cytochalasin-d and Fibrosarcoma

F-actin-stabilizing drugs induce actin aggresome formation. In this study, we found that an actin-depolymerizing drug, latrunculin A (LatA), induced actin aggresomes. Actin stress fibers were retracted and disappeared in minutes, but a large aggresome formed in consequence of LatA treatment. Because cytochalasin D and mycalolide also induced aggresome formation, these results suggest that actin aggresome formation is a common cellular response to actin toxins.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Survival; Cytochalasin D; Fibroblasts; Fibrosarcoma; Humans; Marine Toxins; Microscopy, Fluorescence; Microscopy, Video; Protein Folding; Rats; Stress Fibers; Stress, Physiological; Thiazolidines

Pericellular matrix degradation during cancer invasion and inflammation is dependent on activation of progelatinase A by membrane type 1-matrix metalloproteinase (MT1-MMP); a stoichiometric concentration of tissue inhibitor of metalloproteinase-2 (TIMP-2) is required. Activation of progelatinase A has generally been considered to be a slow process occurring as a result of enhanced expression of MT1-MMP. We herein report that ConA treatment of HT1080 fibrosarcoma cells is followed by MT1-MMP-induced activation of progelatinase A on the cell surface within 1 hour. Cell surface biotinylation, immunohistochemistry, and (125)I-labeled TIMP-2 binding to cell surface MT1-MMP were used to characterize the appearance and function of MT1-MMP on the plasma membrane. Treatment of HT1080 cells with ConA resulted in increased specific binding of (125)I-labeled TIMP-2 to cell surface receptors within 5 minutes. TIMP-2 binds almost exclusively to activated MT1-MMP on the surface of HT1080 cells. MT1-MMP function at the cell surface was also accelerated by treatment of cells with cytochalasin D, an inhibitor of actin filaments, PMA, a stimulator of protein kinase C, and bafilomycin A(1), an inhibitor of lysosome/endosome function. A functional pool of intracellular MT1-MMP available for trafficking to the cell surface was demonstrated by repetitive ConA stimulation. ConA-induced expression of MT1-MMP mRNA (Northern blot analysis) in HT1080 cells was a delayed event (>6 hours). These data suggest that presynthesized MT1-MMP is sorted to a transient storage compartment (trans-Golgi network/endosomes), where it is available for rapid trafficking to the plasma membrane and cell surface proteolytic activity.

Topics: Anti-Bacterial Agents; Cell Membrane; Concanavalin A; Cytochalasin D; Enzyme Activation; Enzyme Precursors; Fibrosarcoma; Fluorescein-5-isothiocyanate; Gelatinases; Humans; Macrolides; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Protein Transport; Receptors, Cell Surface; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured