Iih-Nan Chou

Effects of sodium arsenite on the cytoskeleton and cellular glutathione levels in cultured cells

The effects of As3+ (NaAsO2) on the microtubule and microfilament organization, cytoskeletal protein synthesis, cytoskeletal and cytosolic (soluble) protein sulfhydryls, and cellular glutathione (GSH) levels were examined in Swiss 3T3 mouse cells. Exposure of cells to 2.5 microM As3+ for 16 hr resulted in apparent cell retraction and loss of thick cables of actin filaments. However, the cells still retained numerous thinner microfilaments distributed in a disorganized manner. Microtubule organization was relatively undisturbed. At higher doses (greater than or equal to 20 microM), As3+ treatment caused a severe loss of microtubules and the remaining dense finer actin filaments formed smearing clusters in perinuclear areas. Treatment of cells with As3+ also induced a dose-dependent inhibition of cytoskeletal protein synthesis. Furthermore, As3+ exposure enhanced cellular GSH synthesis since the elevated cellular GSH content in As(3+)-treated cells could be abolished by treatment with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase required for GSH biosynthesis. As determined by the N-[3H]-ethylmaleimide binding assay, As3+ exposure also increased the amount of protein sulfhydryls in both the cytoskeletal and the cytosolic protein fractions. Moreover, a greater increase in protein sulfhydryls occurred in the cytoskeletal fraction than in the soluble fraction. These results indicate that the cytoskeleton could be a cellular target for injury by As3+ exposure. The elevated cellular GSH content induced by As3+ could provide a protective mechanism against further injury from this metal insult.

Hydrogen peroxide‐mediated, lysyl oxidase‐dependent chemotaxis of vascular smooth muscle cells

Lysyl oxidase (LO), an enzyme secreted by vascular smooth muscle cells (VSMC), initiates the covalent crosslinking of polypeptide chains within collagen and elastin. The present study reveals that purified LO strongly induces directional migration of VSMC in an in vitro assay system. LO‐dependent chemotaxis, but not chemokinesis, was abolished by β‐aminopropionitrile, an active site inhibitor of LO, or by catalase, as well as by prior heat denaturation. This indicates that the H2O2 product of amine oxidation by LO is critical to the expression of its chemotactic activity. The results indicate that the chemotactic response requires direct access between LO and a substrate molecule (or molecules) tightly associated with the VSMC. The addition of LO to VSMC elevated the levels of intracellular H2O2, enhanced stress fiber formation, and focal adhesion assembly, is consistent with the induction of the chemotactic response. J. Cell. Biochem. 78:550–557, 2000.

Lysyl oxidase oxidizes basic fibroblast growth factor and inactivates its mitogenic potential

Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6‐1 cells (6‐1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6‐1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras‐induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.

Isolation of plasma membrane fragments from cultured murine melanoma cells

The various manipulations involved in the isolation of membrane fragments from culture fluids of murine melanoma cells were examined to discern their effect on membrane fragment structure. Ultracentrifugation and gel chromatography were compared using the presence of marker enzymes and the sensitivity to a non-ionic detergent (Triton X-100). Fractionation of media by gel chromatography resulted in only one major form of membrane particles, while ultracentrifugation, followed by resuspension, produced at least two major populations from the identical material. These results indicate that the optimal procedure for membrane fragment isolation is fractionation by an agarose-containing gel, followed by concentration using PEG 20,000.

Role of calmodulin in cadmium-induced microtubule disassembly

Micromolar CdCl2 has been shown to cause disassembly of the cytoplasmic microtubule complex in cultured Swiss 3T3 cells. We show in this paper that Cd(II), an environmental and occupational health hazard, induces microtubule disassembly in an in situ cytoskeleton model system, and that the calmodulin inhibitors, trifluoperazine and Compound 48/80, prevent this Cd(II)-induced microtubule disassembly. Our results suggest that Cd(II) affects microtubules by activating calmodulin associated with the cytoskeleton. Furthermore, the fact that these two ions have very similar ionic radii (0.99A vs. 0.97A) supports our conclusion that Cd(II) acts similarly to Ca(II) in inducing microtubule disassembly. This may be relevant to the mechanism of Cd-mediated cellular injury.

Interferon inhibits the release of plasminogen activator from SV3T3 cells.

INTERFERON, as well as inducing an antiviral state, can modify certain other cell characteristics. For example, it has been shown to inhibit cell proliferation1–4 and release of C-type virus5,6, and to enhance the expression of H–2 antigens7,8 and the amount of glycoprotein on the cell surface9. These effects might be explained by decreased release of cell-surface components into the medium. Similarly, the expression of a glycoprotein on the cell surface has been related to the growth state of cells10, which can be explained in part by differential release11,12. We have followed up this suggestion by examining the effect of interferon on the release of plasminogen activator (PA) by a line of simian virus 40 transformed Swiss 3T3 (SV3T3) cells. Our results, reported here, indicate that interferon inhibits the release of PA by SV3T3 cells.

Disruption of cytoplasmic microtubules by ultraviolet radiation

Ultraviolet (UV) irradiation of cultured human skin fibroblasts causes the disassembly of their microtubules. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. Exposure to polychromatic UV radiation inhibits the reassembly of microtubules during cellular recovery from cold treatment. In addition, the ability of taxol to promote microtubule polymerization and bundling is inhibited in UV-irradiated cells. However, UV irradiation of taxol-pretreated cells or in situ detergent-extracted microtubules fails to disrupt the microtubule network. These data suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV-irradiated human skin fibroblasts.

Disruption of keratin intermediate filaments by ultraviolet radiation in cultured human keratinocytes.

Abstract— Fluorescence microscopy has been utilized to investigate the effects of UV irradiation on the organization of keratin intermediate filaments in normal human epidermal keratinocytes. Sun lamp irradiation induced the condensation of keratin intermediate filaments into the perinuclear region and inhibited the reorganization of keratin filaments normally induced by Ca2+. Exposure to UVC appeared to disrupt keratin filaments similarly, whereas UVA had no discernible effect.

Studies on the mechanisms of Ni2+-induced cell injury. I, Effects of Ni2+ on microtubules

Cytoskeletal perturbations have been associated with exposures to a variety of toxic agents as well as a number of human pathological conditions. We have observed dramatic alterations in the organization of microtubules (MT), a major component of the cytoskeleton, in 3T3 cells exposed to Ni2+. Severe perinuclear bundling and aggregation of MT occurred in both a time- and dose-dependent fashion, and this MT damage was reversible upon removal of Ni2+ from the culture media. To understand the mechanism of the Ni2(+)-induced MT change, we investigated the effect of Ni2+ (0.01 to 3.0 mM) on in vitro tubulin polymerization. Ni2+ at lower concentrations (0.01 to 1.0 mM) had little or no significant effect on the kinetics of MT polymerization. In contrast, in the presence of 1.5 to 2.0 mM Ni2+, a significant promoting effect on both the rate and the final extent of polymerization was observed. However, at Ni2+ concentrations higher than 2.0 mM, such stimulatory effect on the rate and the final extent of tubulin polymerization declined. Furthermore, the promoting effects of Ni2+ on MT polymerization were accompanied by a significant decrease in the lag period. Electron microscopic examination of samples of the polymerization product showed that MT, polymerized in the presence of 2.0 mM Ni2+, appeared more numerous and shorter (1.10 +/- 1.02 microns) than those of control (3.81 +/- 2.29 microns; p less than 0.005). This was probably a direct result of an increase in the number of initiation centers in the presence of Ni2+ as a consequence of the decreased critical concentration (7%, p less than 0.05) necessary for polymerization to occur. Our results suggest that Ni2+ may exert its toxic effect on MT in cultured cells by altering the normal kinetics of MT polymerization.

Relationship between 1-chloro2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthiomine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 μM 1-chloro-2,4-dinitrobenzene and 250 μM buthiomine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthiomine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 μM 2-cyclohexene-l-one, which depletes free glutathione by conjugation, resulted in a com