Taxol Pretreatment Research Articles

Taxol stabilizes gap junctions and reduces ischemic ventricular arrhythmias in rats in vivo.

The dynamic movements of connexin 43 (Cx43) are regulated by microtubules and their associated proteins. Dysfunction of Cx43 in the ischemic myocardium is correlated with ventricular arrhythmias (VAs). The present study aimed to determine the effects of microtubules on Cx43 expression and distribution in myocardial tissue, as well as to examine the susceptibility of the heart to VAs during acute myocardial ischemia and reperfusion. Rats were subject to left coronary artery occlusion for 20 min followed by 20 min reperfusion and received taxol at different concentrations (0.1, 0.3 and 0.9 µmol·kg‑1 in 0.5 ml saline) intraperitoneally. Monophasic action potentials at the epicardium were recorded and analyzed using an electrocardiogram. Immunoblots and immunofluorescence staining were used to detect tubulin polymerization and Cx43 expression and distribution. Taxol pretreatment significantly ameliorated the depolymerization of microtubules, improved Cx43 expression and redistribution, reduced the occurrence of VAs, ameliorated shortening of 90% repolarization action potential durations (APD)90 and improved APD dispersion during myocardial ischemia‑reperfusion. The present study demonstrated that taxol reduced ischemic VAs and its mechanism may be correlated with the preservation of Cx43 by stabilizing microtubules.

Effects of taxol on in-vitro matured bovine oocyte vitrification

The experiment was designed to assess the effectiveness of Taxol on cryopreserving bovine MII oocytes using straws as the carrier system for vitrification. Bovine matured oocytes were vitrified in droplets of cryoprotectants with or without 1 µM Taxol (droplet 1: TCM-199, 10% FBS, 5% FCS, 10% ethylene glycol (EG), 10% dimethyl sulfoxide (DMSO) for 45s; droplet 2: TCM-199, 10% FBS, 5% FCS, 20% ethylene glycol (EG), 20% dimethyl sulfoxide (DMSO), 0.5 M sucrose for 25s) on straws. After thawing, the oocytes were cultured in IVM medium in 2 hours and oocytes were assessed by ratio of survival oocytes (SO) to vitrified oocytes (VO); survival oocytes (SO) to colected oocytes (CO) and the proportion of normal nuclear oocytes (NNO). The results showed that the ratio of SO to VO and SO to CO in Taxol groups were slightly higher (62.74 ± 2.74% vs. 59.81 ± 1.75% and 70.65 ± 2.44% vs. 67.66 ± 2.12%, P < 0.05); the ratio of NNO in both taxol-pretreatment groups and non-taxol-pretreatment groups (control) had no significant difference (78.14 ± 4.01% vs. 77.89 ± 2.13%, P > 0.05). The results of this experiment indicated that taxol pretreatment affects the survival ratio of vitrified bovine oocytes but there was no significant evidence on preventing abnormal nuclear oocytes

Chemotherapeutic Agents Targeting the Tubulin Cytoskeleton Modify LPS-induced Cytokine Secretion by Dendritic Cells and Increase Antigen Presentation

Recent studies support the idea that certain chemotherapeutic agents do not act only by cytotoxicity, but also have immunomodulatory activity. Because of their role in mitosis chemotherapeutic agents targeting microtubules are widely used, and this study determined the outcome of disturbing tubulin cytoskeleton dynamics on human dendritic cell function. Dendritic cells (DCs) play a major role in the generation of the adaptive immune response owing to their capacity for antigen presentation leading to T lymphocyte activation and differentiation and there is compelling evidence for their contribution to the antitumoral immune response. Two agents that target the tubulin cytoskeleton were used, taxol and colchicine, and a brief pretreatment with either increased antigen presentation by DCs independently of significant phenotypic change, cell death, or cytokine production. Although taxol and colchicine use different mechanisms to disrupt microtubules, NF-kappabeta was activated by either. We therefore determined whether the cytokine secretion profile in response to lipopolysaccharide (LPS) was modified. LPS stimulation of DCs induced IL-10, IL-12p70, TNFalpha and IL-1beta secretion, and taxol pretreatment modified this response by down-regulating IL-1beta secretion whereas colchicine induced a proinflammatory cytokine profile with reduced IL-10 and increased IL-12p70 and TNFalpha secretion. Taken together, these data reveal new immunomodulatory strategies of microtubule disrupting agents in dendritic cells, that of modifying the cytokine response to LPS and that of increasing T lymphocyte activation without induction of inflammatory cytokine secretion by dendritic cells.

Abstract 4076: Taxanes inhibit the microtubule-dependent androgen receptor trafficking and signaling in prostate cancer

Abstract Androgens (testosterone, DHT) control the development, differentiation and function of the prostate. Their principal action is to regulate gene expression through the androgen receptor (AR), a nuclear steroid hormone receptor, which translocates to the nucleus upon binding to its ligand and activates androgen response elements of target genes. This drives the growth and progression of prostate cancer (PC), the most commonly diagnosed cancer in males. Initial treatment involves androgen ablation, yet, these tumors eventually progress to a castrate-resistant state and taxanes represent the most effective class of anti-tumor agents to improve survival of CRPC patients. However, the molecular basis of taxane activity in prostate cancer is not fully elucidated. Preliminary data from out lab has shown that taxane treatment of PC cells inhibits the ligand-induced nuclear localization of AR and subsequent activation of ARE-containing genes including PSA. We further show that AR colocalizes with the microtubule (MT) cytoskeleton by immunofluorescence followed by confocal microscopy. This result is further supported by co-immunoprecipitation and microtubule co-sedimentation assays that revealed the preferential association of the WT-AR to the MT polymer rather than tubulin dimers. Dynein, a minus-end directed MT motor protein involved in moving cargoes towards the nucleus, co-precipitated with AR and tubulin in untreated PC cells and at higher amounts following ligand induction, further implicating microtubule dynamics in AR trafficking and activity. To investigate whether AR binds MTs directly or via accessory proteins we plan to perform in vitro tubulin binding assays using purified MT protein and purified recombinant AR. We further plan on identifying the minimum AR domain that mediates microtubule binding. To that end we have generated deletion mutants of the AR, exogenously expressed them in PC3 cells stably expressing mCherry-tubulin and are in the process of testing their MT binding ability. To assess the role of microtubule dynamics in AR trafficking and nuclear translocation we performed live-cell confocal microscopy using PC3-mCherry-tubulin cells transfected with full length wild-type GFP-AR. This study revealed AR trafficking along the MTs and rapid AR nuclear accumulation within 30 min of R1881 (synthetic AR ligand) treatment. In contrast, taxol pre-treatment at concentrations that stabilized MTs attenuated the ligand-induced AR nuclear accumulation as well as the rate of AR's translocation to the nucleus, resulting in increased cytoplasmic sequestration of the receptor. Taken together we have identified a new mechanism of action for taxanes that involves the microtubule-dependent regulation of AR trafficking and downstream signalling. This mechanism may help us better understand the molecular basis of clinical response to taxane treatment in CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4076.

Mitochondrial behaviors in the vitrified mouse oocyte and its parthenogenetic embryo: effect of Taxol pretreatment and relationship to competence

To investigate the effect of Taxol pretreatment on mitochondrial behaviors in vitrified mouse mature oocytes and their parthenogenetic embryos. Experimental animal study. University research laboratory and state key laboratory. Sexually mature female Kunming white strain mice. Taxol before vitrification group (Tax). Oocytes were pretreated with M(2) containing 1 mmol/L Taxol for 2 minutes at 37C and then vitrified-warmed using the OPS vitrification procedure. Both ED solution and EDFS30 solution contained 1 mmol/L Taxol. Mitochondrial behaviors examined by fluorescence microscopy technology and fluorescence recovery after photobleaching (FRAP) technology. In the control group, mitochondria were homogeneously distributed, in slow movement in oocytes, and perinuclearly distributed in 42.6% (n = 115) of their parthenogenetic two-cell embryos. Mitochondria from the toxicity group showed similar localization and movement to those of the control group, but not in the vitrification group. The perinuclear mitochondrial localization pattern of two-cell embryos was statistically significantly lower in both the toxicity (27.2%) and vitrification groups (19.8%) than in the control group. After parthenogenetic activation, the blastocyst formation rate of oocytes in the treated groups (28.1 to 48.6%) was statistically significantly lower than that of control (61.2%), but the rate of Taxol group (47.9%) was statistically significantly higher than that in the vitrification group (28.1%). Taxol pretreatment before vitrification helps to reduce the mitochondrial disturbance induced by vitrification in oocytes and their parthenogenetic early-stage embryo.

Non-anti-mitotic concentrations of taxol reduce breast cancer cell invasiveness

Taxol is widely used in breast cancer chemotherapy. Its effects are primarily attributed to its anti-mitotic activity. Microtubule perturbators also exert antimetastatic activities which cannot be explained solely by the inhibition of proliferation. Voltage-dependent sodium channels (NaV) are abnormally expressed in the highly metastatic breast cancer cell line MDA-MB-231 and not in MDA-MB-468 cell line. Inhibiting NaV activity with tetrodotoxin is responsible for an approximately 0.4-fold reduction of MDA-MB-231 cell invasiveness. In this study, we focused on the effect of a single, 2-h application of 10nM taxol on the two cell lines MDA-MB-231 and MDA-MB-468. At this concentration, taxol had no effect on proliferation after 7days and on migration in any cell line. However it led to a 40% reduction of transwell invasion of MDA-MB-231 cells. There was no additive effect when taxol and tetrodotoxin were simultaneously applied. NaV activity, as assessed by patch-clamp, indicates that it was changed by taxol pre-treatment. We conclude that taxol can exert anti-tumoral activities, in cells expressing NaV, at low doses that have no effect on cell proliferation. This effect might be due to a modulation of signalling pathways involving sodium channels.

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher ( P < 0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol + vitrification, 69.98%) and Taxol pretreatment significantly ( P < 0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 μm). The number of lipid droplets (5–10 μm in diameter) in vitrified oocytes pretreated with Taxol was higher ( P < 0.05) than that in the oocytes without Taxol pretreatment (81.87 ± 13.63 vs. 64.27 ± 13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group. In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.

124 TAXOL™ COULD PROMOTE EMBRYO DEVELOPMENT OF BOVINE OOCYTES VITRIFIED BY OPS

Stabilizing the cytoskeleton system during vitrification could be beneficial for improving post-thawed survival and subsequent development of vitrified oocytes. Taxol™, paclitaxel, is a microtubule stabilizer that has been found to improve development competence of vitrified mouse and human oocytes. The objective of this work was to study the effect of a Taxol pretreatment before OPS vitrification on the post-thaw cow and calf oocyte development. Oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-199. Oocytes were randomly assigned to one of 3 experimental groups: (1) control oocytes matured in vitro for 24 h, (2) oocytes matured for 22 h and vitrified by the OPS method (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58), and (3) oocytes matured for 22 h and vitrified by OPS method with 1 µM Taxol. OPS and Taxol–OPS oocytes were transferred back into the maturation dishes and matured for 2 additional h before being subjected to fertilization. Fertilization was performed using frozen–thawed Percoll-selected sperm. At 22 h after insemination, presumptive zygotes were pipetted and then cultured in drops of 25 µL SOF medium and 5% fetal calf serum under paraffin oil at 38.5°C in 5% CO2, 5% O2, 90% N2, and maximum humidity. The Taxol–OPS group provided a significantly higher cleavage rate than the OPS group in cows (41.9% and 34.0%, respectively) or in calves (33.7% and 23.5%, respectively). However, cleavage rate in the experimental groups was significantly lower than in the control group (78.3% and 69.7% for cow and calf control groups, respectively). Blastocyst yield was also higher for the Taxol–OPS group (3.2%) than the OPS group (0%) in cow oocytes. There was no blastocyst development when calf oocytes were vitrified with or without Taxol pretreatment. As expected, cow and calf vitrification groups triggered a significantly lower blastocyst yield when compared with their control (26.7% and 14.9% for cow and calf control groups, respectively). In conclusion, this study showed that supplementation of 1 µM Taxol could promote embryo development after thawing. Further research is indicated to clarify the function of Taxol and its optimal concentration in order to improve the rate of embryo development. Table 1. Effect of Taxol pretreatment on development of cow and calf oocytes vitrified by OPS

Improved development by Taxol pretreatment after vitrification of in vitro matured porcine oocytes

This study was designed to examine the effect of Taxol pretreatment on vitrification of porcine oocytes matured in vitro by an open pulled straw (OPS) method. In the first experiment, the effect of Taxol pretreatment and fluorescein diacetate (FDA) staining on parthenogenetic development of oocytes was evaluated. In the second experiment, viability, microtubule organization and embryo development of oocytes were assessed after oocytes were exposed to vitrification/warming solutions or after vitrification with or without Taxol pretreatment. The results showed that Taxol pretreatment and/or FDA staining did not negatively influence the oocyte's developmental competence after parthenogenetic activation. After being exposed to vitrification/warming solutions, the survival rate (83.3%) of the oocytes was significantly (P < 0.05) reduced as compared with that in the control (100%). Vitrification/warming procedures further reduced the survival rates of oocytes regardless of oocytes being treated with (62.1%) or without (53.8%) Taxol. The proportions of oocytes with normal spindle configuration were significantly reduced after the oocytes were exposed to vitrification/warming solutions (38.5%) or after vitrification with (10.3%) or without (4.1%) Taxol pretreatment as compared with that in control (76.8%). The rates of two-cell-stage (5.6-53.2%) embryos at 48 h and blastocysts (0-3.8%) at 144 h after activation were significantly reduced after exposure to vitrification/warming solutions or after vitrification as compared with control (90.9% and 26.6% respectively). However, the proportion of vitrified oocytes developed to two-cell stage was significantly higher when oocytes were pretreated with (24.3%) than without (5.6%) Taxol. These results indicate that pretreatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and is a potential way to improve the development of vitrified porcine oocytes.

Premeiotic Aster as a Device to Anchor the Germinal Vesicle to the Cell Surface of the Presumptive Animal Pole in Starfish Oocytes

The germinal vesicle (GV) of starfish oocytes stays just beneath the oocyte cortex at the presumptive animal pole during the long period of oogenesis. We subjected oocytes to a centrifugal force field to detach the GV from the cortex. The association between the cortex and the GV persisted and withstood a small amount of centrifugal acceleration at 200g. The GV was eventually separated from the cortex at 700g. The amount of acceleration sufficient for the GV separation was lowered when the oocytes were pretreated with Nocodazole and was increased by Taxol pretreatment. Observation of microtubular structures with an anti-α-tubulin antibody revealed the presence of a complex of spots and radiating arrays as was described by J. J. Otto and T. E. Schroeder (1984, Dev. Biol. 101, 274–281) and called the premeiotic aster. Nocodazole shortened the astral arrays, and Taxol enhanced them. These observations indicate that the premeiotic aster works as a device to hold the GV in an eccentric position just beneath the oocyte cortex.

A novel taxol-induced vimentin phosphorylation and stabilization revealed by studies on stable microtubules and vimentin intermediate filaments.

To understand how protein phosphorylation modulates cytoskeletal organization, we used immunofluorescence microscopy to examine the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor, and taxol, a microtubule-stabilizing agent, on stable (acetylated and detyrosinated) microtubules, vimentin intermediate filaments and other cytoskeletal elements in CV-1 cells. Okadaic acid caused major changes in both stable microtubules and vimentin intermediate filaments, but through independent mechanisms. At 300 nM, okadaic acid caused apparent fragmentation and loss of stable microtubules which was not prevented by prior exposure to K252a. In contrast, major reorganization of vimentin intermediate filaments elicited at 750 nM okadaic acid was prevented by prior exposure to K252a. Taxol pretreatment blocked the effects of okadaic acid on stable microtubules and vimentin intermediate filaments. Recent reports have revealed that taxol can activate cellular signal transduction pathways in addition to its known ability to promote microtubule stabilization, so the possibility that taxol-induced resistance of vimentin intermediate filaments to okadaic acid was through a microtubule-independent mechanism involving direct phosphorylation of intermediate filament proteins was explored. Vimentin immunoprecipitation from cytoskeletal extracts from 32P-labeled cells revealed that taxol (4 microM, 1 or 2 hours) caused about a 2-fold increase in vimentin phosphorylation. This phosphorylation was recovered exclusively in cytoskeletal vimentin, in contrast to the increased phosphorylation of soluble and cytoskeletal vimentin caused by exposure to 750 nM okadaic acid. Phosphorylation of soluble and cytoskeletal vimentin from cells exposed to taxol (4 microM, 1 hour) then okadaic acid (750 nM, 1 hour) was comparable to taxol-treatment alone. These findings demonstrate a novel new activity of taxol, induction of vimentin phosphorylation, that may impact on vimentin organization and stability.

Taxol Pretreatment of Tumor Targets Amplifies Natural Killer Cell Mediated Lysis

Taxol is known to polymerize and stabilize microtubules and thereby alter many cellular functions. Our studies examined the effects of taxol pretreatment of tumor targets and cytotoxic effector cells in an effort to determine whether such treatment would result in increased tumor cell lysis without affecting cytotoxic cell function. Our studies demonstrated that taxol con-centrations of 6–30 ng/ml which induced ∼50% growth inhibition and ±50% block in the G2/M phase of the K562 cell targets did not have any significant effect on the functional ability of NK cells to lyse K562 cells. Pretreatment of K562 cells with taxol (6 and 30 ng/ml) resulted in an increase in K562 cell lysis by NK cells (or NK cells stimulated with 100 units/ml of rIL-2) in 7 out of 9 donors. The amplification of NK cell-mediated lysis of tumor targets due to taxol pretreatment may provide a combination therapeutic approach which includes taxol treatment followed by rIL-2 stimulation of the immune killer cell function.

Complete disintegration of the microtubular cytoskeleton precedes its auxin-mediated reconstruction in postmitotic maize root cells.

The inhibitory action of 0.1 microM auxin (IAA) on maize root growth was closely associated with a rapid and complete disintegration of the microtubular (MT) cytoskeleton, as visualized by indirect immunofluorescence of tubulin, throughout the growth region. After 30 min of this treatment, only fluorescent spots were present in root cells, accumulating either around nuclei or along cell walls. Six h later, in addition to some background fluorescence, dense but partially oriented oblique or longitudinal arrays of cortical MTs (CMTs) were found in most growing cells of the root apex. After 24 h of treatment, maize roots had adapted to the auxin, as inferred from the slowly recovering elongation rate and from the reassembly of a dense and well-ordered MT cytoskeleton which showed only slight deviations from that of the control root cells. Taxol pretreatment (100 microM, 24 h) prevented not only the rapid auxin-mediated disintegration of the MT cytoskeleton but also a reorientation of the CMT arrays, from transversal to longitudinal. The only tissue to show MTs in their cells throughout the auxin treatment was the epidermis. Significant resistance of transverse CMT arrays in these cells towards auxin was confirmed using a higher auxin concentration (100 microM, 24 h). The latter auxin dose also revealed inter-tissue-specific responses to auxin: outer cortical cell files reoriented their CMTs from the transversal to longitudinal orientation, whereas inner cortical cell files lost their MTs. This high auxin-mediated response, associated with the swelling of root apices, was abolished with the pretreatment of maize root with taxol.

Regulation of beta-adrenergic receptor mRNA in rat C6 glioma cells is sensitive to the state of microtubule assembly.

Microtubule disrupter, colchicine, and microtubule stabilizer, taxol, were used to determine whether microtubules play a role in beta-adrenergic receptor mRNA homeostasis and agonist-induced down-regulation in C6 glioma cells. Colchicine treatment had significant, differential, time-dependent effects on constitutive beta 1- and beta 2-adrenergic receptor mRNA levels. These effects stemmed from the action of colchicine on microtubules, because beta-lumicolchicine, an inactive isomer, had no effect, and nocodazole, a structurally unrelated microtubule disrupter, had similar effects. Colchicine treatment had little effect on the total number of beta-adrenergic receptor binding sites as measured by (-)-[125I]iodopindolol binding, but did alter the relative proportion of beta 1- and beta 2-adrenergic receptor subtypes. Colchicine also had no effect on basal cyclic AMP levels. In contrast to colchicine, taxol treatment had little long-term effect on either beta 1- or beta 2-adrenergic receptor mRNA levels. Taxol antagonized the effects of colchicine on total binding and mRNA levels. Taxol treatment increased basal cyclic AMP levels fourfold and potentiated (-)-isoproterenol-induced cyclic AMP production. Colchicine pretreatment completely inhibited (-)-isoproterenol-induced down-regulation of beta 1-adrenergic receptor mRNA, but not that of beta 2-adrenergic receptor mRNA. Taxol pretreatment had little effect on isoproterenol-induced beta-adrenergic receptor mRNA down-regulation. Colchicine pretreatment also attenuated isoproterenol-induced receptor down-regulation and inhibited agonist-stimulated cyclic AMP production. These effects of colchicine were antagonized by taxol. Whereas the effects of taxol and colchicine on isoproterenol-induced down-regulation of beta-adrenergic receptor mRNA are consistent with their effects on cyclic AMP production, those of colchicine in the absence of stimulation must involve other mechanisms. The data demonstrate that the state of microtubule assembly can affect cyclic AMP levels, beta 1- and beta 2-adrenergic receptor mRNA, and binding site levels in C6 glioma cells.

Expression of immediate early genes after treatment of human astrocytoma cells with radiation and taxol

Purpose : The promising chemotherapeutic agent, taxol, has been shown to sensitize the G18 line of human astrocytoma cells to ionizing radiation. The present studies were performed to identify specific changes in gene expression associated with this altered sensitivity. Methods and Materials : The radioresistant, grade 3 human astrocytoma cell line, G18, was exposed for varying periods of time to treatment with taxol, tetradecanoyl phorbol acetate (TPA), serum, isoproterenol, dibutyryl cyclic adenosine monophosphate, or ionizing radiation alone or in combination with taxol pretreatment. Ribonucleic acid samples from the cells were monitored for the expression of a group of immediate early genes (IEGs), including c-fos, c-jun, TIS1, TIS7, TIS8, TIS11 and TIS21, by northern blot hybridization analysis. Results : Transient immediate early gene induction was observed after treatment of G18 cells with tetradecanoyl phorbol acetate, serum, isoproterenol, or ionizing radiation, but not after treatment with taxol. Of the seven immediate early genes analyzed, all but TIS7 were found to be inducible by one or more of the treatments. Only TIS8 (also known as egr-I or zif268) was significantly inducible by radiation, and this transient induction was decreased by at least four-fold by pretreatment for 24 hr with a dose of taxol that was previously shown to block 96.5% of the cells in G2/M and enhance radiosensitivity. Conclusion : The products of immediate early genes, which are induced transiently in cells in response to a variety of treatments, including growth factors, neurotransmitters, and irradiation with UV light or X rays, are thought to initiate a cascade of genetic responses to alterations in cellular environment. The present results demonstrate a dramatic attenuation in one immediate early gene response in association with a treatment that enhances radio-sensitivity in a refractory human brain tumor line.

Activation of microtubule-associated protein kinase by microtubule disruption in quiescent rat 3Y1 cells

Treatment of quiescent rat fibroblastic cells (3Y1) with colchicine, a microtubule-disrupting agent, which could induce the initiation of DNA synthesis [Y. Shinohara, E. Nishida, and H. Sakai (1989)Eur. J. Biochem. 183, 275–280], activated a serine/threonine-specific protein kinase activity in cell extracts that preferentially phosphorylated exogenous microtubule-associated protein 2 (MAP2). Vinblastine treatment also activated the kinase activity, and taxol pretreatment inhibited the colchicine-induced activation of this kinase activity. The detailed biochemical characterization indicated that this microtubule disruption-activated MAP2 kinase was very similar or identical to the mitogen-activated MAP kinase in the substrate specificity and chromatographic behaviors on phosphocellulose, DEAE-cellulose, gel filtration, and phenyl-Sepharose. Pretreatment of the cells with protein synthesis inhibitors did not prevent the MAP2 kinase activation by colchicine. Moreover, phosphatase treatment inactivated the colchicine-activated MAP2 kinase activity. These data suggest that microtubule disruption activates MAP kinase through phosphorylation.

Platelets contain a 21 ok microtubule-associated protein related to a similar protein in hela cells

We have demonstrated the presence of a 210K (K = 10(3) Mr) microtubule-associated protein (MAP) in blood platelets and have studied its relationship to tubulin and to the cytoskeleton, using a well-characterized polyclonal antibody for the analysis. When platelet lysates were enriched for tubulin by an assembly cycle at 37 degrees C, the 210K MAP was also enriched, as detected by Western blotting, while the antigen was not detected in pellets from cold-treated samples that lacked stabilized tubulin. Immunofluorescence of resting platelets showed that the 210K antigen colocalized with the microtubule coil in ring-like structures. On the other hand, in preparations of platelet cytoskeletons, the 210K antigen was present in samples from platelets in which the coil was disassembled (cold-treated without taxol pretreatment) as well as from platelets in which the coil was preserved (at 37 degrees C without taxol, or 4 degrees C with taxol pretreatment). In chilled platelets with disassembled microtubule coils, indirect immunofluorescence using antibodies to 210K or tubulin gave a diffuse signal throughout the platelet cytoplasm. However, immunofluorescence of the 210K antigen in both resting and cold-treated platelets displayed discrete or patchy staining as compared to the continuous staining with antitubulin. We conclude that 210K MAP is present in platelets, that it copurifies with tubulin and that it is localized along the microtubule coil. Our results also suggest that the 210K MAP may interact with some other element(s) of the cytoskeleton, and hence that it might serve as a linking protein.

A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells.

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.