0

.-

N

z ,4e -2.0 E ‘2--1.5

*= ‘s ‘Z6 P .-z *

0.1 Relatwe

Figure

4. Effect

of Proteases

I.0 HF concentrotm

on Z and on Cell Activation

Proteases at the indicated final concentrations were added to the medium of cultures of 2 day old secondary chick embryo fibroblasts as described in Experimental Procedures. For measurement of loss of Z, cultures were iodinated at 4% hr after protease addition. Stimulation of deoxyglucose uptake was determined at 6 hr and increase in cell number at 24 hr. (A) Trypsin; (8) collagenase: (C) human plasmifl, generated from plasminogen by streptokinase as described in Experimental Procedures. Protein concentration is that for the original plasminogen. (D) o-Chymotrypsin; (E) thrombin; (F) crude harvest factor. Concentration is relative to that found in the supernatant from cultures of transformed cells. (0) Fraction Z remaining. (0) Stimulation of SH-deoxyglucose (n) Increase in cell number.

uptake

Activation 141

by Proteases

ty on casein as a substrate, hydrolysis of radioactive E. coli protein was measured. By this assay, the latter batch of Worthington collagenase had the equivalent of only 0.015% tryptic contamination. After reduction and alkylation, this proteolytic activity was further reduced to 0.002%. Such quantities, if the contaminant were any of the proteases examined in this paper, would be much too low to account for the cleavage and activation. On the other hand, the reduced and alkylated collagenase was 4 fold less effective at cleaving 2 than was the starting material while it retained 80% of its collagenase activity. Since the collagenase preparation was a mixture of six collagenases of somewhat different properties (Peterkofsky and Diegelman, 1971), the loss of activity of one that cleaved Z preferentially might account for the results. Until the explanation for the result is determined, however, the specificity of the collagenase preparation should not be accepted without question. Human plasminogen activated to plasmin by streptokinase cleaved Z and stimulated the resting cells (Figure 4C). This activation was not due to the streptokinase. Streptokinase alone at the concentrations in the plasmin preparation had no effect on the cells. Unlike the case of trypsin and collagenase, however, the correlation between cleavage of Z by plasmin and activation of the resting cells was poorer; Z appeared somewhat less sensitive to plasmin than was cell activation. Conversely, whereas n-chymotrypsin both activated the resting cells and cleaved Z, here Z appeared rather more sensitive to the protease (Figure 4D). Examination of the stimulations reveals another unusual aspect of the n-chymotrypsin behavior. Not only is the increase in cell number less than for the other proteases, but the maximum response is displaced toward lower protease concentrations as compared with the stimulation in deoxyglucose transport. Since increase in cell number is measured at 24 hr and the stimulation of deoxyglucose at 6 hr after protease addition, this shift might reflect a long-term toxic effect of a-chymotrypsin on the cells (see below). In contrast to the effects of the above proteases, thrombin activated resting fibroblasts without loss of Z (Figure 4E). Indeed, a 20-40% increase in the level of Z was often observed. Although cleavage of a very small fragment from Z might not have been apparent, loss of more than 1000-2000 daltons would have been detected. Some loss of Z could be seen at concentrations of thrombin considerably higher than those required to induce activation. Such cleavage might well be a secondary effect of the addition of thrombin to the culture medium, possibly arising from activation of some other protease in the serum. Cleavage of Z has been reported not

to occur if thrombin is added to cells in the absence of serum (Teng and Chen, 1975). Transformed cells release into the medium a serine protease, plasminogen activator, which in turn converts plasminogen to the protease plasmin. In initial experiments, a partially purified preparation of this enzyme, “crude harvest factor fraction I” (Unkeless et al., 1974a), was added to resting fibroblasts. Like thrombin, the crude harvest factor preparation induced cell activation without causing cleavage of Z (Figure 4F). Whether the activation resulted from the harvest factor itself or some other component of the crude enzyme preparation, for example overgrowth stimulating factor (Burr and Rubin, 1975), remains to be determined. Prior treatment of the crude harvest factor fraction with an inhibitor of serine proteases, phenylmethane sulfonyl fluoride, had no effect on activation. In the above experiments, cultures were iodinated 4.5 hr after the addition of the protease to the culture medium. The results were essentially the same if the cells were iodinated prior to addition of trypsin to the culture medium (Figure 5). Several minor differences could be noted. First, since Z has a fairly rapid turnover under these culture conditions (Blumberg and Robbins, 1975) the absolute amount of labeling in the control was diminished. Second, Z appeared slightly more sensitive to trypsin than was activation of the cells. One possible explanation is that in this case all of the labeled Z had been exposed to the trypsin over the entire 4.5 hr incubation period, whereas when iodination occurred at the end of the incubation period the

1.0-

08.-P .g ; 0.6 N .E 048

t

0.2 -

0.01

0.1

IO ,w/ml

Figure 5. Effect Cell Activation

of Trypsin

on Z Prelabeled

with

Iodine

and

on

Cells were treated as described in the legend to Figure 4, except that the medium was removed from the plates, the cultures iodinated, and the conditioned medium returned to the plates before addition of the trypsin. The fraction of Z remaining was determined 4% hr after trypsin addition. (0) Fraction Z remaining. (0) Stimulation of 3H-deoxyglucose uptake. (V) Stimulation of 3H-thymidine incorporation.

Cell 142

newly synthesized Z had a shorter exposure to the trypsin. The selection of 4.5 hr after protease addition for measurement of the loss of Z was somewhat arbitrary. To the degree that loss of Z depended on time of incubation as well as on protease concentration, the correlations between cleavage of Z and activation would be distorted. The extent of cleavage of Z by different protease concentrations at 1, 2, 4, and 6 hr was therefore compared (Figure 6). In fact, the sensitivity of Z to proteases showed little time dependence within this time scale. In the case of trypsin, loss of Z was slightly less at early times (l-2 hr) than at later times. In the case of cy-chymotrypsin, no difference with time was apparent. The rapid turnover of Z in chick embryo fibroblasts grown in MEM-2-0-l (Blumberg and Robbins, 1975) can account for these findings. The levels of Z found would reflect both degradation and synthesis. Table 1 summarizes the protease concentrations required for cleavage of Z and for activation. The concentrations for half-maximal activation range from 0.2-0.9 pg/ml. Activation by thrombin or crude harvest factor clearly indicates that cleavage of Z is not a necessary step in cell activation. For those proteases which do cleave Z, half-maximal cleavage and activation occur at concentrations differing by no more than 2.7 fold.

varied somewhat with the batch of cells, serum lot, and so forth. In addition, as was perhaps most apparent for the case of a-chymotrypsin (see Figure 4D), the absolute levels of activation induced by different proteases were not the same. For purposes of comparison, a trypsin stimulated control was therefore always carried out in experiments measuring stimulation by other agents. Table 2 summarizes the levels of activation obtained. Several important features should be noted. First, the cell number of the resting chick embryo fibroblasts, although not absolutely constant, increased an average of only 17% in 24 hr. Activation either by trypsin or thrombin appeared to be comparable and complete: the

Collagenase

1.18

0.85

1.4

Plasmin

2.20

0.82

2.7

Completeness of Activation by Proteases The stimulation of deoxyglucose transport and the increase in cell number induced by proteases

a-chymotrypsin

0.071

0.180

0.39

ID-

Table 1. Relationship Activation

of Cleavage

G@ (Concentration 50% Cleavage (wml)

Protease Thrombin

of Z by Protease

for of Z)

>>4

Crude Harvest Factor* Trypsin

0.63

>>lO

to the activity

GdAw

>>lO 0.88

0.24

relative

for of

>>6

0.94

0.21

*Values are expressed from transformed cells.

A50 (Concentration 50% Stimulation XH-deoxyglucose Uptake) (m/ml)

and Cell

in the supernatant

A

01 0.1

#g/ml Figure Cultures indicated (A--A)

6. Correlation

between

$g /ml

a-chymotrypsrn

addition

the cells

I.0

frypsin Cell Activation

and Cleavage

were treated as described in the legend below. (A) Trypsin. (B) a-Chymotrypsin. Z remaining at 1 hr. (Q-O) Z remaining at 2 hr. (o---O) Z remaining at 4 hr. (V-*-V) Z remaining at 6 hr. (0) Stimulation of deoxyglucose uptake. (A) Increase in cell number.

to Figure

of Z as a Function 4, except

that

of Time after

protease

were

iodinated

at the times

Activation 143

Table

by Proteases

2. Level

of Activation

of Resting Stimulation Deoxyglucose

Blank Thrombin

Chick

Embryo

Fibroblasts

of

Increase Number

Uptake

Absolute

Relative Trypsinb

(1 .oop

(0.00)

to

Number of Experiments

in Cell over 24 Hr

Absolute

Relative Trypsinb

to

Number of Experiments

0.17

27

7.96

1 .27

4

0.99

0.963

3

6.65

1 .lO

2

0.67

0.64

2

Trypsin

6.49

(1 .OO)

40

1 .Ol

(1 .OO)

Plasmin

4.19

,925

2

0.67

0.65

2

Collagenase

4.20

,625

19

0.65

0.65

13

n-chymotrypsin

3.50

,400

6

0.47

0.42

5

Crude

HF

aThis blank corresponds to a mean uptake of 2960 cpm/plate. bSince total activation differs * 15% between experiments, the values experiments rather than the ratio of the averages.

cell number doubled within 24 hr. For some of the other proteases, notably collagenase and a-chymotrypsin, activation of cell division occurred in only a fraction of the cells-65% and 4790, respectively. Stimulation of deoxyglucose transport mirrored the increase in cell number. The stimulation by collagenase was 63% of that by trypsin. The stimulation by cu-chymotrypsin was 40%. Ribonuclease-sensitive phosphate residues have been identified on the surface of CHO cells (Rieber and Bacalao, 1974). Such material has, moreover, been reported to be released by treatment of the cells with trypsin. The effect of deoxyribonuclease I and ribonuclease A on deoxyglucose uptake of chick embryo fibroblasts was therefore examined. Neither treatment had significant effect (Table 3). Effect of Trypsinate on Cell Activation In the above experiments, proteases were added to cells growing in MEM containing 1% chicken serum. It was therefore important to determine whether or not the effects observed arose from the direct action of the added protease on the cell surface. One alternative was that activation was mediated by a product released from cells by the protease and able to activate other cells. A second model was that 2 was merely one of a class of protease-sensitive proteins, and that cleavage of a serum protein of similar protease-sensitivityperhaps the conversion of prothrombin to thrombin-was responsible for the stimulatory effects. Our experimental approach was to compare the stimulation of deoxyglucose uptake by direct trypsin treatment of the cells with the stimulatory effects on the cells of culture medium previously treated with trypsin. In order to differentiate between an effect of trypsin on the serum and the release by trypsin of an activating peptide from the cells, both fi-

relative

to trypsin

are the average

Table 3. Effect of Ribonuclease Deoxyglucose Uptake

and

values

in the individual

Deoxyribonuclease

on SH-

3H-deoxyglucose Uptake (cpm/Plate)

Addition None

2,529

Ribonuclease 40 pg/ml 10 pg/ml 2.5 Pg/ml Deoxyribonuclease 40 pg/ml 10 pg/ml 2.5 gg/ml Trvosin

of the relative

25

2 ualml

A 2,594 2577 2,560 I 3,665 3,013 2,634 13.559

Stimulation of deoxyglucose uptake was measured as described in Experimental Procedures, Ribonuclease A was obtained from Worthington (RAF 388, 4300 units/mg), deoxyribonuclease I from Sigma (DN-100, 1570 Kunitz units/mg).

broblast cultures and isolated conditioned medium were treated with trypsin. After incubation, further trypsin action was blocked by addition of soybean trypsin inhibitor. The medium was then transferred to fresh monolayers for determination of the stimulatory activity. Under such conditions, the medium from the trypsinized cultures caused partial activation of the fibroblasts-about 35% of that induced by trypsin itself (Figure 7). This value reflected (a) the combined effect of the trypsinized conditioned medium plus the mechanical transfer of medium (resulting in 15% activation); (b) an effect specific for the supernatant from trypsinized cells (causing an additional 20% activation). The level of stimulation induced by the supernatant from trypsinized cultures was not increased by varying the period of incubation with trypsin between 1 and 6 hr, or by addition of fresh trypsinized culture supernatant

Cell 144

every hour rather than at the beginning of the 6 hr incubation period. It would thus appear that the trypsin stimulation was largely a nontransferable effect on the cell surface. Protease activation of serum components at best played only a minor role, as did release of an activating factor. Whether this conclusion holds true for the other proteases has not been determined. Inhibitory Effect of a-Chymotrypsin on Trypsin Stimulation The partial activation of cells by cY-chymotrypsin suggested that the enzyme might have inhibitory as well as stimulatory effects. This would in fact appear to be the case (Figure 8). When a-chymotrypsin was added to cultures 4 hr before trypsin stimulation, the trypsin response was greatly inhibited although it remained somewhat higher than that caused by cu-chymotrypsin alone. Much of the inhibitory effect of a-chymotrypsin on trypsin activation requires the joint presence of the two enzymes. Treatment of cells already activat-

V

I I

I 2

I 3 Time

Figure 7. Time Trypsinate

Course

for

Generation

I 4

I 5

I 6

The experiments described above clarify but do not resolve the role of loss of 2 in activation of resting chick embryo fibroblasts. The findings with thrombin and crude harvest factor I strongly indicate that cleavage of Z is not a necessary step in activation. Such a conclusion might have been anticipated from the report by Vaheri et al. (1974) that several nonproteolytic agents, for example insulin and lipid A, exerted a mitogenic effect on chick fibroblasts. A separate question is whether proteolytic cleavage of Z in itself represents a sufficient stimulus for acti-

0.2 Activity

in

Trypsin at a final concentration of 2 pg/ml was added to cultures of secondary chick embryo fibroblasts or to the conditioned medium from such cultures. Incubation was continued for the indicated times, after which soybean trypsin inhibitor (25 fig/ml final concentration) was added. The medium was then added to fresh plates and stimulation of deoxyglucose uptake measured 6 hr later. For comparison, trypsin (2 pg/ml) was added directly to cultures and stimulation of deoxyglucose uptake measured as a function of time. Fractional stimulation was expressed relative to that by 2 pg/ml of trypsin for 6 hr; 1 .OO corresponds to a stimulation of 6.60 in this experiment.

(A) Time course of trypsin stimulation. (0) Stimulation by culture trypsinate. (0) Stimulation by medium trypsinate.

Discussion

I

(hours) of Stimulatory

ed by trypsin led to little inhibition of the tryptic response. Likewise, stimulation by trypsin was unaffected by prior treatment of cells with cu-chymotrypsin followed by removal of the a-chymotrypsin before the trypsin addition. A trivial explanation for the inhibition of the trypsin effect by a-chymotrypsin could have been degradation of the former enzyme. This did not appear to be the case. Tryptic activity measured by tosylarginyl methyl ester hydrolysis (Walsh, 1970) was similar as a function of time in the presence or absence of cY-chymotrypsin.

t

I

I

I

I

I

2

4

6

8

IO

Time (hours 1 Figure 6. Inhibitory Effect of a-Chymotrypsin Deoxyglucose Uptake by Trypsin

on Stimulation

a-Chymotrypsin at a final concentration of to 2 day old cultures of secondary chick the indicated time. Trypsin (1 pg/ml) was at 4 hr and deoxyglucose uptake measured stimulation was expressed relative to that for 6 hr with 2 pg/ml of trypsin (stimulation

0.5 pg/ml was added embryo fibroblasts at added to all cultures at 10 hr. Fractional induced by incubation of 5.91).

(0) Trypsin. (---) ol-chymotrypsin

alone.

of

Activation 145

by Proteases

vation. The evidence presented in this paper is consistent with such a conclusion provided the qualification is made that the result of cleavage of Z may be only partial rather than complete activation. The data at this stage do not exclude the possibility, however, that breakdown of an undetected protein of protease sensitivity similar to Z is actually responsible for the activation. The excellent correlation obtained with trypsin and collagenase between cleavage of Z and cell activation supports a causal relationship. Due to the broad specificity of trypsin, it might be argued that Z is representative of a general class of highly protease-sensitive proteins. In this case cleavage of one of these proteins, either in the serum or in the membrane in amounts too small to be detected by autoradiography of SDS-polyacrylamide gels, would provide the signal for activation. Experimentally, we have shown that activation of a serum component plays no more than a minor role. Could activation be due to concomitant cleavage of a membrane receptor not visualized on the gels? The specificity of the collagenase may make this explanation less likely, although it does not rule it out. How certain is it that the effect seen with the collagenase is actually due to the collagenolytic activity of the enzyme? As described in Results, the contaminating proteolytic activity in the enzyme is very low, equivalent to an 0.3-0.02% contamination by trypsin. In contrast, the concentration of trypsin required to cleave Z is 18% of the concentration for collagenase; none of the other proteases examined is much more active against Z than is trypsin. Were cleavage due to a contaminant, it would thus have to be exceptionally active with respect to both cell activation and cleavage of Z. In the case of plasmin and a-chymotrypsin, the correlation between cleavage of Z and activation is close but not exact. Here, the reason for the discrepancy may be that the response to protease is a complex function of the activating stimulus summed over the time of incubation. Measurement of the cleavage of Z at a given time, 4.5 hr in the present experiments, would thus only be an approximation of this activating stimulus. A reproducible feature of the activation by proteases is that different enzymes give different maximal levels of activation. In particular, collagenase gives 63% and a-chymotrypsin 40% of the activation obtained with trypsin. Such partial activation is by no means unprecedented; Holley and Kiernan (1974) have shown that serum growth factors behave in a similar fashion. In summary then, our present judgement is that cleavage of Z is not a necessary step in cell activation; it may be a sufficient step for induction of the partial activation of the cells (perhaps 60%) such as is observed in the case of collagenase.

In a recent study, in which a rather different experiment protocol was used, Teng and Chen (1975) concluded that cleavage of Z is neither necessary nor sufficient for cell activation. Whereas the experiments reported here used cells exposed over extended periods to proteases in the presence of medium containing serum, Teng and Chen pulsed cells for 10 min with protease in the absence of serum, then measured loss of Z immediately. Further, while increase in cell number (at 24 hr) and stimulation of deoxyglucose transport (at 6 hr) were used as a measure of activation in the present study, Teng and Chen relied on stimulation of thymidine incorporation after incubation for 12 hr in the absence of serum. These differences in procedure presumably account for the contrasting conclusions. One possible model which accounts for the experimental results is as follows. Evidence from this laboratory and elsewhere indicates that much of Z is on the outside of the chick embryo fibroblast membrane. There, as suggested by Hynes (1974) and Wartiovaara et al. (1974), Z may form a cellular exoskeleton, anchoring numerous membrane proteins. Cleavage of Z would bring about a conformational change in one of these proteins, either directly or perhaps by permitting aggregation of the now motile protein. Via one or more intermediates, the cell would then receive an activating stimulus. Other effector substances, for example thrombin, might affect one of the intermediates even in the presence of Z. oc-Chymotrypsin, by cleaving one of these proteins as well as Z, could reduce the level of the activating stimulus. Evidence suggests that cells cycle through different stages of GI, during which they may show altered susceptibility to mitogenie stimulation (Smith and Martin, 1973; Gunther, Wang, and Edelman, 1974). If, for chick embryo fibroblasts, the activation threshold varies in such a fashion, then partial stimulation could result from activation of only those cells having a lower threshold. How do these results with proteases relate to the physiological situations, and in particular the properties of transformed cells? First, confirmation that plasmin at a concentration of 2 pg/ml activates resting chick embryo fibroblasts indicates that it can be responsible for additional cell growth. In chicken plasma, the plasmin precursor plasminogen makes up 0.8% of the total protein (approximately 300-400 pg/ml) (Unkeless et al., 1973). Transformed chick fibroblasts produce high levels of a plasminogen activator, the enzyme which converts plasminogen to plasmin (Unkeless et al., 1974a). Second, if indeed the chick fibroblasts are activated by the loss of Z from their surface, then the absence of Z on transformed cells might have a

Cell 146

stimulatory effect, regardless of whether its loss under these circumstances was due to direct proteolytic action or some other cause. Experimental

Procedures

Chicken serum was from Microbiological Associates; calf serum was from Microbiological Associates or Colorado Serum Company. Powdered medium 199 and Eagle’s minimum essential medium with Earle’s salts (MEM) were purchased from Grand Island Biological Company. Both media were supplemented with 50 pg/ml of streptomycin and 75 units/ml of penicillin G. Purified trypsin (TRL, 2 x recrystallized, 185 units/mg), collagenase (CLSPA > 350 units/mg), a-chymotrypsin (CDI, 3 x recrystallized, 55 units/mg), and human plasminogen were purchased from Worthington Biochemical Company. Thrombin (400 NIH units/mg) soybean trypsin inhibitor, tosyl arginyl methyl ester (TAME), and phenylmethane sulfonyl fluoride (PMSF) were obtained from Sigma Chemical Company. Streptokinase was from Calbiochem, as was the lactoperoxidase (B grade) used in a few of the experiments. The lactoperoxidase used in most of the experiments had been purified by the method of Morrison and Hultquist (1963) and was the generous gift of G. G. Wickus. Crude harvest factor fraction I was prepared according to Unkeless et al. (1974a). IH-deoxyglucose (7.2 or IO Ci/mmole), ‘H-thymidine (51.5 Ci/mmole), Na 1251, L-‘5S-methionine (216 Ci/mmole), and L-IaC-proline (255 mg/mmole) were purchased from New England Nuclear Corporation. Primary chick embryo fibroblasts were plated at 1 X 10’ cells/100 mm dish (Falcon) in medium 199 containing 2% tryptose phosphate broth (Difco). 1% calf serum, and 1% heat inactivated chick serum. Secondary chick embryo fibroblasts were routinely plated at 1.3 x 106 cells/60 mm dish (Falcon) in 5 ml of MEM containing 2% tryptose phosphate broth and 1% chick serum (MEM-2-O-l). All cultures were incubated at 39’C in a 5% CO1 atmosphere. Unless otherwise noted, experiments were performed with 2 day old secondary cultures. For experiments measuring activation by proteases, 0.1 ml of protease dissolved in solution A (PBS minus Ca++ and Mg++) was added directly to the medium of the secondary culture and incubation continued in the presence of the protease without medium change until the end of the experiment. Activation was measured by increase in cell number 24 hr after protease addition or by stimulation of SH-deoxyglucose uptake at 6 hr. Measurement of deoxyglucose uptake was performed as described by Sefton and Rubin (1971) using an incubation time of 10 min. Stimulation of 3Hthymidine incorporation was also monitored occasionally. Measurements were made 18 hr after protease addition with a 1 hr incubation time (Sefton and Rubin, 1971). All determinations were performed in duplicate. Chick embryo fibroblasts were iodinated by the lactoperoxidase method (Sefton. Wickus, and Burge, 1973). Labeled monolayers of cells to be examined by electrophoresis were rinsed 3-4 times with solution A, after which 0.75 or 1 .O ml of boiling SDS gel application buffer was added per plate followed by phenylmethane sulfonyl fluoride (2 mM final concentration). The samples were then subjected to SDS slab gel electrophoresis according to the procedure of Laemmli (1970). Slab gels were dried either according to the procedure of Maize1 (1971) or with a slab gel drier (Model SE540, Hoefer Scientific Instruments). Radioactive bands were detected by autoradiography with Kodak No-screen medical X-ray film. For quantitation, individual bands were cut from the dried gel, eluted into 1% SDS (1 ml per sample), and counted in a liquid scintillation counter, Alternatively, the autoradiograms were scanned with a microdensitometer. and the areas under the peaks determined. Each experimental point represents the average of duplicate determinations. Conversion of plasminogen to plasmin by streptokinase was carried out as follows. Plasminogen, 400 pg, in 200 ~1 of 0.05 M Tris, 0.02 M lysine-HCI, 0.01 M NaCI, 1 mM EDTA (pH 9.0) was added

to 1.80 ml of 0.06 M Tris-HCI (pH 7.5), 0.09 M NaCl containing 400 units of streptokinase. The mixture was incubated for IO min at 22°C. Aliquots were then added to cultures. Caseinolytic activity in the Worthington collagenase preparations was assayed as described (Worthington Enzyme Manual, 1972). General protease activity in the Worthington collagenase was also measured by liberation of trichloroacetic acid (TCA) soluble radioactivity from xs.S-methionine-labeled Escherichia coli protein. To 450 pl of 0.05 M Tris-Cl (pH 7.6), containing 58,000 cpm of heat denatured E. coli supernatant protein was added 50 pl of collagenase (20 pg) dissolved in 0.05 M Tris-Cl (pH 7.6), 5 mM CaC12. After incubation at 37°C for l-2 hr, 500 pg of bovine serum albumin (Armour Pharmaceutical Company) was added, followed by 0.5 ml of 10% TCA (0°C). The samples were incubated for 10 min at 0°C. The precipitate was sedimented by centrifugation, and the radioactivity in an aliquot of the supernatant solution determined. For both assays, the values for general proteolytic activity of the collagenase were compared with the activity of trypsin. Traces of two proteases, inhibited by alkylation with N-ethylmaleimide (NEM) and by reduction respectively, have been reported to be present in Worthington collagenase (Peterkofsky and Diegelman, 1971). To inactivate these enzymes, a batch of collagenase was dissolved in 0.05 M Tris-Cl (pH 7.6), 5 mM CaCI? at a concentration of 0.4 mg/ml. The mixture was incubated for 30 min at 37°C with NEM. Dithiothreitol (5.5 mM final concentration) was added and incubation continued for 30 min. The enzyme was realkylated by incubation with NEM (12 mM final concentration) for 30 min at 37OC. The enzyme was then dialyzed against three changes of 0.05 M Tris-Cl (pH 7.6), 5 mM CaCIZ. Acknowledgments We wish to thank E. Andrews and Lin Huey-Chen for technical assistance. This research was supported by a grant from the National Institutes of Health. PMB is a postdoctoral fellow of the Helen Hay Whitney Foundation. Received

May 30. 1975;

revised

July 3, 1975

References Blumberg, Symposium Brunette,

P. M.. and Robbins, P. W. (1975). Cold Spring Harbor on Proteases and Biological Control, in press. D. M., and Till, J. E. (1971).

Burger,

M. M. (1970).

Nature

J. Membrane

Biol. 5, 215.

227, 170.

Burr, J. G., and Rubin, H. (1975). Cold Spring Harbor on Proteases and Biological Control, in press. Gahmberg, 248, 413.

C. G., Kiehn,

D., and

Hakomori,

S-l.

Glynn R. D., Thrash, C. R., and Cunningham, Nat. Acad. Sci. USA 70, 2676. Goldberg,

A. R. (1974).

Gunther, G. R., Wang, Biology 62, 366. Hogg,

N. M. (1974).

Proc.

Edelman.

Nat. Acad.

R. W.. and Kiernan,

Hynes.

R. 0. (1973).

Proc.

Hynes,

R. 0. (1974).

Cell 7, 147.

Hynes,

R. O., and Humphryes, U. K. (1970).

Nature

D. D. (1973).

Proc.

Nat. Acad.

PNAS

M., and Hultquist,

J. Cell

77, 2908.

Sci. USA

K. C. (1974).

Nature

G. M. (1974).

Sci. USA 77, 489.

J. A. (1974).

70, 3170.

J. Cell Biol. 62, 438.

227, 680.

Maizel, J. V. (1971). In Methods in Virology, and H. Kaprowski eds. (New York: Academic Morrison,

(1974).

Cell 2, 95. J. L., and

Halley,

Laemmli,

Symposium

D. E. (1963).

Ossowski, L.. Quigley, J. P., Kellerman. (1973a). J. Exp. Med. 138, 1056.

5, K. Maramorosch Press), p. 179.

J. Biol. Chem. G. M., and

238, 2847. Reich,

E.

Activation 147

by Proteases

Ossowski, L., Unkeless. J. C.. Tobia, A., Quigley, J. P., Rifkin. D. B. and Reich, E. (1973b). J. Exp. Med. 737, 112. Ossowski, L., Quigley, 249, 4312. Peterkofsky,

J. P., and Reich,

B., and Diegelman,

Quigley. J. P., Ossowski, 249, 4306.

E. (1974).

R. (1971).

L., and Reich,

Biochemistry

E. (1974).

Reich, E. (1974). In Control of Proliferation Clarkson and R. Baserga eds. (Cold Spring p. 351, Rieber, 4960.

M., and Bacalao,

J. (1974).

J. Biol. Chem.

Proc.

70, 988.

J. Biol. Chem.

of Animal Cells, B. Harbor Laboratory),

Nat. Acad.

Sci. USA 71,

Robbins, P. W., Wickus, G. G., Branton. P. E., Gaffney. B. J., Hirschberg, C. B., Fuchs, P.. Blumberg, P. M. (1975). Cold Spring Harbor Symposium on Quantitative Biology 39, 1173. Ruoslahti,

E., and Vaheri,

A. (1974).

Ruoslahti. E., Vaheri, A., Kuusela, chim. Biophys. Acta 322, 352. Sefton,

B., and Rubin,

Sefton, 8. M., and 68, 3154.

H. (1970).

Rubin,

Sefton, 8. M., Wickus, 11, 730.

Nature

Nature

H. (1971).

J. A., and Martin,

Temin.

H. M. (1971).

L. (1973).

L. B. (1975).

Nat.

Acad.

B. W. (1973).

Proc.

J. Cell. Physiology

Teng, N N. H. and Chen, 72, 413.

E. (1973).

Nat. Acad.

Unkeless, 139, 834.

J. C., Gordon,

Sci. USA J. Virology

Sci. USA 70,

78, 161. Proc.

Nat. Acad.

Unkeless. J. C., Tobia. A., Ossowski, L., Quigley, D. B., and Reich, E. (1973). J. Exp. Med. 737, 85. Unkeless, J., Dano, K., Kellerman, J. Biol. Chem. 249, 4295.

Bio-

227, 843. Proc.

G. G., and Burge.

Smith, 1263.

248, 789.

P.. and Linder,

G. M.. and

S., and Reich,

J. P., Rifkin,

Reich,

E. (1974b).

Sci. USA

E. (1974a). J. Exp.

Med.

Vaheri, A., Ruoslahti, E., and Hovi. T. (1974). In Control of Proliferation in Animal Cells, B. Clarkson and R. Baserga eds. (Cold Spring Harbor Laboratory), p, 305. Walsh, K. A. (1970). In Methods in Enzymology, and N. 0. Kaplan eds. (New York: Academic Wartiovaara, J. Exp. Med. Wickus, 65.

J.. Linder, 140. 1522.

E., Ruoslahti,

G. G., and Robbins,

79, S. P. Colowick Press), p, 41.

E., and Vaheri,

P. W. (1973).

Nature

A. (1974).

New Biol. 245,

Wickus, G. G., Branton, P. E., and Robbins. P. W. (1974). In Control of Proliferation in Animal Cells, B. Clarkson and R. Baserga, eds. (Cold Spring Harbor Laboratory), p. 541. Worthington Enzyme Manual(1972). poration, Freehold, N. J., p. 138.

Worthington

Biochemical

Cor-