Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3594-3598, September 1975

Cell Biology

Plasma membrane alteration associated with malignant transformation in culture (chick embryo fibroblasts/temperature-sensitive Rous sarcoma virus/3T3 mouse cell line/SVT2/freeze-fracturing/glycerol/ intramembrane particles)

NORTON B. GILULA, RICHARD R. EGER, AND DANIEL B. RIFKIN The Rockefeller University, New York, N.Y. 10021

Communicated by James G. Hirsch, July 7, 1975

ABSTRACT The intramembrane organization of the plasma membranes of nonmalignant cells in culture has been compared by freeze-fracturing with that of virally-transformed malignant cells. No dramatic differences are present in the distribution of intramembrane particles in the plasma membranes of these cells when the cells are examined without fixation or with mild fixation (glutaraldehyde treatment) prior to freezing. However, a redistribution of intramembrane particles into aggregates occurs in the membranes of nontransformed cells after treatment with glycerol. The aggregation of particles is extensive in normal chick embryo fibroblasts, and less extensive in mouse 3T3 cells. The glycerol-induced particle redistribution is not inhibited at 40, but it is inhibited by pretreatment with 2.5% glutaraldehyde. A significant number of the cells remain viable after the glycerol treatment, and the process is reversible. Particle aggregation does not appear to be related to either growth rate or cell density. Transformed Rous sarcoma virus/chick embryo fibroblasts and simian virus 40/3T3 cells have few particle aggregates after glycerol treatment. The plasma membranes of chick embryo fibroblasts transformed with a mutant of Rous sarcoma virus (TS-68) that is temperature sensitive for transformation, have few particle aggregates when grown at the permissive temperature (37°). Extremely prominent particle aggregates are present in the plasma membranes ofcells grown at the nonpermissive temperature (410). These observations indicate that there is an alteration in the plasma membrane associated with viral transformation which is related to a glycerol-sensitive mechanism that controls the distribution of intramembrane particles.

ol, as well as dimethyl sulfoxide, can induce particle aggregates in the plasma membranes of lymphoid cells (12, 13). (ii) Were the observed differences related to transformation or cell growth? Only one cell system was used, 3T3 and simian virus 40 (SV40)-transformed 3T3, and the 3T3 line used may not necessarily represent a "normal" cell (14). In this study we have examined the plasma membrane structure of Balb 3T3 cells and an SV40 transformant of these 3T3 cells, chick embryo fibroblasts (CEF) and chick embryo fibroblasts transformed by Rous sarcoma virus (RSV), as well as chick embryo fibroblasts infected with a virus that is temperature sensitive for transformation. We have found that the particle aggregates are definitely present in normal populations as a result of glycerol treatment. Furthermore, our data indicate that the inability of glycerol to induce aggregation is related to transformation. All of the transformed cell membranes (three different types) are relatively insensitive to glycerol-induced particle aggregation, whereas the parental or nontransformed counterparts of these cells have membranes that are sensitive to glycerol-induced particle aggregation. MATERIALS AND METHODS Growth of cell cultures 3T3 Mouse Cell Lines. Cells of the A31 clone from the mouse Balb/3T3 cell line were kindly provided by Dr. George Todaro, National Institutes of Health, Bethesda, Md. These cells were grown on 150-mm petri dishes in 30 ml of Dulbecco's medium with 10% fetal bovine serum. Cells were maintained at 370 in a 10% CO2 environment. A simian virus 40 transformant of A31 (SVT2) was also obtained from Dr. George Todaro. For these studies, the A31 cells were plated at 5 X 104 cells/dish, and the populations were examined at low density (1 X 106 cells/dish), near confluency (4 X 106 cells/dish), and above confluency or at their normal saturation density (9.5 X 106 cells/dish). At high densities the number of SVT2 cells was approximately 2.5 X 107 cells/ dish. Chick Embryo Fibroblasts (CEF). Chick embryo fibroblasts were prepared from COFAL negative eggs (Spafas, Inc.) as described (15). The cells were grown on 150-mm petri dishes in 30 ml of Eagle's medium with 10% fetal bovine serum. Normal cells were maintained at either 360 or 410. The cells were plated at a low density (106 cells/dish), and samples were taken for observation at low density (2 X 106 cells/dish), high density (7 X 106 cells/dish), and confluency (2.2 X 107 cells/dish). Transformed cells were ob-

Malignant transformation of cells in culture has been associated with several changes, such as increased protease production (1), biochemical changes in cell surface components (2-6), increased lectin agglutinability (6, 7), and cell shape changes (8, 9). All of these changes may be directly or indirectly related to modification of the cell surface membrane. Recently, Scott et al. (10, 11) reported that differences exist in the internal structure of the membranes of normal and transformed cells. With the freeze-fracture technique, these workers found that at high cell densities the intramembrane particles were aggregated in the membranes of normal cells, while at similar densities, the intramembrane particles were dispersed in transformed cell plasma membranes. Several questions were raised from the data in this earlier study. (i) Were intramembrane particle aggregates a characteristic feature of nontransformed cell membranes, or were they induced by the glycerol treatment that was used for cryoprotection during the preparation of the cells? In other studies on intact cells, it has been demonstrated that glycerAbbreviations: CEF, chick embryo fibroblasts; RSV, Rous sarcoma virus; TS-68, temperature-sensitive Rous sarcoma virus; 3T3, 3T3 mouse cell line; SV40, simian virus 40. 3594

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FIGS. 1-9. Fracture faces (inner halves) of plasma membranes from CEF cells and RSV transformants. Fig. 1. Normal CEF, fixed. Fig. 2. Normal CEF, glycerinated. Fig. 3. CEF/RSV, fixed. Fig. 4. CEF/RSV, glycerinated. Fig. 5. CEF/RSV (TS-68), 360, fixed. Fig. 6. CEF/ RSV (TS-68), 360, glycerinated. Fig. 7. CEF/RSV (TS-68), 410, fixed. Fig. 8. CEF/RSV (TS-68), 410, glycerinated. Fig. 9. Normal CEF, trypsin-treated, glycerinated. Figs. 1-8: all X65,000. Fig. 9: X64,890.

tained by infection with the Schmidt-Ruppin strain of Rous virus (RSV) of the subgroup A, and transformed populations were examined at 360 and 41°. A temperaturesensitive nontransforming mutant of RSV (TS-68) originally obtained from Dr. S. Kawai (Rockefeller University) was also used in these studies. sarcoma

Preparation of cell cultures for freeze-fracture observations Unfixed Preparations. The cells were rinsed twice in phosphate-buffered saline, without Ca++ or Mg and then

gently scraped from the petri dishes. The cells were collected, centrifuged into a pellet, and frozen rapidly in Freon 22. Glycerinated Preparations. The cells were rinsed twice in phosphate/saline and then treated with the following graded series of glycerol solutions: 5% glycerol in phosphate/ saline for 5 min; 10% glycerol in phosphate/saline for 10 min; 20% glycerol in phosphate/saline for 20-30 min. During the 20% glycerol treatment, the cells were removed from the petri dishes by gentle pipetting or by scraping with a coverslip. This procedure was used at both room temperature and at 4°. After the 20% glycerol treatment, the cells were centrifuged and the pellet was rapidly frozen in Freon

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Proc. Nat. Acad. Sci. USA 72 (1975)

Table 1. Quantitation of particle aggregation

Classification of fracture faces (%)

Sample CEF-fixed (360 )[111 CEF-glycerol (36°)[123] RSV/CEF-fixed (360 )[102] RSV/CEF-glycerol (360 )[122] TS-68-glycerol (41°)[134] TS-68-glycerol (360)[126] CEF/trypsin-glycerol (360)a [116] CEF/trypsin-glycerol (360 )b [92] 3T3-fixed (36°)[111] 3T3-glycerol (36°)[110] SVT2-fixed (36°)[69] SVT2-glycerol (360 )[114]

Dis- Inter- Aggrepersed mediate gated 92.8 11.3 92.4 76.2 25.4 57.1 22.4 42.7 92.8 12.7 97.0 70.2

4.5 9.9 5.4 10.7 7.5 17.5 9.5 11.0 5.4 20.0 1.5 13.2

2.7 78.8 2.2 13.1 67.2 25.4 68.1 46.3 1.8 67.3 1.5 16.6

Cells were prepared as described in Materials and Methods. The numbers in brackets indicate the number of inner plasma membrane fracture faces that were used to characterize the particle distribution in each population. The particle populations on each of these fracture faces were scored as: (i) Dispersed-no detectable particle aggregation; (ii) Intermediate-aggregates of 10 particles or fewer; and (iii) Aggregated-aggregates of 10 or more particles. In this manuscript, Figs. 1, 3, 5, 6, 7, 10, and 12 would be scored as Dispersed; Figs. 4 and 13 would be scored as Intermediate; and Figs. 2, 8, 9, and 11 would be scored as Aggregated. a Cells were treated with 0.05 Ag/ml of TPCK-trypsin for 5 min at 370 prior to glycerination. b Cells were treated with 1 jg/ml of TPCK-trypsin for 15 min at 370 prior to glycerination.

22. The glycerination process may be reversed by treatment with 10% glycerol in phosphate/saline, 5% glycerol in phosphate/saline, and phosphate/saline alone. Also, the glycerination process can be "fixed" at any point by treatment of the cells with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 15-20 min at room temperature. Fixed Preparations. The medium was removed and replaced with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 15-20 min at room temperature. After fixation the cells were rinsed thoroughly with 0.1 M cacodylate buffer (pH 7.4) prior to removing the cells from the petri dishes. In some samples, the cells were subsequently treated with 25% glycerol in 0.1 M cacodylate buffer (pH 7.4) for 2 hr at room temperature in order to reduce the freezing damage of cytoplasmic components. Protease treatment CEF cells were treated with trypsin (Worthington, TPCKtrypsin) at 0.05 Ag/ml in phosphate/saline for 5 min at 370 or at 1 gg/ml in phosphate/saline for 15 min at 37°. The protease treatment was stopped by the addition of soybean trypsin inhibitor (Miles) at 20 ,g/ml in phosphate/saline. The protease-treated samples were then either fixed or glycerinated prior to freeze-fracturing. Freeze-fracturing and electron microscopy Small samples of the cell pellets were mounted on cardboard disks and rapidly frozen in Freon 22, prior to storage in liquid nitrogen. Freeze-fracturing was performed at -115° in a Balzers BM 360 apparatus. The carbon-platinum replicas were cleaned in bleach and water prior to mounting on uncoated copper grids. Electron microscopic observations were

made with a Philips 300. All freeze-fracture images have been mounted so that the shadow direction is from the bottom to the top of the micrograph. RESULTS Chicken embryo fibroblasts Unfixed and Fixed Cells. In general, the fracture faces of plasma membranes of "fixed" cell populations contain randomly distributed intramembrane particles (Figs. 1, 3, 5, and 7). These particles vary in size from 60 to 110 A in diameter, and they are found primarily on the inner membrane halves (fracture face A). The outer membrane halves (fracture face B) contain relatively few particles. Thus, all of the data presented have been selected to represent the characteristic features of the inner membrane halves (A fracture face). The "unfixed" cells are severely damaged during freezing; however, the fracture faces are virtually identical to those of fixed cells with regard to particle distribution. All of the control images represent "fixed" cell populations. Glycerinated Cells. While there are no marked differences in particle distributions between "fixed" and "unfixed" cell populations, there are considerable differences in the particle arrangements in "glycerinated" and "nonglycerinated" plasma membranes. The fracture faces of normal "glycerinated", CEF plasma membranes contain large aggregates of particles (Fig. 2). These aggregates vary in size, shape, and distribution. Some of the largest may contain as many as 300-400 particles. The precise quantitation of particles in the aggregates is difficult because particles appear to fuse and coalesce. In some instances, 80-90% of the intramembrane particles in an exposed field are present in the aggregated regions with the remaining particles dispersed in smooth regions of the membrane. The effect of glycerination upon the distribution of the intramembrane particles in CEF cultures appears to be independent of cell density and temperature. Treatment of cells at a density of 2 X 106 cells per dish or 2 X 107 cells per dish yields a similar redistribution of particles. The treatment with glycerol is effective at 40, at 240, and at 37'. Furthermore, cells remain viable after this treatment. The viability can be demonstrated by gradually removing the glycerol and adding fresh medium. Within 24 hr, the cells begin growing again with less than a 15% decrease in cell number when compared to control untreated cultures. The redistribution of intramembrane particles occurs only when unfixed cells are glycerinated. If cells are fixed prior to glycerination, no particle redistribution can be seen (Fig. 1). Conversely, particle aggregates are maintained in cells that are glycerinated and then fixed prior to freezing (Fig. 2, Table 1). In cells transformed by Rous sarcoma virus and subsequently glycerinated (Fig. 4), the particles are slightly redistributed when compared with nonglycerinated cells (Fig. 3, Table 1). However, the particle redistribution in the glycerinated transformed cells is significantly less than in membranes from glycerinated nontransformed cells (see Fig. 2, Table 1). It should be noted that there is a striking change in particle densities in the nonglycerinated and the glycerinated samples. This is particularly apparent on comparing Fig. 3 with Fig. 4, and Fig. 5 with Fig. 6. Fewer particles are present after glycerol treatment. At present, we have no precise quantitation for the alteration in particle densities. In order to

and

relate

RSV/CEF

to

the

apparent difference

between

transformation rather than

CEF

to virus pro-

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Proc. Nat. Acad. Sci. USA 72 (1975)

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FIGS. 10-13. Fracture faces (inner halves) of plasma membrane from 3T3 cells and SV4O transformants (SVT2). Fig. 10. Normal 3T3,

fixed. Fig. 11. Normal 3T3, glycerinated. Fig. 12. SVT2, fixed. Fig. 13. SVT2, glycerinated. Figs. 10 and 11: X64,890. Figs. 12 and 13: X65,000

duction, plasma membranes were examined from cells infected with the temperature-sensitive nontransforming mutant of RSV, TS-68 (16), and grown at both permissive (360) and nonpermissive (41°) temperatures. TS-68 multiplies equally well at both temperatures; however, at 410 the infected cells appear normal by a number of criteria, while at 360 they appear to be transformed by the same criteria. The plasma membranes of glycerinated cells grown at the nonpermissive temperature (Fig. 8) closely resemble those from normal cells (Fig. 2, Table 1). However, the particle distributions of cells grown at the permissive temperature (Fig. 6) are similar to those present in cells transformed by wild-type RSV (Table 1). Protease-Treated Cells. The glycerol-induced particle aggregation can be effectively reduced by prior treatment of the cells with trypsin (Fig. 9, Table 1). Treatment of the cells with low concentrations of trypsin (0.05 gg/ml) for a short period of time detectably alters the glycerol effect. Trypsin treatment (0.05 Aig/ml) prior to glycerination slightly decreased the "aggregated" CEF population from 78.8% to 68.1%. However, treatment with a higher concentration of trypsin (1 Ag/ml) further reduces the "aggregated" population to 46.3% (Table 1). It should be indicated that while the overall distribution of particles in Fig. 9 is clearly different from that present in Fig. 2, particle aggregates are still present in the trypsin-treated sample. In fact, the fracture

face in Fig. 9 would be classified as "aggregated" according to the scheme presented in Table 1. 3T3 and transformed 3T3 cells Unfixed and Fixed Cells. The intramembrane particles are randomly distributed in both fixed and unfixed plasma membranes. The spatial arrangements of particles in A31 cells (Fig. 10) and SVT2 cells (Fig. 12) are strikingly similar. The distribution patterns of cells at low density, high density, saturation density, or at an elevated saturation density (stimulated by a change of medium) are identical, as was found for the chick embryo fibroblasts. Glycerinated Cells. The intramembrane particles in the plasma membranes of the A31 cells are significantly aggregated by treatment with glycerol (Fig. 11, Table 1). However, the degree of aggregation as well as the size of the aggregates seen in the AS1 membranes is rarely as extensive as that in CEF cells. Unlike Scott et al. (10, 11), we have been unable to detect a significant variation in the amount of aggregation with changes in cell density. The reasons for this discrepancy are not obvious and may be related to differences in handling of the data or differences in cell lines. The effect of glycerination on particle redistribution in SVT2 cell membranes (Fig. 13) is considerably reduced

when compared with the glycerinated AS1 cells (Table 1). The extent of glycerol-induced redistribution in the plasma

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membranes of SVT2 cells is roughly equivalent to that in the glycerinated RSV/CEF membranes (Table 1). DISCUSSION The results in this study indicate that glycerol induces the aggregation of intramembrane particles in normal cells, but not in transformed cells. Furthermore, normal and transformed cells that are fixed prior to glycerol exposure contain a dispersed distribution of particles within their plasma membranes. This work helps to clarify certain discrepancies in previous studies on particle aggregation in 3T3 cells (10, 17), in which either fixed cells or glycerinated cells were examined, but not both. In contrast to Scott et al. (10, 11), we have seen only marginal effects of cell density upon the degree of particle aggregation. While these differences may reflect differences in cell lines or experimental protocols, they may also be related to the difficulty of interpreting the particle distributions. We have attempted to quantitate this phenomenon by classifying the distribution patterns into three groups based on the size of particle aggregates. However, this method has definite limitations, for it does not account for changes in particle densities, nor does it deal with the distribution of aggregate sizes within a fracture face. For example, the distribution of particles in the protease-treated sample in Fig. 9 is clearly different from the distribution in Fig. 2. However, in our scoring system, they would both be grouped together. Additional work is necessary to describe the glycerol effect in more quantitative terms as well as the effect of agents such as proteases that appear to reduce the glycerol effect. Even with the above reservations, it appears that there is a distinct difference between the glycerol-induced distributions of particles in the plasma membranes of normal and transformed cells. Approximately 79% of the CEF cells show membranes with aggregated particles, while only 13% of the RSV transformants are affected. Since only two cell types have been examined, it would be premature to generalize these observations. One report already indicates that certain "normal" cell types (erythrocytes and macrophages) are insensitive to glycerol-induced particle redistributions (13). More work will be required to determine if this phenomenon extends to other cell types and/or species. In addition to the glycerol-induced particle aggregation, there is also an apparent glycerol-induced alteration in particle densities. Although we have not attempted to quantitate density changes in this study, there are several possible ex-

Proc. Nat. Acad. Sci. USA 72 (1975)

planations. For example, glycerol may induce a global redistribution of particles that is not detected, or glycerol may induce changes in membrane structure or composition that result in the loss of membrane particles. Since trypsin treatment of normal cells decreases the amount of particle aggregation induced by glycerol, it is tempting to relate the lack of particle aggregation seen in transformed cells to the known production of protease by these cells (1). In these studies, the observed membrane alteration is a direct consequence of the glycerol treatment. Therefore, glycerol appears to be a useful probe for examining the mechanism that controls the mobilities of intramembrane particles in normal and transformed cells. We thank Eleana Sphicas and Lucy Palmer for their excellent technical assistance. This work was supported by USPHS Grants HL 16507 and CA 13138. N.B.G. holds an Irma T. Hirschl Career Scientist Award and D.B.R. holds a Faculty Research Award (PRA99A) from the American Cancer Society. 1. Reich, E., Shaw, E., & Rifkin, D. B. (1975) Proteases and Cel-

lular Proliferation (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). 2. Hynes, R. 0. (1973) Proc. Nat. Acad. Sci. USA 70,3170-3174. 3. Vaheri, A. & Ruoslahti, E. (1974) Int. J. Cancer 13,579-586. 4. Wickus, G. G. & Robbins, P. W. (1973) Nature New Biol. 245, 65-67. 5. Stone, K. R., Smith, R. E. & Joklik, W. K. (1974) Virology 58, 86-100. 6. Burger, M. M. & Goldberg, A. R. (1967) Proc. Nat. Acad. Sci. USA 57,359-366. 7. Inbar, M. & Sachs, L. (1969) Proc. Nat. Acad. Sci. USA 63, 1418-1425. 8. Porter, K. R., Puck, T. T., Hsie, A. W. & Kelley, D. (1974) Cell 2, 145-162. 9. Pollack, R., Osborn, M. & Weber, K. (1975) Proc. Nat. Acad. Sci. USA 72,994-998. 10. Scott, R. E., Furcht, L. T. & Kersey, J. H. (1973) Proc. Nat. Acad. Sci. USA 73,3631-3635. 11. Furcht, L. T. & Scott, R. E. (1974) Exp. Cell Res. 88,311-318. 12. McIntyre, J. A., Karnovsky, M. J. & Gilula, N. B. (1973) Nature New Biol. 245, 147-148. 13. McIntyre, J. A., Gilula, N. B. & Karnovsky, M. J. (1974) J. Cell Biol. 60, 192-203. 14. Boone, C. W. (1975) Science 188, 68-70. 15. Rifkin, D. B. & Reich, E. (1971) Virology 45, 172-181. 16. Kawai, S. & Hanafusa, H. (1971) Virology 46,470-479. 17. Pinto da Silva, P. & Martinez-Palomo, A. (1975) Proc. Nat. Acad. Sci. USA 72,572-576.

Plasma membrane alteration associated with malignant transformation in culture.

Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3594-3598, September 1975 Cell Biology Plasma membrane alteration associated with malignant transforma...
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