cytochalasin-d and Cell-Transformation--Neoplastic

Increased expression and alteration of intracellular trafficking of lysosomal cathepsins have been reported in malignant tumors, or in cells transformed by the transfection with the ras oncogene. In the present study, immortal MCF-10A human breast epithelial cells were transformed with the mutated ras oncogene. Both cell lines were investigated for changes in the intracellular localization of lysosomal cathepsin D and lamp-1 (lysosome-associated membrane protein) employing specific antibodies and confocal immunofluorescence microscopy. The results revealed that staining for cathepsin D along with for lamp-1 was mostly localized in the perinuclear region of MCF-10A cells. In contrast, the staining for these proteins was found to be widely distributed throughout the cytoplasm and at the cell periphery in MCF-10AneoT cells. The organization of microtubules, but not actin, appeared to differ between MCF-10A cells and their oncogenic ras transfectants. When the microtubules were depolymerized by treatment of MCF-10A cells with nocodazole, vesicles containing the lysosomal cathepsin D were dispersed in the cytoplasm and translocation of these vesicles to the cell periphery was observed. The intracellular localization of cathepsin D in the nocodazole-treated MCF-10A cells seemed to be similar to that observed in the oncogenic ras transfectants of these cells. When taxol, which inhibits microtubule depolymerization, was added to the culture medium of neoT cells, a polymerized microtubule network was observed, and the reclustering of cathepsin D and lamp-1 occurred in an unidirectional manner towards the perinuclear region. These findings support a model in which cytoskeletal microtubule organization is closely related to the trafficking of lysosomes/endosomes, and in which oncogenic ras interferes with such organization in human breast epithelial cells.

Topics: Animals; Antigens, CD; Biological Transport; Breast; Cathepsin D; Cell Line, Transformed; Cell Transformation, Neoplastic; Cytochalasin D; Epithelial Cells; Female; Fibrocystic Breast Disease; Humans; Immunoglobulin G; Lysosomal Membrane Proteins; Lysosomes; Male; Membrane Glycoproteins; Microscopy, Confocal; Microscopy, Fluorescence; Microtubules; Nocodazole; Oncogene Protein p21(ras); Paclitaxel; Proto-Oncogene Proteins p21(ras); Rabbits; Rats; Recombinant Fusion Proteins; Transfection

The overall coordination of cell structure and function that results in gene expression requires a spatial and temporal precision that would be unobtainable in the absence of structural order within the cell. Cells contain extensive and elaborate three-dimensional skeletal networks that form integral structural components of the plasma membrane, cytoplasm, and nucleus. These skeletal networks form a dynamic tissue matrix are composed of the nuclear matrix, cytoskeleton, and extracellular matrix. The tissue matrix is an interactive network which undergoes dynamic changes as cells move and change shape. Pathologists have long recognized cancer in pathologic specimens based on the altered morphology of tumor cells compared to their normal counterparts. The structural order of cells appears to be altered in transformed cells. This structural order is reflected in the altered morphology and motility observed in transformed cells compared to their normal counterparts, however, it is unclear whether the structural changes observed in cancer cells have any functional significance. We report here on the nature of the physical connections between the nucleus and cell periphery in nontransformed cells and demonstrate that the nucleus is dynamically coupled to the cell periphery via actin microfilaments. We also demonstrate that the dynamic coupling of the nucleus to the cell periphery via actin microfilaments is altered in Kirsten-ras transformed rat kidney epithelial cells. This loss of structure-function relationship may be an important factor in the process of cell transformation.

Topics: Acrylamide; Acrylamides; Actins; Animals; Cell Line; Cell Line, Transformed; Cell Movement; Cell Nucleus; Cell Transformation, Neoplastic; Colchicine; Cytochalasin D; Cytoskeleton; Rats

A quantitative analysis based on centrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3-K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3-K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well-formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2-deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation. It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.

Topics: Actins; Animals; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Centrifugation; Cytochalasin B; Cytochalasin D; Cytochalasins; Fibronectins; Fluorescent Antibody Technique; Mice; Monensin

The relationships between cytoskeletal network organization and cellular response to cytochalasin D (CD) in a normal rat fibroblast cell line (Hmf-n) and its spontaneous transformant (tHmf-e), with markedly different cytoskeletal phenotypes, were compared (using immunofluorescence, electron microscopy, and DNAse I assay for actin content). Hmf-n have prominent, polar stress fiber (SF) arrays terminating in vinculin adhesion plaques whereas tHmf-e, which are apolar, epithelioid cells with dense plasma membrane-associated actin networks, lack SF and adhesion plaques. Hmf-n exposed to CD become markedly retracted and dendritic, SF-derived actin aggregates form large endoplasmic masses, and discrete tabular aggregates at the distal ends of retraction processes. Prolonged exposure leads to recession of process, cellular rounding, and development of large cystic vacuoles. tHmf-e cells exposed to similar doses of CD display a diagnostically different response; retraction is less drastic, cells retain broad processes containing scattered actin aggregates in discrete foci often associated with plasma membrane, large tabular aggregates are never found and processes persist throughout long exposure, vacuolation is uncommon. The CD-induced microfilamentous aggregates in Hmf-n are composed of short, kinky filament fragments forming a felt-like skein, often aggregates contain a more ordered array of roughly parallel fragments, while those of tHmf-e are very short, kinky, randomly orientated filaments imparting a distinctly granular nature to the mass. Total actin content and the amount of actin associated with detergent-resistant cytoskeletons increase following CD exposure in both cell types. Throughout exposure to CD, the actin-associated contractile proteins tropomyosin, myosin, and alpha-actinin co-localize within the actin aggregates in both cell types. Fodrin, the protein linking cortical actin to membrane, co-localizes with actin aggregates in tHmf-e cells and most, but not all, such aggregates in Hmf-n cells, consistent with their stress fiber derivation. Vinculin is lost from the tabular aggregates at the distal ends of retraction processes in Hmf-n cells concomitant with the fragmentation and contraction of SF. The aborized processes in both cells types contain strikingly similar axial cores of bundled vimentin filaments associated with passively compressed microtubules. The characteristic CD-induced distribution of actin filament aggregates and redistributio

Topics: Actin Cytoskeleton; Actins; Animals; Carrier Proteins; Cell Adhesion; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cytochalasin D; Cytochalasins; Cytoskeletal Proteins; Cytoskeleton; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Fluorescent Antibody Technique; Hydrocortisone; Intermediate Filaments; Microfilament Proteins; Microscopy, Electron; Microtubules; Muscle Proteins; Phenotype; Rats; Vinculin

The effect of cytochalasin D, which is known to disrupt specifically actin cytoskeleton, on DNA replication was studied. The incubation of cultured mouse embryonic fibroblasts (MEF), cells of Balb/3T3 line and cells of minimally transformed clones 12 MC and 6 st/T CAK-7 line with cytochalasin D leads to inhibition of DNA synthesis. A complete inhibition of labeled index in MEF culture was observed after an 8 day incubation in cytochalasin D. Part of cells of clones 12 MC and 6 st/T were insensitive to cytochalasin D and continued to enter to S-phase even after a 10 day incubation. The transfer of cells into a fresh medium leads to a rapid restoration of DNA synthesis. Strongly transformed L cells were almost insensitive to cytochalasin D. Thus, the reorganization of actin cytoskeleton caused by cytochalasin D can inhibit the cycle of normal and minimally transformed cells. In the course of neoplastic progression, in the transformed cells there is a loss of dependence of cell proliferation on microfilament system.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cytochalasin D; Cytochalasins; DNA; DNA, Neoplasm; Fibroblasts; L Cells; Mice; Mice, Inbred BALB C; Thymidine

Treatment of a variety of mesenchymal cells (normal and transformed rat fibroblasts, bovine aortic endothelial cells, rabbit smooth muscle cells), exhibiting different cytoskeletal organizations and derived from several species, with doses of cytochalasin D (CD, 2-6 microM for 20 h) sufficient to induce cytoskeletal rearrangement and altered cellular morphology results in an increase in the relative content and rate of synthesis of actin. These data extend our previous findings for HEp-2 cells to other cell types and provide further evidence for our hypothesis that the CD-induced cytoskeletal reorganization triggers stimulation of actin synthesis and the resulting increase in actin content.

Topics: Actins; Animals; Cattle; Cell Transformation, Neoplastic; Cells, Cultured; Cytochalasin D; Cytochalasins; Cytoskeleton; Rabbits; Rats; Structure-Activity Relationship

Epithelial hamster lens cells, transformed by SV40 can be grown in suspension culture. Triton X-100 extraction of these cells grown under conditions when ribosome run off is blocked releases about 40% of the total amount of polyribosomes, designated as free- and loosely-bound polyribosomes. The Triton ghosts retain the remaining polysomal population which can be released by a combined treatment with deoxycholate and Nonidet P 40. Electron microscopic examination of the ghosts reveals microfilament-associated ribosome clusters next to a fraction of polysomes still attached to membranes. Preincubation of the cells with cytochalasin D prior to polyribosome isolation enables us to discriminate between these two latter polysome populations. The experiments indicate that about 25% of the polyribosomes are attached to microfilaments, while the remaining 35% are tightly bound to the membranes of the endoplasmic reticulum. When the different polyribosome classes were translated in a reticulocyte lysate, no significant differences could be observed in the patterns of the newly synthesized polypeptides. In all cases actin was one of the major products synthesized de novo.

Topics: Animals; Cell Fractionation; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Cytochalasin D; Cytochalasins; Cytoskeleton; Epithelium; Lens, Crystalline; Mesocricetus; Microscopy, Electron; Polyribosomes; Protein Biosynthesis; Reticulocytes; Simian virus 40

When primary baby mouse skin (BMS) cultures were subcultured for 48 hours into media containing 10(-6) to 10(-7) M colchicine or demecolcine, the number of altered cell foci appearing after 3-4 weeks' maintenance at 36 degrees C was substantially enhanced over drug-free controls. This applied whether or not the primary cultures had been irradiated with white fluorescent light. The additional presence of cytochalasin D and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) sometimes improved and sometimes partly suppressed the enhancing effect of the antitubulin drugs, and these drugs were omitted for reproducible focus enhancement. The enhancement depended on passage through DNA synthesis in presence of colchicine, which did not prevent concurrent or subsequent DNA synthesis but induced a substantial proportion (greater than 33%) to replicate in the tetraploid (4n to 8n) chromosome configuration. Another effect was to induce widespread asymmetric nuclear division, allowing the potential for chromosome loss. All these effects occurred within the first one or few cell cycles after removal of the antitubulin drugs. The results suggest that the generation of tetraploidy perhaps followed by chromosome loss may be an important factor in the rapid induction of altered cell foci. Pre-existing DNA damage is another important factor.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Colchicine; Cytochalasin D; Cytochalasins; Demecolcine; DNA; Light; Mice; Oxygen; Polyploidy; Skin; Tetradecanoylphorbol Acetate