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I N Chou

Publications and source records attributed to I N Chou.

At least 37 records · Page 2Linked to original sources
PubMed1988

Rapid phosphorylation of microtubule-associated proteins through distinct mitogenic pathways.

Mitogenic stimulation of sparse quiescent Swiss 3T3 cells with serum induces a transient reorganization of microtubules which may be necessary for generation or transduction of the mitogenic signal(s). Recently, several studies have shown that microtubule-associated proteins (MAPs) modulate microtubule-mediated functions in vitro and in vivo. We have analyzed, by two-dimensional electrophoresis, the molecular changes in MAPs associated with microtubules in situ following cell activation. By as early as 15 min after addition of serum, several of the MAPs present in quiescent cells are lost from the assembled microtubule fraction while one additional MAP becomes evident. This new MAP is a phosphoprotein whose appearance is independent of protein synthesis. Four additional MAPs also become phosphorylated, and this phosphorylation is accompanied by a partial redistribution of MAPs into the unassembled soluble fraction. Stimulation of cells with purified platelet-derived growth factor or phorbol tumor promoter, a direct activator of protein kinase C, also induces phosphorylation of the same MAPs and DNA synthesis. These results demonstrate that activation of the protein kinase C pathway is sufficient to promote the phosphorylation of MAPs and mitogenesis. However, epidermal growth factor, which does not activate protein kinase C, also stimulates phosphorylation of MAPs and DNA replication. Furthermore, down-regulation of the protein kinase C pathway does not prevent these responses. We conclude that phosphorylation of MAPs and mitogenesis can proceed through protein kinase C-dependent and -independent pathways in 3T3 cells.

Animals↗
PubMed1987

Paraquat-induced cytoskeletal injury in cultured cells.

Although the redox cycling of paraquat (PQ) and the resultant "activated oxygen" generation are important in toxicity development, the intracellular events leading to cell injury remain unclear. To understand the mechanism of PQ-induced cell injury, we have studied the effects of PQ on DNA synthesis, cell proliferation, the cytoskeletal organization, particularly microtubules (MT) and microfilaments (MF), and the synthesis and composition of cytoskeletal proteins in mouse 3T3 cells. PQ treatment produced a dose-dependent inhibition of DNA synthesis and cell growth. Exposure of cells to PQ (313 microM, 20 hr) resulted in MT aggregation and bundling as well as MF redistribution in the perinuclear area as revealed by fluorescence microscopy. Although this PQ dose inhibited DNA synthesis by 95%, it caused only a 22% decrease in protein synthesis of the cytoskeletal fraction. Higher doses of PQ (1250 microM, 20 hr) caused (a) dramatic thinning out and loss of MT and (b) marked loss of MF cables and the appearance of numerous pine needle-like structures much finer and shorter than normal MF. Under these conditions, the synthesis of cytoskeletal proteins was decreased by about 83%. Further analysis of the cytoskeletal fraction from PQ-treated cells by sodium dodecyl sulfate gel electrophoresis showed (a) that tubulin was greatly diminished, in agreement with microscopic observations; (b) that two new protein bands appeared; and (c) another protein band which was also reduced considerably. These results indicate that the PQ-induced dose-dependent cytoskeletal injury may be important to the mechanism of cytotoxicity of this herbicide.

Actin Cytoskeleton↗
PubMed1987

Cytoskeletal perturbation induced by herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).

To understand the mechanisms of toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), we have studied their effects on the cytoskeletal organization, particularly microtubules (MT) and microfilaments (MF), DNA synthesis, and the synthesis and composition of cytoskeletal proteins in mouse 3T3 cells. Exposure of cells to 2,4-D or 2,4,5-T resulted in a dose-dependent inhibition of DNA synthesis; 50% inhibition occurred at 2.21 mM and 0.90 mM for 2,4-D and 2,4,5-T, respectively. Furthermore, a strong synergistic inhibition of DNA synthesis was produced by mixtures (each having a total concentration of 1.25 mM) of 2,4-D with 2,4,5-T. Similarly, 2,4,5-T is more potent than 2,4-D in causing cytoskeletal perturbation as revealed by fluorescence microscopy. Treatment of cells with 2,4-D (2.5 mM) or 2,4,5-T (1.25 mM) for 20 h resulted in severe MT aggregation and the appearance of large bundles, which were organized in a rope-like structure in the former and a dramatic octopus-like pattern in the latter. Further, MT bundling is particularly severe in the cell center. Under these conditions, marked changes in MF organization also occurred as evidenced by clustering and crisscrossing of MF in the perinuclear region. A 1:1 mixture (final = 1.25 mM) of 2,4-D and 2,4,5-T, a formulation equivalent to Agent Orange composition, also induced a dramatic perturbation to the organization of MT and MF, resulting in the formation of ring-like structures. MT bundling is still apparent, especially around the outer edge of the "rings." MF are localized predominantly along the cell periphery, where they appear to be aggregated tightly forming patches. Surprisingly, the synthesis and composition of cytoskeletal proteins, which are resistant to detergent extraction but released by CaCl2, are essentially unaffected by 2,4-D or 2,4,5-T. These results suggest that the dramatic perturbation of the cytoskeletal morphology caused by these herbicides probably only results from a structural reorganization and redistribution of MT and MF.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1987

Environmental wavelengths of ultraviolet light induce cytoskeletal damage.

The ultraviolet component of sunlight is the major cause of skin cancer and is responsible for accelerating the aging of human skin. It is therefore important to determine the mechanisms by which ultraviolet light alters normal cellular functions. The potential importance of ultraviolet light-induced damage to non-DNA targets has received little attention. Since the cytoskeleton is an important participant in the control of normal cell growth, the microfilaments and microtubules of UV irradiated human skin fibroblasts have been studied using fluorescence microscopy. Polychromatic ultraviolet light, composed of environmentally relevant wavelengths, was found to disrupt the cytoplasmic microtubule complex in a dose dependent manner. The induction of microtubule disassembly did not correlate with the cytotoxicity of ultraviolet light of varying composition.

Actin Cytoskeleton↗
PubMed1986

Elevation of intracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts.

The relationship between total glutathione (GSH) content and cell growth was examined in 3T3 fibroblasts. The intracellular GSH level of actively growing cultures gradually decreases as these cells become quiescent by either serum deprivation or high cell density. Upon mitogenic stimulation of sparse, quiescent (G0/G1) cultures with serum, there is a rapid 2.3-fold elevation in intracellular GSH levels which is maximal by 1 h and returns to baseline by 2 h. This is followed by a more gradual increase in GSH content as cells enter the S phase. In addition, the elevation in GSH content is required for maximum induction of DNA synthesis. Treatments that prevent the early increase in intracellular GSH levels do not affect protein synthesis but result in a reversible dose-dependent decrease in the percent of cells capable of entering S phase. These results indicate that GSH may be important in the regulation of cellular proliferation.

Animals↗
PubMed1986

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.

Animals↗
PubMed1984

Microtubule disassembly and morphologic alterations induced by 1-chloro-2,4-dinitrobenzene, a substrate for glutathione S-transferase.

1-chloro-2,4-dinitrobenzene (CDNB), a potent substrate for glutathione S-transferase, is known to rapidly deplete cellular glutathione (GSH) via conjugate formation. Treatment of quiescent 3T3 cells with 5 uM CDNB results in disassembly of microtubules (MT) within 1 hr as revealed by indirect immunofluorescence microscopy. In addition, CDNB treatment also induces dramatic morphologic alterations similar to those mediated by colchicine. Furthermore, taxol prevents both MT disassembly and morphologic changes normally occurring in CDNB as well as colchicine-treated cells. The mechanism of CDNB-mediated MT disassembly in vivo and its possible relationship to cellular GSH metabolism are under current studies.

Animals↗
PubMed1984

Imbalance of total cellular nucleotide pools and mechanism of the colchicine-induced cell activation.

Treatment with colchicine or vinblastine, both inhibitors of microtubule assembly, renders quiescent 3T3 cells in an "activated state" as evidenced by induction of DNA synthesis and other criteria. Microtubule disassembly caused by colchicine or vinblastine brings about a dramatic expansion of total cellular UTP pools with a concomitant diminution in total cellular ATP pools, thus resulting in a marked imbalance in total cellular nucleotide pools. Colchicine and vinblastine also stimulate total cellular RNA synthesis without enhancing uridine phosphorylation, suggesting that these drugs affect the G1 phase of the cell cycle at a point beyond the enhancement of uridine phosphorylation that usually accompanies mitogenic stimulation of quiescent mammalian cells. The markedly expanded cellular UTP pools appear to be necessary for initiation of the colchicine-stimulated DNA synthesis because decreasing cellular UTP pools by addition of D-glucosamine results in a selective inhibition of DNA synthesis in the colchicine-stimulated, but not control, cells. Furthermore, D-glucosamine exerts its inhibitory effect only when it is present in the cultures within the first 14 hr after colchicine treatment. When added at 21 hr, D-glucosamine still decreases cellular UTP pools, but it is no longer inhibitory for DNA synthesis, which commences 14-16 hr after colchicine stimulation. Taxol, an antitumor drug, prevents microtubule disassembly and also blocks such events as expansion of total cellular UTP pools and stimulation of RNA and DNA synthesis, indicating that microtubule depolymerization acts as a primary event initiating the process of cell activation induced by colchicine.

Adenosine Triphosphate↗
PubMed1983

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.

Animals↗
PubMed1980

Effects of retinoic acid on plasminogen activator and mitogenic responses of cultured mouse cells.

Treatment of simian virus 40-transformed 3T3 cells with 1 microM beta-all-trans-retinoic acid (RA) resulted in a 5- to 6-fold enhancement of plasminogen activator (PA) release. Intracellular PA levels rose to twice control levels. Confluent 3T3 cells were less responsive to RA. In 6 of 10 experiments, no increase in 3T3 cell PA levels was noted, although up to a 2.5-fold enhancement of PA elaboration has been observed in some experiments at a dose of 10 microM RA. Simultaneous treatments of 3T3 cells with 10 microM RA and 2.1 to 9.3 mM Ca2+, 5 to 40 ng phorbol myristate acetate per ml, or 150 to 600 ng Fraction I from lactalbumin hydrolysate (Fl) protein per ml indicated that RA potentiated the PA stimulatory activities of these agents. Extracellular PA levels of RA-treated cells increased by 4 to 10 times the amount of increase observed for Ca2+, PMA, or Fl alone. A potentiating activity of RA was also evident when quiescent 3T3 cells were pretreated with 10 microM RA and then stimulated to synthesize DNA with Ca2+, PMA, or Fl. For RA-pretreated cells, an increased percentage of nuclei was labeled with [3H]thymidine (24 hr) in response to doses of the three mitogens which were ineffective without RA pretreatment (2.4mM Ca2+, 5 ng PMA per ml, or 150 ng Fl protein per ml). Additional experiments have indicated that, like platelet extracts, Ra renders quiescent 3T3 cells competent to synthesize DNA in response to the progression factors of human plasma as defined by Pledger et al. (Proc. Natl. Acad. Sci. U. S. A., 74: 4481-4485, 1977). Pretreatment of quiescent 3T3 cells for 6 hr with 10 microM RA resulted in a greater than 4-fold increase in the percentage of cells which incorporated [3H]thymidine in response to a 36-hr treatment with 10% human plasma, as compared to cells treated with human plasma alone. Thus, under certain conditions, RA may have cell-activating properties, and caution should be exercised with regard to its suggested use as an antitumor agent.

Animals↗
PubMed1979

Isolation from lactalbumin hydrolysate of a high molecular weight mitogenic factor.

A new mitogenic factor has been isolated from tissue culture grade lactalbumin hydrolysate. Incubation of postconfluent Swiss 3T3 cells in serum-free medium containing lactalbumin hydrolysate resulted in enhanced synthesis and release of plasminogen activator. Sephadex G-100 gel filtration of concentrated, dialyzed lactalbumin hydrolysate revealed two fractions, LH-FI and LH-FII. LH-FI elutes at the void volume, indicative of high molecular weight, and contains all plasminogen activator stimulatory activity of the original lactalbumin hydrolysate, whereas LH-FII has no activity. In addition, LH-FI also induces DNA synthesis in Swiss 3T3 cells in a dose-dependent fashion, 1 to 2 microgram/ml being equivalent to 10% fetal calf serum. Again, LH-FII is without effect. Induction of DNA synthesis in LH-FI or serum-stimulated quiescent sparse cells followed essentially identical kinetics at least through the first 20 h. Furthermore, LH-FI also enhances 3T3 cell growth. Preliminary results of Sepharose 4B filtration of LH-FI reveal the presence of five subfractions each with plasminogen activator stimulatory as well as mitogenic activity.

Animals↗
PubMed1979

Studies on the mechanism of Ca2+ stimulation of plasminogen activator synthesis/release by Swiss 3T3 cells.

Stimulation of postconfluent Swiss 3T3 cells in serum-free medium with 4.3 mM Ca2+ results in marked increases in both released and cell-associated plasminogen activator (PA). Increased release of PA commenced approximately 10 to 12 hours post-stimulation and continued to increase steadily until 48 hours at which time the stimulates cells (4.3 mM Ca2+) released approximately 14 times more PA than control cells (1.8 mM Ca2+). Sr2+, like Ca2+, also stimulates PA synthesis/release either in the presence or in the absence of 1.8 mM Ca2+ whereas an excess of Mg2+ inhibits Ca2+ stimulation. Supranormal [Pi] in the medium stimulates PA synthesis/release in the presence of 1.8 mM mM Ca2+. Further, optimal stimulation by 4.3 mM Ca2+ requires a normal level of Pi (1.0 mM). Elevation of medium [Ca2+] or [Pi] results in an enhanced uptake of Ca2+. The facts that cycloheximide treatment completely abolishes the Ca2+ stimulatory effect and that an increase in cell associated PA precedes release indicate that PA release is coupled to synthesis of new PA. Ca2+ stimulation of PA synthesis/release also requires continuous energy production and RNA as well as protein synthesis. A hypothesis is proposed to explain the relationship between stimulation of PA production and its enhanced release from cells stimulated by elevated [Ca2+] or [Pi] in the media. The possibility that PA release may be an example of the phenomenon of membrane shedding as opposed to secretion is discussed.

Animals↗
PubMed1977

Calcium stimulation of plasminogen activator secretion/production by swiss 3T3 cells.

Actively growing Swiss 3T3 cells secret high levels of plasminogen activator which decreases after the cells become confluent. In contrast, simian virus 40-transformed 3T3 cells secrete large amounts of plasminogen activator independent of cell density (Chou, I.-N., O'Donnel, S.P., Black, P.H., and Roblin, R.O. (1977) J. Cell. Physiol. 91, 31-38). These results suggest a correlation between active cell multiplication and plasminogen activator secretion in both 3T3 and simian virus-transformed 3T3 cells. The data reported herein indicate that treatment of both subconfluent and confluent Swiss 3T3 cells with high concentrations of Ca2+ (final 3.0 to 4.9 mM) increases the amounts of both secreted and cell-associated plasminogen activator in a dose-dependent manner. In addition, the ionophore A23187 (19 to 95 nM) in the presence of a normal level of Ca2+ (1.8 mM) stimulates both production and secretion of plasminogen activator from growing 3T3 cells. The Ca2+ stimulation of plasminogen activator production/secretion may be related to the mitogenic effect of Ca2+.

Calcimycin↗
PubMed1977

Cell density-dependent secretion of plasminogen activator by 3T3 cells.

The expression of extracellular fibrinolytic activity in untransformed 3T3 cell cultures depends on the growth state of the cells. Actively growing 3T3 cultures exhibit a relatively high level of fibrinolysis, which decreases progressively as the cells become confluent and density-inhibited. The low level of fibrinolytic activity in confluent 3T3 cultures is due to a diminution in secretion of plasminogen activator since the intracellular level of plasminogen activator remains high. The amount of plasminogen activator observed in growing 3T3 cultures varies depending upon whether the cells are passaged with trypsin/EDTA solution, or with Ca++ selective chelating agent, ethylene-bis (oxyethylenenitrilo) tetraacetic acid (EGTA). However, in cells passaged using either agent, the amount of plasminogen activator secreted is always greatest when the cells are actively growing and decreases thereafter. In contrast to confluent 3T3 cultures, dense cultures of SV40-virus transformed 3T3 cells continued to secrete relatively large amounts of plasminogen activator. The ability to decrease secretion of plasminogen activator as cells become dense may be an important characteristic of cells which demonstrate density-dependent inhibition of cell multiplication in vitro.

Cell Division↗