cytochalasin-d and Carcinoma--Ehrlich-Tumor

Cytochalasins have been used extensively to probe the role of F-actin in different aspects of cellular function. Most of the data obtained are interpreted on the basis of the well-established depolymerizing effects of cytochalasins on F-actin preparations in vitro. However, some evidence indicates that, in intact cells, different cytochalasins can have varying effects on cell morphology and F-actin content and organization. To examine this problem in more detail, we analyzed the effects of cytochalasins on the cell morphology of and F-actin content and organization in Ehrlich ascites tumor (EAT) cells. After a 3-min exposure to 0.5 microM cytochalasin D, B, or E, F-actin content was equally reduced in all cases and this correlated with a reduction in the amount of cortical F-actin associated with the EAT cell membrane. However, only with CE was cell morphology markedly altered, with the appearance of numerous blebs. At 10 microM, blebbing was present in all conditions and the organization of cortical F-actin was disrupted. F-actin content, however, was not further reduced by this higher concentration and in CD it was identical to control levels. Exposure of EAT cells to similar concentrations of cheatoglobosin C, an analog of the cytochalasins that has little to no affinity for F-actin, resulted in a loss of F-actin content, a reduction in F-actin fluorescence, but no change in cell morphology, including a complete lack of bleb formation. Myosin II immunoreactivity, concentrated in the cortical cytoplasm colocalized with F-actin and in an area associated with the Golgi, was reduced by the high-dose cytochalasin. These results demonstrate that caution must be exercised in the use of cytochalasins to probe the role of F-actin in cellular function and that several parameters must be analyzed to obtain an accurate assessment of the effect of cytochalasin on the actin filament system.

Topics: Actins; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane; Cytochalasin B; Cytochalasin D; Cytochalasins; Mice; Mice, Inbred Strains; Myosins; Organelles; Tumor Cells, Cultured

Effects of cytochalasins on actin polymerization state in living cells were measured using fluorimetry of TRITC-phalloidin bound to F-actin. Normal (3T3) and tumour (SV-3T3, B16 melanoma, and Ehrlich ascites) cells were treated with cytochalasin B and cytochalasin D (1 microgram/ml). Three effects of cytochalasins were demonstrated--depolymerization of F-actin, promotion of polymerization, and redistribution of actin without change in polymerization state. Occurrence of a given effect was dependent on cell type, cell density, cytochalasin concentration and type. This indicates that cells from different lines, and even the same cells in different culture conditions may differ significantly in their state of actin polymerization, which we suppose is the cause of their different reactions to cytochalasins. Accordingly, caution should be taken in generalizing the results concerning the effect of cytochalasis on the polymerization state of actin.

Topics: 3T3 Cells; Actins; Animals; Carcinoma, Ehrlich Tumor; Cell Line, Transformed; Cytochalasin B; Cytochalasin D; Melanoma; Mice; Microscopy, Fluorescence; Polymers; Simian virus 40; Tumor Cells, Cultured

Evidence presented previously indicated that shape changes in circulating cancer cells within the microvasculature may lead to rapid, lethal rupture of the cell surface membranes, thereby contributing to the inefficiency of this phase of hematogenous metastasis. As the cytoskeleton is known to regulate cell shape and, in at least some cases, to be attached to the surface membrane, we have determined whether or not it plays a role in inhibiting lethal, deformation-associated, mechanically induced surface membrane trauma. Thus, Ehrlich ascites tumor (EAT) cells were treated with different cytoskeleton-perturbing agents, and their susceptibility to filtration trauma on passage through Nuclepore membranes was determined. In contrast to the involvement of the cytoskeleton in nonlethal cell deformation, agents which disrupt intracellular networks of microtubules, microfilaments and intermediate filaments had little or no direct effect on EAT cell susceptibility to rapid, mechanically induced, lethal trauma.

Topics: Acrylamide; Acrylamides; Actins; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane; Colchicine; Cytochalasin D; Cytoskeleton; Ethanol; Filtration; Fluorescent Antibody Technique; In Vitro Techniques; Keratins; Mice; Microtubules; Tumor Cells, Cultured; Vimentin

The effect of cytochalasins B or D on the surface topography of mouse neoplastic fibroblasts of L line (detached from glass by trypsin-EDTA solution) or of its LS subline (adapted to the growth in suspension in vitro), as well as on that of Ehrlich's ascites tumor cells was investigated by screening electron microscopy. Incubation of suspended cells with cytochalasin B (2 micrograms/ml) or cytochalasin D (0.2 microgram/ml) for 30-180 min led to the following changes: (I) progressive decrease of the proportion of the cells with a microvillous surface relief and simultaneous increase in the percentage of the cells with a blebbed microrelief; (2) shortening of the microvilli and decrease of their density on the cell surface; (3) appearance of surface areas with a rough folded relief; (4) formation of very large blebs on the LS or L cell surfaces; (5) unusual "polar" distribution of blebs on Ehrlich's tumor and L cells: the blebs were concentrated in one locus on the cell surface. The data show that normal organization of the actin microfilament system in the cell cortex is necessary for formation of the microvilli but not for the blebs.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cytochalasin B; Cytochalasin D; Cytochalasins; L Cells; Mice; Microvilli

A series of halogenated and related analogues of cytochalasin C (CC) and D (CD) has been synthesized, and the biological activities of the analogues as inhibitors in a cell-free contractility model system obtained from Ehrlich ascites tumor cells were evaluated. The reaction sequence involved treatment of CD with phenyltrimethylammonium perbromide to give 6,12-dibromo-CD (2), dehydrohalogenation of 2 to 12-bromo-CC (3), and the subsequent conversions of 3 to 12-azido- (4), 12-iodo- (5), and 12-cyano-CC (6). The ID50 values for 5, 3, 4, 2, and 6 are 6.0, 7.4, 8.8, 45, and 77 X 10(-7) M, respectively, in comparison to ca. 2.8 X 10(-7) M for the parental compounds. The potential cell and molecular biological applications of these compounds are delineated.

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Chemical Phenomena; Chemistry; Cytochalasin D; Cytochalasins; Structure-Activity Relationship

Cell fusion-inducing (fusogenic) proteoliposomes of defined chemical composition were reconstituted from purified glycoproteins of hemagglutinating virus of Japan (Sendai virus) either with lipids extracted from the virus particles or with a chemically defined lipid mixture. Cell fusion reactions induced by the reconstituted system have several important characteristics similar to the virus-induced fusion reaction: fusogenic activity of the proteoliposomes depends on the presence of active fusion protein in the vesicles and, in the case of Ehrlich tumor cells, the fusion is almost completely inhibited by adding cytochalasin D to a final concentration of 4 microgram/ml. The only known difference between the original and reconstituted systems is that a greater amount of the latter is necessary for the same degree of fusogenic activity. Thus, the reconstituted system can be used as a model for the Sendai virus-induced fusion reaction. A lipid mixture (phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine:sphingomyelin = 1:2:1:1, by weight, and cholesterol equimolar to the total phospholipids) similar to that of the virion was active for reconstitution, whereas a mixture containing the same composition of phospholipids but no cholesterol, and ones containing cholesterol with only a single species of phospholipid were not reconstitutively active.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Fusion; Cholesterol; Cytochalasin D; Cytochalasins; Erythrocytes; Glycoproteins; HN Protein; Humans; Liposomes; Mice; Muscle Proteins; Oxidoreductases; Parainfluenza Virus 1, Human; Phosphofructokinase-1, Muscle Type; Phosphofructokinases; Proteins; Viral Proteins