Transformation-dependent increase in endogenous cytochalasin-like

Transformation of chicken embryo fibroblasts by infection with Rous sarcoma virus has been shown to cause disruption of actin filament organization as seen with fluorescence staining techniques. This study is an attempt to use quantitative biochemical techniques to compare actin-related parameters in normal and transformed cells. Normal cells and cells infected with a temperature-sensitive mutant virus (NY68) and grown at the restrictive temperature of41.50C have normal bundles of actin filaments, or F-actin; these cells also have about the same number of high-affmity cytochalasin binding sites at the ends of F-actin (=5 pmol of sites per mg of cellular protein; Kd, 20 nM). In contrast, infected cells grown at the permissive temperature of37C have a more diffuse pattern of actin ifiaments, and the number of cytochalasin binding sites in these transformed cells was below the level of detection. DNase I inhibition assays showed that the percent of unpolymerized actin, or G-actin, in cell extracts was not significantly different between normal and transformed cells (:50%). In assays of cell extracts for endogenous cytochalasin-like activity on actin filaments (i.e., retardation of ifiament assembly at the fastgrowing end, inhibition of cytochalasin binding to actin "nuclei," and decrease of low-shear viscosity of solutions of actin filaments), infected cells at 3rC showed a higher level of activity per mg of protein than did uninfected cells or infected cells at 41.50C. These results suggest that the increase in endogenous cytochalasin-ilke activity in transformed cells may relate to the decrease in measurable cytochalasin binding sites and the abnormal distribution of actin ifiaments previously seen by fluorescence staining techniques. Normal fibroblasts in culture have sheaths of actin filament bundles (also called actin cables or stress fibers) that often run the length ofthe cell (1) as shown by immunofluorescence studies. In contrast, virally transformed cells lack this ordered type of actin structure (2-5); the diffuse fluorescence staining in these cells shows cytoplasmic actin to be disorganized. Experiments with temperature-sensitive mutants of tumor viruses demonstrated the disappearance of bundles of actin filaments or filamentous actin (F-actin), in virusinfected cells at the permissive but not at the restrictive temperature, which would indicate that a functional product of the transforming gene of the virus (e.g., the src gene of Rous sarcoma virus) is producing this effect. Furthermore, studies of these virus mutants in combination with inhibitors of protein synthesis suggest that the viral gene product does not act on the actin cytoskeleton through newly synthesized proteins (6). Much progress in understanding the molecular actions of proteins encoded by transforming genes of tumor viruses has occurred over the past several years. In the case of the Rous sarcoma virus, the product of the src gene (protein pp6Osrc) appears to be a protein kinase that catalyzes phosphorylation of tyrosine residues of many cellular proteins (7, 8). However, little molecular information exists at present concerning the linkage of this kinase activity to the disorganization ofthe actin cytoskeleton in the transformed cell. This study is an attempt to use quantitative biochemical techniques to examine several actin-related parameters in normal and transformed chicken embryo fibroblasts (CEF) infected with a temperature-sensitive mutant of Rous sarcoma virus (NY68RSV). Our results suggest that the transformation-dependent changes in actin organization may relate to an altered activity of the cellular proteins that affect actin assembly in a manner similar to that of the cytochalasins, the fungal alkaloid metabolites that have potent activity on actin-related cytoskeletal and motile functions. MATERIALS AND) METHODS Materials. Triton X-100, ATP (grade II), phenylmethylsulfonyl fluoride and 2-mercaptoethanol were obtained from Sigma. DNase and calf thymus DNA were supplied by Millipore (Bedford, MA). Unlabeled cytochalasin B (CB) and cytochalasin D (CD) were purchased from Aldrich. [3HJCB was obtained from New England Nuclear. The Sephacryl S-200 was a Pharmacia product. Cell Culture. Fibroblasts were isolated from chicken embryo as described by Rubin (9) and grown in Ham's F10 medium (GIBCO or MA Bioproducts) supplemented with 10% trypase phosphate broth (GIBCO or Scott Laboratories, Richmond, CA)/10% heat-inactivated calf serum (GIBCO or Colorado Serum, Denver, CO)/0.5% beef embryo extract (GIBCO)/1 x penicillin/streptomycin/fungizone mixture (GIBCO or MA Bioproducts). All experiments were done on secondary or tertiary cell cultures. Virus Stocks. Our initial stocks ofwild-type (Schmidt-Ruppin B) and the temperature-sensitive mutant ofRous sarcoma virus (NY68, originally from the laboratory of H. Hanafusa, Rockefeller University) were generously provided by K. Yamada of the National Cancer Institute. We prepared our own stocks by the following procedure. One milliliter of virus was added to a 100-mm dish offreshly isolated CEF. The cells were then grown at 370C and subcultured twice. Growth medium of confluent tertiary cultures was replaced with fresh growth medium containing 1% (vol/vol) dimethyl sulfoxide, and the cells were incubated at 370C for 2 hr. The resulting virus-containing medium was collected, centrifuged to reAbbreviations: CEF, chicken embryo fibroblasts; G-actin, monomeric actin; F-actin, filamentous actin; CB, cytochalasin B; CD, cytochalasin D; PBS, Dulbecco's phosphate-buffered saline; NY68RSV, temperature-sensitive mutant of Rous sarcoma virus. *Present address: Hypertension Research Program, Division of Cardiology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294. 8201 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8202 Cell Biology: Magargal and Lin move cells, and frozen in 1.5-ml aliquots at -700C. To obtain infected cells, we added 1.5 ml of virus stock to each 100-mm dish of freshly isolated CEF. The cells were grown at 370C and split at a 1:3 ratio. Secondary and tertiary cell cultures grown at the nonpermissive temperature (41.50C) were used only after growing for at least 24 hr. Binding of [3H]CB to Cultured Cells. Cells used for CB binding experiments were plated on 60-mm dishes and grown to confluence. After the plated cells had been rinsed three times with L15 medium (GIBCO), one ml of L15 containing [3H]CB with or without 20 AM CD was added to each plate. The cells were incubated at 370C for 15 min, and the medium was removed for scintillation counting to determine the concentration of free [3H]CB. The cell layer was dissolved in 1 M NaOH and then neutralized with acetic acid. Aliquots of this solution were used for determining protein content and bound [3H]CB by scintillation counting. Specific binding is defined as binding that can be eliminated by the presence of 20 AtM CD in the assay medium. Preparation of Cell Extracts. Extracts of CEF were prepared by modification of the method of McClain et al. (6). Confluent cells were rinsed twice with phosphate-buffered saline (PBS; pH 7.4) and gently scraped into fresh buffer. After centrifugation at 1000 rpm in an International Equipment HN-S centrifuge for 10 min, the supernatant fraction was aspirated, and the cell pellet was quickly rinsed with ice-cold buffer A (0.2 mM CaCl2/0.5 mM 2-mercaptoethanol/0.2 mM ATP/0.2 mM phenylmethylsulfonyl fluoride/5 mM Tris HCl, pH 8.0 at 220C). All subsequent procedures were done on ice or at 40C. The cell pellet was resuspended in buffer A with a loose-fitting (B-type) Dounce homogenizer and then sonicated for 20 sec with a Branson Sonifier fitted with a microprobe at the lowest power setting and 50% duty cycle. The homogenate was centrifuged at 62,000 rpm for 35 min in a Beckman type 65 rotor. The supernatant fraction to which was added 100 units/ml of aprotinin was designated as the cell extract. Actin Preparations. Actin was prepared from acetone powder of rabbit skeletal muscle by the method of Spudich and Watt (10) and further purified over a Sephacryl S-200 column. The peak fractions were pooled, and actin concentration was measured by absorption at 290 nm (E, 0.637 ,ul/,ug cm). Stable actin "nuclei" in the form of spectrin-band 4.1-actin complex were extracted from erythrocyte membranes at low ionic strength and used without further purification (11). DNase I Assay. The methods of Blikstad et al. (12) and Carlsson et al. (13) were used to measure monomeric actin (G-actin) by the inhibition of DNase. DNA was dissolved in 0.1 M Tris HCl, pH 7.5/2.5 mM MnCl2. The substitution of MnCl2 for CaCl2 and MgCl2 resulted in slightly higher DNase activity and gave a more linear response. Other Procedures. Protein concentrations of extracts and cell lysates were determined with the method of Hartree (14). Binding of CB to erythrocyte spectrin-band 4.1-actin complex was measured with the isoelectric precipitation method (15). Actin filament assembly was monitored with an Ostwald-type viscometer (11). Low-shear viscosity of filamentous actin (F-actin) was determined with the falling-ball method described by MacLean-Fletcher and Pollard (16). Measurements were made at an angle of 600, and glycerol standards were used to compute apparent viscosity of the samples. RESULTS AND DISCUSSION Binding of CB to Fibroblasts. CB, a fungal metabolite that affects many different forms of cytoskeletal and motile functions in nonmuscle cells (17), can bind with high affinity to the fast-growing end (i.e., the "barbed end" after decoration with heavy meromyosin) of actin filaments (18, 19). Because the affinity of this compound is much higher for F-actin than for G-actin (19), we have devised a cytochalasin binding assay as a way to study the state of polymerization of F-actin in intact cells. For instance, when human platelets are activated by thrombin or other agents, an increase in actin filaments-i.e., F-actin-can be observed with electron microscopy (20), and a shift of actin in cell lysate from the monomeric to the polymeric state can be measured with the DNase assay for G-actin and a sedimentation assay for