Cell, Vol. 37, 60-614, June 1984, Copyright 0 1984 by MIT 0092.8674/84/060609-06 $02.00/O

A Variant Form of @-Actin in a Mutant of KB Cells Resistant to Cytochalasin B

Sakuji Toyama and Sumi Toyama Institute for Virus Research Kyoto University Kyoto 606, Japan

Summary

Lines of KB cells resistant to cytochalasin B have been isolated and characterized. The mutant Cyt 1 exhibits increased cross-resistance to cytochalasin E. Cyf 7 cells bind less cytochalasin B than parental KB cells. Two-dimensional gel analysis shows that Cyt 1 cells carry an alteration in @actin. This is further confirmed by one-dimensional peptide anal- ysis. The altered @-actin @‘-actin) is synthesized when poly(A)+ RNA from Cyf 1 cells is translated in a reticulocyte-cell-free translation system. These re- sults suggest that the primary site of action of cy- tochalasin B on cellular motility processes is @actin.

Introduction

The cytochalasins are a group of fungal metabolites that inhibit a wide variety of cell movements, including cytoki- nesis, cell locomotion, exocytosis, and endocytosis (re- viewed by Tannenbaum, 1978). The mechanism of this effect on cell movements is not known because the mo- lecular nature of the cytochalasin binding sites is not completely understood. Cytochalasin B binds to and in- hibits the uptake of several different classes of small molecules (i.e., sugars, nucleosides, and purines) into certain types of cultured mammalian cells (Estensen and Plagemann, 1972; Graff et al., 1977). However, this does not seem to account for the effect on cellular motility, since dihydrocytochalasin B, which differs from cytochalasin B by the absence of a single double bond, retains the ability to inhibit cell motility but not sugar transport (Atlas and Lin, 1978; Lin et al., 1978).

Recently, several reports demonstrated that cytochal- asins inhibit the rate of actin-filament polymerization (Bren- ner and Korn, 1979; Brown and Spudich, 1979; Flanagan and Lin, 1980; Lin et al., 1980; MacLean-Fletcher and Pollard, 1980). This drug action seems to be due to inhibition of the addition of actin monomer to the rapidly growing barbed end of actin filament (Lin et al., 1980; MacLean-Fletcher and Pollard, 1980; Brown and Spudich, 1981; Pollard and Mooseker, 1981). Hartwig and Stossel (1976) Weihing (1976) and Pollard (1976) showed that low concentrations of cytochalasin B inhibit the formation of actin gels in cytoplasmic extracts. In addition, Yahara et al. (1982) compared the effect of 24 cytochalasin deriva- tives on the cellular level with those on the molecular level. They showed that a correlation coefficient between inhibi- tion of capping and inhibition of actin-filament elongation is calculated to be 0.87. Although these results suggest that the inhibition of cellular motility processes by cyto-

chalasins may be attributed to the binding of this drug to actin, proof of the intracellular location of biologically active binding sites for cytochalasins awaits genetic analysis.

In this report, we describe the method for isolation of a cytochalasin B-resistant mutant from KB cells and their morphological and biochemical characterization. Aquisition of resistance to cytochalasin B was found to be associated with alteration in P-actin polypeptide.

Results

Isolation of the Cytochalasin B-Resistant Mutant Cytochalasin B affects normal and transformed or normal and tumor cells in very different ways (for review, see Tannenbaum, 1978). This drug markedly inhibits DNA synthesis of normal cells so that most of the cells become binucleated but Qo not undergo further nuclear division. The viability of the cells is not markedly affected during prolonged drug treatment and most of the cells undergo mitosis upon removal of this drug. In transformed and tumor cells, DNA synthesis continues and the cells become highly multinucleated. The ability of these cells to form colonies is drastically reduced after removal of this drug. Based on these findings, we have attempted to isolate a cytochalasin B-resistant mutant from tumor-derived KB cells.

Cytochalasin-resistant mutant was selected from un- mutagenized KB cells by plating 1 X lo6 cells/l00 mm plastic dish with 25 ml of medium containing 2.5 pg/ml of cytochalasin B. Two weeks later, the medium with the drug was removed and replaced by fresh medium without the drug. The medium was changed every 5-7 days. When surviving cells formed colonies, the cells were removed by trypsinization and transferred to a new culture dish with fresh medium containing 2.5 pg/ml of cytochalasin B. This selection cycle was repeated until the cells continued to grow in the presence of 2.5 pg/ml of cytochalasin B. Successive selections were then carried out using increas- ing concentrations of the drug. The cells that were able to grow in the presence 6 pg/ml of cytochalasin B were cloned by distributing the cells at limiting dilution into the wells of a 96-well microtest plate, and one clone, termed Cyt 1, was selected for further study. The phenotype of the Cyt ? mutant was stable in the absence of selecting agent for at least two years in serial culture.

The effects of treatment with various concentrations of cytochalasin B on the colony-forming abilities of KB and Cyt 1 cells were compared. The results are shown in Figure 1. Based upon their DIO values (concentrations of the drug that reduce relative plating efficiency of a cell line to IO%), the Cyt 1 cells are about five times more resistant to cytochalasin B than the parental KB cells,

Cross-Resistance of Cyf 1 Cells to Other Cytochalasins The cross-resistance of Cyt 7 cells toward cytochalasin E was also examined, because earlier studies indicated that cytochalasin E has little or no effect on carrier-mediated

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Figure 1. Effect of Different Concentrations of Cytochalasin B on the Growth of KB and Cyt 1 Cells

The colony-forming ability of KB (U) and Cfl 1 (O----O) cells was determined as described in Experimental Procedures.

and nonmediated transport systems of several different classes of small molecules (for review, see Tannenbaum, 1978). The results of these studies are shown in Figure 2, which shows that the Cyt 7 mutant cells exhibit increased cross-resistance to cytochalasin E. The Cyt 7 cells also exhibited increased resistance toward cytochalasin D and dihydrocytochalasin B (results not shown). The Cyt 7 mutant did not exhibit any cross-resistance toward unre- lated drugs, including actinomycin D and ouabain. These results indicate that the resistance of Cyt 7 cells to cyto- chalasin B is not due to reduced sensitivity of the transport system to this drug.

Effect of Cytochalasin B on Cytokinesis The difference in the sensitivity of KB and Cyt 7 cells to cytokinesis by cytochalasin B is shown in Figure 3. In the parental KB cells, the vast majority of cells are binucleated after 24 hr exposure to cytochalasin B. There is no net increase in the total number of cells during the cytochalasin treatment. On the other hand, the Cyt 7 cells continue to divide and appear as a virtually homogeneous population of mononucleate cells in the presence of cytochalasin B.

Binding of Cytochalasin B to KB and Cyf 1 Cells Binding of cytochalasin B to KB and Cyt 7 cells was determined over a wide range of drug concentrations. Typical binding curves are shown in Figure 4. The Scat- chard plots (Scatchard, 1949) of these data curved, sug- gesting the presence of two or more classes of binding sites with different affinities for cytochalasin B. Although the complexity of curves makes it difficult to calculate an accurate association constant and total number of binding sites, approximate values can be obtained. The high- affinity binding has an apparent association constant of about -10’ M for both KB and Cyt 7 cells. The approximate number of high-affinity binding sites per cell is 3 X IO6 for KB and 1.5 x IO6 for Cyt 7 cells, indicating that the Cyt 7 mutant cells possess about one-half the number of binding sites in the parental KB cells for cytochalasin B. Binding of

Figure 2. Resistance of KB and Cyt 7 Cells to Cytochalasin E

The experimental procedures and symbols are as described in the legend to Figure 1.

1 2 3 1 2 3 Number of nuclei per cell

Figure 3. Effect of Cytochalasin B on Cytokinesis

A total of 1 x 105 ceils were seeded onto 60 mm plastrc petri plates. When the culture was 60% confluent, the medium was replaced with new medium containing cytochalasin B at a concentration of 3 pg/ml. The cells were fixed in methanol and stained with Giemsa’s solution. Random fields were photographed and the number of mononucleate and multinucleate cells were counted. The incubation time in the presence of cytochalasin B was: (A) 0 hr; (B) 24 hr.

3H-cytochalasin B was inhibited by the addition of a high concentration of unlabeled cytochalasin B, but D-glucose at concentrations less than 0.5 M did not inhibit binding of 3H-cytochalasin B. In addition, the reduction in cytochalasin binding in Cyt 7 cells was also observed when the cells were permeabilized by freezing and thawing.

Altered pActin in Cyf 1 Cells To find out the possible biochemical alteration that may have occurred in Cyt 7 mutant cells, the patterns of peptides present in parental KB and Cyt 7 cells were compared by two-dimensional gel electrophoresis. Figure 5 shows the gel patterns of acidic polypeptides from the parental KB and the Cyt 7 cells. The only reproducible difference in the polypeptide pattern between the KB and the Cyt 7 cells is detected in the position of the p-actin polypeptide. The Cyt 7 mutant lacks p-actin polypeptide and displays a new spot, designated p’, on the two- dimensional gel. The 73’ spot appears at the same molec-

,!U/in Mutant of KB Cells

Figure 4. Scatchard Plot of Cytochalasin B Binding to KB and Cyt I Cells

The dotted lines represent the least square fit of the first six data points. The inset shows a standard plot of the data. (O---C) KB cells; (O----O) Cyt I cells.

Figure 5. Two-Dimensional Gel Electrophoretic Patterns of Proteins from KB and Cyt 7 Cells

Total cellular proteins were prepared from monolayers of cells. The mono- layers were washed three times with phosphate-buffered saline. The cells were harvested by gentle pipetting and were washed twice with phosphate- buffered saline. The final pellet was dissolved directly in O’Farrel’s lysis buffer. About 20 pg of cell proteins were analyzed by two-dimensional gel electrophoresis. The gels were stained with Bio-Bad Silver Stain. The position of o-, B’-, and y-actin polypeptides are indicated. Polypeptide pattern of KB (A) and Cyt I (B) cells.

Frgure 6. Isoelectric Focustng Analysis of Purified Actins from KB and Cyt I Cells

The gel was run as described in Experimental Procedures and stained with Coomassie Brilliant Blue. Gel 1, KB cell actin; gel 2, Cyt 1 cell actin; gel 3, mixture of KB ceil actin with Cyt 1 cell actin.

ular-weight position as the wild-type P-actin spot, but at a more acidic isoelectric focusing position.

To confirm that the p peptide was actin, actin was purified from KB and Cyt 1 cells. Figure 6 shows the isoelectric focusing pattern of actins purified from the KB and the Cyt 1 cells. Two different actin species can be distinguished in the Cyt 1 cells, one of which has the same isoelectric point as y-actin of the KB cells. The second actin species has a more acidic isoelectric point than p- actin of the KB cells.

Further evidence supporting the contention that p’ spot is an actin species was provided by one-dimensional peptide mapping (Figure 7). The peptide map of /I’ spot is very similar to that of p-actin, and slightly different from that derived from y-actin.

In Vitro Translation of Poly(A)+ RNA from KB and Cyt 7 Cells To exclude the possibility that P’-actin polypeptide was the product of a post-translational modification of wild-type p-actin polypeptide, poly(A)+ RNA from both wild-type and Cyt 1 cells was translated in a reticulocyte-cell-free trans- lation system. Products were analyzed by two-dimensional gel electrophoresis (Figure 8). The poly(A)+ RNA from the KB cells directs the synthesis of p- and y-actins, while the poly(A)+ RNA from the Cyt ? cells directs the synthesis of p’- and y-actins. Tne more basic 6- and t-actins, which are unacetylated precursors of p- and y-actins, are found in the products of poly(A)+ RNA from the KB cells. Similarly, the analogous unacetylated forms of p’- and y-actins (b- and t-actins) are detected in the products of poly(A)+ RNA

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Figure 7. One-Dimensional Peptide Maps of Polypeptides Of P-, 8’., and y-Actin

A ffuorogram of the gel is shown. (p) @actin from KB cells; (0’) 8’.actin from Cyf 7 cells; (7) y-actin from KB cells. The band with an arrow in 8- actin is considered to be derived from contaminating y-aCtin because of insufficrent resolution in two-dimensional gel.

from the Cyt 7 cells. Thus the p’-actin does not arise as a result of a post-translational modification.

Discussion

Recent evidence suggests that the primary site of actin of cytochalasin B over a wide variety of cellular movement is actin (Brenner and Korn, 1979; Brown and Spudich, 1979; Hartwig and Stossel, 1979; Lin and Lin, 1979; Low et al., 1979; Flanagan and Lin, 1980; Lin et al., 1980; MacLean- Fletcher and Pollard, 1980; Fox and Phillips, 1981; Casella et al., 1981; Yahara et al., 1982). Since mammalian cells have two or more classes of binding sites with different affinities for cytochalasin B (Lin et al., 1974; Czech, 1976; Plageman et al., 1977; Atlas and Lin, 1978; Lin and Lin, 1978) a conclusive causal relationship between binding of cytochalasin B to actin and impairment of cellular motility processes has yet to be established. One approach that should be capable of proving this relationship is to look for actin alterations among mutants selected for resistance to cytochalasin B. In this report, we describe the isolation of a cytochalasin B-resistant mutant. This mutant displays reduced cytochalasin B-binding ability and shows an alter- ation in P-actin polypeptide on two-dimensional gels.

The alteration in P-actin is the result of: a mutation of the gene coding for P-actin; a post-translational modifica- tion of p-actin; and an activation of a silent gene for p’-

IEF- h

Figure 8. Two-Dimensional Gel Analysis of In Vitro Translation Products Directed by Poly(A)+ RNA from KB and Cyt 1 Cells

Translation products of poly(A)+ RNA from KB cells (A) and Cyf I cells (B). The arrows indicate the position of fi-, @‘-, y-, 6-, c-, and b-actins.

actin. The second possibility could be ruled out by the finding that p’-actin polypeptide is synthesized in a retic- ulocyte-cell-free system in the presence of poly(A)+ RNA from Cyt 7 cells but not from KB cells. This is further supported by the observation that no labeled p’-actin is found when nonlabeled extracts of Cyt 7 cells are mixed with 35S-methionine-labeled extracts of KB cells (data not shown). The most likely interpretation is that @‘-actin results from a mutation in the structural gene for p-actin. However, we could not exclude the possibility that a silent gene is expressed for p’-actin. Regardless of the mechanism of this alteration, the linked appearance of the p’-actin poly- peptide and cytochalasin resistance provides evidence that cytochalasin inhibits cellular motility processes by interacting with /?-actin. In addition, the differential sensitiv- ity of p- and r-actin to cytochalasin suggests that the /3- and y-actin isomers have functionally different roles.

The most probable explanation for the mechanism by- which an alteration in p-actin can overcome inhibition of cellular motility processes by cytochalasin B is that the altered fi’-actin no longer binds the drug, or binds the drug less strongly. This is supported by the finding that Cyt 7

p-Actrn Mutant of KB Cells 613

cells bind less cytochalasin 6 than parental KB cells. This explanation is still speculative and must await further ex- periments for confirmation, using purified P’-actin.

Cyt I cells are missing P-actin polypeptide. In vitro studies using purified actin demonstrate that cytochalasins bind to actin-filament ends (Brown and Spudich, 1979; Flanagan and Lin, 1980; Lin et al., 1980) and inhibit the addition of actin monomer to the barbed end of the filament (McLean-Fletcher and Pollard, 1980; Brown and Spudich, 1981; Pollard and Mooseker, 1981). If this is the case and cytochalasin binds to the barbed end, actin polymerization would not be inhibited in the presence of cytochalasin B when fl’-actin locates at the barbed end of actin filament. The further addition of p-actin to this terminal p’-actin causes inhibition of the elongation of the actin filament. Therefore, the absence of p-actin is advantageous for Cyt 1 cells to express resistance to cytochalasins. The ab- sence of p-actin polypeptide in Cyt 1 cells can be simply explained by assuming that the P-actin locus is functionally hemizygous (Siminovitch, 1976). However, this mechanism is improbable because the Cyt 1 mutant cannot be ob- tained in a single isolation procedure, and the isolation of the mutant requires extended growth in the presence of the drug. In addition, there is evidence for the codominant expression of both mutant and wild-type actin in mutants with altered p-actin polypeptide (Leavitt and Kakunaga, 1980; Vandekerckhove et al., 1980; Bravo et al., 1981). The other possible explanations are that the absence of p- actin polypeptide is the result of: occurrence of multiple mutations in p-actin genes; loss of the corresponding chromosomes on which @-actin genes are located; and deletion or inactivation of a region including the wild-type p-actin allele. The first possibility seems unlikely, since multiple P-actin genes would all mutate to produce the identical p’-actin polypeptide. Further studies are needed to determine the validity of the latter two possibilities.

The ratio of /3’- and y-actin in Cyt I cells is about 2:l and almost identical with that of p- and y-actin in KB cells. This suggests that the relative amounts of fl- and y-actin are strictly controlled. However, this molecular mechanism remains unknown.

Kelly and Sambrook (1975) have reported the isolation of variant mouse cells resistant to cytochalasin B. However, none of these variants display an ability to divide in the presence of the drug.

The use of the Cyt I mutant should make it possible to draw conclusions concerning the nature of actin-cyto- chalasin interaction and to design experiments that will elucidate the role of ,& and y-actins in a variety of cellular motility processes.

Experimental Procedures

Cell Line and Cell Culture The KB 100 cell line used in thus study has been described (Toyama et al., 1977). Cells were routinely grown in monolayers in Eagle’s minimum essential medium (MEM) supplemented with 10% newborn calf serum and 60 @g/ml kanamycin.

Assay for Cytochalasin Resistance The resistance of cells to the growth-inhibitory effects of cytochalasins was determined by plating 150 ceils at various drug concentrations in 60 mm diameter plastic Petri dishes containing 5 ml MEM. After 10 days of incubation at 37°C in a CO? incubator, the cells were fixed with methanol and stained with Giemsa’s solution.

Assay for Cytochalasin Binding to Cells Cells from monolayers were harvested by trypsinization. washed once with Hepes-buffered salrne (20 mM Hepes, 107 mM NaCI, 6.8 mM KCI, pH 7.2) and resuspended in the same buffer. ‘H-cytochalasin B (New England Nuclear) was incubated with 2 X IO6 cells (KB and Cyt 1 cells have the same size and protein content) in the Hepes-buffered saline. After incuba- tion at 37°C for 30 min, the cells were decanted onto HA Millipore filters and the filters were washed two times each with 5 ml of ice-cold Hepes- buffered saline. The dried filters were counted for radioactivity in a toluene- based scintillation solution using a Beckman LS-7000 liquid scintillation spectrometer.

Purification of Actin Actin was isolated from KB and Cyf 1 cells usrng the procedure described by Weber et al. (1977). The purity of actin preparation was better than 95% as judged by sodium dodecylsulfate-polyacrylamide gel electrophoresis. However, we failed to isolate tubulin from the same batch of these cells.

Isoelectric Focusing and Two-Dimensional Gel Electrophoresis Isoelectric focusing was carried out on 2.5 mm cylindrical polyacrylamide gel, 13 cm long, in a pH 5-7 gradient according to O’Farrel(i975). Sodium dodecylsuflate-polyacrylamide gel electrophoresis was carried out in 10% polyacrylamide slab gel by the method of O’Farrel(1975). The polypeptides were vrsualized by staining with the Bio-Rad Silver stain. The gels containing radioactive material were prepared for fluorography as described by Laskey and Mills (1975).

One-Dimensional Peptide Analysis Cells grown in 12.well multiwell plate were labeled with 1 ml of MEM lacking amino acids and containing 10% dialyzed calf serum and 100 pCi of a mixture of 16 “‘C-amino acids (New England Nuclear). After labeling for 16 hr, the cells were washed four times with phosphate-buffered saline and dissolved in 0.3 ml of lysis buffer (O’Farrel, 1975). One-dimensional peptide mapping was performed essentially as described (Cleveland et al., 1977) with the following modification. Two-dimensional gels were stained with Coomassie Brilliant Blue for 15 min and then destained for 30 min. Gel chips containing actins were cut from the gels, washed three times with 10% methanol, and lyophilized. The lyophilized gel chips were taken up in 50 ~1 of load buffer (Fey et al., 1981) mashed with a glass rod in a microtube (Eppendorf), and boiled for 5 min. The samples were placed in a sample well and overlayed with IO HI of the load buffer containing 10 ag/ ml of V8 protease (Miles). Following electrophoresis, the gels were visual- rzed by fluorography (Laskey and Mills, 1975).

Preparation of Poly(A)+ RNA and In Vitro Translation RNA was extracted using the technique of Chirgwin et al. (1979). Poly(A)+ RNA was isolated by chromatography on oligo(dT) cellulose (P-L Biochem- icals) as described by Aviv and Leder (1972). Translation of poly(A)+ RNA by using reticulocyte lysate (Amersham) with 35S-methionine (Amersham) was carried out in 25 ~1 reaction mixture containing 1.25 pg of poly(A)+ RNA. The mixtures were incubated at 37°C for 80 min. The cell-free products were analyzed by two-dimensional gel electrophoresis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received December 30, 1983; revised March 7, 1984

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