Fraction Of Tubulin Research Articles

The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.

The role of MAPRE2 and microtubules in Brugada syndrome pathogenesis

Abstract Background Brugada syndrome (BrS) is associated with loss-of-function variants in SCN5A (encoding NaV1.5), which are found in only ∼20% of patients. Recent genome-wide association studies identified a novel locus within an intron of MAPRE2 (encoding microtubule end-binding protein 2, EB2), implicating microtubule (MT) involvement in BrS. Purpose To understand the role of MAPRE2 and MT in BrS pathogenesis. Methods A mapre2 knock-out (KO) zebrafish model was generated using CRISPR/Cas9 and validated by western blot. Larval hearts at 5 day-post-fertilization (dpf) were isolated for voltage mapping and immunohistochemistry with confocal imaging. Morpholino targeting mapre2 or ttl (encoding tubulin tyrosine ligase) were injected into zebrafish embryos at the one-cell stage. A transgenic zebrafish line with cdh2 (encoding N-cadherin) tandem fluorescent timer (tFT) was used to study adherens junction. MT plus-end tracking experiments were performed in iPSC-derived cardiomyocytes (CM) with MAPRE2 knockdown (KD) using siRNA. ECG was recorded in adult fish. Results Voltage mapping of larval hearts (14 WTs vs. 19 KOs) showed a significant decrease in ventricular conduction velocity (17.4±1.5 vs. 13.4±0.8 mm/s; p<0.05) and action potential upstroke velocity (Vmax; 86.1±3.6 vs. 73.9±3.2 1/s; p<0.05), suggesting loss of cardiac NaV function. Correspondingly, ECG of adult fish (11 WTs vs. 10 KOs) showed a significant increase in QRS duration (27.8±0.5 vs. 32.3±0.7 ms; p<0.0001). Confocal imaging of KO hearts showed disorganized adherens junction based on N-cadherin staining. Similarly, morpholino KD of mapre2 in the cdh2 tFT line showed ∼40% mislocalization of mature N-cadherin relative to ZO-1 staining in larval ventricles (8 KDs vs 9 control hearts; p<0.05), suggesting disruption of adherens junction. In terms of MT, immunohistochemistry showed a 23% decrease of detyrosinated tubulin relative to total alpha tubulin in KO hearts (16 vs. 8 WT hearts; p<0.01). MAPRE2 KD in iPSC-CM (337 control vs. 285 KD MT in 4 sets of cells) showed an increase in MT velocity (7.9±0.2 vs. 8.8±0.2 µm/min; p<0.001), distance (5.8±0.2 vs. 7.1±0.2 µm; p<0.0001), and duration (46.2±1.4 vs. 52.2±1.6 s; p<0.001), suggesting changes to MT dynamics. Finally, morpholino KD of ttl in mapre2 KO embryos restored the fraction of detyrosinated tubulin (46% increase; 16 controls vs. 16 KDs; p<0.01) as well as Vmax (74.3±3.0 vs. 88.3±2.1 1/s; 13 controls vs. 9 KDs; p<0.01) to WT levels. Conclusion Genetic ablation of mapre2 led to a decrease in cardiac NaV function, a hallmark of BrS. This is associated with disruption of adherens junction, a decrease of detyrosinated tubulin as a marker of MT stability, and changes in MT dynamics. Restoration of detyrosinated tubulin fraction with ttl KD led to a rescue of NaV function in zebrafish larval hearts. Taken together, MAPRE2 loss-of-function may contribute to BrS pathogenesis by disruption of adherens junction via changes in MT dynamics.

Mechanism of Tubulin Oligomers and Single-Ring Disassembly Catastrophe.

Cold tubulin dimers coexist with tubulin oligomers and single rings. These structures are involved in microtubule assembly; however, their dynamics are poorly understood. Using state-of-the-art solution synchrotron time-resolved small-angle X-ray scattering, we discovered a disassembly catastrophe (half-life of ∼0.1 s) of tubulin rings and oligomers upon dilution or addition of guanosine triphosphate. A slower disassembly (half-life of ∼38 s) was observed following an increase in temperature. Our analysis showed that the assembly and disassembly processes were consistent with an isodesmic mechanism, involving a sequence of reversible reactions in which dimers were rapidly added or removed one at a time, terminated by a 2 order-of-magnitude slower ring-closing/opening step. We revealed how assembly conditions varied the mass fraction of tubulin in each of the coexisting structures, the rate constants, and the standard Helmholtz free energies for closing a ring and for longitudinal dimer–dimer associations.

Efficacy of Tubulin Polymerization Inhibitor in Myelodysplastic Syndrome

Background: Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Tubulin belongs to protein superfamily of globular proteins. Monomeric tubulin can polymerize into microtubules, which play an important role in the attachment and segregation of chromosomes in various phases of cell division. Therefore, the targeting of microtubules represents a therapeutic strategy against both solid and hematological cancers. The first approved tubulin binding agent by the FDA was vincristine, which has been clinically used to treat multiple types of cancers, particularly hematological malignancies. Over the past few decades, additional tubulin binding agents have been developed and received FDA approval, mostly for applications to cancer therapy. These agents have been classified by their binding sites on tubulin, which influences their roles in the inhibition or stabilization of polymerized microtubules. We herein investigated the efficacy of PTC-028, a novel microtubule polymerization inhibitor for MDS. Method: Anti-MDS efficacy of PTC-028 was studied using human MDS cell lines and primary MDS cells in vitro. The efficacy of PTC-028 was also assessed in a xenograft mouse model of MDS using an MDS cell lines. PTC-028 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometry and RNA sequencing. Results: A previous study reported that PTC596 suppressed cell proliferation and induced apoptosis in AML cell lines. Since MDS is regarded as a pre-leukemic stage, we examined the effects of PTC-028, another novel microtubule polymerization inhibitor, on MDS cells. PTC-028 induced a dose-dependent inhibition of cell proliferation on MDS cell lines, such as MDS-L and SKM-1 cells. Caspase 3/7 activity was also significantly induced in MDS-L and SKM-1 cells in the presence of PTC-028, suggesting the induction of apoptotic cell death by PTC-028. We then isolated CD34+ cells from primary MDS BM samples and investigated the efficacy of PTC-028 on primary MDS cells. The efficacy of PTC-028 in CD34+ MDS cells was also confirmed by cell proliferation assays. To enhance the therapeutic benefit of PTC-028 on MDS cells, we investigated synergism between PTC-028 and DNA hypomethylating agents. We treated MDS-L and SKM-1 cells with PTC-028 in combination with DNA hypomethylating agents. After 3 days of culture, cell growth was analyzed by MTS and Annexin V assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit the growth and induce apoptosis of MDS cells. We assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using an MDS cell line. Recipient mice were treated with PTC-028 for 7 weeks. PTC-028 significantly inhibited the growth of MDS-L cells and prolonged the overall survival of recipient mice. Furthermore, a significant synergistic effect was observed between PTC-028 and decitabine. Mice that received combination therapy showed moderate weight loss 11 days after the initiation of the treatment, but subsequently recovered. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We then investigated the effect of PTC-028 on the levels of soluble (unpolymerized) versus polymerized tubulin in MDS-L cells. Cells were treated with PTC-028 and paclitaxel for 4 hours, and cell lysates were then separated into soluble and polymerized fractions by centrifugation. The visualization of tubulin fractions by Western blotting demonstrated that the PTC-028 treatment for 4 hours resulted in the near-complete loss of polymerized microtubules. In contrast, polymerized microtubules increased in cells treated with paclitaxel, which stabilizes microtubules against depolymerization. These result indicates that PTC-028 also acts as a microtubule polymerization inhibitor. Conclusion: Our data reveal a possible chemotherapeutic strategy for PTC-028 and PTC-596 in MDS by disruption of microtubule dynamics as a single agent and in combination with hypomethylating agents. The present study provides a preclinical framework for the clinical evaluation of this promising therapeutic approach to improve outcomes in MDS patients. Disclosures Lennox: PTC therapeutics: Current Employment. Sheedy:PTC therapeutics: Current Employment. Weetall:PTC Therapeutic: Current Employment.

Mechanics and kinetics of dynamic instability.

During dynamic instability, self-assembling microtubules (MTs) stochastically alternate between phases of growth and shrinkage. This process is driven by the presence of two distinct states of MT subunits, GTP- and GDP-bound tubulin dimers, that have different structural properties. Here, we use a combination of analysis and computer simulations to study the mechanical and kinetic regulation of dynamic instability in three-dimensional (3D) self-assembling MTs. Our model quantifies how the 3D structure and kinetics of the distinct states of tubulin dimers determine the mechanical stability of MTs. We further show that dynamic instability is influenced by the presence of quenched disorder in the state of the tubulin subunit as reflected in the fraction of non-hydrolysed tubulin. Our results connect the 3D geometry, kinetics and statistical mechanics of these tubular assemblies within a single framework, and may be applicable to other self-assembled systems where these same processes are at play.

Interaction of tubulin and protein kinase CK2 in Trypanosoma equiperdum.

A polypeptide band with an apparent molecular weight of 55,000 was phosphorylated in vitro in whole-cell lysates of Trypanosoma equiperdum. This band corresponds to tubulin as demonstrated by immunoprecipitation of the phosphorylated polypeptide from T. equiperdum extracts when anti-α and anti-β tubulin monoclonal antibodies were employed. A parasite protein kinase CK2 was in charge of modifying tubulin given that common mammalian CK2 inhibitors such as emodin and GTP, hindered the phosphorylation of tubulin and exogenously added casein. Interestingly, a divalent cation-dependent translocation of the T. equiperdum tubulin and the CK2 responsible for its phosphorylation was noticed, suggesting a direct interaction between these two proteins. Additionally, this fraction of tubulin and its kinase coeluted using separations based on parameters as different as charge (DEAE-Sepharose anion-exchange chromatography) and size (Sephacryl S-300 gel filtration chromatography). Analyses by non-denaturing polyacrylamide gel electrophoresis and immunoblot of the purified and radioactively labeled fraction containing both tubulin and the CK2 enzyme, established the phosphorylation of a single band that was recognized by anti-CK2 α-subunit and anti-tubulin antibodies. All these findings revealed a physical association between a pool of tubulin and a CK2 in T. equiperdum.

Novel taxane derivatives from Taxus wallichiana with high anticancer potency on tumor cells.

Four novel taxane derivatives, N-debenzoyl-N-methyl-N-heptanoyl-taxol (1), N-debenzoyl-N-me-thyl-N-octanoyl-taxol (2), N-debenzoyl-N-methyl-N-(4-methylhexanoyl)-taxol (3), and N-debenzoyl-N-methyl-N-[(4Z)-1-oxo-4-tenenoyl]-taxol (4), were isolated from the ethanol extract of the whole plant of Taxus wallichiana.var.mairer. These structures were identified on the basis of extensive spectroscopic analysis, and their antitumor activity was evaluated against MCF-7, A549, and 3-AO cancer cell lines by the MTT method. Compound 3, the most promising one, exhibited encouraging effect with IC50 of 77nm in MCF-7 cells, which was almost matching that of positive control Taxol. In further mechanism study, the tubulin polymerization assay demonstrated that four compounds caused shifts from the soluble tubulin (depolymerized tubulin) to the particulate tubulin fraction (polymerized) which was similar to Taxol. These results revealed novel natural products with paclitaxel-likemicrotu-bule-stabilizing activity owned their major importance in the clinical treatment of cancer.

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes

Ca2+ oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca2+ homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Matertm/tm (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Matertm/tm oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca2+ oscillations was altered in Matertm/tm oocytes after fertilization in vitro. Intriguingly, Ca2+ oscillations in Matertm/tm oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca2+ oscillation defect in Matertm/tm oocytes was likely caused by a reduced amount of Ca2+ in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca2+ homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.

Inhibition of HDAC6 Deacetylase Activity Increases Its Binding with Microtubules and Suppresses Microtubule Dynamic Instability in MCF-7 Cells

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ε-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.

Microtubule Stability in the Axon: New Answers to an Old Mystery

It has been known for decades that a fraction of neuronal tubulin is insoluble in cold and also resistant to calcium as well as drugs that depolymerize microtubules. In this issue of Neuron, Song etal. (2013) suggest that this unusual stability results from the polyamination of tubulin by transglutaminase.

Centaurin-α₂ interacts with β-tubulin and stabilizes microtubules.

Centaurin-α2 is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α2 can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α2 interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α2 overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α2 mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α2 remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α2 and tubulin confirmed that Centaurin-α2 promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α2 overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α2 role on MT stabilization. Centaurin-α2 interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α2 as a new microtubule-associated protein (MAP) increasing MT stability.

An in vitro study of the role of β-boswellic acid in the microtubule assembly dynamics

Structural integrity of microtubule protein (MTP) is pivotal for its physiological roles. Disruption of the MTP network is known to be involved in neurodegenerative disorders. The gum resin of plants of the boswellia species, with β-boswellic acid (BBA) as the major component, has long been used in Ayurveda and Oriental Medicine to prevent amnesia. In the current study, we addressed the question whether BBA affects assembly dynamics behavior of tubulin. Our in vitro results revealed that BBA increases MTP length distribution and the polymerization rate of tubulin, moderately stabilizing it and diminishing both the critical concentration (Cc) and the fraction of inactive tubulin (Fi).

Microtubule destabilization is shared by genetic and idiopathic Parkinson's disease patient fibroblasts.

Data from both toxin-based and gene-based models suggest that dysfunction of the microtubule system contributes to the pathogenesis of Parkinson’s disease, even if, at present, no evidence of alterations of microtubules in vivo or in patients is available. Here we analyze cytoskeleton organization in primary fibroblasts deriving from patients with idiopathic or genetic Parkinson’s disease, focusing on mutations in parkin and leucine-rich repeat kinase 2. Our analyses reveal that genetic and likely idiopathic pathology affects cytoskeletal organization and stability, without any activation of autophagy or apoptosis. All parkinsonian fibroblasts have a reduced microtubule mass, represented by a higher fraction of unpolymerized tubulin in respect to control cells, and display significant changes in microtubule stability-related signaling pathways. Furthermore, we show that the reduction of microtubule mass is so closely related to the alteration of cell morphology and behavior that both pharmacological treatment with microtubule-targeted drugs, and genetic approaches, by transfecting the wild type parkin or leucine-rich repeat kinase 2, restore the proper microtubule stability and are able to rescue cell architecture. Taken together, our results suggest that microtubule destabilization is a point of convergence of genetic and idiopathic forms of parkinsonism and highlight, for the first time, that microtubule dysfunction occurs in patients and not only in experimental models of Parkinson’s disease. Therefore, these data contribute to the knowledge on molecular and cellular events underlying Parkinson’s disease and, revealing that correction of microtubule defects restores control phenotype, may offer a new therapeutic target for the management of the disease.

Localized accumulation of tubulin during semi-open mitosis in theCaenorhabditis elegansembryo

The assembly of microtubules inside the cell is controlled both spatially and temporally. During mitosis, microtubule assembly must be activated locally at the nascent spindle region for mitotic spindle assembly to occur efficiently. In this paper, we report that mitotic spindle components, such as free tubulin subunits, accumulated in the nascent spindle region, independent of spindle formation in the Caenorhabditis elegans embryo. This accumulation coincided with nuclear envelope permeabilization, suggesting that permeabilization might trigger the accumulation. When permeabilization was induced earlier by knockdown of lamin, tubulin also accumulated earlier. The boundaries of the region of accumulation coincided with the remnant nuclear envelope, which remains after nuclear envelope breakdown in cells that undergo semi-open mitosis, such as those of C. elegans. Ran, a small GTPase protein, was required for tubulin accumulation. Fluorescence recovery after photobleaching analysis revealed that the accumulation was accompanied by an increase in the immobile fraction of free tubulin inside the remnant nuclear envelope. We propose that this newly identified mechanism of accumulation of free tubulin-and probably of other molecules-at the nascent spindle region contributes to efficient assembly of the mitotic spindle in the C. elegans embryo.

Abstract 2768: Development of a tubulin fractionation assay for the evaluation of “on target” activity of tubulin targeting agents in clinical PBMC samples

Abstract The novel agent, BNC105, is a tubulin polymerization inhibitor which acts as a vascular disruption agent (VDA). BNC105 exhibits 100-fold selectivity for activated endothelial cells compared to quiescent endothelial cells, providing a large differential between effects on cancer vasculature and normal vasculature. We developed a tubulin fractionation assay that enables evaluation of tubulin polymerisation changes in peripheral blood mononuclear cell samples (PBMCs) obtained from patients treated with BNC105. The assay capitalizes on the mass differences between polymerized and depolymerised tubulin to separate each fraction in a density gradient by ultracentrifugation. Experimental conditions during the extraction process were optimised to maintain the polymerisation state of tubulin. This optimisation was carried out in cell lines and PBMCs from healthy volunteers. Critical parameters include temperature, GTP concentration, DMSO concentration, pH, and buffer density. Tubulin monomer and polymer extracts were resolved using SDS-PAGE and transferred onto nitrocellulose membranes followed by detection using βI tubulin specific antibodies. The tubulin bands were analysed by densitometry and normalized to an actin loading control. Following exposure of cell lines or PBMCs to tubulin polymerisation inhibitors for a period of 60 minutes, the depolymerised tubulin fraction increased, while the polymerised tubulin fraction decreased. Conversely, exposure to tubulin polymerizing agents caused a greater proportion of tubulin to appear in the polymerized fraction. We used this method to evaluate the tubulin polymerisation status in PBMC samples obtained from cancer patients treated with BNC105. Samples were obtained prior to dosing and at 1, 2, 3-5, 7, and 24 hours post-dosing. Based on the data obtained, BNC105 treatment caused an 80% reduction in the polymerised tubulin fraction at the 1, 2 and 3-5 hour sampling time points. The amount of tubulin in the polymerised fraction returned to pre-dose levels by the 7 hour post-dose time point. The pharmacokinetic profile of BNC105 shows a steep curve reaching Cmax at around 20 minutes post-administration, followed by plasma clearance with no BNC105 detectable after the 7 hour time point. The pharmacokinetics of BNC105 supports our tubulin depolymerisation data with respect to response time and the subsequent recovery period. These results were reproducible, and have shown a similar pattern in all patients tested to date. 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 2768.

Tubulin binding cofactor C (TBCC) suppresses tumor growth and enhances chemosensitivity in human breast cancer cells

BackgroundMicrotubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC), a crucial protein for the proper folding of α and β tubulins into polymerization-competent tubulin heterodimers.MethodsWe developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells.ResultsTBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to antimicrotubule agents both in vitro and in xenografts.ConclusionThese results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterisation of resistant phenotypes.

Ubiquitin editing enzyme UCH L1 and microtubule dynamics: Implication in mitosis

Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, Western blot analysis of tubulin fractions after polymerization revealed the presence of a specific ~50 kDa band of UCH L1 (not the normal ~25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. The inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the ~50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins.

Antimitotic effect of the retinoid 4-oxo-fenretinide through inhibition of tubulin polymerization: a novel mechanism of retinoid growth–inhibitory activity

The retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR), a metabolite of fenretinide (4-HPR) present in plasma of 4-HPR-treated patients, is very effective in inducing growth inhibition and apoptosis in several cancer cell lines. 4-Oxo-4-HPR and 4-HPR have different mechanisms of action because 4-oxo-4-HPR, unlike 4-HPR, causes marked cell accumulation in G2-M phase. Here, we investigated the molecular events involving 4-oxo-4-HPR-induced cell cycle perturbation in ovarian (A2780 and IGROV-1) and breast (T47D, estrogen receptor+ and BT-20, estrogen receptor-) cancer cells. 4-Oxo-4-HPR induced a delay of mitosis (with mitotic index increasing 5- to 6-fold in all cell lines) without progression beyond the anaphase, as shown by cyclin B1 expression. 4-Oxo-4-HPR induced multipolar spindle formation and phosphorylation of BUBR1, resulting in activation of the spindle checkpoint. Multipolar spindles were not due to impairment of pole-focusing process, loss of centrosome integrity, or modulation of the expression levels of molecules associated with spindle aberrations (Kif 1C, Kif 2A, Eg5, Tara, tankyrase-1, centractin, and TOGp). We show here that 4-oxo-4-HPR targets microtubules because, in treated cells, it interfered with the reassembly of cold-depolymerized spindle microtubules and decreased the polymerized tubulin fraction. In cell-free assays, 4-oxo-4-HPR inhibited tubulin polymerization (50% inhibition of microtubule assembly at 5.9 micromol/L), suggesting a direct molecular interaction with tubulin. In conclusion, by showing that 4-oxo-4-HPR causes mitotic arrest through antimicrotubule activities, we delineate a new molecular mechanism for a retinoid.

Adenosine regulation of microtubule dynamics in cardiac hypertrophy

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.

Effect of Pin1 or Microtubule Binding on Dephosphorylation of FTDP-17 Mutant Tau

Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wild-type Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidyl-prolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wild-type Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.