Kirsten murine sarcoma virus-coded p21ras may act on multiple targets to effect pleiotropic changes in transformed cells

VIROLOGY

121,

327-344

(1982)

Kirsten Murine Sarcoma Virus-Coded p2 1’“” May Act on Multiple Targets to Effect Pleiotropic Changes in Transformed Cells MICHAEL

*Department of Biology, McGill University,

W. DEVOUGE,*r’ AND SERGIO

BARID B. MUKHERJEE,*+ D. J. PENA*++

TCentre for Human Genetics, and $Department of Neurology/Neurosurgery, 1205 avenue Docteur Pen&e& Montreal, Quebec H3A lB1, Canada Received

October

13, 19X1; accepted

May

31, 19&?

Kirsten murine sarcoma virus (Ki-MSV) is a replication-defective recombinant retrovirus capable of transforming cells in culture. A Ki-MSV coded, 21,000-dalton protein (~21’““) is required for the maintenance of cellular transformation. It is unknown whether the ~21’“” of Ki-MSV induces transformation by acting on multiple targets, as has been suggested for Rous sarcoma virus transformed cells, or by a single target mechanism. In order to resolve this question, we have used a normal rat kidney cell line transformed by a temperature-sensitive mutant strain of Ki-MSV (tsKNRK), which codes for a therthe correlative aspects of the expression of several transmolabile ~21’“” to investigate formation-related cellular properties upon shifting from the permissive (32”) to nonpermissive (39”) temperature. Except for an altered morphology, an organized cytoskeleton, and increased adhesion to substratum, tsKNRK cells at 32” displayed similar properties to those of wild-type Ki-MSV transformants at either temperature. Upon shifting to 39”, anchorageand density-dependent growth were restored, although the growth rate and glucose uptake were unaffected. The cells assumed a more flattened morphology, although adhesiveness did not increase significantly. Increased levels of cell surface fibronectin were observed within 48 hr post-temperature shift, although fibronectin levels comparable to that of normal rat kidney cells (NRK) were not observed until later. Epidermal growth factor (EGF) binding increased only slightly at 48 hr post-temperature shift but did not approach EGF-binding levels of NRK cells. Cytoskeletal organization was invariant between the two temperatures. Although our results suggest a multiple target model for Ki-MSV-mediated transformation, a single target mechanism cannot totally be ruled out.

in order to phosphorylate tyrosine residues of specific protein substrates (Collett et al., 1980; Hunter and Sefton, 1980). A number of proteins which appear to serve as substrates for the pp60”‘” protein kinase have been identified. These include the 36K and 50K polypeptides and vinculin-a 130K protein located at junctions of the plasma membrane and microfilament bundle termini, as well as in focal adhesion plaques (Hunter and Sefton, 1980; Radke and Martin, 1979; Sefton et al., 1980). The Harvey and Kirsten strains of murine sarcoma virus (Ha-MSV and Ki-MSV, respectively) are replication-defective retroviruses derived from the recombination of murine leukemia virus sequences and specific rat cellular sequences (Scolnick et

INTRODUCTION

A significant development in understanding the mechanism of retrovirusmediated cell transformation is the identification and characterization of those retrovirus-coded gene products required for the maintenance of the transformed state. The most extensively characterized of these transforming proteins is a 60,000dalton polypeptide (pp60”‘“) of Rous sarcoma virus (RSV) (Brugge and Erikson, 1977). This protein possesses a protein kinase activity (Collett and Erikson, 1978) and utilizes the gamma phosphate of ATP ’ To whom all correspondence prints should be addressed.

and requests

for re-

327

0042-6822/82/120327-18$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

328

DEVOUGE,

MUKHERJEE,

al, 1973; Scolnick and Parks, 1974). These two viruses each code for a 21,000-dalton polypeptide (~21’““) which are immunologically cross-reactive (Shih et aZ., 19’79a), although the rat cellular sequences which code for the Ha-MSV and Ki-MSV ~21’“” species are not identical (Ellis et aZ., 1980). The Ha-MSV ~21’~” possesses a guanine nucleotide binding activity and is capable of transferring the gamma phosphate of GTP to a threonine residue on the ~21’“” (Shih et aZ., 1980). This autophosphorylating activity has not, however, been demonstrated in v&o, nor have any other in vivo or in vitro substrates for ~21’“” yet been identified. Studies using a mutant KiMSV (h-371), temperature-sensitive for the maintenance of the transformed state (Scolnick et al., 1974), have shown that the immunoprecipitability of the mutant ~21’” is thermolabile in vitro (Shih et ab, 197913). The Ha-MSV p21’““, like the pp60”‘” of RSV, is associated with cellular membranes, in particular, the plasma membrane (Willingham et ab, 1979, 1980). Despite an abundance of information with respect to the biochemical properties and localization of retrovirus-coded transforming proteins, little is known of the mechanism(s) by which a transforming protein may act to effect pleiotropic changes encompassing morphological, biochemical, and growth-related properties of the transformed cell. Loss of cell surface fibronectin, decreased adhesion to substratum, disruption of cytoskeletal elements, increased hexose uptake, loss of density-dependent growth, decreased binding of epidermal growth factor (EGF), and loss of anchorage-dependent growth are some of the many changes often associated with cell transformation. In the present study, we have used a normal rat kidney cell line transformed by the ts-371 mutant strain of Ki-MSV (tsKNRK) to study the correlative aspects of the expression of several transformation-related properties after shifting from the permissive to the nonpermissive temperature. Since genetic analysis of molecularly cloned Ki-MSV sequences (Ellis et al, 1981), as well as studies using b-371transformed cells have shown that a func-

AND

PENA

tional ~21’“” is required for the maintenance of the transformed state in tsKNRK cells (Shih et al., 1979b), the present study may aid in understanding the mechanism of p21ra”-mediated cellular transformation. The results obtained from this study suggest that the ~21’“” may act on multiple targets to induce various transformation-specific properties in Ki-MSV-transformed cells. MATERIALS

AND

METHODS

Cells and viruses. Normal rat kidney cells (NRK), Kirsten murine sarcoma virus (Ki-MSV), transformed NRK cells (KNRK), and NRK cells transformed by a mutant strain of Ki-MSV (ts-371, clone 5) temperature-sensitive in a viral function required for the maintenance of transformation (tsKNRK), were obtained as a gift from Dr. E. M. Scolnick, NIH, Bethesda, Md. Cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% newborn calf serum (NCS). Stock cultures of KNRK and NRK cells were maintained at 37” whereas tsKNRK cells were kept at the permissive temperature (32”). Cell growth, saturation density, and marphology. Replicate cultures of NRK, KNRK,

and tsKNRK cells were prepared by seeding 3 X lo5 cells into 25-cm2 plastic flasks and incubating at 32” for 24 hr in a CO2 incubator. At this point, half the cultures were maintained at 32” and the other half of the cultures were shifted to the nonpermissive temperature (39”). All cultures were fed daily with DMEM supplemented with 10% newborn calf serum. The harvest protocol was as follows: every 24 or 48 hr duplicate cultures were gently washed with versene and trypsinized with 0.25% trypsin in Tyrode’s solution. Cells were pelleted by centrifugation and resuspended in 1 ml phosphate-buffered saline (PBS), pH 7.2. Cell counts were made by a Coulter counter. For morphological examination NRK, KNRK, and tsKNRK cells were grown to confluency at either 32 or 39’, after preincubation at 32” for 24 hr. Cultures were photographed with Kodak Panatomic-X

PLEIOTROPIC

film on a Zeiss inverted microscope equipped with phase contrast attachment. Soft agar assay. Culturing of cells in soft agar is based on the technique of Macpherson and Montagnier (1964). Cells from each line were harvested by trypsinization and suspended in complete medium at a concentration of 3 X lo5 cells/ml. The suspension was mixed with 0.5% agar in DMEM containing 10% NCS in a 12 ratio to yield a 0.33% agar medium containing 1 X lo5 cells/ml. One and a half milliliters of cell suspension was pipetted onto a solidified base layer of 7 ml 0.5% agar in DMEM with 10% NCS in 60-mm plastic culture dishes. After 24 hr of incubation at 32” half the cultures were shifted to 39”, whereas the other half were maintained at 32”. Cultures were fed with 1 ml 0.3% agar in DMEM with 10% NCS every 5 days. Photographs were taken at 7 and 14 days after plating, using Kodak Panatomic-X film on a Zeiss inverted microscope. 1”51-labeled EGF binding assay. Replicate cultures of each cell line were plated at a density of 3 X lo5 cells in 60-mm plastic culture dishes and incubated at 32” for 24 hr prior to the temperature shift. The assay, slightly modified from that of Lee and Weinstein (1979), was carried out at 24 and 48 hr post-temperature shift. After cultures were washed twice with 2.5 ml Hanks’ balanced salt solution (HBSS), duplicate cultures were incubated for 25, 50, 75, and 100 min with 0.60 ng 1251-labeled EGF (100-200 &i/pg) (Collaborative Research, Waltham, Mass.) in 1.3 ml binding buffer consisting of 1 mg/ml bovine serum albumin (BSA) (Sigma), 50 mM, NJ-bis[Z-hydroxyethyll-2 aminoethane sulfonic acid (BES) (pH 6.8) in DMEM. Following incubation, cultures were washed three times with 5 ml cold HBSS and solubilized in a solution of 1% Triton X-100 and 1% sodium dodecyl sulfate (TX-SDS). Cell lysates were pipetted into scintillation vials containing 10 ml Biofluor (NEN). Dishes were further rinsed with 0.5 ml TX-SDS solution and pooled. Samples were counted in a scintillation counter. Duplicate cultures were also harvested and cells were counted in a Coulter counter to determine

~21’~

329

cell number. Input counts were obtained by counting 20 ~1 of binding buffer containing 1251-labeled EGF (0.46 rig/ml) in the presence of cell lysate in order to simulate quenching conditions during the assay. Nonspecific binding, which represented 0.5-1.2 pg/106 cells, was measured by incubation of cultures of each cell line with a 104-fold excess of unlabeled EGF EGF. (6 a) Over ‘=I-labeled Adhesion assay. Replicate cultures were seeded with 3 X lo5 cells per 60-mm plastic culture dish, grown at 32” for 24 hr, and either maintained at 32” or shifted to 39”. One culture of each cell line at each temperature was used per assay and two assays were performed consecutively. After washing cultures with PBS (pH ‘7.2), 1.5 ml of 0.01% trypsin in Ca2+, M2+-free PBS was applied to each dish. After 5 min incubation at 37” the dishes were rocked once and decanted into tubes containing 0.5 ml complete medium. This process was repeated at either 5- or lo-min intervals for a maximum of 30 min. Samples were pelleted by centrifugation, resuspended in 1 ml cold PBS (pH 7.2), and counted in a Coulter counter. Cells remaining in each dish were harvested with 0.25% trypsin and counted. Cell count,s were expressed as the cumulative percentage of total cells detached at each time point. Visualixaticm of actin cables. Cells from each line were stained specifically for actin filaments with nitrobenzooxadiazole-phallacidin (fluorescent derivative of phallacidin), a F-actin-specific phallotoxin isolated from Amanita phalloides (Barak et al., 1980). Sparse cover slip cultures of each cell line were prepared, incubated at 32” for 24 hr, and were either shifted to 39” or maintained at 32”. Cover slips were washed once in PBS (pH 7.2) and monolayers extracted in 0.2% Triton X-100 in buffer consisting of 1 mM ethylene-glycolbis-NJ-tetraacetic acid (EGTA), 4% polyethyleneglycol-6000 in 0.1 M piperazine-N,N-bis [2-ethane sulfonic acid] (PIPES), pH 6.9 for 40 min. The cells were fixed for 20 min in 3.7% formaldehyde in PBS, washed twice with PBS, rinsed quickly with water, and air-dried. Fifty microliters of NBD-phallacidin (Molecular

330

DEVOUGE,MUKHERJEE,ANDPENA

Probes Inc., Plano, Texas) (20 p&f) in PBS was applied and staining was allowed to proceed for 30 min. Cells were quickly washed twice in PBS, mounted in 1:l mixture of glycerol and PBS, sealed, and photographed on an Olympus BH-2 fluorescence microscope equipped with 455-nm barrier filter, using Kodak Technical Pan film. Immunojbrescent detection of cell surface fdmmectin. An initial assessment of fibronectin levels was performed by growing cultures of KNRK, tsKNRK, and NRK cells at either 32 or 39” for 5 days. Monolayers grown on cover slips were rinsed with HEPES-buffered saline, pH 7.4 (HBS), fixed with 3.7% formaldehyde in HBS for 15 min and stained by indirect immunofluorescence as previously described (Mukherjee et ab, 1982). After washing with HBS three times, 50 ~1 of rabbit anti-human fibronectin (Collaborative Research, Waltham, Mass.) diluted 1:lO was applied and incubation proceeded for 30 min. After several washes with HBS, 50 ~1 of FITC conjugated goat antirabbit antibody diluted 1:5 was applied and cover slips were incubated for 30 min. Cover slips were washed in HBS, mounted in Tris-buffered glycerol (50 mMTris-HCl, pH 7.0, 90% glycerol) and photographed, using Kodak Technical Pan film. In order to study the time course of fibronectin appearance after shifting tsKNRK cells from 32 to 39”, monolayers were prepared and stained as described, except that cultures were shifted only after they reached confluency. Immunochemical detection of jibronectin in geZs. Cultures of each cell line were grown at 32” to confluency before shifting to 39”. Monolayers were then solubilized in a hot solution of 2% sodium dodecyl sulfate (SDS), 10% glycerol, 2% 2-mercaptoethanol in 0.125 M Tris-HCl, pH 6.8, and stored frozen at -70” until used. SDSgel electrophoresis was performed using the method of Hubbard and Lazarides (1979) in slab gels containing 10% acrylamide and 0.13% tis-acrylamide. Duplicate gels were run simultaneously. One of the gels was stained with Coomassie blue R250. The proteins in the second gel were transferred to nitrocellulose sheets (Bio-

Rad Laboratories, Toronto, Ont.) essentially as described by Towbin et al. (1979), but using a Bio-Rad Trans-blot apparatus. They were then reacted with a rabbit antihuman fibronectin serum (Collaborative Research, Waltham, Mass.) diluted 1:lOO for 30 min at 37”, washed extensively in PBS, and then treated sequentially with a biotinylated goat anti-rabbit serum and avidin-biotin-peroxidase complex as described by Hsu et al. (1981). The nitrocellulose sheets were finally stained with 3,3’diaminobenzidine as described by Graham and Karnowsky (1966). Details of this procedure will be the subject of a future publication (Pena, in preparation). Glucose uptake assay. Cells from each line were seeded at 3 X lo5 per 60-mm2 culture dish and incubated at 32” for 24 hr prior to the temperature shift. At the indicated times after the temperature shift, triplicate plates from each line at each temperature were washed twice with 3 ml Hanks’ balanced salt solution without glucose (HBSS), containing 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 7.2, prewarmed to either 32 or 39”. The cells were then incubated for 20 min at 32 or 39” in 1.5 ml HBSS-HEPES containing 1 yCi of 2-deoxyD-glucose (15.2 Ci/mmol, Amersham). After washing the cells three times with 5 ml ice-cold HBSS-HEPES, the cells were trypsinized, harvested, resuspended in PBS, and an aliquot counted by Coulter counter. The remaining cells were solubilized by addition of Triton X-100 to a final concentration of 1% (v/v) and the radioactivity of the cell lysates were counted with 10 ml Biofluor (NEN). Results are expressed as cpm/106 cells. RESULTS

Morphological Alterations of NRK, KNRK, and tsKNRK Cells at 32 and 39’ Transformed cells in culture exhibit a randomly oriented, multilayered growth consisting of rounded or polygonal cells arranged in dense foci (Allred and Porter, 1979). The morphology of confluent cultures of NRK, KNRK, and tsKNRK cells is shown in Fig. 1. NRK cells exhibited a bipolar or triangular structure in sparse

PLEIOTROPIC

FIG. 1. Phase contrast micrographs of maintained at either the permissive (32”) grown at 32”; (B) KNRK cells shifted to shifted to 39”; (E) NRK cells grown at 32”; photographed on an inverted microscope.

confluent cultures of KNRK, tsKNRK, and NRK or nonpermissive temperature (39”). (A) KNRK 39O; (C) tsKNRK cells grown at 32O; (I)) tsKNRK (F) NRK cells shifted to 39”. Unfixed monolayers Magnification X150.

culture at both 32 and 39” and upon reaching confluency formed a contact inhibited monolayer characterized by tight junc-

331

~21’”

cells cells cells were

tions between individual cells. Although not evident in Fig. 1, NRK cells, as well as KNRK and tsKNRK cells, displayed a

332

DEVOUGE,

MUKHERJEE,

AND

PENA

FIG. 2. Colony formation of KNRK, tsKNRK, and NRK cells after 7 days of growth in 0.5% agar medium at either 32 or 39”. Cells were seeded at a density of 1.5 X lo6 per 60-mm culture dish and fed every 5 days with 0.3% agar medium. (A) KNRK cells maintained at 32’; (B) KNRK cells shifted to 39”; (C) tsKNRK cells maintained at 32”; (D) kKNRK cells shifted to 39”; (E) NRK cells maintained at 32”; (F) NRK cells shifted to 39”. Unfixed colonies were photographed on an inverted microscope. Magnification X40.

less refractile morphology at 39 than at 32”. KNRK cells were multipolar or spindle shaped, and formed numerous foci of

rounded cells at both temperatures, especially in confluent cultures. The tsKNRK cells, when grown to confluency at 32”,

PLEIOTROPIC

displayed a somewhat random orientation with numerous rounded cells and fewer foci than in KNRK cells. However, when shifted to 39” and grown to confluency, a more highly ordered, less refractile monolayer was observed, with fewer rounded cells. These changes took place within 24 hr post-temperature shift. Growth of NRK, KNRK, in Soft Agur

and tsKNRK

Cells

Possibly the most significant characteristic of transformed cells is their ability to grow in a semisolid medium (Shin et al., 1975). As seen in Fig. 2, tsKNRK cells at the permissive temperature formed irregularly shaped colonies, as did KNRK cells at both temperatures. However, at the nonpermissive temperature, no colonies of fsKNRK cells were evident. Some small colonies consisting of two to four cells were observed within 24 hr of the temperature shift, but complete inhibition of further growth of these colonies took place soon after. Such colonies probably resulted either from cells which entered the DNA synthetic phase before the temperature shift, or from the presence of growth fa.ctors secreted into the medium before the temperature shift. The viability of the TABLE GROWTH

333

~21’“”

growth arrested cells was tested by shifting cultures of tsKNRK cells back to 32”. Colonies were formed within 5-7 days. NRK cells demonstrated an inability to grow in semisolid agar medium at either temperature even when plated at high cell densities. The KNRK cells in semisolid medium showed accelerated colony formation at 39”. Since anchorage-independent growth has been correlated with cellular transformation (Shin et ah, 1975) the above observations demonstrate the reversion of tsKNRK cells to a nontransformed state within 24 hr post-temperature shift. Growth, Saturation Uptake

Density,

A high correlation exists between increased saturation density and the neoplastic state (Hynes et al, 1979). As shown in Table 1, the growth rate of tsKNRK cells was intermediate between that of KNRK and NRK cells. The growth rate of tsKNRK cells did not vary significantly between 32 and 39”, whereas NRK cells showed increased growth at 39” as compared to 32”. The decreased growth rate of KNRK cells at 39” may be due to increased death or sloughing off of these 1

DENSITY OF KNRK, NRK, AND tsKNRK CELLS AT THE PERMISSIVE(~~~) OR NONPERMISSIVETEMPERATURE (39")

RATE AND SATURATION

Saturation Cells

Growth (doublings/24

hr)”

(X106 cells maintained

shifted

Time

(hr)

8.1 + 2.6b 8.2 f 0.2 2.6 k 0.1

180 341 193

8.2 f 0.6b 4.4 +- 0.1 2.6 f 0.3

140 140 193

to 39”

1.3 1.0 0.6

U The number of doublings/24 hr was calculated from shift onward. ‘Represents number of cells able to adhere to culture off cells were found in culture supernatants with time.

* SE)

at 32”

1.6 0.9 0.3 Cells

KNRK tsKNRK NRK

density

rate

Cells KNRK tsKNRK NRK

and Glucose

cell counts flask

obtained

substratum;

from increasing

48 hr post-temperature numbers

of sloughed

334

DEVOUGE,MUKHERJEE,ANDPENA TABLE2 TIME COURSEOF GLUCOSE UPTAKE OF KNRK, tsKNRK, AND NRK CELLS AT PERMISSIVE (32”) OR NONPERMISSIVE (39”) TEMPERATURE

THE

Glucose uptake

Cell line KNRK 32" 39” tsKNRK 32" 39” NRK 32" 39" a All cells were maintained

6 hr postshifY’ (X104 cpm per lo6 cells * SE)

24 hr postshifF (X104 cpm per lo6 cells + SE)

48 hr postshift’ (X104 cpm per lo6 cells ? SE)

8.2 f 0.4 8.9 + 1.7

6.4 f 0.1 7.8 AZ0.G

5.7 f 0.9 12.4 f 0.6

14.9 + 1.1 16.7 k 1.2

11.0 f 0.7 20.4 -i 4.3

11.3 f 1.1 27.6 f 1.7

4.9 k 0.6 5.2 + 0.7

4.0 k 0.2 4.9 f 0.2

4.5 f 0.6 4.8 + 0.2

at 32” for 24-48 hr prior to the temperature

cells from the growth substratum at 39” relative to 32’. KNRK cells reached a maximum density of 8.1 X lo6 cells at 32” and 8.2 X lo6 cells at 39” before sloughing off. Increased numbers of sloughed off cells after saturation indicated that growth of KNRK cells was density-independent at either temperature. The tsKNRK cells at 32” reached a similar density to KNRK cells, although the increased time required to reach this density reflects both the decreased growth rate of tsKNRK cells relative to KNRK cells and the incomplete transformation exhibited by tsKNRK cells at the permissive temperature. At 39”, tsKNRK cells saturated at a substantially decreased density relative to that at 32”, although saturation occurred at a higher density than that of NRK cells at either temperature. Glucose uptake of the three cell lines at the permissive and nonpermissive temperatures is shown in Table 2. KNRK cells showed an approximately twofold increase in glucose uptake compared to KNRK cells. Although glucose uptake is dependent on the number of cells per culture dish, tsKNRK cells at 32” exhibited an enhanced rate of glucose uptake relative to either KNRK or NRK cells, even when similar numbers of cells per dish were assayed. However, this uptake was not significantly decreased upon shifting tsKNRK cells to 39”. Neither the KNRK nor

shift.

the NRK cell lines showed significant differences in glucose uptake at 39” as compared to 32”. These data indicate that saturation density is a transformation-specific cellular activity dissociable from both growth rate and glucose uptake in tsKNRK cells. “‘I-Labeled EGF Binding of NRK, KNRK, and tsKNRK Cells The decreased binding of ““I-labeled EGF to transformed cells has been reported (Todaro et al, 1976; Blomberg et al., 1980) and such inhibition was attributed to a decrease in the number of functional EGF receptors on the cell surface (Guinivan and Ladda, 1979; Pratt and Pastan, 1978), as well as to the production of polypeptide growth factors by sarcoma virustransformed cells which compete for EGF receptors (Todaro and DeLarco, 1978; DeLarco and Todaro, 1979; Todaro et al., 1980). Subconfluent, actively growing cultures of KNRK, tsKNRK, and NRK cells were incubated with lz51-labeled EGF for various times, and the results are shown in Fig. 3. KNRK cells did not bind EGF at either temperature nor did tsKNRK cells at 32”. At 24 hr post-temperature shift to 39” tsKNRK cells showed a negligible increase in EGF binding, i.e., 1.2% of EGF binding observed for NRK cells at 39”. Binding of lz51-labeled EGF to ts-

PLEIOTROPIC

KNRK cells at 39” increased further at 48 hr post-temperature shift, but still only represented 7.2% of EGF-binding levels exhibited by NRK cells at 39”. NRK cells exhibited a two- to threefold higher le51labeled EGF binding at 39” than at 32”, a result which may be attributed to the increased synthesis of EGF receptors at the higher temperature, since NRK cells also exhibited an increased growth rate at 39”.

p21’=”

335

25

1 A

15

Effects on Cgtoskeleton Transformed cells generally display a disorganization of the various cytoskeletal elements, i.e., microtubules, lo-nm filaments, and microfilaments (Allred and Porter, 1979). As shown in Fig. 4, KNRK cells grown at 32” showed essentially no cytoskeletal detail. When shifted to 39” for 24 hr, these cells flattened out and adhered more strongly to the growth substratum. This accounts for the increased organization of actin at the nonpermissive temperature, although few discernible microfilaments were found in these cells at either temperature. NRK and tsKNRK cells, however, displayed extensive arrays of actin cables arranged in a parallel fashion at either temperature. These observations were further confirmed by staining Triton X-loo-extracted cover slip cultures by indirect immunofluorescence using anti-actin antibody (Mukherjee et al., 1982), as well as with Coomassie blue R250 by the method of Pena (1980) (data not shown). Staining of tsKNRK cells with Coomassie blue R250 at intervals up to 72 hr posttemperature shift indicated that cytoskeletal detail remained unchanged with length of time in culture.

Adhesion to Substratum In this study, adhesiveness of NRK, KNRK, and tsKNRK cells was examined in subconfluent cultures within 48 hr of the shift from permissive to nonpermissive temperature in order to minimize cellto-cell attachment and to measure adhesion of the cells to the growth substratum only. In Fig. 5, the cumulative percentage of cells detached is shown. The adhesive-

1 IO-

Or

0 25

8 50 75 TIME (rnin)

100

FIG. 3. Time course of ‘Z51-labelled epidermal growth factor binding to subconfluent, actively growing cultures of KNRK (O), tsKNRK cells at 24 hr (0) and 48 hr (A) post-temperature shift, and NRK (A) cells. Cells were seeded at a density of 3 X 10” per 60-mm culture dish and after 24 hr at 32” were either shifted to 39” (A) or maintained at 32” (B). Binding assays took place 24 hr after the temperature shift unless indicated (see Materials and Methods for details). Nonspecific binding (0.5-1.2 pg/106 cells) was subtracted from all values obtained. Each point represents the mean of duplicate values.

ness of KNRK cells grown at 39” varied considerably but, in general, these cells were significantly more adherent at 39 than at 32” (P < 0.1). This trend was not observed with either tsKNRK or with NRK cells. Both tsKNRK and NRK cells were significantly more adherent than KNRK cells only at 32” (P < 0.005 and P < 0.025, respectively). NRK and tsKNRK cell lines exhibited a similar degree of adhesiveness at either temperature. An initial period of incubation of all three cell lines at 32” was required for all studies to enable the cells to adhere to substratum. Cells seeded and incubated directly at 39” usually did not survive.

336

DEVOUGE,

MUKHE~EE,

AND

PENA

FIG. 4. Visualization of intracellular actin filaments by staining of 0.2% Triton X-loo-extracted coverslip cultures with NBD-phallacidin. Cultures were seeded sparsely and after 24 hr at 32” were either shifted to 39” or maintained at 32” for 24 hr (see Materials and Methods for details). (A) KNRK cells maintained at 32’: (B) KNRK shifted to 39”; (C) taKNRK cells maintained at 32”; (D) tsKNRK cells shifted to 39O; (E) NRK cells maintained at 32”; (F) NRK cells shifted to 39”. Photographs were taken on au Olympus BH-2 fluorescence microscope with 45%nm barrier filter (magnification X250). Similar distributions of filaments were observed when cells were stained with rabbit anti-actin antibody, or with Coomassie blue R250.

PLEIOTROPIC

~21’~

337

80a g

70-

U

3 z

60-

i

10

2b

30

5

10

20

30

5

10

20

30

TIME (min)

FIG. 5. Cumulative percentage of total KNRK (A), tiKNRK (B), and NRK (C) cells detached from growth substratum. Cells were seeded at a density of 3 X lo5 per 60-mm culture dish. After maintaining at 32” for 24 hr, cells were either maintained at 32” (0) or shifted to 39” (0). Assays were performed 48 hr after temperature shift (see Materials and Methods for details). Each point represents the mean of duplicate samples assayed consecutively. Bars denote SE.

Cell Surface Fibronectin

Levels

Cell surface fibronectin was initially examined in cultures of each cell line grown for 5 days to confluency at either the permissive or nonpermissive temperature by indirect immunofluorescence, as shown in Fig. 6. NRK cells at either temperature and tsKNRK cells at 39” were shown to have an elaborate fibrillar network of fibronectin, particularly in areas of cell to cell contact. The tsKNRK cells grown at 32” and KNRK cells at either temperature showed markedly decreased levels of fibronectin on their surfaces, even when the cells formed dense foci. Immunofluorescent staining of coverslip cultures grown to confluency at 32” and shifted to 39” for 24 or 48 hr was performed to determine whether or not the appearance of fibronectin on the cell surface was an early event. Fibronectin was detected on the cell surface within 24 hr, although extensive networks of filaments were not observed within 48 hr post-temperature shift. Further studies of cell surface fibronectin levels were carried out by immunochemical means, in order to compare more directly the changes in fibronectin levels with time. Cellular polypeptides were sep-

arated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and reacted sequentially with rabbit anti-human fibronectin antibodies, biotinyl-goat anti-rabbit antiserum, and avidin-biotin-peroxidase complex. When provided with the chromogenic substrate 3,3’-diaminobenzidine the fibronectin-specific bands alone were visualized. The results of these experiments are shown in Fig. 7. NRK cells grown at 32 or 39” for 5 days (lanes 1, 2, Fig. ‘7A) show heavy staining of the 240,000-dalton fibronectin component and also of some smaller, but still very high-molecular-weight polypeptides, which probably represent partially cleaved molecules of fibronectin (Pena et al., 1980). tsKNRK cells at the nonpermissive temperature (lane 3) gave a pattern essentially identical to that of NRK cells. On the other hand, tsKNRK cells grown at 32” (lane 4) and KNRK cells at either temperature (lanes 5, 6) gave a strikingly different pattern. Significantly decreased amounts of immunoreactive material were evident in the high-molecular-weight region of the gel, accompanied by the appearance of several bands of lower molecular weight (30,000-40,000) which probably represent degradation products of

338

DEVOUGE,

MUKHERJEE,

AND

PENA

FIG. 6. Indirect immunofluorescence micrographs of surface fibronectin of KNRK, tsKNRK, and NRK cells. Cells were seeded heavily, incubated at 32” for 24 hr, and either maintained at 32” or shifted to 39”. Unless indicated all micrographs were taken at 5 days post-temperature shift. (A) KNRK cells grown at 32”; (B) KNRK cells shifted to 39”; (C) tsKNRK cells grown at 32”; (D) tsKNRK cells at 24 hr post-temperature shift; (E) tsKNRK cells at 48 hr post-temperature shift; (F) tsKNRK cells at 5 days post-temperature shift; (G) NRK cells grown at 32”; (H) NRK cells shifted to 39”. Photographs were taken on a Leitz Orthoplan fluorescence microscope. Magnification X250.

PLEIOTROPIC

'

339

p21””

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FIG. 7. Visualization of polypeptides reacting specifically with rabbit anti-human fibronectin antiserum. Monolayers were grown as indicated, solubilized in boiling SDS buffer, and separated by SDS-polyacrylamide gel electrophoresis. Transfer to nitrocellulose sheets, as well as staining of fibronectin-specific bands are as described under Materials and Methods. (A) Assessment of the temperature sensitivity of fibronectin levels in tsKNRK cells. All monolayers were maintained either at 32 or 39” for 5 days. Lanes 1 and 2: NRK cells at 39 and 32”, respectively; lanes 3 and 4: kKNRK cells at 39 and 32”, respectively; lanes 5 and 6: KNRK cells at 39 and 32”, respectively. (B) Time course of fibronectin appearance in kKNRK cells. Lanes 2 and 4: tsKNRK cells maintained at 32” for 24 hr and shifted to 39” for 24 and 48 hr, respectively; lanes 1 and 3: tsKNRK cells maintained at 32’ for 48 and 72 hr, respectively. Constant amounts of protein were added to each lane of individual gels. Fb denotes intact fibronectin (240K daltons).

fibronectin. In Fig. 7B, the time course of fibronectin appearance is shown within 48 hr of shifting tsKNRK cells from 32 to 39”. Intact fibronectin was detected in LsKNRK cells until 48 hr at 32” (lane l), but by ‘72 hr at the permissive temperature, only immunoreactive material of about 40,000 daltons was detected; little or no intact fibronectin could be detected (lane 3). The levels of cell surface fibronectin observed in tsKNRK cells at 24 and 48 hr post-temperature shift were similar (lanes 2,4, respectively); however, at 48 hr postshift, little or no immunoreactive degradation products of fibronectin were detected. These findings indicate that decreased levels of cell surface fibronectin observed in KNRK and tsKNRK cells may be caused

either by degradation of fibronectin after synthesis by secreted proteases, or by increased turnover of fibronectin (Olden and Yamada, 1974; Robbins et al., 1974; Hynes et al, 1975). DISCUSSION

We have used a normal rat kidney cell line transformed by the ls-371 mutant of Ki-MSV in order to correlate the expression of several transformation-related cellular properties with a shift in temperature from permissive to nonpermissive. Our data show that anchorage- and density-dependent growth are restored in tsKNRK cells upon shifting from 32 to 39”. It has been well established that anchor-

340

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age-independent growth is the property which best correlates with tumorigenicity (Freedman and Shin, 1974; Shin et aZ., 1975). Similarly, density-dependent inhibition of growth has also been associated with tumorigenicity (Pollack et al., 1968), although a tumorigenic cell line which is density-dependent has been isolated (Shin et ak, 1975). It is conceivable, therefore, that the properties of anchorage- and density-independent growth may be functionally related to the development of the neoplastic state in Ki-MSV-transformed cells. Cells transformed by murine and feline sarcoma viruses secrete sarcoma growth factors (SGF), peptides which compete for epidermal growth factor (EGF) receptors (Todaro and DeLarco, 1978). It is assumed, therefore, that cells producing SGF would show reduced binding of lz51-labeled EGF. It has previously been shown that EGFbinding by tsKNRK cells shifted from 36 to 39” was enhanced to normal levels within 24 hr of the temperature shift (DeLarco and Todaro, 1979). Our data show a slight enhancement of EGF binding only after 48 hr post-temperature shift from 32 to 39”. Since these data are in agreement with the study of Guinivan and Ladda (1979), the discrepancy between our data and that of DeLarco and Todaro may be attributed to the difference in the temperatures at which the tsKNRK cells were maintained. At 36”, an intermediate temperature, only low levels of SGF were secreted, as evidenced by intermediate levels of EGF binding (DeLarco and Todaro, 1979). tsKNRK cells maintained at 32”, as in our study, required a much longer period of incubation at 39” before EGF binding was significantly enhanced, indicating that high levels of SGF were secreted at 32”. This would account for the difference in time required at 39” before EGF binding is enhanced. This has been verified in our laboratory by further experiments utilizing tsKNRK cells maintained at 36” (unpublished results). Treatment of NRK cells with SGF is known to induce anchorage-independent growth (DeLarco and Todaro, 1979). It may therefore be speculated that SGF secretion, as reflected by decreased levels of EGF binding, and an-

AND

PENA

chorage-independent growth may be linked to the ~21’“” by a common pathway. It has been proposed that decreased adhesion to substratum caused by decreased fibronectin levels is responsible for the rounded morphology and disorganized cytoskeleton in transformed cell lines (Willingham et ah, 1977), as well as for the invasive behavior exhibited by tumorigenic cells in vivo (Hynes et aZ., 1979; Chen et al., 1976). Our study has demonstrated that while tsKNRK cells exhibit increased levels of cell surface fibronectin within 48 hr of a shift to the nonpermissive temperature, cellular adhesion is not significantly altered in these cells. These data indicate that adhesion is dissociable from fibronectin levels in tsKNRK cells. This is in agreement with studies demonstrating the absence of fibronectin from focal adhesion plaques (Chen and Singer, 1980; Birchmeier et aZ., 1980). In the present study, we were unable to dissociate decreased fibronectin levels from growthrelated parameters of transformation, i.e., anchorage- and density-independent growth. The increased levels of fibronectin observed in the tsKNRK cells by 48 hr post-temperature shift clearly suggest the existence of an association between fibronectin levels and cellular transformation. However, fibronectin levels are not always found to be associated with anchorageand density-independent growth (Kahn and Shin, 1979; Lau et ah, 1979). Tumorigenie cell lines which possess normal levels of fibronectin are known (Pearlstein et aZ., 1976). Such evidence casts doubt on the necessity of decreased levels of fibronectin in cellular transformation. A number of studies have linked cytoskeletal disorganization to tumorigenicity. A reversible, temperature-dependent organization and disorganization of cytoskeleton has been demonstrated in chick embryo fibroblasts and rat kidney cells transformed by ts mutants of RSV (Edelman and Yahara, 1976; Ash et al., 1976), as well as in rat embryo cells transformed by tsA mutants of SV40 (Pollack et aZ., 1975). However, morphological revertants of RSV-transformed vole cells, as well as chick embryo fibroblasts transformed by

PLEIOTROPIC

a ts mutant of RSV, have been shown to possess normal cytoskeleton, yet are tumorigenic (Lau et ab, 1979; Fujita et al., 1981). Our data further substantiate previous reports (Willingham et ab, 1977; Lau et al., 1979) that altered cytoskeleton is not a prerequisite for cellular transformation, since the integrity of actin filaments in tsKNRK cells is preserved at either temperature. More importantly, the presence of an intact cytoskeleton in tsKNRK cells, as well as other data obtained from this study, i.e., increased adhesion and decreased growth rate of tsKNRK cells at 32” relative to KNRK cells at the same temperature, indicate that cells transformed by the ts-371 mutant of Ki-MSV are incompletely transformed at the permissive temperature. Moreover, the inability of tsKNRK cells to saturate at a normal density at 39” suggests that there is a residual expression of the ~21’“” at this temperature. If it is presumed that the presence of a mutated ~21’“” has rendered it incapable of inducing certain transformation-specific parameters in tsKNRK cells even at 32”, then we may, in fact, assign a hierarchy of parameters based on the degree to which a wild-type ~21’“” is required for induction of such changes. Thus, increased saturation density, increased growth rate, and decreased EGF binding are most susceptible to the action of the ~21’“” since we have observed an increased saturation density, as well as enhanced growth and decreased EGF binding relative to normal cells at 39”. Depletion of fibronectin, altered morphology, and induction of anchorage-independent growth would be the next most susceptible parameters to ~21’“” action since these parameters are temperature-dependent and display a normal phenotype at 39”. Finally, adhesion and cytoskeletal organization may be considered as least susceptible to ~21’“” action, since normal adhesion and cytoskeletal organization are evident at 32”. It must be emphasized, however, that this classification is highly dependent on the number of steps required to revert these transformation-specific changes, Further clarification of this

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scheme would require studies of tsKNRK cells at different temperatures, as well as time course studies over an extended period. Classification of glucose uptake in this scheme is not possible, since tsKNRK cells display elevated levels of glucose uptake relative to wild type Ki-MSV transformants. It may be speculated that (1) a conformational change in the ts-371 ~21’“” which renders it unable to induce certain parameters of transformation may induce other parameters more efficiently that the wild-type Ki-MSV, or (2) enhancement of the rate of hexose uptake is dependent on a separate function of the Ki-MSV ~21’““. From studies of transformation-specific cellular properties of several thermosensitive RSV src mutants, Weber and Friis (1979) proposed a model whereby the ~~60”‘” acts at more than one target to induce RSV-mediated transformation. Subsequent identification of several putative substrates for the pp60”” kinase has substantiated such a model. Further characterization of partial “transformation-deficient” mutants of RSV has demonstrated that the pp60”‘” kinase activity in each of these mutants is reduced relative to that measured for wild-type pp60”‘” (Anderson et al., 1981). Moreover, growth-related parameters of transformation were dissociated from morphological alterations in cells transformed by a temperature-sensitive mutant of RSV (Fujita et ah, 1981). Taken together, these studies strongly suggest that the pp60”‘” kinase may act on at least two different substrates. Such substrates may either serve as an initial step in the induction of growth-related alterations in transformation, or may be responsible for induction of a morphologically transformed phenotype. Although our results suggest such a model for KiMSV-mediated cellular transformation, we are unable to rule out a single target mechanism, since one may envisage a model whereupon an incompletely transforming virus may act on a single target and induce a partially transformed phenotype. Implicit in this model is the assumption that expression of a given transformation parameter requires a threshold level of expression of the p21ras of Ki-MSV.

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If such a mechanism were operative, all incompletely transforming mutants of KiMSV would show a similar relative hierarchy of transformation-specific parameters. Studies using other mutants of KiMSV would be required to conclusively resolve the question of whether a single target mechanism or a multiple target mechanism is in operation during transformation by Ki-MSV. The in vivo mechanism of action of the Ki-MSV ~21’“” remains largely unknown. Infection of cultured cells by Ki-MSV is known to result in elevated expression of a Ki-MSV-specific lactate dehydrogenase (LDH,J (Anderson et al, 1979), which is thermolabile in vitro when extracted from cells infected by the ts-371 mutant of KiMSV (Anderson et al., 1981). This indicates that the LDHk is most probably coded by the viral genome. The ~21’~” may conceivably be involved in the regulation of the LDHk, since immunoprecipitation of the LDHk with a specific antiserum will also precipitate a 22,000-dalton protein (Anderson et ah, 1981). Although the ~21’“” of Ki-MSV has been shown to bind guanine containing nucleotides and is capable of autophosphorylation in vitro (Shih et al., 1980), these characteristics have not been shown in vivo. Furthermore, the unexpected finding that the Ki-MSV and HaMSV ~21’” species contain domains unique to each (Ellis et al., 1981) indicates that the Ki-MSV ~21’“” may possess functions distinct from that of Ha-MSV. In any case, further characterization of the functions of the Ki-MSV ~21’“” in vivo is required before a mechanism for Ki-MSV-mediated transformation may be elucidated. ACKNOWLEDGMENTS The authors wish to express their thanks to Drs. Y. Nishioka and G. Brown for their critical evaluation of the manuscript; Ms. P. Mobry and Mr. C. Guerin for technical assistance, Mr. R. Lamarche and Mr. G. L’Heureux for photographic services, and Ms. R. Bayreuther, Ms. J. Smith, and Ms. F. Langton for their assistance in preparation of the manuscript. This study was funded by grants from the Medical Research Council of Canada to B.B.M. (MT-2169) and S.D.J.P. (MT-6668) and by a grant from the Muscular Dystrophy Association of Canada to S.D.J.P. S.D.J.P is a Medical Research Council of Canada Scholar.

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