cytochalasin-d and Leukemia--Myeloid

Mechanical stiffness is closely related to cell adhesion and rounding in some cells. In leukocytes, dephosphorylation of ezrin/radixin/moesin (ERM) proteins is linked to cell adhesion events. To elucidate the relationship between surface stiffness, cell adhesion, and ERM dephosphorylation in leukocytes, we examined the relationship in the myelogenous leukemia line, KG-1, by treatment with modulation drugs. KG-1 cells have ring-shaped cortical actin with microvilli as the only F-actin cytoskeleton, and the actin structure constructs the mechanical stiffness of the cells. Phorbol 12-myristate 13-acetate and staurosporine, which induced cell adhesion to fibronectin surface and ERM dephosphorylation, caused a decrease in surface stiffness in KG-1 cells. Calyculin A, which inhibited ERM dephosphorylation and had no effect on cell adhesion, did not affect surface stiffness. To clarify whether decreasing cell surface stiffness and inducing cell adhesion are equivalent, we examined KG-1 cell adhesion by treatment with actin-attenuated cell softening reagents. Cytochalasin D clearly diminished cell adhesion, and high concentrations of Y27632 slightly induced cell adhesion. Only Y27632 slightly decreased ERM phosphorylation in KG-1 cells. Thus, decreasing cell surface stiffness and inducing cell adhesion are not equivalent, but these phenomena are coordinately regulated by ERM dephosphorylation in KG-1 cells.

Topics: Actins; Amides; Cell Adhesion; Cell Line, Tumor; Cytochalasin D; Cytoskeletal Proteins; Elasticity; Fibronectins; Humans; Leukemia, Myeloid; Leukocytes; Membrane Proteins; Microfilament Proteins; Microvilli; Phorbol Esters; Phosphorylation; Pyridines; Staurosporine

Sodium influx is tightly regulated in the cells of blood origin. Amiloride-insensitive sodium channels were identified as one of the main sodium-transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics. Here, to search for physiological mechanisms controlling epithelial Na

Topics: Actin Cytoskeleton; Actins; Amiloride; Cell Membrane; Cell Membrane Permeability; Cytochalasin D; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels; Humans; K562 Cells; Leukemia, Myeloid; Membrane Potentials; Microscopy, Fluorescence; Models, Biological; Patch-Clamp Techniques; Phalloidine; Sodium; Trypsin; Trypsin Inhibitors

With the use of the patch clamp technique, the role of cytoskeleton in the regulation of ion channels in plasma membrane of leukemic K562 cells was examined. Single-channel measurements have indicated that disruption of actin filaments with cytochalasin D (CD) resulted in a considerable increase of the activity of non-voltage-gated sodium-permeable channels of 12 pS unitary conductance. Background activity of these channels was low; open probability (po) did not exceed 0.01-0.02. After CD, po grew at least 10-20 times. Cell-attached and whole-cell recordings showed that activation of sodium channels was elicited within 1-3 min after the addition of 10-20 micrograms/ml CD to the bath extracellular solution or in the presence of 5 micrograms/ml CD in the intracellular pipette solution. Preincubation of K562 cells with CD during 1 h also increased drastically the activity of 12 pS sodium channels. Whole-cell measurements confirmed that CD-activated channels were permeable to monovalent cations (preferentially to Na+ and Li+), but not to bivalent cations (Ca2+, Ba2+). Colchicine (1 microM), which affect microtubules, did not alter background channel activity. Our data indicate that actin filaments organization plays an important role in the regulation of sodium-permeable channels which may participate in providing passive Na+ influx in red blood cells.

Topics: Actin Cytoskeleton; Actins; Cell Membrane Permeability; Cytochalasin D; Electrophysiology; Humans; Leukemia, Myeloid; Patch-Clamp Techniques; Sodium Channels; Tumor Cells, Cultured

A protein phosphatase inhibitor, tautomycin induces blebs on the surface of human myeloid leukemia K562 cells within 10 min. In this paper we examined the cytoskeleton of tautomycin-treated cells. In the presence of tautomycin, cells with blebs turned into segmented forms with smooth surfaces after 15 min and into smooth round shapes without microprotuberance after 60 min. Observation with fluorescence microscopy showed that F-actin detached from the plasma membrane at the site of the blebs. Further treatment with tautomycin induced the accumulation of F-actin at the segmentation centers. Under electron microscopy, an electron dense ring-structure was detected at the segmentation center. Tautomycin did not induce major changes of the microtubule network although F-actin accumulated near the microtubule organizing center. The amount of F-actin increased in tautomycin-treated cells. These results indicate that the morphological changes are induced by reorganization of actinfilaments.

Topics: Actin Cytoskeleton; Actins; Antifungal Agents; Cell Membrane; Cytochalasin D; Humans; Intermediate Filaments; Leukemia, Myeloid; Microscopy, Electron; Microscopy, Fluorescence; Microtubules; Phosphoprotein Phosphatases; Pyrans; Spiro Compounds; Tumor Cells, Cultured

A novel antibiotic tautomycin induced many blebs on the surface of K562 human chronic myeloid leukemia cells, similar to the morphological changes induced by phorbol esters. However, tautomycin did not induce nitroblue tetrazolium reducing activity, when HL60 human promyelocytic leukemia cells were caused to differentiate by quinomycin into mature granulocytes. It did not induce spread of HL60 cells, one of the phenotypes of mature macrophages. In addition, it did not compete with phorbol dibutyrate to bind to the cell surface of K562 cells. However, tautomycin significantly activated protein kinase C (PKC) extracted from K562 cells. These results indicate that tautomycin is a new activator of PKC, distinct from phorbol esters.

Topics: Anti-Bacterial Agents; Antifungal Agents; Cell Differentiation; Cytochalasin D; Cytochalasins; Enzyme Activation; Humans; Leukemia, Myeloid; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Pyrans; Spiro Compounds; Tumor Cells, Cultured