Cell, Vol. 11.957-969,

August

1977, Copyright

0 1977 by MIT

Mechanism of the Decrease in the Major Cell Surface Protein of Chick Embryo Fibroblasts after Transformation Kenneth Olden and Kenneth M. Yamada Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20014

Summary The major cell surface glycoprotein of cultured chick embryo fibroblasts (CSP, a LETS protein) is substantially decreased after neoplastic transformation. We investigated the regulation of this glycoprotein by determining the kinetics of CSP biosynthesis, transit to the cell surface, and degradation before and after transformation by Rous sarcoma virus. CSP synthesis, as measured by immunoprecipitation after pulse-labeling with “C-leucine, is decreased 3-6 fold after transformation by the Bryan high titer, Schmidt-Ruppin and temperature-sensitive ts68 and T5 strains of Rous sarcoma virus. Steady state quantities of CSP in intracellular pools are also decreased 4-5 fold after transformation. However, the rate at which newly synthesized CSP is processed and exported to the cell surface is similar before and after transformation. Degradation and release of CSP from cells were measured after labeling for 24 hr. The half-life of CSP on normal cells is 36 hr and is decreased to 16-26 hr after transformation. The absolute amount of intact CSP released into the culture medium is decreased 3 fold after transformation; these amounts, however, represent losses of approximately 20 and 40% of the total CSP synthesized by normal and transformed cells, respectively. These results indicate that the major mechanism for the decrease in CSP after transformation is reduction in its biosynthesis, although increased degradation and loss from the cell surface also contribute significantly. These changes can account for the observed 5-6 fold decreases in cell-associated CSP after transformation of chick embryo fibroblasts. Introduction The major cell surface glycoprotein of cultured chick embryo fibroblasts (CSP, a LETS protein) is considerably reduced after transformation of these cells by oncogenic viruses (Stone, Smith and Joklik, 1974; Vaheri and Ruoslahti, 1974; Robbins et al., 1974). Similar LETS proteins are decreased after transformation of many other fibroblastic cells (reviewed by Hynes, 1976), and their loss generally correlates with tumorigenicity (Chen, Gallimore and McDougall, 1976). CSP has been isolated in purified form, and it can be reattached to the cell

surface of a number of transformed cells. Such reconstitution experiments indicate that CSP is an adhesive protein, and they also suggest that loss of this class of glycoprotein from cells after transformation is at least partially responsible for the decreased adhesiveness, altered morphology and cell surface architecture, decreased contact inhibition of movement, and loss of microfilament bundles of many neoplastic cells (Yamada, Yamada and Pastan, 1975; Yamada, Yamada and Pastan, 1976a; Yamada, Ohanian and Pastan, 1976b; Willingham et al., 1977; reviewed by Yamada and Pastan, 1976). Previous studies have suggested that this glycoprotein could be decreased after transformaton due to increased degradation (Robbins et al., 1974; Hynes and Wyke, 1975), to decreased synthesis or loss of association with plasma membranes (Stone et al., 1974), or to faulty retention by cells, resulting in loss into the culture medium (Vaheri and Ruoslahti, 1975). To determine the precise regulatory mechanisms, we have examined sizes of intracellular and extracellular CSP pools, rates of CSP biosynthesis, transit times to the cell surface, and degradation rates before and after transformation. Our results indicate that the principal mechanism responsible for the decrease in CSP after transformation is decreased biosynthesis, although other factors also contribute. Results Alterations in CSP after Transformation Steady state levels of CSP before and after transformation were measured by labeling cells to constant specific activity with 14C-leucine for 72 hr, homogenizing, then immunoprecipitating with anti-CSP, electrophoresing in SDS gels and determining counts in the CSP band. Figure 1A shows that cellular CSP was decreased 5-6 fold after transformation of CEF by the Bryan high titer, Schmidt-Ruppin and temperature-sensitive mutant T5 strains of Rous sarcoma virus. These decreases are similar in magnitude to the changes found in the CSP band using Coomassie blue staining for protein in SDS gels of whole cell homogenates or immunoprecipitates (Figure 1 B), although we consider the labeling and immunoprecipitation procedure to be more accurate. The distribution of CSP in CEF before and after transformation is shown in Figure 2 and Tables 1 and 2. Cells were labeled for 24 hr with 14C-leucine, and then the quantities of CSP on the cell surface, in intracellular pools or secreted into the culture medium were quantitated as described in Experimental Procedures. Extracellular trypsin-sensitive CSP constituted 70% of total CSP and was de-

Cell 958

6 A

abcdef

g

,:”

Figure

1. Amounts

of CSP after

Transformation

Quantitated

by lmmunoprecipitation

Normal and transformed chick embryo fibroblasts (CEF) were labeled at 37°C with 1 &i/ml “C-leucine for 72 hr. The cells were washed 3 times with PBS, homogenized in 2 ml of 50 mM sodium phosphate buffer (pH 11 .O) containing l%Triton X-100, centrifuged at 100,000 g for 1 hr and neutralized by dilution to 10 ml with 100 mM sodium phosphate (pH 7.0) also containing 1% Triton X-100. Then the CSP in 2 ml fractions were isolated by immunoprecipitation with anti-CSP and anti-goat globulin. An amount of anti-CRP equivalent to anti-CSP protein was incubated with homogenates to control for specificity. The resulting precipitates were sedimented, washed 3 times in PBS and homogenized in 2% SDS, and 25 (~1 aliquots were electrophoresed in a 5% slab gel. The CSP band was cut from the gel, dissolved in 1.5 ml 30% hydrogen peroxide, diluted to 20 ml with Aquasol (New England Nuclear) and counted by liquid scintillation spectrophotometry. The quantitative results of four separate experiments are presented in (A), expressed as the percentage of total TCA-insoluble cpm found in the immunoprecipitated CSP band. The error bar indicates standard error of the mean for each determination. In a representative experiment, radioactivity in CSP was 33,054 cpm, CEF; 4250 cpm, BH-RSV; 6662 cpm, SR-RSV; 5595 cpm, T5-RSV; and 207 cpm, nonspecific precipitation with anticyclic AMP receptor protein. The total TCA-insoluble cpm were 961,494, CEF; 1,072,232, BH-RSV; 1,129,781, SR-RSV; and 1,108,115, TI-RSV. Total cpmlmg total protein was 506,049, CEF; 517,986, BHRSV; 535,441, SR-RSV; and 554,058, T5-RSV. The Coomassie blue-stained precipitates are shown in (B). The 220,000 dalton CSP band is indicated by arrows immediately above myosin standard. (a) protein standards: (b) normal CEF; (c) BH-RSV CEF; (d) SR-RSV CEF; (e) TS-SR-RSV CEF; (f) ts68-SR-RSV; and (g) CEF with anticyclic AMP receptor protein antibody control for specificity: small amounts of myosin precipitate nonspecifically.

creased approximately 5 fold after transformation (Table 1). Intracellular immunoprecipitable CSP (CSP not hydrolyzed after trypsinizing the cells) constituted 10% of total CSP and was also decreased about 5 fold after transformation (Figure 2). The percentage of CSP in the culture medium varied from 15-20% of total CSP for normal cells and from 20-40% of total CSP for the transformed cells. The amount of CSP found in the culture medium, however, was decreased approximately 3 fold after transformation (Table 2). The decreases in all CSP pools suggested that transformation could be decreasing the synthesis of CSP. CSP Biosynthesis Normal and transformed

CEF cultures

were

pulse-

labeled with IO $Xml “Gleucine for 20-80 min, and at fixed intervals, they were homogenized and CSP was isolated by immunoprecipitation. A representative experiment is shown in Figure 3. Incorporation of ‘YXeucine into CSP is linear with time up to 80 min for CEF and for each of the WV transformants. In this experiment, the rate of CSP synthesis is decreased about 5 fold after transformation by a Schmidt-Ruppin and Bryan high titer strains of RSV. In three experiments, the average decreases in CSP synthesis determined using 15 min pulses of “C-leucine and immunoprecipitation were 3.4-6.5 fold (Table 1). The expression of the transformed phenotype in CEF can be readily controlled using temperaturesensitive mutants of RSV (Kawai and Hanafusa,

Mechanism 959

of Decrease

in CSP after

Transformation

CEF when cultured at the temperature (415°C) which is nonpermissive for expression of the transformed phenotype (Table 3). When these ceils were maintained at the permissive temperature (37”C), they synthesized 4 fold less CSP. The rate of CSP synthesis in the wild-type transformant (SchmidtRuppin), however, was not significantly altered by the shift from 37 to 415°C. These decreases in biosynthesis after transformation are found whether the cells are sparse or confluent. For example, the rate of incorporation of “C-leucine into immunoprecipitated CSP by CEF transformed by the Bryan high titer stain of Rous sarcoma virus is 3 fold lower than the rate for normal CEF when both were examined at a final cell density of 4 x IO6 cells per 100 mm dish (6163 cpm as opposed to 2049 cpm), 3 fold lower when at 6 x IO6 cells per dish (9121 cpm as opposed to 2678 cpm), and 5 fold lower when at 15 x IO6 cells per dish (19,899 cpm as opposed to 4108 cpm). These results indicate that the reduction of CSP in transformed CEF is primarily due its decreased biosynthesis.

1971; Martin, 1971). The rate of CSP biosynthesis in CEF infected with these temperature-sensitive strains was found to be similar to that of normal

1 A

A

A B B

c

j

C

C

'SR-ASV

Figure

2. Distribution

of CSP in Normal

TS-SA-RSV

and Transformed

Transport to the Cell Surface Immediately after biosynthesis, CSP is not accessible to hydrolysis by extracellular proteases such as trypsin. The time at which CSP becomes trypsinsensitive provides a measure of the rate at which it appears on the cell surface. Figure 4 shows that after biosynthesis, the rates at which CSP becomes accessible to trypsinization are similar for untransformed and transformed CEF. Cells were continuously labeled with ‘%-leucine; then at fixed intervals, parallel cultures were either homogenized in SDS immediately or homogenized after treatment with 10 ,ug/ml trypsin for 5 min at 37°C. The homogenates were electrophoresed, and the band corresponding to CSP was cut from the gel, dissolved and counted in a scintillation counter. On the average, trypsin-sensitive counts can be detected in the CSP band in about 30-40 min in CEF

CEF

Cultures were labeled with 2 ,&i/ml ‘%-leucine for 24 hr, and half were treated with IO pg/ml trypsin in PBS containing Ca++, Mg++ for IO min at 37°C. The reaction was stopped by the addition of soybean ttypsin inhibitorto a final concentration of 15 pg/ml. The cells were collected by centrifugation and washed twice with PBS containing the same concentration of trypsin inhibitor and 2 mM PMSF. The treated and untreated control cells were homogenized and immunoprecipitated, and cpm in CSP were quantitated as described in the legend to Figure 1. cpm per dish in cell surface CSP were calculated as the difference between the cpm in immunoprecipitated CSP bands of untreated and trypsinized cells. The culture medium from these cell cultures was centrifuged at 12,000 g at 4°C for 10 min. The CSP in 1.0 ml of the supernatant was immunoprecipitated by the addition of 20 r.d of anti-CSP and incubation for 60 min at room temperature. Then 100 ~1 of antigoat globulin were added and incubated for 30 min. The precipitate was washed, homogenized and electrophoresed as described above. Anticyclic AMP receptor protein also served as a control for specificity. (A) cell surface CSP; (B) CSP in culture medium: (C) intracellular CSP.

Table

1. Amount

Cell Type Normal

of CSP on the Cell Surface

Experiment CEF

(Percentage

1

of Total

TCA-Insoluble

Experiment

2

Counts)

Experiment

3

I% plus Standard Error of Mean

2.9

3.1

2.65

2.88

+ 0.03

BH-RSV

0.58

0.67

0.45

0.67

t 0.01

SR-RSV

0.54

0.67

0.58

0.60

t 0.03

ts68-SR-RSV

0.62

0.68

0.57

0.62

i 0.03

Cells were labeled for24-hr at 36°C with 2 &i/ml ‘%-leucine and rinsed twice with PBS, and half were treated with 10 Kg/ml trypsin in PBS containing Ca++, Mg++ for IO min at 37°C. These conditions of trypsinization are adequate to remove all CSP from the cell surface as detected by Iactoperoxidase-mediated iodination (Yamada and Weston, 1974). The reaction was stopped by the addition of soybean trypsin inhibitor to a final concentration of 15 pglml. The cells were collected by centrifugation and washed twice with PBS containing the same concentration of trypsin inhibitor and 2 mM PMSF. The treated and untreated controls were homogenized in SDS and electrophoresed. The CSP band was cut from the gel, solubilized and counted. Radioactivity per dish in ceil surface CSP was calculated as the difference between the cpm in CSP bands of untreated and trypsinized cells.

Cell 960

Table 2. Amount of CSP Released (Percentage of Total TCA-insoluble

into Culture Counts)

Medium

in 24 Hr

Cell Type 0.66 f

0.16

BH-RSV

0.26

f

0.14

Normal

CEF

SR-RSV

0.27

+ 0.17

TS-SR-RSV

0.20

+ 0.09

ts68-SR-RSV

0.25

+ 0.08

‘*r

0 NORMALCEF 0 BH-RSV 0 SRRSV A TSSR-Rsv

8c

The cells were labeled at 37°C for 24 hr with 2 $J/ml j4C-leucine, and then the CSP released into the culture medium was immunoprecipitated with anti-CSP. The antibody precipitates were washed, homogenized in SDS, electrophoresed and counted. The results presented here represent the average of four experiments.

or in CEF transformed by BH-RSV, SR-RSV or ts68 at the permissive temperature. This suggests that the reduction in CSP on the cell surface is not due to’an impairment in the intracellular processing or export mechanism of this glycoprotein. The findings that intracellular CSP pools are decreased in size in the same proportion to the decreases in CSP biosynthesis (4-5 fold; Figures 2 and 3) are also consistent with unimpaired transport of CSP to the exterior of the cell. Protein Turnover Since the decreases in CSP biosynthesis did not entirely account for the reduced levels of CSP found in RSV-transformed CEF, we investigated whether increased losses from the cell surface also contribute. CEF cultures were prelabeled with ‘%leucine for 24 hr, then placed in chase medium containing excess unlabeled leucine for 24 hr more to determine the rate at which I%-leucine disappeared from labeled proteins. In one series of experiments examining the rate of CSP degradation, cultres were homogenized with SDS at fixed intervals during the chase, and equal amounts of radioactivity were applied to each lane of SDS gels; hence increasing amounts of protein were applied as the cells were maintained longer in culture. Figures 5A-5C show gels stained for protein with Coomassie blue; the relative increase in protein relative to the constant counts applied is due to a combination of protein turnover and cell growth over the 24 hr chase period. Autoradiograms of these gels in Figures 5D-5F show that the CSP band of CEF homogenates does not change significantly over the 24 hr chase, indicating that its degradation rate is similar to that of total labeled protein. In contrast, the CSP band in autoradiograms of transformed cells decreases over time, indicating an accelerated rate of degradation. We quantitated these degradation rates in subsequent experiments by determining trypsin-sensitive

m

40

60

80

MINUTES Figure

3. Rates

of CSP Synthesis

Normal and transformed CEF were pulse-labeled at 37°C with IO &i/ml j4C-leucine for 20, 40, 60 and 80 min. CSP was immunoprecipitated, electrophoresed and quantitated as described in the legend to Figure. I.

Table 3. CSP Biosynthesis Incorporation)

(Percentage

of Total

Cellular

(B) Comparison of Biosynthesis at:

Cell Type

(A) R of Three Experiments at 37°C

37°C

41°C

Normal

3.0

3.1

3.4

i 0.20’

BH-RSV

CEF

0.52

+ 0.01

SR-RSV

0.68

i 0.02

0.63

0.60

T5-SR-RSV

1.01 + 0.15

0.77

3.3

ts68-SR-RSV

0.84

0.71

2.9

+ 0.04

+ Indicates the standard error of mean for three experiments. Cells were maintained at the indicated temperatures for 72 hr before initiation of the experiment. Cells were pulse-labeled with 10 &i/ml ‘+C-leucine for 15 min (A) or 30 min (B). Incorporation was determined by precipitation of protein with 10% TCA and antibody. The TCA precipitates were solubilized in Protosol (New England Nuclear) and counted. The antibody precipitates were electrophoresed and quantitated as described in the legend to Figure 1.

radioactivity in the CSP band. This procedure decreased the error introduced by the radioactivity in minor intracellular protein subunits co-migrating with CSP in this gel system. Figure 6 compares the rate of disappearance of counts from this cell surface CSP fraction of CEF with rates in CEF transformed by SR-RSV, BH-RSV and ts68-RSV at 37°C (the temperature permissive for transformation). The half-life of CSP on the surface of normal CEF is 35 hr. This rate is comparable to the 48 hr half-life for CSP (and total protein) previously estimated for CEF grown in different media (Yamada and Wes-

Mechanism 961

of Decrease

in CSP after

Transformation

r Normal CEF

A

I / 40

1 al

I 120

MINUTES 6

Bryan Transformed CEF

B

MINUTES 6

Schmidt-RuppinTransformed CEF

C

ton, 1974). Aftertransformation, the half-life of CSP is decreased to 16-23 hr (Figure 6). Similar rates of degradation were obtained when total cell-associated CSP was isolated from cell homogenates by immunoprecipitation with antibody to CSP (see legend to Figure 8). To explore the relationship of this apparent increase in degradation to transformation, we used the ts68 temperature-sensitive mutant of RSV (Kawai and Hanfusa, 1971). CEF cultures infected with ts68 were labeled for 24 hr at the nonpermissive temperature (415”C), then rinsed with culture medium preequilibrated to 415°C. The cultures were then incubated in chase medium at either 41.5 or 36°C (the temperature permissive for transformation). Figure 7 compares the rate of disappearance of cpm in cell surface trypsin-sensitive CSP of ts68infected CEF at the permissive and nonpermissive temperatures, and the turnover rates on normal cells at these temperatures. The turnover rate of CSP is twice as fast at 36”C, the permissive temperature, as a 41.5%. In contrast, the turnover of CSP in uninfected CEF is not significantly affected by the same shift in temperature. The increase in turnover of CSP after shift to the permissive temperature appears to be relatively rapid, since an increased rate of turnover is evident by 6 hr. It is also important to note that CSP turnover in ts68 at 415”C, the nonpermissive temperature, was slightly faster than that for infected normal cells. This slightly higher turnover rate may account for the finding that the steady state level of CSP in ts68 at 41.5% is always slightly lower than that in uninfected normal CEF. When ts68 was cultured for 72 hr at 41.5”C, the morphology of the cells appeared normal, but the level of CSP was about 70-95% normal. These turnover results indicate that most of the increased rate of CSP degradation is associated with transformation and is not simply due to the presence of the viral genome or to virus production. Figure 4. Rate Surface

MINUTES I

7s x b

t&B Schmidt-RuppinTraasfoned CEF

D

of Export

of Newly

Synthesized

CSP to the Cell

Normal and transformed CEF were continuously labeled with 10 &i/ml “C-leucine for up to 120 min. At the indicated intervals, parallel cultures were homogenized either in 2% SDS solution directly or after treatment with 10 pg/ml trypsin for 5 min at 37°C. The reaction was stopped by the addition of 15 kg/ml soybean trypsin inhibitor, and the cells were then homogenized in SDS solution. 40 pg protein aliquots were electrophoresed in 5% polyacrylamide gels. The radioactivity in the CSP band in treated and untreated control cultures was determined by cutting this band from the gel and counting as described in the legend to Figure 1 and expressed as cpm per 100 mm dish. The relative differences in rates of biosynthesis after transformation shown in this figure are smaller than those described earlier using immunoprecipitation (Figure 3; Table 3) because we have not corrected for differences in uptake of label and because of incorporation into other minor proteins that migrate in this region of the gel.-

I

-

Mechanism 963

of Decrease

in CSP after

Transformation

In contrast, the overall rate of protein degradation was not increased after transformation. The average half-life for degradation of total protein, as determined by the rate of loss of acid-insoluble radioactivity from cells, was 37 hr for both normal CEF and cells transformed by SR-RSV, BH-RSV and t&&RSV. In SDS gels of CEF homogenates, the major protein band that migrates adjacent to the CSP band appears to be the intracellular protein filamin (Wang, Ash and Singer, 1975; Shizuta et al., 1976b). This band has an apparent molecular weight of 240,000-245,000 daltons, and it co-migrates with the chicken gizzard filamin molecular weight marker. Antibody to filamin stains only this band in CEF homogenates (Olden and Yamada, 1977; unpublished results). The turnover of this intracellular protein band was also unchanged after transformation. The half-life of incorporated leucine was about 33 hr in CEF and in CEF transformed by Bryan high titer, Schmidt-Ruppin and T5 temperature-sensitive strains of Rous sarcoma virus. The 1.5-2 fold increases in CSP turnover in RSVtransformed CEF that we find in these experiments, combined with the 3-6 fold decrease in biosynthesis, can account for the 5-6 fold decrease in cellular CSP levels. CSP Turnover: Degradation versus Release into Medium These data did not indicate whether the observed turnover of “‘C-labeled CSP is due to direct degradation involving proteolytic digestion or to release of intact CSP into the culture medium. In addition, the decreased half-lives after transformation could be explained by either an increase in the rates of proteolysis or of release into the medium or a combination of both. To decide between these alternatives, we prelabeled cell cultures with 14C-leucine, placed them in chase medium and followed the rate of release of intact CSP into the culture medium. At designated intervals, the culture medium was withdrawn, centrifuged and immunoprecipitated with antibody to CSP. The cells were also homogenized and immunoprecipitated as described above. The rates of loss of labeled CSP from normal and RSVtransformed CEF are compared with its rates of appearance in the culture medium in Figures 8A and 8B. CSP was released into the culture medium at a rate of 14% of the original CSP cpm per 24 hr Figure

5. Decrease

in Radioactivity

Found

1

ial664,.

t t b z

3-

2-

b

l-

0 NormalCEF A !3ltRsv q sFl-Rsv 0 l5$8-sRRSVl37~)

I 6

I 12

I

I 24

16

I 30

I 36

HOURS Figure 6. Rates Transformants

of Degradation

of CSP

in Normal

and Wild-Type

Normal and transformed CEF were labeled for 24 hr with 2 @J/ml ‘Gleucine at 37°C. The labeling medium was removed, washed 3 times with serum-free culture medium equilibrated to 37°C and replaced with culture medium supplemented with 2 mg/ml unlabeled leucine for up to 36 hr. Fresh chase medium was added at 12 hr. At designated intervals, parallel cultures were directly homogenized either in 2% SDS or after treatment with 10 /*g/ml trypsin for 10 min at 37°C. Equal amounts of protein (40 /*g) were applied to each lane of SDS-polyacrylamide gel, electrophoresed and quantitated by determining the number of trypsin sensitive counts per dish remaining at each time point as indicated in Experimental Procedures.

for normal cells and at 19-24% for the transformed cells. Since CSP disappears from the cell surfaces (turnover) at a rate of 33% per 24 hr for normal cells and at 4052% per 24 hr transformed cells, turnover cannot be explained solely on the basis of release into the culture medium. Release of intact CSP into the culture medium can account for only about half the CSP turnover in both normal and transformed cells. Fate of CSP Released into Medium CSP released into the culture medium

was identical

in the CSP Band

Normal and transformed CEF were labeled for 24 hr with 2 &i/ml YYeucine. The labeling medium was withdrawn, and the cultures were washed 3 times with serum-free medium equilibrated to 37°C and then placed in medium supplemented with 2 mg/ml unlabeled leucine for 24-36 hr. The cells were homogenized in 2% SDS at the designated intervals, and equal amounts of radioactivity (25,000 cpm) were applied to each lane of the SDS-polyacrylamide gel: the amount of protein applied to each lane increased as the cells were longer in culture. The Coomassie blue-stained gels are shown in (A-C), and the autoradiograms are shown in (D-F).

Cell 964

,”

A

9 8 7

0 0 A 0

6 7 0s.

l Normal CEF LI BH-RSV

NORMALCEF 418 lt112= 37 hr) NOFMAL CEF 37Oltl/.?= 37 hd tsWtSV - 41.5’ (tl/Z =23 hrl t&LRSV - JI” ttlR = 17hrl

ill SR-WV x T5-SR-RSV o ts68-SR-RSV

1L

I 6

I

I

I

12

18

24

HOURS Figure 7. Rates of Degradation of CSP ture-sensitive Transformed Cells

in Normal

and Tempera-

Normal and transformed CEF in culture for 46 hr at 41.5% were labeled for 24 hr with 2 &i/ml ‘%-leucine. The labeling medium was withdrawn, and cultures were rinsed 3 times with serum-free culture medium equilibrated to 41.5”C, then placed in culture medium supplemented with 2 mg/ml unlabeled leucine and incubated for up to 24 hr at 41.5 or 36°C. Fresh chase medium was added at 12 hr. The remainder of the procedure is the same as that described in the legend to Figure 6.

IL

LT

HOURS

to cell-associated CSP with respect to migration in SDS gels, immunoprecipitation by anti-CSP and labeling with glucosamine (data not shown; see also Vaheri and Ruoslahti, 1975; Critchley, Wyke and Hynes, 1976). We investigated whether this released pool of CSP was in equilibrium with the CSP remaining on the cell surface. If the CSP in these two locations were in equilibrium, increasing the volume of medium would be expected to result in an increase in the total quantity of CSP in the medium to maintain a constant ratio of CSP concentrations. Increasing the volume of GM over monolayers of CEF 10 fold, however, did not alter the total quantity of CSP released into medium (12.5 pg in 10 ml as opposed to about 11.8 pg in 100 ml) in 24 hr. In addition, CSP continued to accumulate if the culture medium was not changed. The pools are therefore not in equilibrium. The rate of exchange of the CSP in medium back onto cells was estimated by combining prelabeled CSP in conditioned medium with unlabeled CEF cultures. Monolayer cultures of CEF were labeled 24 hr with 3 &i/ml “C-leucine in GM, and 50 ml of medium from 100 mm dishes were collected, centrifuged and dialyzed 12 hr against three changes of fresh medium. The dialyzed medium was added to untransformed CEF with or without 2 pg/ml cycloheximide, which inhibited protein synthesis by 98%. After incubation for 24 hr, the cells were homogenized in SDS, the culture medium was immunoprecipitated with anti-CSP and both were run on SDS gels. We found that 6.3% (5% per mg cell

Figure 6. Rates Medium

of CSP Turnover

and

Release

into the

Culture

Normal and transformed cells were labeled at 37°C for 24 hr with 3 &i/ml ‘%-leucine. The labeling medium was withdrawn, and cells were washed as described in the legend to Figure 6. Fresh medium containing 2 mg/ml unlabeled leucine was added and incubated at 37°C for 24 hr. Cells and 1 ml aliquots of culture medium were immunoprecipitated with anti-CSP. The total counts in cell-associated CSP were determined as described in the legend to Figure 1 and in Experimental Procedures. The percentage of total cell-associated CSP counts remaining at 0, 12 and 24 hr is shown in (A), and the percentage of total CSP counts released into the culture medium is shown in (8). The half-life of CSP in normal CEF was 42 hr; BH-RSV, 34 hr; SR-RSV, 27 hr; T5-RSV, 25 hr; and ts66, 23 hr.

protein) of the total CSP counts bound to the cycloheximide-inhibited cells and 10.7% (4.6% per mg cell protein) to uninhibited cultures. We recovered 96% of the total counts added to each culture, and autoradiography of the gels of cells and culture medium proteins showed that all the counts were localized in the CSP band. These results indicate that once CSP is released into culture medium, it is not degraded significantly, and that only a small proportion of the CSP released into medium becomes reassociated with cells. Discussion The principal findings of these studies on the mechanism of the decrease in CSP after transformation are summarized in Figure 9. First, the rate of CSP synthesis in transformed cells is 3-6 times

Mechanism 965

of Decrease

in CSP after

CSP IN CULTURE I t

Transformation

MEDIUM:

1520%

50% RELEASED INTACT INTO MEDIUM 50% DEGRADED 2% PER 24 hr.

I/CSP TURNOVER: t%=36

hr.

t

i I I

CELL

SURFACE

CSP:7O%v

I

I’““““” ---_

TIME TO CELL SURFACE: 30-40 min. 4

INTRACELLULAR

CSP POOL:

10%

T

CSP SYNTHESIS: 3% TOTAL CELLULAR

NORMAL CSP IN CULTURE t

t

AMOUNT

CEF MEDIUM

i 2.5-3-

fold

RELEASED INTACT INTO MEDIUM1 1.5 fc’d

AMOUNT DEGRADED 1 1.5 fold k CSP TURNOVER:! 1.5-2 fold t

CSP

ICELL I I

SURFACE

~ANSIT ---_

TIME TO CELL SURFACE: n0 change

t 5 fold

t -I

INTRACELLULAR

CSP POOL:

4 5 fold

t

CSP SYNTHESIS: 13-6 fold TOTAL CELLULAR

TRANSFORMED Figure Kinetics

9. Summary

incorporation into the 200,000-250,000 region of gels of CEF plasma membranes was decreased 5080% after transformation. They concluded that the peptides in this region were either synthesized less or not associated appropriately with the plasma membrane. This decrease was probably due to the decrease in CSP biosynthesis, although their measurements may have also included decreases in membrane-associated myosin (Shizuta et al., 1976a).

CEF

Transit to the Cell Surface Our data on the rate of appearance of newly synthesized CSP on the cell surface do not support an earlier hypothesis that CSP is synthesized at normal rates in transformed CEF but is not expressed at the cell surface because of faulty processing (Hynes et al., 1975). While it is conceivable that such an intracellular CSP precursor would not immunologically cross-react with our antibody and hence would not be precipitated, it is highly improbable that we would be unable to detect its accumulation by radioactivity since it is a major cellular protein (about 3% of total protein; Figure 1; Yamada and Weston, 1974). In addition, intercellular pools of CSP are decreased in the same proportion to the decrease in biosynthesis after transformation (Figure 2). The decrease in intracellular CSP pools after transformation and the absence of any immunologically detectable accumulation of a CSP precursor pool in trypsinized cells were confirmed using a new procedure for detecting antigens in SDS gels (Olden and Yamada, 1977).

of Data

and pool sizes of CSP in normal

and transformed

cells

lower than that of untransformed CEF. Second, the intracellular pools of CSP in transformed cells is 45 times smaller than in normal cells. Third, the rate of export of newly synthesized CSP to the cell surface is not changed after transformation. Fourth, the rate of CSP degradation in transformed cells is 1.5-2 times greater than normal cells. The alterations in biosynthesis and degradation are sufficient to account for the differences in CSP levels between normal and transformed cells. Biosynthesis The 3-6 fold decrease in CSP synthesis after RSV infection is due to transformation in that it occurs after infection by all four RSV strains and is found only at the temperature permissive for transformation in the temperature-sensitive mutants. The decrease is not due to altered growth rates, since CEF and transformants grew at approximately equal rates according to cell counts, protein determinations and incorporation of “C-leucine. Stone et al. (1974) previously reported that valine

CSP Degradation and Loss into Culture Medium The 1.5-2 fold increases in turnover of CSP that we find in RSV-transformed CEF, taken together with the 3-6 fold decrease in biosynthesis, will account for the 5-6 fold decrease in cellular CSP levels. Our degradation results using an incorporated labeled amino acid confirm and extend earlier studies of CSP turnover using external labeling procedures which suggested that the rate of degradation was increased after transformation (Robbins et al., 1974; Hynes and Wyke, 1975; Hynes et al., 1975; Rieber, Bacalao and Alonso, 1975). It is possible that the wide variability in turnover rates which these investigators noted could be due to variable loss into culture media of a more labile subpopulation of CSP labeled by iodination. This increase in apparent degradation rate can be attributed only partly to increased loss into the medium. In addition, there is a 3 fold decrease in the absolute amount of CSP lost into the culture medium after transformation, which is a larger decrease than previously reported by Vaheri and Ruoslahti (1975) for human fibroblasts. In contrast to their results, our direct measurements of the

Cell 966

total amount of CSP indicate that the cell-associated CSP pool is substantially larger than the amount released into media. These results and the finding that exogenous CSP will reattach to many transformed cells (Yamada et al., 1976a, 1976b) are not consistent with the hypothesis that LETS proteins are decreased on the cell surface due to inability to attach to the plasma membrane after trans+. formation. The increased degradation of CSP is relatively specific in that the turnover of the intracellular protein filamin, as well as of total cellular protein, is not increased after transformation. The increased degradation may be due to the increased protease activity at the cell surface following transformation, which has been investigated extensively (Reich, Shaw and Rifkin, 1975). Another possible mechanism would be alterations in CSP itself. Previous studies in both normal and transformed cells (Goldberg and Dice, 1974; Goldberg, Olden and Prouty, 1975; Bradley, Hayflick and Schimke, 1976) have shown that abnormal proteins are degraded more rapidly than normal protein, and that abnormal proteins are more sensitive to proteases in vitro. It is improbable that the accelerated rate of CSP turnover seen after shifting cells infected by the temperature-sensitive virus ts68 to the temperature permissive for transformation is due to altered primary structure, since the CSP had been prelabeled at the nonpermissive temperature. It is possible that CSP is modified in some other manner after transformation-for example, in glycosylation (Critchley et al., 1976). CSP in Conditioned Medium The CSP released into culture media is identical to that on the cell surface by the criteria of immunoprecipitation by anti-CSP, co-migration with cellassociated CSP and labeling with glucosamine. This pool of CSP, however, is not in equilibrium with cell-associated CSP and does not rapidly exchange back onto the cell surface. We speculate that the CSP in media is due to variable sloughing of extracellular CSP from cells. CSP or other LETS proteins may account for the increase in cell-tosubstratum adhesion of transformed cells reported after exposure to conditioned media from untransformed cells (Moore, 1976). In conclusion, our results indicate that the decrease in CSP and the accompanying morphological effects due to its loss are due primarily to a specific decrease in its biosynthesis. Other alterations, such as increased loss into the culture medium, and increased degradation, possibly due to general modifications of the cell surface, appear to have lesser roles. The decreased biosynthesis we observe appears to be ,due to decreased transcrip-

tion of CSP mRNA after transformation, since a similar specific decrease in CSP synthesis is seen using isolated RNAs in an in vitro translation system (S. Adams et al., manuscript in preparation). Experimental

Procedures

Cell Culture Primary chick embryo fibroblasts (CEF) were prepared from 10 day old White Leghorn embryos and infected by Bryan high titer, Schmidt-Ruppin or temperature-sensitive strains ts66 (Kawai and Hanafusa, 1971) or T5 (Martin, 1971) of Rous sarcoma virus according to the procedures of Vogt (1969). Cells were cultured in Ham’s FIO medium supplemented with 10% tryptose phosphate broth, 5% heat-inactivated calf serum, 0.5% beef embryo extract (Grand Island Biological), 0.056% sodium bicarbonate, 50 units per ml penicillin, 50 pg/ml streptomycin and 2 mM glutamine (modified GM; Vogt, 1969). Cells were maintained in a humidified 37°C incubator with an atmosphere of 5% CO, and 95% air, and were fed daily (15 ml per 100 mm dish). Cells in the fourth or fifth passage were subcultured using 0.25% trypsin, plated at 1 x lo6 cells per 100 mm diameter plastic tissue culture dish (Falcon) and cultured at least 48 hr before an experimental treatment; experiments were terminated before the cells became heavily confluent (~1 x 10’ per dish). The extent of transformation was monitored routinely by phase-contrast microscopy to evaluate cellular morphology, overlapping and alignment, and quantitated by the rate of uptake of “C-2-deoxyglucose using previously described methods (Yamada and Pastan, 1976). The RSV-infected cells appeared fully transformed by these criteria. In one experiment, for example, the 10 min uptake of 0.5 @/ml of “C-2-deoxy-D-glucose (52.5 mCi/ mmol; New England Nuclear) was 75,400 2 2500 cpm/mg protein for CEF; 457,600 c 5,600 cpm/mg for CEF transformed by the Schmidt-Ruppin strain of RSV; and 411,800 i 4,300 cpm/mg for CEF infected by the ts66 strain of RSV and maintained for 48 hr at the temperature permissive for transformation. Protein Synthesis Protein synthesis was measured by the rate of incorporation of “C-leucine (10 &i/ml) into trichloroacetic acid (lo%)-insoluble cellular material or into specific proteins. Subconfluent monolayer cultures were incubated with j4C-leucine in GM for 15-60 min, and rinsed twice with Dulbecco’s phosphate-buffered saline (PBS) and once with PBS minus Ca++ and Mg++. Parallel cultures were either immunoprecipitated as described below, or homogenized in 2% SDS and divided into aliquots for determination of total protein, total acid-insoluble incorporation by precipitation with 10% TCA or SDS gel electrophoresis. lmmunoprecipitation Because CSP is insoluble at physiological pH even in the presence of nonionic detergents, we developed a protocol in which CSP is first solubilized at pH 11 in Triton X-100, and then diluted 10 fold to minimize nonspecific precipitation and neutralized to permit immunoprecipitation. Each dish of cells was homogenized in 2 ml of 50 mM sodium phosphate (pH 11 .O) containing 1% Triton X-100, and the pH was readjusted to 11 .O with NaOH. CSP is solubilized at this pH (Yamada et al., 1976a; see below). The homogenate was centrifuged at 100,000 g at 4°C for 1 hr to sediment insoluble material. The supernatant was diluted 10 fold with 100 mM Na-phosphate (pH 7.0) containing 1% Triton X-100 and 50 mM NaCI. CSP was immunoprecipitated by the addition of 90 ~1 of affinity-purified anti-CSP (1 mg/ml) to 2 ml of the diluted supernatant and incubated for 60 min at room temperature. A parallel sample incubated with an equal amount of goat affinity-purified antibody against the cyclic AMP receptor protein of E. coli (anti-CRP; provided by Drs. B. Howard, B. decrombrugghe and I. Pastan) served as a control for

Mechanism 967

of Decrease

in CSP after

Transformation

specificity. 0.7 ml of rabbit anti-goat IgG (Miles Laboratories) were then added, and incubation was continued for an additional 30 min. The resulting precipitate was sedimented by centrifugation at 12,000 g at 4°C for 10 min. The pellet was disrupted with a glass stirring rod, resuspended in PBS containing 1% Triton X-100 and again sedimented. This was repeated twice and then once more using PBS without Triton X-100 or Ca++ and Mg++. The pellet was then homogenized in 2% SDS, and 25 pi aliquots were electrophoresed in SDS slab gels as described below. The amount of CSP synthesized was quantitated by cutting out the CSP band, solubilizing for 48 hr in 30% hydrogen peroxide and counting in a liquid scintillation counter. All buffers contained 2 mM phenylmethyl sulfonyl fluoride (PMSF) to inhibit proteases. Evaluation of the lmmunoprecipitation Procedure The proportion of CSP solubilized in the first step at pH 11 .O was determined using cultures labeled with “C-leucine for 15 min or 24 hr as described for the biosynthesis or pool-size experiments. After homogenization and centrifugation, the 100,000 g supernatants and pellets were neutralized, homogenized in 2% SDS and electrophoresed on SDS-polyacrylamide gels. In cultures labeled for 15 min with “C-leucine (for determination of CSP synthesis rates), the radioactivity in the CSP region of the pH 11 .O supernatants constituted more than 99% of the total counts in that region for each cell type (CEF, BH-RSV, SR-ASV, T5-SR-RSV and ts68SR-RSV). In cultures labeled for 24 hr with “Z-leucine, the radioactivity in the CSP region of the pH 11 supernatants constituted 97% of the total for CEF, 89% for BH-RSV, 89% for SR-RSV, 87% for T5-SR-RSV and 85% for ts68-SR-RSV. The apparent decreases in recovery were due to a constant background of radioactivity in insoluble material in the CSP region of SDS-polyacrylamide gels of all these cultures (for example, in one experiment, 614-786 cpm in the 100,000 g pellets as opposed to 4384-25,824 cpm in the supernatants). Nonspecific precipitation of CSP after reneutralizing was minimized by diluting samples 10 fold prior to immunoprecipitation. The nonspecific precipitation of CSP using anticyclic AMP receptor protein antibody was ~5% of total CSP (for example, see Figure 1 and legend). In this procedure, small amounts of cellular myosin also precipitate nonspecifically (Figure 1B). This necessitates SDS gel electrophoresis and counting radioactivity in the CSP band. The immunoprecipitation procedure recovered CSP quantitatively as determined by precipitation of ‘251-labeled carrier CSP added to homogenates. For example, 2000 cpm of CSP were added and 2020 cpm were recovered. We performed a mixing experiment to rule out the possibility that the apparent decreases in CSP biosynthesis after transformation were due to degradation of CSP by the proteases of transformed cells during the 1.5 hr immunoprecipitation procedure. After’iabeling fdr 15 min with 14C-16Licine, 2 ml of CEF supernatant, 2 ml of supernatant from CEF transformed by Bryan higi+titer RSV and 1 ml of each mixed together were immunoprecipitated in parallel. The radioactivity found in CSP in the mixed samples equaled the average of the values of samples from normal and transformed cells that had been immunoprecipitated separately: CEF, 21,500 f 1500 cpm; BH-RSV, 1750 2 500 cpm; and CEF plus BH-RSV, 11,900 + 1200 cpm (mean t- standard error for three samples). In addition, it is possible to check the conclusions of the immunoprecipitation experiments against data derived using other methods such as protein staining or determinations of trypsin-sensitive radioactivity in the CSP band. These agree in all cases. For example, total cellular CSP is decreased 5-6 fold by immunoprecipitation (Figure IA) and by protein staining (Figures IB and 5); CSP in the cell surface pool is decreased 5 fold by immunoprecipitation (Figure 2) or by determination of trypsinsensitive cell surface radioactivity in the CSP band (Table 1); and the rates and decreases after transformation in CSP turnover as determined by these two procedures are also similar (Figures 6-

8).

Protein Degradation The rate of protein degradation was measured by the disappearance of previously incorporated labeled amino acid. Monolayer cultures were incubated for 24 hr in medium containing 2 &i/ml “C-leucine. The labeling medium was removed, cells were rinsed 4 times with serum-free medium supplemented with 2 mg/ml unlabeled leucine, and the cultures were incubated in 30 ml fresh GM medium supplemented with an additional 2 mg/ml unlabeled leucine (“chase medium”) for a24 hr period, during which label in TCA- or anti-CSP-precipitable material decreased. The chase medium was replaced with fresh chase medium after 12 hr. If the additional leucine was omitted, the apparent half-life of total protein became greatly prolonged (>50 hr) due to reutilization of label. The addition of 2 pglml cycloheximide, a concentration that inhibits 14C-leucine incorporation by 98%, to the leucinesupplemented chase medium did not further shorten the apparent half-life of total protein, indicating minimal reutilization of label. The degradation of CSP was monitored by two alternate procedures. With the first procedure, cells were homogenized at designated intervals, and labeled CSP was isolated by immunoprecipitation and quantitated as described above. With the second procedure, at fixed intervals, monolayer cultures were treated with IO pg/ml trypsin for 5 min at 37°C. The trypsin solution contained 2 pg/ml cycloheximide to block incorporation of labeled amino acids released during proteolysis. The trypsinized and untreated control cultures were washed twice with PBS, once with PBS minus Ca++, Mg ++ , solubilized in 2% SDS and electrophoresed. All buffers contained 2 mM PMSF. The rate of loss of trypsinsensitive counts from the CSP band was determined by comparison to untreated controls by the following calculations: CSP cpm per dish = CSP cpm per lane x (total volume of homogenate per dish/volume applied per lane); trypsin-sensitive CSP cpm per dish = CSP cpm per dish (untrypsinized) minus CSP cpm per dish (trypsinized). In comparisons of normal and transformed cells, we normalized for diffferences in uptake and incorporation of j4C-leucine during the initial labeling period. For example, if the total acidprecipitable cpm per dish were 1 .O x lo4 for CEF, 1.2 x IO4 for BH-RSV and 1 .I x IO4 for SR-RSV, we would divide all BH-RSV cpm by 1.2 and all SR-RSV cpm by 1 .l_ ‘I. Release into Culture Medium The release of CSP into the culture medium .was determined after incubating cells with 1 pCilml “C-leucine for 24 hr and precipitation of protein with 10% TCA or antibody. At fixed intervals, an aliquot of the culture medium was removed and centrifuged at 12,000 g at 4°C for 10 min to remove floating cells and debris. 1 ml of the supernatant was precipitated with 10% TCA, washed twice with 5% TCA and homogenized in 2% SDS. For immunoprecipitation, 20 ~1 of anti-CSP were added to 1 ml of the supernatant and incubatedJ,for 60 min at room temperature. The addition of an equal amount of anticyclic AMP receptor protein and antimyosih served as controls for antibody specificity. 100 ~1 of rabbit antigoat globulin were then added, and incubation was continued for 30 min. The precipitate was sedimented and washed with PBS minus Triton X-100 as described earlier. Gel Electrophoresis Cells or precipitated protein were homogenized in 10 mM sodium phosphate buffer (pH 7.0) containing 2% SDS and 2 mM PMSF to inhibit proteolysis, and heated in a boiling water bath for 3 min. Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin standards. The homogenate was reduced with 0.1 M dithiothreitol and electrophoresed in 5% polyacrylamide 1 mM thick slab gels as described previously (Studier, 1973; Yamada and Weston, 1974). Autoradiograms were prepared by drying the gels onto filter paper and exposing them to Kodak RP Royal X-ray film. Gels were calibrated for molecular weight using the following standards: chicken gizzard filamin, 240,000 daltons (a gift from Dr. P. Davies); rabbit skeletal muscle myosin, 200,000 daltons; RNA polymerase, 150,000 and 160,000 daltons;

Celt,, 966

phosphorylase a, 94,000 daltons; and ovalbumin,

daltons; bovine 43,000 daltons.

serum

albumin,

68,000

Other Procedures Antibodies to fibroblast myosin (Willingham, Ostlund and Pastan, 1974; Olden, Willingham and Pastan, 1976), CSP (Yamada et al., 1975) and cyclic AMP receptor protein (CRP) (Pastan. Gallo and Anderson, 1974) were purified by affinity chromatography (Axen, Porath and Emback, 1967; Shapiro et al., 1974). CSP was isolated and fractionated by ammonium sulfate as described previously (Yamada et al., 1976a). Glycoproteins were labeled by growing the ceils in Dulbecco’s modified Eagle’s medium with glucose reduced to 9 mM previously described (Olden et al., 1976). Chemicals Phenylmethylsulfonyl fluoride (PMSF) and Triton X-100 were purchased from Calbiochem; electrophoresis reagents from Bio-Rad laboratories; cycloheximide from Sigma Chemical; twice-crystallized trypsin and soybean trypsin inhibitor from Worthington Biochemicals. The radiochemicals l%(U)-L-leucine (spec. act. 325 (spec. act. 238 mCi/ mCi/mmole) and “C(U)-D-glucosamine mmole) were obtained from New England Nuclear. Acknowledgments We thank Drs. Ira Pastan and Benoit decrombrugghe for valuable advice and discussions; Ms. Elizabeth Lovelace for aid in cell culture; Mr. Ellis Neufeld for his assistance in preparing affinitypurified antimyosin antibodies; Dr. Bruce Howard for the generous gift of affinity-purified anti-E. coli cyclic AMP receptor protein antibodies; and Drs. Sadaaki Kawai and Hidesaburo Hanafusa for the gift of tsNY68 virus. We also thank Mr. Raymond Steinberg for photographic services and Mrs. Wilma Davis for typing the manuscript. Received

March

1, 1977;

revised

April

19, 1977

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Transformation

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Cell

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