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PubMed2003

Agmatine inhibits the proliferation of rat hepatoma cells by modulation of polyamine metabolism.

BACKGROUND/AIMS: Previous experiments have shown that agmatine, the product of arginine decarboxylase, is transported in competition with putrescine into quiescent rat hepatocytes, where it promotes several effects, including marked decrease of intracellular polyamines and induction of apoptosis. The primary aim of the present study was to assess the action of agmatine on transformed and proliferating hepatic rat cells. METHODS: To assess the effect of agmatine on hepatoma cells, analysis by flow cytometry, Western blotting, reverse transcription-polymerase chain reaction, scanning and transmission electron microscopy, immunofluorescence detection of beta-actin and alpha-tubulin were performed. RESULTS: The results showed that agmatine has antiproliferative effects on the cell lines studied (HTC, JM2, HepG2). Further experiments were performed on HTC cells. The effect was proportional to agmatine concentration (in a range between 50 and 500 microM). It was not correlated with induction of necrosis or apoptosis and was accompanied by accumulation in G(2)/M cell cycle phase and by dramatic modification of cell morphology. Spermidine reversed these effects, suggesting that the marked decrease of the polyamine pool is the main target of agmatine . CONCLUSIONS: The results obtained show a relationship between the decrease of intracellular polyamine content, the rate of cell growth and the cytoskeleton organization.

Actins↗
PubMed1998

A six-module human nebulin fragment bundles actin filaments and induces actin polymerization.

We have investigated the interaction of a 6-repeat recombinant human nebulin fragment (S6R2R7) with F-actin, with Mg2+-induced actin paracrystals, and G-actin, respectively. This fragment corresponds to super-repeat 6, repeat 2 to 7 of human nebulin, and is located in the N-terminal part of the super-repeat region of the nebulin molecule. The S6R2R7 fragment included an immuno-tag of three amino-acid residues (EEF) at one end which was detectable by a monoclonal anti-tubulin YL1/2. By a cosedimentation assay, interaction between F-actin and S6R2R7 was observed. Electron microscopy revealed the formation of large bundle-like aggregates containing highly parallelized actin filaments, apparently caused by actin bundling of the nebulin fragment. Compared with Mg2+-induced actin paracrystals where the helices of the actin filaments are arranged in register, the filaments in the actin-nebulin bundles seem to be packed in a different way and show no obvious periodicity. The bundles were also visible in the light microscope, and immunofluorescence microscopy revealed binding of the nebulin fragment S6R2R7 to both preformed Mg2+ paracrystals and to F-actin. We also analyzed the effect of S6R2R7 on actin under non-polymerizing conditions by cosedimentation assays and pyrene actin fluorimetry, as well as fluorescence microscopy and electron microscopy. Nebulin-induced actin polymerization was observed with an enhancement of the nucleation step indicating a stabilization of actin nuclei by S6R2R7. Light and electron microscopy revealed bundle-like actin-nebulin aggregates similar to those formed by pre-assembled F-actin and S6R2R7. Thus, even in the absence of salt, S6R2R7 promotes actin polymerization and induces formation of tightly packed actin filament bundles. We assume that the actin filaments are crosslinked by the nebulin fragments, indicating a rather low cooperativity of binding to a single filament.

Actins↗
PubMed1995

Regulation of microtubule dynamic instability by tubulin-GDP.

The regulation of the spontaneous transitions between growth and shortening of microtubules is central to the biological function of dynamic instability. Here we examine the effects of controlled amounts of tubulin-GDP (Tu-GDP) on the dynamic properties of microtubules in vitro. The transphosphorylation equilibrium between GTP, GDP, UTP, and UDP in the presence of nucleoside-5'-diphosphate kinase (NDPK) was used to fix the ratio chi D = [Tu-GDP]/([Tu-GTP]) + [Tu-GDP]) in solution. Lower levels of Tu-GDP (chi D < 0.6) produce only a small increase in the apparent critical concentration, Cc'. However, at chi D > 0.6 a dramatic increase in Cc is observed. At steady state of assembly, low levels of Tu-GDP (chi D < 0.5) cause a significant reduction of the dynamic length redistribution of the microtubule population. The principal observable effect of Tu-GDP on the empirical parameters of microtubule dynamic instability is to decrease the duration of individual phases of microtubule growth and shortening, with relatively little effect on the intrinsic rates of growth and shortening. Observations in dark-field video microscopy reveal that the irregularities in the rates of growth (and shortening) are increased in the presence of Tu-GDP. At elevated levels of Tu-GDP, pauses occur frequently during the growth phase, microtubule dynamics cease to conform to a clear two-phase process, and the extents of growth and shortening excursions are strongly attenuated. The experimental results are well reproduced by computer simulation, using mechanisms defined in the lateral cap model for dynamic instability [Martin, S. R., Schilstra, M. J., & Bayley, P. M. (1993) Biophys. J. 65, 578-596], which includes the binding of Tu-GDP to the microtubule end in competition with Tu-GTP. In the presence of Tu-GDP, the growing-state lifetime is significantly attenuated, and the microtubule length versus time excursions simulated by the model show irregularities and complex multistate behavior, including pauses, as observed experimentally. These results suggest that Tu-GDP can modulate microtubule dynamics significantly under conditions where little bulk microtubule disassembly is induced. Tu-GDP therefore appears to exemplify the action of a relatively simple factor with the potential capability for regulation of microtubule dynamics in a cellular environment.

Computer Simulation↗
PubMed1996

Phase II data on paclitaxel and docetaxel in gastrointestinal malignancies.

From the contributions in this volume, it is apparent that in spite of promising chemotherapy combinations and new multidisciplinary approaches, the treatment of advanced gastrointestinal malignancies remains a difficult task. New agents have recently been developed with either a better therapeutic ratio (fluoropyrimidine analogs or modulators of the metabolic pathway of those agents) or a better activity due to a different mechanism of action. Among the latter agents, the topoisomerase I inhibitors (especially irinotecan, also known as CPT-11) and new antimetabolities (difluorodeoxycytidine, also known as gemcitabine) are of interest for difficult tumors, such as colon and pancreatic cancer. This short review describes the phase II studies on the activity of taxoids, agents that promote tubulin assembly in microtubules and inhibit their depolymerization.

Antineoplastic Agents, Phytogenic↗
PubMed1989

Association of casein kinase II with microtubules.

A magnesium-dependent heparin-inhibited protein kinase activity associated with brain microtubule preparations has been identified as casein kinase II using a monospecific polyclonal antibody. This enzyme appears enriched in cold-stable microtubule fractions. By immunofluorescence microscopy using an antiserum against casein kinase II, the in situ immunolabeling of some microtubule assays has been observed. Thus, mitotic spindles are stained by the anti-casein kinase II antibody in fibroblast cells. In neuroblastoma cells induced to differentiate, the labeling of microtubule arrays inside developing axon-like processes is also seen. These results support the view that casein kinase II can modulate cytoskeletal assembly and dynamics through phosphorylation of microtubule proteins.

Animals↗
PubMed2001

Modulated polarization microscopy: a promising new approach to visualizing cytoskeletal dynamics in living cells.

In an effort to visualize cytoskeletal filaments in living cells, we have developed modulated polarization microscopy. Modulated polarization microscopy visualizes cytoskeletal filaments based on their birefringence but differs from the standard polarization microscopy by exploiting the angle dependence of birefringence. A prototype instrument has been developed using two Faraday rotators under computer control to change the angle of plane polarized light at a known rate. By placing one Faraday rotator before and one after the specimen, rotation produced by the first Faraday rotator is cancelled by the second. This allows the use of fixed polarizer and analyzer in a crossed configuration and continuous imaging of the specimen between crossed polarizers. The variation in polarization angle of light illuminating the specimen causes birefringent elements to oscillate in brightness. Images acquired as polarization angle is varied are then processed by a Fourier filter image-processing algorithm. The Fourier filtering algorithm isolates those signals that vary at the proper rate, whereas static or random signals are removed. Here we show that the modulated polarization microscope can reveal cytoskeletal elements including stress fibers and microtubules in living cells.

Animals↗
PubMed1993

In vitro regulation of rat prolactin messenger ribonucleic acid poly(A) tail length: modulation by bromocriptine.

Recent analysis of endocrine gene transcripts has revealed that several hormone mRNAs exhibit regulated size changes (due to alterations in length of the 3' poly(A) tail) which may function as an additional level of control in the determination of gene expression. We have now shown, through the novel application of an organ culture technique, that prolactin mRNA exhibits a similar regulated change in poly(A) tail length when rat anterior pituitary glands are explanted. The effect is observed in glands of either male or female rats and is specific with respect to growth hormone and alpha-tubulin mRNAs. Furthermore, we have also found that the size change in prolactin mRNA is attenuated in the presence of bromocriptine, indicating regulation through a dopaminergic pathway.

Animals↗
PubMed2005

Multisite protein modification and intramolecular signaling.

Post-translational modification is a major mechanism by which protein function is regulated in eukaryotes. Instead of single-site action, many proteins such as histones, p53, RNA polymerase II, tubulin, Cdc25C and tyrosine kinases are modified at multiple sites by modifications like phosphorylation, acetylation, methylation, ubiquitination, sumoylation and citrullination. Multisite modification on a protein constitutes a complex regulatory program that resembles a dynamic 'molecular barcode' and transduces molecular information to and from signaling pathways. This program imparts effects through 'loss-of-function' and 'gain-of-function' mechanisms. Among the latter, covalent modifications specifically recruit a diverse array of modules, including the SH2 domain, 14-3-3, WW domain, Polo box, BRCT repeat, bromodomain, chromodomain, Tudor domain and motifs binding to ubiquitin and other protein modifiers. Such recruitments are often modulated by modifications occurred at neighboring and distant sites. Multisite modification thus coordinates intermolecular and intramolecular signaling for the qualitative and quantitative control of protein function in vivo.

Acetylation↗
PubMedDate not supplied

Low levels of ethanol stimulate and high levels decrease phosphorylation in microtubule-associated proteins in rat brain: an in vitro study.

Phosphorylation and dephosphorylation of proteins associated with microtubules (MAPs) modulate the functional properties of microtubules (MT). A study was designed to test the hypothesis that ethanol at pharmacologically relevant levels affects phosphorylation of MAPs. Low (6, 12, 24, and 48 mM) and high (96, 384, and 768 mM) levels of ethanol were used in the study. MT prepared from rat brain by successive cycles of assembly-disassembly were found to contain two high molecular weight proteins (MAP2 and MAP1), tubulin, and 70-kDa neurofilament. The kinase activity was determined using [gamma(32)P]ATP as a phosphate donor. The results showed that ethanol primarily stimulated MAP2 phosphorylation. Low levels of ethanol stimulated, whereas high levels decreased, the kinase activity. MAP1 was phosphorylated to a lesser extent. 70-kDa neurofilament and tubulin were phosphorylated, however, the dose-dependent biphasic effect of ethanol on phosphorylation was not found in these cytoskeleton proteins. To determine whether the ethanol-induced kinase activity was cAMP-dependent, the catalytic subunit of cAMP-dependent protein kinase was isolated, purified, and kinase activity was determined with and without ethanol. The results showed that cAMP was not involved in ethanol-induced kinase activity. We conclude that ethanol predominantly stimulates phosphorylation of MAP2 in a dose-dependent manner.

Animals↗
PubMed2003

A novel interaction between kinesin and p120 modulates p120 localization and function.

p120-catenin exists in a membrane-associated cadherin-bound pool, a cytosolic pool that affects Rho GTPases, and a nuclear pool that is thought to associate with the methylation-relevant transcriptional repressor Kaiso. We show here that cytoplasmic p120 can also associate both directly and indirectly with the microtubule network, and that p120 traffics along microtubules toward their plus ends. The direct binding required most of the armadillo repeats and was mutually exclusive for interaction with E-cadherin. Perturbing the p120-microtubule interaction with nocodazole or taxol markedly affected both the tubulin interaction and the balance between cytoplasmic and nuclear p120. The indirect binding occurred via a novel interaction between a segment of the p120 N-terminal domain and conventional kinesin heavy chains. Selective uncoupling of the p120-kinesin interaction by overexpression of the respective p120 and kinesin-binding fragments promoted nuclear p120 accumulation. In addition, expression of full-length kinesin reduced the nuclear accumulation of p120 and blocked the branching phenotype associated with p120 overexpression. Taken together, the data suggest that kinesin affects both the targeting and activity of p120 at several cellular locations.

Animals↗
PubMed1997

Oxidized and phosphorylated synthetic peptides corresponding to the second and third tubulin-binding repeats of the tau protein reveal structural features of paired helical filament assembly.

The microtubule-associated protein tau of normal brains is attached to tubulin through its 18-amino-acid repeat units. In the paired helical filaments (PHF) of Alzheimer's disease, however, tau is oligomerized in an abnormally hyperphosphorylated from (PHF-tau). tau contains two cysteine residues in repeat units 2 and 3, but only the R3-R3 homodimer is present in PHF-tau. A serine residue two amino acids downstream of the R3 cysteine is a major phosphate acceptor site for protein kinase C. In the work repeated here, we used synthetic peptides corresponding to R2, R3 and phosphorylated R3 to determine the binding of the tau repeat peptides to a peptide fragment corresponding to the C-terminal domain of beta-tubulin and to study the kinetics of homo- and heterodimer formation. Additionally, we studied two major biochemical properties of the peptides that distinguish between normal tau and PHF-tau: conformation and metabolic stability. All R2 and R3 peptides bound specifically to the tubulin peptide regardless of the state of phosphorylation or dimerization. The reverse-turn conformation of the tau repeat peptides in the presence of the tubulin peptide remained unaffected. Phosphorylation slightly loosened the turn structure of the monomeric and dimeric peptides, and did not univocally affect the serum stability of the peptides or the ability of the peptides to form dimers. The isolated R2 and R3 units formed homodimers approximately in the same rate. When the two peptides were mixed, however, the R2-R3 heterodimer was formed preferentially over the homodimers. The dimers were generally more stable in human serum than the monomers. Our results with the synthetic peptide fragments of tau indicate that neither oxidation nor phosphorylation of the repeat units is able to generate extended structure such as that found in PHF-tau. Additionally, phosphorylation of Ser324 does not appear to modulate the kinetics of oligomerization of tau, and in general biochemistry terms, does not affect disulfide bridge formation nearby. In agreement with studies at the full-protein level, the formation of homodimers of the peptides, a model of the self-association of tau, is not preferred. If the dimers are formed, however, their clearance is considerably slower than that of the monomers, explaining the remarkable protease resistance of PHF-tau in the affected brains.

Alzheimer Disease↗
PubMedDate not supplied

Regulation of the subcellular distribution and gene expression of GABA(A) receptor by microtubules and microfilaments in cultured brain neurons.

Mechanisms underlying the intracellular transport of gamma-aminobutyric acid(A) receptor (GABA(A)R) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and (3)H-flunitrazepam (FNZ) binding assay were employed to determine the cell surface and intracellular distribution of the receptor. A 3-h treatment of the cells with 1 microM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABA(A)R density by about 36% and decreased that of the cell surface receptors by 18% from respective control values, whereas the 3-h incubation with 2 microM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the (3)H-FNZ binding sites of the neurons and the affinity of the ligand. Moreover, the exposure to colchicine seemed to produce a stronger cytoplasmic immunostaining of the GABA(A)R alpha subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular (3)H-FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of alpha(1)+51kDa proteins. In addition, the colchicine or cytochalasin D treatment inhibited approximately 21 or 18% of the rate of general protein synthesis in the culture. Notably, in situ hybridization assay showed that the GABA(A)R alpha(1) or alpha(2) mRNA was present in 92 +/- 2% or 94 +/- 2% of the cytochalasin D-treated neurons, both of which were higher than 71 +/- 2-74 +/- 3% of the control and colchicine-treated cells. The data suggest that by regulating the intracellular transport, the microtubular system participates in the maintenance of normal subcellular distribution of GABA(A)R in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABA(A)R subunits.

Actin Cytoskeleton↗
PubMed1987

Defective distal regulatory element at the 5' upstream of rat prolactin gene of steroid-nonresponsive GH-subclone.

The prolactin-nonproducing (PRL-) GH cell strains (rat pituitary tumor cells in culture). GH12C1 and F1BGH12C1, do not respond to steroid hormones estradiol or hydrocortisone (HC). However, the stimulatory effect of estradiol and the inhibitory effect of hydrocortisone on prolactin synthesis can be demonstrated in the prolactin-producing GH cell strain, GH4C1. In this investigation we have examined the 5' end flanking region of rat prolactin (rat PRL) gene of steroid-responsive, GH4C1 cells to identify the positive and negative regulatory elements and to verify the status of these elements in steroid-nonresponsive F1BGH12C1 cells. Results presented in this report demonstrate that the basel level expression of the co-transferred Neo gene (neomycin phosphoribosyl transferase) is modulated by the distal upstream regulatory elements of rat PRL gene in response to steroid hormones. The expression of adjacent Neo gene is inhibited by dexamethasone and is stimulated by estradiol in transfectants carrying distal regulatory elements (SRE) of steroid-responsive cells. These responses are not observed in transfectants with the rat PRL upstream sequences derived from steroid-nonresponsive cells. The basal level expression of the host cell alpha-2 tubulin gene is not affected by dexamethasone. We report here the identification of the distal steroid regulatory element (SRE) located between 3.8 and 7.8 kb upstream of the transcription initiation site of rat PRL gene. Both the positive and the negative effects of steroid hormones can be identified within this upstream sequence. This distal SRE appears to be nonfunctional in steroid-nonresponsive cells. Though the proximal SRE is functional, the defect in the distal SRE makes the GH substrain nonresponsive to steroid hormones. These results suggest that both the proximal and the distal SREs are essential for the mediation of action of steroid hormones in GH cells.

Animals↗
PubMed2005

Conformational analysis of rhazinilam and three-dimensional quantitative structure-activity relationships of rhazinilam analogues.

3D QSAR of rhazinilam (1), an alkaloid isolated from Rhazya stricta (Apocynaceae) with an activity involving disassembly of microtubules and its derivatives was investigated by using the comparative molecular field analysis (CoMFA). In an effort to get a better understanding of the correlation between conformation and antitubulin activity of 1, most probable minimum energy conformation in solution of 1 was analyzed on the basis of NMR data of 1 in solution. The results indicated a correlation between the antitubulin activity of these alkaloids and the steric and electrostatic factors, which modulate their biological activity, and accounted for the potent activities of 1 with suitable relationship for the overall conformation.

Alkaloids↗
PubMed2003

Alteration of gene expressions by the overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx).

To determine the effect on gene expression of trace levels of reactive oxygen species from mitochondria, we used the mRNA differential display technique to compare gene expression in two cell lines: M15, which overexpresses mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx), in rat basophilic leukemia RBL-2H3 cells, and a control cell line, S1. We isolated 27 differentially expressed genes, including 10 previously unreported sequences. These genes included cytoskeletal proteins (beta-tubulin, nonmuscle myosin alkali light chain, and vimentin), growth or proliferation regulators [growth differentiation factor 1 (Gdf-1), Rap1a, and inhibitor of growth 3 (Ing3)], and others. Although the expression of most of the isolated genes did not respond to ROS (hydrogen peroxide) or antioxidant (pyrolidine dithiocarbamate) treatment, the expression of Gdf-1 was downregulated by hydrogen peroxide treatment. Thus, low levels of ROS produced in mitochondria during normal cellular metabolism can modulate gene expression.

Animals↗
PubMed1990

Alteration of differentiation state of human hepatocytes cultured with novobiocin and butyrate.

Chang liver cells cultured in the simultaneous presence of novobiocin and butyrate stopped proliferating and changed into fibroblast-like cells with remarkably elongated cytoplasm. In these fibroblast-like cells, the cellular content of both protein and DNA was increased 2- to 3-fold. In addition, the production of specific proteins such as type III procollagen, actin, and tubulin was increased and the expression of proliferation-associated nuclear antigen which was reactive with Ki-67 monoclonal antibody was reduced remarkably. Therefore, Chang liver cells cultured with novobiocin and butyrate were considered to be arrested at the premitotic G2 phase of the cell cycle and then enter into the noncycling resting state. These actions of novobiocin and butyrate were not mediated by the pathway of epidermal growth factor action or modulated by the diacylglycerol agonist 1-oleoyl-2-acetylglycerol and the C kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. On the other hand, novobiocin was disclosed to be a stimulator of [3H]acetate uptake and acted synergistically with butyrate in enhancing nuclear protein acetylation. From these results it can be speculated that novobiocin and butyrate chemically modulate nuclear proteins and thereby alter the gene expression and the differentiation state of Chang liver cells.

Acetylation↗
PubMed2003

Insulin promotes formation of polymerized microtubules by a phosphatidylinositol 3-kinase-independent, actin-dependent pathway in 3T3-L1 adipocytes.

Direct demonstrations implicating the microtubule cytoskeleton in insulin-mediated adipose/muscle-specific glucose transporter (GLUT4) translocation are beginning to emerge, and one role of the microtubule network appears to be the provision of a solid support for GLUT4 vesicle movement. In the current study we show that insulin treatment increases total polymerized alpha-tubulin in microtubules in a time- and dose-dependent manner that coincides with established insulin-mediated changes in GLUT4 translocation. Insulin stimulates the growth of microtubules through a pathway that requires tyrosine kinase activity, as indicated by inhibition of the effect after treatment with genistein. Insulin-mediated growth was not inhibited by treatment with the MAPK kinase (MEK) inhibitor, PD98059 or by wortmannin, indicating that the effect does not require activation of extracellular signal-regulated kinase 1/2 or phosphatidylinositide 3-kinase. Depolymerization of the actin cytoskeleton with latrunculin B abrogated the effect of insulin on microtubule polymerization, indicating that an intact actin network is a requirement for insulin-dependent modulation of microtubules. Using methods that measure insulin-dependent GLUT4 translocation in populations of adipocytes as opposed to individual cells, we show a statistically significant reduction in translocation (30% inhibition) in the presence of low concentrations of nocodazole (2 mum). This concentration incompletely depolymerizes the microtubule network, revealing that partial depolymerization of microtubules is sufficient to inhibit GLUT4 translocation. It is likely that stabilization of the microtubule network contributes to insulin stimulation of GLUT4 translocation.

3T3 Cells↗
PubMed2006

Distinct roles of doublecortin modulating the microtubule cytoskeleton.

Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules. However, it is a potent anti-catastrophe factor that stabilises microtubules by linking adjacent protofilaments and counteracting their outward bending in depolymerising microtubules. We show that doublecortin-stabilised microtubules are substrates for kinesin translocase motors and for depolymerase kinesins. In addition, doublecortin does not itself oligomerise and does not bind to tubulin heterodimers but does nucleate microtubules. In cells, doublecortin is enriched at the distal ends of neuronal processes and our data raise the possibility that the function of doublecortin in neurons is to drive assembly and stabilisation of non-centrosomal microtubules in these doublecortin-enriched distal zones. These distinct properties combine to give doublecortin a unique function in microtubule regulation, a role that cannot be compensated for by other microtubule-stabilising proteins and nucleating factors.

Animals↗