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At least 91 records · Page 5Linked to original sources
PubMed2005

A formal total synthesis of eleutherobin using the ring-closing metathesis (RCM) reaction of a densely functionalized diene as the key step: investigation of the unusual kinetically controlled RCM stereochemistry.

Asymmetric oxyallylation reactions and ring-closing metathesis have been used to synthesize compound 3, a key advanced intermediate used in the total synthesis of eleutherobin reported by Danishefsky and co-workers. The aldehyde 6, which is readily prepared from commercially available R-(-)-carvone in six steps in 30 % overall yield on multigram quantities, was converted into the diene 5 utilizing two stereoselective titanium-mediated Hafner-Duthaler oxyallylation reactions. The reactions gave the desired products (8 and 12) in high yields (73 and 83 %, respectively) as single diastereoisomers, with the allylic alcohol already protected as the p-methoxyphenyl (PMP) ether, which previous work has demonstrated actually aids ring-closing metathesis compared to other protective groups and the corresponding free alcohol. Cyclization under forcing conditions, using Grubbs' second-generation catalyst 13, gave the ten-membered carbocycle (E)-14 in 64 % yield. This result is in sharp contrast to similar, but less functionalized, dienes, which have all undergone cyclization to give the Z stereoisomers exclusively. A detailed investigation of this unusual cyclization stereochemistry by computational methods has shown that the E isomer of the ten-membered carbocycle is indeed less thermodynamically stable than the corresponding Z isomer. In fact, the selectivity is believed to be due to the dense functionality around the ruthenacyclobutane intermediate that favors the trans-ruthenacycle, which ultimately leads to the less stable E isomer of the ten-membered carbocycle under kinetic control. During the final synthetic manipulations the double bond of enedione (E)-16 isomerized to the more thermodynamically stable enedione (Z)-4, giving access to the advanced key-intermediate 3, which was spectroscopically and analytically identical to the data reported by Danishefsky and co-workers, and thereby completing the formal synthesis of eleutherobin.

Catalysis↗
PubMed2003

The ATP-dependent helicase RUVBL1/TIP49a associates with tubulin during mitosis.

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Glutamylated and glycylated tubulin isoforms in the aberrant sperm axoneme of the gall-midge fly, Asphondylia ruebsaameni.

The axonemal organization expressed in the sperm flagella of the cecidomyiid dipteran Asphondylia ruebsaameni is unconventional, being characterized by the presence of an exceedingly high number of microtubular doublets and by the absence of both the inner dynein arms and the central pair/radial spoke complex. Consequently, its motility, both in vivo and in vitro, is also peculiar. Using monoclonal antibodies directed against posttranslational modifications, we have analyzed the presence and distribution of glutamylated and glycylated tubulin isoforms in this aberrant axonemal structure, and compared them with those of a reference insect species (Apis mellifera), endowed with a conventional axoneme. Our results have shown that the unorthodox structure and motility of the Asphondylia axoneme are concomitant with: (1). a very low glutamylation extent in the alpha-tubulin subunit, (2). a high level of glutamylation in the beta-subunit, (3). an extremely low total extent of glycylation, with regard to both monoglycylated and polyglycylated sites, either in alpha- or in beta-tubulin, (4). the presence of a strong labeling of glutamylated tubulin isoforms at the proximal end of the axoneme, and (5). a uniform distribution of glutamylated as well as glycylated isoforms along the rest of the axoneme. Thus, our data indicate that tubulin molecular heterogeneity is much lower in the Asphondylia axoneme than in the conventional 9+2 axoneme with regard to both isoform content and isoform distribution along the axoneme.

Adenosine Triphosphate↗
PubMed1992

End-stabilized microtubules observed in vitro: stability, subunit, interchange, and breakage.

We report a reliable method to prepare, in vitro, microtubules that are stabilized at both ends by axonemal structures, and report studies of their properties. Such "end-stabilized" microtubules neither grow nor shorten over times of several hours when tubulin subunits are present in the surrounding solution. When subunits are removed, the microtubules eventually break. Breakage occurs within a sinuous and flexible region, a few microns in length, that begins at a single point on the microtubule and grows. When breakage does occur, the resulting two free ends shorten very rapidly until the flexible part has depolymerized and the region of straight microtubule is reached. The remainder of the microtubule then shortens at rates comparable to those ordinarily observed in dynamic instability. Formation of the flexible region can be reversed if subunits are added to the buffer prior to breakage. End-stabilized microtubules are a useful tool for studying interactions of molecules with the microtubular wall. They may be a good model for interpreting stabilizing events that happen in the cell. A preliminary study of the effects of microtubule poisons on the wall is presented.

Alkaloids↗
PubMed2006

Synthesis and biological evaluation of boronic acid containing cis-stilbenes as apoptotic tubulin polymerization inhibitors.

A series of boronic acid containing cis-stilbenes as potent inhibitors of tubulin polymerization was synthesized by the introduction of boronic acid as an acceptor-type functional group into the aromatic ring B of the combretastatin framework. High cell-growth inhibition was observed with boron compounds 13 c and 13 d, in which a hydroxy group on the aromatic ring B of combretastatin A-4 was replaced with boronic acid; IC50 values toward B-16 and 1-87 cell lines are 0.48-2.1 microM. Compounds 13 c and 13 d exhibited significant inhibitory activity toward tubulin polymerization (IC50=21-22 microM). The carboxylic acid derivative 17, which can be considered as a mimic of boronic acid 13 c, did not show significant inhibition of cell growth or tubulin polymerization. According to the FACScan analysis using Jurkat cells, apoptosis was induced after incubation for 8 h with 13 c at a concentration of >10(-8) M. Growth inhibitory experiments against a panel of 39 human cancer cell lines revealed 13 c to inhibit growth differently than combretastatin A-4; the correlation coefficient (r) between the two compounds was 0.553 in the COMPARE analysis.

Animals↗
PubMed2006

Structure-activity relationship studies of 3-aroylindoles as potent antimitotic agents.

The concise synthesis and structure-activity relationship (SAR) studies of 3-aroylindoles were carried out in an effort to improve the potency and solubility of anticancer drug candidate BPR0L075 (8) by exploring structure modifications through three regimens: substitution of the B ring, at the N1 position, and of the 3-carbonyl linker. The SAR information revealed that the methoxy group of the B ring could be replaced with an electron-donating group such as methyl (in compound 9) or N,N-dimethylamino (in compound 13) while retaining both strong cytotoxic and antitubulin activities. The introduction of amide (compounds 30-33) and carbamate (compounds 34-37) functionalities at the N1 position of 8 gave analogues with potent antiproliferative activities. The cytotoxic potency of 8 was improved by replacing the carbonyl group with sulfide (compound 41) or oxygen (compound 43), indicating that the carbonyl moiety is important but not essential. The N,N-dimethylamino derivative 13 not only displayed potent cytotoxicity and antitubulin activity, but also showed a markedly improved physicochemical profile relative to the parent compound.

Animals↗
PubMed2005

Genotoxicity of inorganic lead salts and disturbance of microtubule function.

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 microM lead chloride and 0.05 microM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 microM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 microM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 microM and reached half-maximal inhibition of motility at about 50 microM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.

Animals↗
PubMed2003

Resveratrol analog (Z)-3,5,4'-trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin polymerization.

Resveratrol (3,5,4'-trihydroxystilbene) a natural polyphenol present in medicinal plants, grapes and wines, has potent chemopreventive properties on intestinal carcinogenesis. A methylated derivative (Z-3,5,4'-trimethoxystilbene: R3) was synthesized. R3 at 0.3 microM exerted a 80% growth inhibition of human colon cancer Caco-2 cells and arrested growth completely at 0.4 microM (R3 was 100-fold more active than resveratrol). The cis conformation of R3 was also 100-fold more potent than the trans isomer. R3 (0.3 microM) caused cell cycle arrest at the G2/M phase transition. The drug inhibited tubulin polymerization in a dose-dependent manner (IC50=4 microM), and it reduced also by 2-fold ornithine decarboxylase and s-adenosylmethionine decarboxylase activities. This caused the depletion of the polyamines, putrescine and spermidine, which are growth factors for cancer cells. R3 inhibited partially colchicine binding to its binding site on tubulin, indicating that R3 either partially overlaps with colchicine binding or that R3 binds to a specific site of tubulin that is not identical with the colchicine binding site modifying colchicine binding by allosteric influences. The resveratrol derivative (Z)-3,5,4'-trimethoxystilbene (R3) is an interesting anti-mitotic drug that exerts cytotoxic effects by depleting the intracellular pool of polyamines and by altering microtubule polymerization. Such a drug may be useful for the treatment of neoplastic diseases.

Adenocarcinoma↗
PubMed2006

Different signaling responses to anti-proliferative agents in human aortic and venous smooth muscle cells.

Proliferation of smooth muscle cells (SMCs) contributes to the stenosis of coronary arteries and vascular grafts. Local delivery of anti-proliferative drugs can prevent vascular stenosis. To understand the cellular responses to anti-proliferative agents, we investigated the signaling events in cultured human aortic SMCs (ASMCs), saphenous venous SMCs (VSMCs), and dermal fibroblasts (DFs) in response to paclitaxel or etoposide. Cellular mitochondrial and proliferative activities were examined with the methylthiazoletetrazolium (MTT) dye reduction and the bromodeoxyuridine (BrdU) incorporation assay, respectively. Cell proliferation was almost completely suppressed by paclitaxel or etoposide, but apoptosis was achieved in only about 50% of cells at the highest drug concentrations, suggesting the presence of compensatory mechanisms to prevent apoptosis. Examination of three important signaling pathways revealed significant differences between ASMCs, VSMCs, and DFs. Treatment with either paclitaxel or etoposide caused a transient phosphorylation/activation of p42 MAPK in ASMCs and DFs, but had no effect on phospho-p42/44 MAPK in VSMCs. High-dose etoposide enhanced p38 MAPK activation in ASMCs, but not in VSMCs. The p38 inhibitor, PD169316, partially inhibited etoposide-induced ASMC apoptosis, but induced apoptosis in VSMCs. The effects of etoposide and paclitaxel on Akt also differed between ASMCs and VSMCs. These observations indicate that ASMCs and VSMCs differ in the response of signaling pathways to anti-proliferative agents. In ASMCs, p42/44 MAPK appears to serve a pro-survival role, whereas p38 MAPK is a pro-apoptotic regulator. In contrast, p38 MAPK is an important pro-survival regulator in VSMCs and p42/44 MAPK appears to play a minor role in responding to anti-proliferative drugs.

Animals↗
PubMed1984

Cystamine augments the stimulation of DNA synthesis by peptide growth factors and microtubule-disrupting agents in cultures of 3T3 mouse fibroblasts.

Cystamine together with colchicine markedly enhanced the uptake of [3H]-thymidine into DNA of quiescent cultures of insulin-stimulated Swiss 3T3 mouse fibroblasts. Flow cytofluorometric analyses showed an increased rate of transition of cells from G0/G1----S + G2 in response to combinations of insulin, colchicine, and cystamine. Cystamine, the most effective of several thiol compounds, gave maximal augmentation at 200 microM and was toxic at 300-500 microM. Amplification of DNA synthesis by cystamine was also obtained with epidermal growth factor, vasopressin, and 0.5% fetal bovine serum. Combinations of cystamine and other microtubule-disrupting agents such as nocodazole, maytansine, and podophyllotoxin enhanced DNA synthesis in insulin-stimulated cells. In experiments involving sequential addition of agents, significant enhancement of DNA synthesis was observed when the addition of colchicine to cystamine-treated cells was delayed or conversely when the addition of cystamine to colchicine-treated cultures was delayed. This reciprocal interaction between cystamine and colchicine suggests that a prereplicative intermediate accumulates in response to the action of these dissimilar compounds. We consider the possibility that cystamine may act by forming mixed disulfides with thiol groups of unknown protein(s) that regulate DNA replication.

Animals↗
PubMed1984

Effects of prolonged antitubulin culture on the ultrastructure of anterior limb bud cells of the chick embryo.

The anterior limb bud mesenchyme cells of stage 24 chick embryos were dissociated by trypsinization followed by gentle pipetting, and placed in a tissue culture medium of F12 containing 10% fetal calf serum and antibiotics. As the cells became nearly confluent, some of them were exposed to colchicine or vinblastine sulfate for durations as long as 48 hr. The control and antitubulin-treated cells were processed for transmission electron microscopy and the ultrastructure of the cells was compared. Annulate lamellae (AL) were observed in small amounts in both control and antitubulin-treated cells. The amount of AL did not markedly differ in the control versus antitubulin-treated cells. Furthermore, few multinucleated cells were observed in antitubulin-treated cultures. These results indicate that prolonged culture of cells in antitubulins need not, in itself, lead to a condition of enhanced AL development as reported in several other studies using various cell types.

Animals↗
PubMed1984

Effects of prolonged antitubulin culture on annulate lamellae in mouse alpha L929 fibroblasts.

The fine structure of alpha L929 fibroblasts cultured in colchicine or vinblastine sulfate for periods as long as 48 hr was compared to control cells not exposed to antitubulins . In response to prolonged antitubulin culture, several changes in cell ultrastructure were noted: Control fibroblasts contain cytoplasmic annulate lamellae (AL), but prolonged exposure to either vinblastine sulfate or colchicine results in enhanced development of AL. Single pore complexes are present in the rough-surfaced endoplasmic reticulum (rER) in both control and antitubulin-treated cells, but stacked porous cytomembranes also occur under both conditions. Polyribosomes often are closely associated or continuous with the pore complexes. Many antitubulin-treated cells become multinucleate. Some nuclei in both control and antitubulin-treated cells contain large and multiple nucleoli. The large and multiple nucleoli are either attached directly to the inner membrane of the nuclear envelope or to infoldings of the nuclear envelope. Antitubulin-treated cells, after 48-hr exposure, appear also to contain enhanced quantities of smooth-surfaced endoplasmic reticulum (sER) and cytoplasmic filaments (and in some cells, lysosomes and rER as well) when compared to untreated cells. In both control and colchicine-treated cells, AL can exhibit continuity with either rER or sER. Further, all three membrane systems may at times be continuous, but the quantity of these membranes appears to be greater in colchicine-treated cells than in control cells. The results are discussed with respect to possible functional significance.

Animals↗
PubMed1995

Expression and distribution of phosphorylated MAP1B in growing axons of cultured hippocampal neurons.

Microtubule associated proteins (MAPs) interact with tubulin to modulate neurite stability and growth during development. The phosphorylated form of one of these MAPs, MAP1B (MAP1B-P) is hypothesized to be of particular importance for the regulation of neurite outgrowth. To investigate the mechanisms by which MAP1B and MAP1B-P contribute to this regulation, we used a new antibody against an isoform of MAP1B-P to determine its pattern of expression during neuronal development in vitro. We examined cultured hippocampal neurons because these provide a well-established system to evaluate the development of axons and dendrites. MAP1B, MAP1B-P and MAP2 colocalized to the cell bodies and minor processes during the first 24 hours of culture, but MAP1B-P also extended well into the growth cones. As neurite outgrowth and differentiation proceeded, MAP1B and MAP1B-P became localized to the cell bodies and axons, and MAP2 to the cell bodies and dendrites. After 3 days, MAP1B-P declined in the cell body and was segregated to the distal axon; MAP1B remained in the cell body, but was also concentrated in the distal axon. Over 5-9 days in culture, MAP1B-P levels decreased and became undetectable; MAP1B levels decreased later (19-23 days). MAP2 levels, however, remained high through the entire culture period in cell bodies and dendrites. These results are consistent with the hypothesis that MAP1B-P plays an important role in the initiation and elongation of axons by regulating the dynamics of microtubules near the growth cone: MAP1B-P expression is greatest during the period of active neurite extension, is particularly prominent in growth cones where axon outgrowth is most active, and decreases along with the decline in active axon extension.

Animals↗
PubMed1997

Differential effects of colchicine and its B-ring modified analog MTPT on the assembly-independent GTPase activity of purified beta-tubulin isoforms from bovine brain.

Tubulin occurs as several isoforms whose physiological significance is not clear. The binding of colchicine to tubulin inhibits its assembly and is associated with a stimulation of the intrinsic GTPase activity of tubulin. In an effort to study the conformational states of different beta-tubulin isoforms in the drug-bound form, the intrinsic GTPase activity was studied in the presence of colchicine and its B-ring-modified analog MTPT. The results show that the GTPase activities of alpha beta II, alpha beta III, and alpha beta IV are 3.1, 2.8, and 2.6 nmole/h/ml. In the presence of colchicine, the values were increased to 4.5, 9.6, and 3 nmole/h/ml for alpha beta II, alpha beta III, and alpha beta IV respectively. In the case of MTPT, which lacks the B-ring, the GTPase activities were 5.8, 11.5, and 7.3 nmole/h/ml, respectively, for alpha beta II, alpha beta III, and alpha beta IV. The results show that the B-ring of colchicine contributes differentially to the conformational changes in the beta-tubulin isoforms.

Animals↗
PubMed1996

Collagen synthesis and cell growth in chick embryo fibroblasts: influence of colchicine, cytochalasin B and concanavalin A.

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases alpha 2 more than a1 chain production. 3H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.

Actins↗
PubMed1994

Induction of apoptosis by the anti-tubulin drug colcemid: relationship of mitotic checkpoint control to the induction of apoptosis in HeLa S3 cells.

We have studied the relationship between apoptosis and drug-induced cell cycle perturbation in HeLa S3 cells when treated with the anti-tubulin drug colcemid. We found that at least two distinct mechanisms contributed to colcemid cytotoxicity and apoptosis. Continuous exposure to concentrations of colcemid sufficient to block cells at the mitotic checkpoint led to the appearance of apoptotic cells approximately one cell cycle after their initial accumulation in mitosis. Continuous exposure to concentrations sufficient to delay mitotic progression but insufficient to cause mitotic arrest, or pulse exposure to concentrations of colcemid sufficient to induce mitotic block, led to the generation of multipolar mitoses and genetically deficient hypodiploid daughter cells which underwent apoptosis while in interphase. The fact that aberrant spindle function delayed but did not block cells at the mitotic checkpoint indicates that the mitotic checkpoint senses the presence or absence of the spindle but not spindle abnormalities. In both mitotic and interphase cells, colcemid-induced apoptosis occurred after a period of cell cycle stasis during which cells failed to complete an initiated cell cycle. These results are discussed with reference to understanding the relationship between apoptosis and the regulation of cell cycle progression.

Apoptosis↗
PubMed1996

Antitumor activity of combretastatin-A4 phosphate, a natural product tubulin inhibitor.

The tubulin-binding natural product combretastatin A-4 (CA-4) was tested for antitumor activity against fresh human tumors in vitro and 2 mouse tumors, both in vitro and in vivo. In colony forming assays using 10% fetal bovine serum, CA-4 was inhibitory in 27/40 human ovary cancers with a mean IC50 of 3.18 micrograms/mL for a 1-hour exposure (n = 35 specimens) and 0.27 microgramf1p4for a continuous exposure to CA-4 for 11-14 days (n = 5 specimens). Murine B-16 melanoma and P-388 leukemia were also highly sensitive to CA-4 in vitro with an identical IC50 value of 0.0007 micrograms/mL for continuous drug exposure for 8 days. Comparable in vitro cell culture studies performed in serum concentrations higher than 10%, revealed a significant loss of cytotoxic potency. Using the same reversed-phase HPLC technique as developed for paclitaxel, CA-4 was shown to bind to serum proteins (> or = 30,000 mw) > 99% and to albumin approximately 70%. CA-4 was only marginally active (25% increased lifespan) in DBA/2 mice bearing P-388 leukemia who were given doses of 100 mg/kg IP on either days, 1, 5 and 9 (p = 0.075 by Wilcoxon analysis) or on consecutive days 1-9 (p = 0.19 compared to control). A higher IP dose of 150 mg/kg on days 1, 5 and 9 did not delay subcutaneous B-16 melanoma tumor growth in C57/B1 mice. These findings demonstrate a substantial loss of antitumor efficacy for CA-4 in physiologic serum concentrations in vitro. No consistent antitumor activity was observed in two murine tumor models in vivo.

Animals↗