Cytologia 39: 709-727 , 1974 The Cellular Response to Cytochalasin B: A Critical Overview Michelle Copeland Children'sCancer Research Foundation , 35 BinneyStreet, Boston, Massachusetts02115 , U. S. A. ReceivedApril 3, 1973 The cytochalasins comprise a novel class of fungal metabolites which have attracted a great deal of recent interest as a result of the numerous apparently di versecytological effects which are elicited by their presence, both in vivo and in vitro. Six cytochalasins have been isolated, to date, from the cultures of Helminthospor ium dematioidem, Metarrhizium anisopliae, and Rosellina necatrix, although some what similar biological activity has been associated with the fermentation products of several additional fungi (Aldridge et al. 1967). The six currently isolable cyto chalasins are designated A, B, C, D, E, and F. In terms of their biological activity, i nsofar as this is known at present, these six cytochalasins appear qualitatively simi lar to one another, although the potencies of the isomeric cytochalasins C and D Aldridge et al., 1967 Chem. Comm. 1: 26 Fig. 1. The chemical structure of cytochalasin B. exceedthose of cytochalasins A and B by a factor of approximately ten (Carter 1967). Themost readily isolated of these is the B variant , which has been shown to be iden tical with the antibiotic "phomin" (Aldrige and Turner 1968) (Fig. 1). The addi tional features of appropriate potency , essential nontoxicity at low concentrations and reversibility of action have made cytochalasin B the subject of most laboratory studies of the biological phenomena produced by the cytochalasins. Preliminary investigators employed the term"cytochalasin" (from the Greek cytos: cell;+chalasis: relaxation) to denote the cytological changes induced by these metabolic end-products . These substances appeared to "relax" the cell 710 Michelle Copeland Cytologia 39 through a mechanism which may have involved an inhibition of the normal process of cell motility or a structural variation of the cytoskeleton. A more thorough consideration of this phenomenon will be presented later in this discussion. A reasonable approach to the problem of formulating a realistic mechanism for the action of cytochalasin B is to attempt to establish a functional relationship between the apparently diverse cytological effects which it has been demonstrated to elicit. Among the more prominent of these effects are several which are chara acterized as morphological in nature, such as cytoskeletal variation, multinucleation, Fig. 2. Large quantities of granular endoplasmic reticulum and annulate lamellae are seen in the cytoplasm of this multinucleated cell; this fibroblast cell was exposed to cytochalasin B (1ƒÊg/ml) for 6-7 days. Scale line is 1um. Courtesy of Dr. Awtar Krishan. diffuse organelle alteration, nuclear extrusion, and surface phenomena of diminished membrane ruffling; other effects are described as functional or physiological, and include inhibition of cell motility, of mitotic reproduction and of phagocytosis and pinocytosis, as well as alteration of surface adhesion properties and of micro filaments and depression of synthesis or uptake of membrane components. One rather convincing element which is consistent with some of these processes is an alteration in the properties of the cell plasma membrane and it is not without evi dence that this avenue may be viewed as central to the activity of cytochalasin B. 1974 Cytochalasin B 711 Alteration of cell morphology The diverse cellular effects of cytochalasin B were first systematically noted by Carter (1967) in experiments with fibroblasts in culture . His initial observation of these cells treated with cytochalasin B included diminished levels of both normal motile activity and characteristic membrane ruffling . At concentrations of 1 .0 ƒÊ g/ml of cytochalasin B these fibroblasts were also noted to assume a flattened configuration, and to occupy a larger surface area on glass after treatment . Kro shan (1971) corroborated this early evidence for membrane involvement and sup plemented it with the observation that fibroblasts exposed to similar concentra tions of cytochalasin B transformed from the normal spindle into flat , epithelioid shapes through contraction of elongated pseudopodia. These discoid fibroblasts became multinucleated and accumulated Golgi membranes, rough endoplasmic reticulum and annulate lamellae such that after fifteen days of culture in medium Fig. 3. A large cytochalasin-induced multinucleated fibroblast cell with 9 nuclei . Individual variation in the nuclear size is evident. Exposed to cytochalasin B (1ƒÊg/ml) for 6-7 days. Scale line is 1ƒÊm. Courtesy of Dr. Awtar Krishan. containing cytochalasin B, the cells attain diameters of approximately 300 microns and possessed eight or nine nuclei each (Figs. 2 and 3). The possibility that these alterations in cell structure might be due to an augmentation by cytochalasin B of the synthesis of cellular elements has been effectively eliminated by the demonstra tion that the rates of DNA, RNA and protein syntheses are unaffected by cyto chalasin B (Estensen 1971). Krishan's data are consonant with a process in which the drug influences merely the plasma membrane such that the maintenance of even normal metabolic activities results in large-scaled cytoeconomics. That is, quite simply, that cells behave metabolically as if they had been separated from one another following normal mitosis, and that each member "cell" of the cytochalasin B-induced conglomerate contributed to the mass that quantity of synthetic pro ducts which it would have formed in the independent state. This consideration points out an important aspect of the analysis of the activities of cytochalasin B; 712 Michelle Copeland Cytologia 39 that is, the drug may have a single direct action on a component of, say, the plasma membrane and yet produce in vitro variations which suggest a diversity of roles. An additional example of the application of this principal has been provided by Krishan in an explanation of the conspicuously large number of RNA virus-like particles found within the giant fibroblasts produced by cytochalasin B. Krishan has realistically suggested that the larger fibroblast cells were preferentially suited to the proliferation of endogenous L-cell virus-like particles as well as exogenously introduced virus-like reo and Herpes viruses in that the hosts contained large cyto plasmic masses, unusually large nuclei, relatively hypertrophied endoplasmic reti culum, and numerous other cell organelles in high numbers. Arborization and cellular adhesion Insight into the processes resulting in the observations of Carter and Krishan have been provided by Sanger and Holtzer (1972), who have validated an alteration by cytochalasin B of the cytoskeleton. Exposure of myoblasts, fibroblasts and stellate chondroblasts for 48 hours to 5ƒÊg/m1 of cytochalasin B caused the cells to assume a branched gross morphology and induced a virtual immotility. Reversal of this "arborization" could be achieved by withdrawal of cytochalasin B from the medium. It was noted by these authors that the resumption of normalized cell morphology and considerable recovery of cellular motility did not entail retraction of the extruded processes, the overall dimensions of which remain unchanged. The cells apparently lose their arborized morphology by gradually filling the area between the processes with outflowing lobopodia, the forms of which are indis tinguishable from the advancing tip of amoebae, and no membrane "ruffling" pheno mena were associated with the lobopodial protrusion. Additional experiments were conducted by these researchers in which specific metabolic inhibitors were employed to assess the role of cytochalasin B in the arborization process. Ar borized cells incubated with cycloheximide (an inhibitor of protein synthesis) and 5ƒÊg/ml of cytochalasin B for 30 minutes and then transferred to a medium con taining only cycloheximide underwent normal reversal of shape. From this datum it can be concluded that the motile lobopodial activity responsible for normaliza tion of shape is not dependent upon protein synthesis. On the other hand, during exposure of arborized cells to sodium cyanide, an inhibitor of oxidative phos phorylation, the branched conformation is maintained until this inhibitor is with drawn. It is apparent, therefore, that protrusion of lobopodia is dependent upon energy production and independent of protein synthetic activity. In addition, it should be mentioned that not all of the cell types treated with cytochalasin B ex hibited arborization under the conditions employed. Replicating chick embryo erthrocytes undergoing nuclear division show neither cytokinesis nor arborization; whereas presumptive myoblasts arborize upon treatment with cytochalasin B, their fusion products, myotubes, do not; furthermore, the nonfunctional chondrocyte, lacking a maturation capsule, does exhibit arborization, while the functional chon droblast encapsulated in chondroitin sulfate does not branch. It has been sug gested that these differing responses to cytochalasin B may reflect fundamental differences in cell surfaces. Myoblasts, for example, are thought to undergo con siderable surface alteration following fusion into multinucleate myotubes, and 1974 Cytochalasin B 713 this change may effect variation in the cellular response to cytochalasin B . Simi larly, while the nonfunctional chondrocyte is freely exposed to the drug , the en capsulated chondroblast may not