cytochalasin-b and Fibrosarcoma

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of alpha3beta1-TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of alpha3beta1-TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B alpha3beta1-TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.

Topics: Actins; Antigens, CD; Breast Neoplasms; Cell Adhesion; Cell Adhesion Molecules; Culture Media, Serum-Free; Cytochalasin B; Cytoskeleton; Extracellular Matrix Proteins; Fibrosarcoma; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha3beta1; Integrins; Intercellular Junctions; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Nocodazole; Organelles; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Signal Transduction; Solubility; Talin; Tetraspanin 29; Tumor Cells, Cultured; Vinculin

The present paper describes an in vivo micronucleus assay using Cytochalasin B (CyB). Mice bearing three different tumours, fibrosarcoma (Swiss albino mice), B16 F1 melanoma (C57 BL) and Ehrlich ascites carcinoma (Swiss albino mice), were injected with repeated doses of CyB at different time intervals and binucleate cells were scored at 24, 36, 48, 60 and 72 h after CyB injection. It was found that three doses of 3+2+2 mg/kg CyB administered intraperitoneally (i.p.) at 12-h intervals effectively blocked cytokinesis. The maximum number of binucleated cells (BNC) was scored at 60 h after the last CyB dose. This dose schedule was also effective in scoring micronuclei in BNC after irradiation.

Topics: Analysis of Variance; Animals; Carcinoma, Ehrlich Tumor; Cell Division; Cobalt Radioisotopes; Cytochalasin B; Female; Fibrosarcoma; Gamma Rays; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Micronucleus Tests; Mitotic Index

Matrix metalloproteinase-2 (MMP-2) is activated on the cell surface by membrane type 1-MMP (MT1-MMP). Activation of proMMP-2 is induced in vitro by concanavalin A (ConA). The regulation of proMMP-2 activation is, however, not yet fully understood. We investigated the effect of plant lectins, carbohydrates and inhibitors of the cytoskeleton on proMMP-2 activation in normal (HLF1) and malignant fibroblast (HT1080) cells. Native ConA induced proMMP-2 activation in both cell types while dimeric succinyl-ConA had no effect, suggesting that receptor clustering is involved in activation. Wheat germ agglutinin (WGA) also induced proMMP-2 activation. N-acetyl-D-glucosamine (GlcNac) inhibited the effects of ConA and WGA while mannose only inhibited ConA-induced proMMP-2 activation. Mannose also inhibited the expression of MT1-MMP mRNA induced by ConA. Cytochalasin B and colchicine had no effect on the ConA induction of proMMP-2 activation. These studies help to define some of the cellular and molecular mechanisms for the induction of proMMP-2 activation.

Topics: Acetylgalactosamine; Acetylglucosamine; Carbohydrates; Cell Line; Colchicine; Concanavalin A; Cytochalasin B; Cytoskeleton; Enzyme Activation; Enzyme Precursors; Fibroblasts; Fibrosarcoma; Gelatinases; Humans; Lectins; Lung; Mannose; Matrix Metalloproteinase 2; Metalloendopeptidases; Protein Processing, Post-Translational; Tumor Cells, Cultured

The transition of a stationary cell to a motile cell is associated with the breakdown of the cytoskeleton and expression of enzymes responsible for the degradation of the extracellular matrix. Reverse transcription PCR was used to address the question of whether disruption of the microfilament and microtubule systems is a signal for the increased expression of the urokinase plasminogen activator receptor (uPAR), a key molecule involved in extracellular matrix degradation. Disruption of the cytoskeleton of human fibroblasts with cytochalasin B (1 x 10(-5) M or 1 x 10(-7) M), colchicine (1 x 10(-5) M or 5 x 10(-7) M), or nocodazole (1 x 10(-6) M) resulted in major increases in the levels of uPAR mRNA with smaller increases in uPAR protein levels. Both cycloheximide and alpha-amanitin inhibited the increase in uPAR mRNA levels, demonstrating the requirement for the de novo synthesis of a short-lived protein for transcriptional activation. In contrast to normal fibroblasts, treatment of the metastatic cell line HT1080 with either cytochalasin B or colchicine produced little change in the high levels of uPAR mRNA expressed in these cells. These data suggest that cytoskeletal disruption may contribute to cell migration by signaling an increase in uPAR synthesis.

Topics: Amanitins; Base Sequence; Cell Size; Cells, Cultured; Colchicine; Cycloheximide; Cytochalasin B; Cytoskeleton; Fibroblasts; Fibrosarcoma; Gene Expression Regulation; Humans; Molecular Sequence Data; Nocodazole; Polymerase Chain Reaction; Protein Synthesis Inhibitors; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Transcriptional Activation; Tumor Cells, Cultured

The possible relations between cell volume, microfilaments and microtubules networks have been studied in cultured mice fibrosarcoma cells of line T2 and rat pheochromocytoma cells of line PC12. The obtained results show that: 1. Changes in volume induced by application of hypo-osmotic medium are concomitant with a modification in the organization of the microfilaments network as visualized by immunocytochemistry. The microtubules lattice is not affected in these conditions. 2. Disruption of the microfilaments network by cytochalasin B causes a significant decrease in cell volume in isosmotic conditions. It also deeply affects the volume regulation response of cells swollen in hypo-osmotic media. 3. Disruption of the microtubules lattice by colchicine has no effect on volume in isosmotic conditions nor on the volume regulation that follows application of hypo-osmotic shock. The possible role of microfilaments in cell volume control is discussed.

Topics: Actin Cytoskeleton; Adrenal Gland Neoplasms; Animals; Cell Line; Colchicine; Cytochalasin B; Cytoskeleton; Fibrosarcoma; Mice; Microtubules; Osmotic Pressure; Pheochromocytoma; Rats

Topics: Animals; Chick Embryo; Cycloheximide; Cytochalasin B; Cytoskeleton; Fibrosarcoma; Heart; Mice; Microscopy, Electron; Microtubules; Neoplasm Invasiveness; Potassium Cyanide; Sarcoma, Experimental; Temperature