62 results on '"Luduena, R."'
Search Results
2. Over‐Expression of the β II Isotype of Tubulin and Especially Its Localization in Cell Nuclei Correlates with Poorer Outcomes in Colorectal Cancer
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Ruksha, K., Mezheyeuski, Artur, Nerovnya, A., Bich, T., Tur, G., Gorgun, J., Luduena, R. F., Portyanko, Anna, Ruksha, K., Mezheyeuski, Artur, Nerovnya, A., Bich, T., Tur, G., Gorgun, J., Luduena, R. F., and Portyanko, Anna
- Abstract
See: Supplementary Material, 2017 ASCB-EMBO Meeting-Poster Abstracts, p. Tuesday‐176, Meeting Abstract: P3000.
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- 2017
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3. Expression of class III beta tubulin in non-small cell lung cancer is correlated with resistance to taxane chemotherapy
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Charles Dumontet, Isaac, S., Souquet, P. J., Bejui-Thivolet, F., Pacheco, Y., Peloux, N., Frankfurter, A., Luduena, R., Perol, M., Institut National de la Santé et de la Recherche Médicale (INSERM), Hospices Civils de Lyon (HCL), Rétrovirus et Pathologie Comparée (RPC), Institut National de la Recherche Agronomique (INRA)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL), University of Virginia, University of Texas Health Science Center, The University of Texas Health Science Center at Houston (UTHealth), and ProdInra, Migration
- Subjects
[SDV] Life Sciences [q-bio] ,NON-SMALL CELL LUNG ,BETA TUBULIN ,[SDV]Life Sciences [q-bio] ,CHEMOTHERAPY ,TAXANE ,RESISTANCE - Abstract
International audience; This study determined the prevalence and the prognostic value of the expression of microtubule components in tumors of 19 patients with non small cell lung cancer receiving taxane-based regimens. Patient samples were stained with antibodies directed against total β tubulin, classes I, II and III β tubulin isotypes, δ2 alpha tubulin, τ protein, and the P-gp protein involved in the classical multidrug resistance phenotype. All tumors were stained with pan-β tubulin antibody and class I tubulin isotype. A majority of the tumor samples expressed class II and class III, although the percentage of positive cells varied significantly between tumors. δ2 alpha tubulin, τ protein and Pgp protein were found in only one tumor sample each. Progression-free survival was shorter (41 days) in patients whose tumors expressed high levels of class III tubulin isotype in comparison to patients with low levels (288 days, p = 0.02). There were 2 responses to chemotherapy among 9 patients (22%) with high levels of class III tubulin vs. 6 among 10 patients (60%) with low levels of expression (Fisher exact test: p = 0.11). These data suggest that high expression of class III tubulin by tumor cells is associated with poor prognosis in patients with NSCLC receiving a taxane-based regimen.
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- 2005
4. Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.
- Author
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Hoffman, P N, primary, Lopata, M A, additional, Watson, D F, additional, and Luduena, R F, additional
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- 1992
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5. Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of beta-tubulin
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Banerjee, A, primary and Luduena, R F, additional
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- 1991
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6. Differential expression of b tubulin isotypes in the adult gerbil cochlea
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Hallworth, R. and Luduena, R. F.
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- 2000
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7. Bis(1,8-anilinonaphthalenesulfonate). A novel and potent inhibitor of microtubule assembly.
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Horowitz, P, Prasad, V, and Luduena, R F
- Abstract
Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.
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- 1984
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8. Investigation of the mechanism of the interaction of tubulin with derivatives of 2-styrylquinazolin-4(3H)-one.
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Lin, C M, Kang, G J, Roach, M C, Jiang, J B, Hesson, D P, Luduena, R F, and Hamel, E
- Abstract
A new class of antimitotic agents, derivatives of 2-styrylquinazolin-4(3H)-one (SQZ), was recently described [J. Med. Chem. 33:1721-1728 (1990)]. Because they appeared to interact at a new ligand binding site on tubulin, we attempted to determine their mechanism of action as inhibitors of tubulin polymerization. Although in initial studies inhibition of colchicine binding was negligible, substantial and competitive inhibition of this reaction could be demonstrated with very short incubation times (less than 5 min), provided that a relatively low colchicine to tubulin ratio was used. The initial apparent failure to inhibit colchicine binding resulted from extremely rapid binding to tubulin and dissociation from tubulin by the SQZ derivatives, in comparison with the slow, temperature-dependent, poorly reversible binding of colchicine. The most inhibitory of the SQZ derivatives in the colchicine binding assay was 6-methyl-2-styrylquinazolin-4(3H)-one (NSC 379310), and its interaction with tubulin, particularly as an inhibitor of colchicine binding, was compared with that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (MTPT), because the binding parameters of MTPT with tubulin have been well described. The data indicate that NSC 379310 binds to tubulin and dissociates from the protein about 3 times as rapidly as MTPT. The other SQZ derivatives with equal or greater potency as inhibitors of tubulin polymerization but apparently less potency as inhibitors of colchicine binding presumably bind to and/or dissociate from tubulin even more rapidly than does NSC 379310.
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- 1991
9. The effects of 6-benzyl-1,3-benzodioxole derivatives on the alkylation of tubulin.
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Roach, M C, Trcka, P P, Jurd, L, and Luduena, R F
- Abstract
Derivatives of 6-benzyl-1,3-benzodioxole are known to bind to tubulin and inhibit tubulin polymerization. For a better understanding of the mechanism of action of the 6-benzyl-1,3-benzodioxole derivatives, we have examined their effect on the alkylation of tubulin sulfhydryls by iodo[14C]acetamide and N,N'-ethylene(bis)iodoacetamide. We have found that the 6-benzyl-1,3-benzodioxole derivatives with an intact dioxole ring affect alkylation to an extent proportional to their ability to inhibit tubulin polymerization. Those derivatives with the strongest resemblance to podophyllotoxin have the weakest effects. However, derivatives with a disrupted dioxole ring show little or no ability to inhibit polymerization, but their effect on alkylation is directly related to the degree of resemblance they bear to the trimethoxy ring of podophyllotoxin. It thus appears that the relatively simple approach of using alkylating agents can generate a significant amount of information on the mechanism by which various drugs interact with tubulin.
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- 1987
10. Griseofulvin: A Novel Interaction with Bovine Brain Tubulin
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Chaudhuri, A. R. and Luduena, R. F.
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- 1996
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11. Water ordering during the cell cycle: Nuclear magnetic resonance studies of the sea-urchin egg
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Zimmerman, S., Zimmerman, A. M., Fullerton, G.D., Luduena, R. F., and Cameron, I. L.
- Abstract
Nuclear magnetic resonance was used to measure spin-lattice water proton relaxation times (T1) during the first cell cycle in sea-urchin zygotes of packed Strongylocentrotus purpuratus. Following insemination there was a 90% increase in the T1 value. The increase in T1 at fertilization could be accounted for by the accumulation of extracellular fluid between the egg surface and the fertilization envelope. The T1 value then remained without change during the first cell cycle, except at metaphase when there was a significant 13% decrease. The lowered T1 values measured at metaphase were not related to a change in the water content of the packed cells, which remained fairly constant throughout the cell cycle. High hydrostatic pressure, low temperature and colchicine (agents that depolymerize mitotic apparatus microtubules) did not affect the T1 values in fertilized eggs. Treatment in vitro of a microtubule protein preparation with low temperature and colchicine resulted in an increased T1, which accompanied the depolymerization of microtubule protein. Since depolymerization of the microtubules associated with the mitotic apparatus by high pressure, colchicine or low temperature does not alter the T1 of water protons in the cell, it is proposed that the increased state of ordered water molecules at metaphase is maintained by non-microtubular factor(s) of the metaphase egg.
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- 1985
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12. Isolation and partial characterization of flagellar tubulin from
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LUDUENA, R, primary, MYLES, D, additional, and PFEFFER, T, additional
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- 1980
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13. ChemInform Abstract: Synthesis and Antitumor Activity of Tropolone Derivatives. Part 6. Structure‐Activity Relationships of Antitumor‐Active Tropolone and 8‐Hydroxyquinoline Derivatives.
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YAMATO, M., primary, HASHIGAKI, K., additional, YASUMOTO, Y., additional, SAKAI, J., additional, LUDUENA, R. F., additional, BANERJEE, A., additional, TSUKAGOSHI, S., additional, TASHIRO, T., additional, and TSURUO, T., additional
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- 1988
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14. The tubulins of animals, plants, fungi and protists--implications for metazoan evolution
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Morejohn, L. C., Little, M., Hoffman, E., Asnes, C., and Luduena, R. F.
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- 1983
15. Editorial: The isotypes of α , β and γ tubulin: From evolutionary origins to roles in metazoan development and ligand binding differences.
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Moore J, Luduena R, and Tuszynski JA
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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- 2023
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16. Cost-Effectiveness and Burden of Disease for Adjuvanted Quadrivalent Influenza Vaccines Compared to High-Dose Quadrivalent Influenza Vaccines in Elderly Patients in Spain.
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Ruiz-Aragón J, Márquez-Peláez S, Gani R, Alvarez P, and Guerrero-Luduena R
- Abstract
Influenza is a contagious respiratory disease that causes severe illness and death, particularly in elderly populations. Two enhanced formulations of quadrivalent influenza vaccine (QIV) are available in Spain. Adjuvanted QIV (aQIV) is available for those aged 65+ and high-dose QIV (HD-QIV) for those aged 60+. In this study, we used a health economic model to assess the costs and outcomes associated with using aQIV or HD-QIV in subjects aged 65+. Using aQIV instead of HD-QIV to vaccinate an estimated 5,126,343 elderly people results in reductions of 5405 symptomatic cases, 760 primary care visits, 171 emergency room visits, 442 hospitalizations, and 26 deaths in Spain each year. Life-years (LYs) and quality-adjusted LYs (QALYs) increases by 260 and 206, respectively, each year. Savings from a direct medical payer perspective are EUR 63.6 million, driven by the lower aQIV vaccine price and a minor advantage in effectiveness. From a societal perspective, savings increase to EUR 64.2 million. Results are supported by scenario and sensitivity analyses. When vaccine prices are assumed equal, aQIV remains dominant compared to HD-QIV. Potential savings are estimated at over EUR 61 million in vaccine costs alone. Therefore, aQIV provides a highly cost-effective alternative to HD-QIV for people aged 65+ in Spain.
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- 2022
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17. Head home: a prospective cohort study of a nurse-led paediatric head injury clinical decision tool at a district general hospital.
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Aldridge P, Castle H, Phillips C, Russell E, Guerrero-Luduena R, and Rout R
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- Adolescent, Child, Child, Preschool, England, Female, Humans, Infant, Infant, Newborn, Male, Predictive Value of Tests, Prospective Studies, Triage, Craniocerebral Trauma diagnostic imaging, Decision Support Techniques, Emergency Service, Hospital, Nursing Assessment, Tomography, X-Ray Computed
- Abstract
Objectives: To assess if a nurse-led application of a paediatric head injury clinical decision tool would be safe compared with current practice., Methods: All paediatric (<17 years) patients with head injuries presenting to Frimley Park Emergency Department (ED), England from 1 May to 31 October 2018 were prospectively screened by a nurse using a mandated electronic 'Head Injury Discharge At Triage' questionnaire (HIDATq). We determined which patients underwent CT of brain and whether there was a clinically important intracranial injury or re-presentation to the ED. The negative predictive value of the screening tool was assessed. We determined what proportion of patients could have been sent home from triage using this tool., Results: Of the 1739 patients screened, 61 had CTs performed due to head injury (six abnormal) with a CT rate of 3.5% and 2% re-presentations. Of the entire cohort, 1052 screened negative. 1 CT occurred in this group showing no abnormalities. Of those screened negative, 349 (33%)/1052 had 'no other injuries' and 543 (52%)/1052 had 'abrasions or lacerations'. HIDATq's negative predictive value for CT was 99.9% (95% CI 99.4% to 99.9%) and 100% (95% CI 99.0% to 100%) for intracranial injury. The positive predictive value of the tool was low. Five patients screened negative and re-presented within 72 hours but did not require CT imaging., Conclusion: A negative HIDATq appears safe in our ED. Potentially 20% (349/1739) of all patients with head injuries presenting to our department could be discharged by nurses at triage with adequate safety netting advice. This increases to 50% (543/1739), if patients with lacerations or abrasions were given advice and discharged at triage. A large multicentre study is required to validate the tool., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2020
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18. Over-Expression of βII-Tubulin and Especially Its Localization in Cell Nuclei Correlates with Poorer Outcomes in Colorectal Cancer.
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Ruksha K, Mezheyeuski A, Nerovnya A, Bich T, Tur G, Gorgun J, Luduena R, and Portyanko A
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- Aged, Disease Progression, Female, Humans, Male, Microtubules metabolism, Middle Aged, Prognosis, Biomarkers, Tumor metabolism, Cell Nucleus metabolism, Colorectal Neoplasms pathology, Tubulin metabolism
- Abstract
Tubulin is a heterodimer of α and β subunits, both existing as isotypes differing in amino acid sequence encoded by different genes. Specific isotypes of tubulin have associations with cancer that are not well understood. Previous studies found that βII-tubulin is expressed in a number of transformed cells and that this isotype is found in cell nuclei in non-microtubule form. The association of βII expression and its nuclear localization with cancer progression has not previously been addressed. We here used a monoclonal antibody to βII to examine patients with colorectal cancer and found that patients whose tumors over-express βII have a greatly decreased life expectancy which is even shorter in those patients with nuclear βII. Our results suggest that βII-tubulin may facilitate cancer growth and metastasis and, to accomplish this, may not need to be in microtubule form. Furthermore, βII expression and localization could be a useful prognostic marker. We also found that βII appears in the nuclei of otherwise normal cells adjacent to the tumor. It is possible therefore that cancer cells expressing βII influence nearby cells to do the same and to localize βII in their nuclei by an as yet uncharacterized regulatory pathway.
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- 2019
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19. Novel Colchicine Derivatives and their Anti-cancer Activity.
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Johnson L, Goping IS, Rieger A, Mane JY, Huzil T, Banerjee A, Luduena R, Hassani B, Winter P, and Tuszynski JA
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- Animals, Antineoplastic Agents chemical synthesis, Antineoplastic Agents chemistry, Cell Proliferation drug effects, Colchicine chemical synthesis, Colchicine chemistry, Drug Screening Assays, Antitumor, Humans, Neoplasms metabolism, Neoplasms pathology, Tubulin genetics, Tubulin metabolism, Antineoplastic Agents pharmacology, Colchicine pharmacology, Neoplasms drug therapy
- Abstract
In this paper we provide an overview of the status of various colchicine derivatives in preclinical development with special focus on their anti-cancer activity. We discuss several groups of compounds that have been designed to differentially bind with specific affinities for tubulin β isotypes, especially in regard to βIII, which is commonly over-expressed in cancer. Computational prediction, protein-based and cell-based assays are summarized as well as some animal tests conducted on these compounds. It is concluded that an untapped potential exists for exploiting the colchicine scaffold as a pharmacophore with the possibility of increasing its affinity for tubulin isotypes overexpressed in cancer and decreasing it for normal cells thereby widening the therapeutic window., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)
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- 2017
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20. Mechanism of action of the microtubule-targeted antimitotic depsipeptide tasidotin (formerly ILX651) and its major metabolite tasidotin C-carboxylate.
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Ray A, Okouneva T, Manna T, Miller HP, Schmid S, Arthaud L, Luduena R, Jordan MA, and Wilson L
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- Animals, Breast Neoplasms drug therapy, Breast Neoplasms pathology, Carboxylic Acids pharmacokinetics, Carboxylic Acids pharmacology, Cattle, Cell Growth Processes drug effects, Cell Line, Tumor, Humans, Microtubules drug effects, Microtubules metabolism, Mitosis drug effects, Oligopeptides pharmacokinetics, Prodrugs pharmacokinetics, Prodrugs pharmacology, Oligopeptides pharmacology
- Abstract
Tasidotin (ILX-651), an orally active synthetic microtubule-targeted derivative of the marine depsipeptide dolastatin-15, is currently undergoing clinical evaluation for cancer treatment. Tasidotin inhibited proliferation of MCF7/GFP breast cancer cells with an IC(50) of 63 nmol/L and inhibited mitosis with an IC(50) of 72 nmol/L in the absence of detectable effects on spindle microtubule polymer mass. Tasidotin inhibited the polymerization of purified tubulin into microtubules weakly (IC(50) approximately 30 micromol/L). However, it strongly suppressed the dynamic instability behavior of the microtubules at their plus ends at concentrations approximately 5 to 10 times below those required to inhibit polymerization. Its major actions were to reduce the shortening rate, the switching frequency from growth to shortening (catastrophe frequency), and the fraction of time the microtubules grew. In contrast with all other microtubule-targeted drugs thus far examined that can inhibit polymerization, tasidotin did not inhibit the growth rate. In contrast to stabilizing plus ends, tasidotin enhanced microtubule dynamic instability at minus ends, increasing the shortening length, the fraction of time the microtubules shortened, and the catastrophe frequency and reducing the rescue frequency. Tasidotin C-carboxylate, the major intracellular metabolite of tasidotin, altered dynamic instability of purified microtubules in a qualitatively similar manner to tasidotin but was 10 to 30 times more potent. The results suggest that the principal mechanism by which tasidotin inhibits cell proliferation is by suppressing spindle microtubule dynamics. Tasidotin may be a relatively weak prodrug for the functionally active tasidotin C-carboxylate.
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- 2007
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21. Expression of class III beta tubulin in non-small cell lung cancer is correlated with resistance to taxane chemotherapy.
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Dumontet C, Isaac S, Souquet PJ, Bejui-Thivolet F, Pacheco Y, Peloux N, Frankfurter A, Luduena R, and Perol M
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- Aged, Carcinoma, Non-Small-Cell Lung metabolism, Disease-Free Survival, Female, Humans, Lung Neoplasms metabolism, Male, Middle Aged, Tubulin metabolism, Antineoplastic Agents therapeutic use, Bridged-Ring Compounds therapeutic use, Carcinoma, Non-Small-Cell Lung drug therapy, Drug Resistance, Neoplasm, Lung Neoplasms drug therapy, Microtubule-Associated Proteins metabolism, Neoplasm Proteins metabolism, Taxoids therapeutic use
- Abstract
This study determined the prevalence and the prognostic value of the expression of microtubule components in tumors of 19 patients with non small cell lung cancer receiving taxane-based regimens. Patient samples were stained with antibodies directed against total beta tubulin, classes I, II and III beta tubulin isotypes, delta2 alpha tubulin, tau protein, and the P-gp protein involved in the classical multidrug resistance phenotype. All tumors were stained with pan-beta tubulin antibody and class I tubulin isotype. A majority of the tumor samples expressed class II and class III, although the percentage of positive cells varied significantly between tumors. delta2 alpha tubulin, tau protein and Pgp protein were found in only one tumor sample each. Progression-free survival was shorter (41 days) in patients whose tumors expressed high levels of class III tubulin isotype in comparison to patients with low levels (288 days, p = 0.02). There were 2 responses to chemotherapy among 9 patients (22%) with high levels of class III tubulin vs. 6 among 10 patients (60%) with low levels of expression (Fisher exact test: p = 0.11). These data suggest that high expression of class III tubulin by tumor cells is associated with poor prognosis in patients with NSCLC receiving a taxane-based regimen.
- Published
- 2005
22. Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified beta-tubulin isotypes.
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Derry WB, Wilson L, Khan IA, Luduena RF, and Jordan MA
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- Animals, Antineoplastic Agents, Phytogenic administration & dosage, Brain Chemistry, Cattle, Kinetics, Microscopy, Video, Microtubules metabolism, Paclitaxel administration & dosage, Polymers metabolism, Structure-Activity Relationship, Time Factors, Tubulin chemistry, Tubulin isolation & purification, Antineoplastic Agents, Phytogenic pharmacology, Microtubules drug effects, Paclitaxel pharmacology, Tubulin metabolism
- Abstract
Substoichiometric binding of taxol to tubulin in microtubules potently suppresses microtubule dynamics, which appears to be the most sensitive antiproliferative mechanism of taxol. To determine whether the beta-tubulin isotype composition of a microtubule can modulate sensitivity to taxol, we measured the effects of substoichiometric ratios of taxol bound to tubulin in microtubules on the dynamics of microtubules composed of purified alphabeta(II)-, alphabeta(III)-, or alphabeta(IV)-tubulin isotypes and compared the results with the effects of taxol on microtubules assembled from unfractionated tubulin. Substoichiometric ratios of bound taxol in microtubules assembled from purified beta-tubulin isotypes or unfractionated tubulin potently suppressed the shortening rates and the lengths shortened per shortening event. Correlation of the suppression of the shortening rate with the stoichiometry of bound taxol revealed that microtubules composed of purified alphabeta(II)-, alphabeta(III)-, and alphabeta(IV)-tubulin were, respectively, 1.6-, 7.4-, and 7.2-fold less sensitive to the effects of bound taxol than microtubules assembled from unfractionated tubulin. These results indicate that taxol differentially modulates microtubule dynamics depending upon the beta-tubulin isotype composition. The results are consistent with recent studies correlating taxol resistance in tumor cells with increased levels of beta(III0- and beta(IV)-tubulin expression and suggest that altered cellular expression of beta-tubulin isotypes can be an important mechanism by which tumor cells develop resistance to taxol.
- Published
- 1997
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23. Changes in the isotype composition of beta-tubulin delivered to regenerating sensory axons by slow axonal transport.
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Hoffman PN and Luduena RF
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- Animals, Biological Transport physiology, Ganglia, Spinal metabolism, Male, Rats, Rats, Sprague-Dawley, Axons metabolism, Ganglia, Spinal cytology, Tubulin metabolism
- Abstract
beta-Tubulin is encoded by a family of genes that produces at least five distinct polypeptide isotypes in neurons. Two of these isotypes (i.e., classes II and III) preferentially accumulate in axons, and the expression of one of them (i.e., class II) correlates closely with axonal outgrowth during development and regeneration. In dorsal root ganglion (DRG) neurons, expression of the class II isotype declines to relatively low levels during early postnatal development, and increases dramatically in mature neurons during axon regeneration (i.e., to a level comparable to that in developing neurons). In contrast, expression of the class III isotype, which rises slightly during postnatal development, increases much less than the class II isotype during regeneration. We now document that these changes in gene expression are associated with an increase in the relative amount of class II as compared to class III beta-tubulin delivered to regenerating sensory axons of rat sciatic nerve by slow axonal transport. In this study, the tubulin transported in sensory axons was labeled by injecting [35S]methionine into the L5 DRG either 7 or 14 days after crushing the sciatic nerve; pulse-labeled class II and class III beta-tubulin were identified using immunoprecipitation. This change in the isotype composition of beta-tubulin transported in regenerating axons may influence outgrowth by altering the assembly and dynamic properties of axonal microtubules.
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- 1996
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24. The axonal transport of beta III-tubulin is altered in both branches of sensory axons after injury of the rat sciatic nerve.
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Hoffman PN and Luduena RF
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- Animals, Axonal Transport, Cytoskeletal Proteins metabolism, Male, Models, Neurological, Nerve Crush, Rats, Rats, Sprague-Dawley, Sciatic Nerve injuries, Time Factors, Axons physiology, Ganglia, Spinal physiology, Neurons, Afferent physiology, Sciatic Nerve physiology, Tubulin metabolism
- Abstract
We have analyzed the axonal transport of beta III-tubulin in the central (dorsal root) and peripheral (sciatic nerve) branches of sensory axons after injury of the sciatic nerve. Our finding that the relative amount of beta III-tubulin transported in slow component b (SCb) is increased in both axonal branches does not support the generally accepted hypothesis that the transport of cytoskeletal proteins is altered in the peripheral, but not the central branch after injury of the sciatic nerve.
- Published
- 1996
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25. Interaction of bovine brain tubulin with the 4(1H)-pyrizinone derivative IKP104, an antimitotic drug with a complex set of effects on the conformational stability of the tubulin molecule.
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Luduena RF, Roach MC, Prasad V, Chaudhuri AR, Tomita I, Mizuhashi F, and Murata K
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- Alkylation, Animals, Antineoplastic Agents pharmacology, Carbon Radioisotopes, Cattle, Colchicine metabolism, Iodoacetamide chemistry, Mitosis drug effects, Protein Binding, Protein Conformation drug effects, Pyridones pharmacology, Sulfhydryl Compounds chemistry, Tritium, Tubulin chemistry, Antineoplastic Agents metabolism, Brain metabolism, Pyridones metabolism, Tubulin metabolism
- Abstract
The ligands of tubulin have proved to be excellent probes for the conformation of the tubulin molecule. The most varied in their effects on tubulin are those ligands which are competitive or noncompetitive inhibitors of vinblastine binding. The 4(H)-pyrizinone derivative 2-(4-fluorophenyl)-1-(2-chloro- 3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone [sequence: see text] (IKP104) is a novel antimitotic drug which inhibits microtubule assembly in vitro and in vivo and polymerizes tubulin into spiral filaments. Using a fluorescence assay, we found that IKP104 appears to bind to tubulin at two classes of site, differing in affinity. IKP104 also blocks formation of an intrachain cross-link in beta-tubulin, induced by N,N"-ethylenebis(iodoacetamide), linking Cys12 to either Cys201 or Cys211. IKP104 appears to belong to that group of tubulin ligands which includes vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, and halichondrin B. An unusual effect of IKP104 is that it greatly enhances the decay or apparent unfolding or opening of the tubulin molecule. The sulfhydryl titer of tubulin is doubled and the exposure of hydrophobic areas on the tubulin molecule is tripled by IKP104. These effects of IKP104 are counteracted by vinblastine, maytansine, and phomopsin A, suggesting that IKP104 may be competing with these other drugs for binding to tubulin. However, the effects are also counteracted by colchicine and podophyllotoxin, implying a more complex effect, namely, that IKP104 and colchicine, even when both are bound to tubulin, are competing for their effects on the same domain of tubulin. Surprisingly, when IKP104 is used in conjunction with colchicine, binding of colchicine to tubulin is strongly stabilized.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1995
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26. In vitro analysis of microtubule assembly of isotypically pure tubulin dimers. Intrinsic differences in the assembly properties of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers in the absence of microtubule-associated proteins.
- Author
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Lu Q and Luduena RF
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- Animals, Brain metabolism, Cattle, Glycerol, Kinetics, Macromolecular Substances, Magnesium, Microscopy, Electron, Microtubules metabolism, Peptide Fragments isolation & purification, Subtilisins, Tubulin isolation & purification, Tubulin metabolism, Microtubules ultrastructure, Tubulin ultrastructure
- Abstract
Microtubule assembly of different beta tubulin isotypes in the presence of 4 M glycerol and 6 mM magnesium ion demonstrates significantly different characteristics. alpha beta II and alpha beta IV assembled faster and to a greater extent than did unfractionated phosphocellulose-purified tubulin (PC-tubulin). Microtubule assembly from alpha beta III showed a distinctive delay in nucleation, proceeded at a slower rate than those of the other beta tubulin isotypes, and had the highest critical concentration. However, treatment of beta tubulin isotypes with subtilisin to remove the C-terminal domain of the tubulin dimer abolished these differences in microtubule assembly pattern and enhanced self-assembly. The kinetic analysis of microtubule elongation of different beta tubulin isotypes also showed significant differences. Elongation of alpha beta III from microtubule seeds had a lower apparent K alpha and a lower apparent Kd than did alpha beta II and alpha beta IV. The dynamic behaviors of different beta tubulin isotypes were qualitatively similar to each other and fit the dynamic instability model. However, microtubules formed from alpha beta III appeared to be less dynamic than microtubules formed from other beta tubulin isotypes. Our results suggest that the beta III isotype might have a different conformation than do the other beta tubulin isotypes. The distinctive nucleation and elongation behaviors of the alpha beta III dimers demonstrated in vitro may have a significant influence on microtubule functions in vivo.
- Published
- 1994
27. Removal of beta III isotype enhances taxol induced microtubule assembly.
- Author
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Lu Q and Luduena RF
- Subjects
- Animals, Brain, Cattle, Colchicine pharmacology, Cold Temperature, In Vitro Techniques, Microscopy, Electron, Microtubules metabolism, Microtubules ultrastructure, Podophyllotoxin pharmacology, Tubulin metabolism, Microtubules drug effects, Paclitaxel pharmacology, Tubulin drug effects
- Abstract
The interaction of beta III-depleted tubulin with taxol was investigated. A monoclonal antibody against the beta III tubulin isotype was immobilized on a sepharose 4B column and used to remove the beta III tubulin isotype from unfractionated tubulin. The assembly of beta III-depleted tubulin in the presence of taxol was enhanced compared to unfractionated tubulin. The critical concentration of unfractionated tubulin in the presence of 10 microM taxol is 0.4 mg/ml, while the critical concentration of beta III-depleted tubulin is 0.16 mg/ml. At different concentration of taxol, the assembly of beta III-depleted tubulin is increased relative to that of unfractionated tubulin and reaches the maximum at about a 1:1 ratio of tubulin and taxol. The assembly of unfractionated tubulin and beta III-depleted tubulin has also been studied by electron microscopy. After 2 minutes at 37 degrees C, unfractionated tubulin assembly in the presence of 10 microM taxol results only in ribbon-like and ring structures; there are no visible microtubules. By 5 minutes, microtubules appear and increase in length. The assembly of beta III-depleted tubulin in the presence of 10 microM taxol occurs more quickly. In contrast to the case with unfractionated tubulin, beta III-depleted tubulin assembles within 2 minutes into microtubules which increase in length with time. At 30 minutes, microtubules assembled from beta III-depleted tubulin are shorter than the microtubules assembled from unfractionated tubulin. There is no visible difference between the microtubules assembled from unfractionated tubulin and beta III-depleted tubulin. Taxol-induced beta III-depleted tubulin assembly is more resistant to the inhibiting effect of podophyllotoxin and colchicine. It is also less sensitive to the inhibiting effect of cold temperature.
- Published
- 1993
- Full Text
- View/download PDF
28. Kinetics of colchicine binding to purified beta-tubulin isotypes from bovine brain.
- Author
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Banerjee A and Luduena RF
- Subjects
- Animals, Antibodies, Monoclonal, Cattle, Chromatography, Affinity, Kinetics, Tubulin immunology, Brain metabolism, Colchicine metabolism, Tubulin metabolism
- Abstract
Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.
- Published
- 1992
29. Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.
- Author
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Banerjee A, Roach MC, Trcka P, and Luduena RF
- Subjects
- Amino Acid Sequence, Animals, Cattle, Electrophoresis, Polyacrylamide Gel, Kinetics, Macromolecular Substances, Molecular Sequence Data, Peptides chemical synthesis, Peptides immunology, Tubulin analysis, Tubulin isolation & purification, Antibodies, Monoclonal, Brain metabolism, Tubulin metabolism
- Abstract
Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.
- Published
- 1992
30. Tubulin sulfhydryl groups as probes and targets for antimitotic and antimicrotubule agents.
- Author
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Luduena RF and Roach MC
- Subjects
- Animals, Humans, Tubulin drug effects, Antineoplastic Agents pharmacology, Microtubules drug effects, Sulfhydryl Compounds metabolism, Tubulin metabolism
- Abstract
The sulfhydryl groups of tubulin are highly reactive entities. The reactivity of the sulfhydryl groups is sensitive to the presence of tubulin ligands, making these groups excellent probes for the interaction of tubulin with ligands. When tubulin is reacted with N,N'-ethylenebis-(iodoacetamide), two intrachain cross-links form in the beta subunit. Formation of one of these cross-links is completely blocked by colchicine, podophyllotoxin, and nocodazole; formation of the other is blocked completely by maytansine, phomopsin A and GTP and partly by Vinca alkaloids. Different ligands also differ in their effect on the rate of alkylation of tubulin with iodo[14C]acetamide, with vinblastine and phomopsin A being strong inhibitors and maytansine having very little effect. Oxidation of certain key sulfhydryl groups can inhibit microtubule assembly. One of these sulfhydryl groups appears to be cys239, but there are others not yet identified. Sulfhydryl-oxidizing agents also interfere with microtubule-mediated processes in vivo, raising the question of the existence of a physiological regulator of microtubule assembly. Potential physiological regulators have been examined to see if they can control microtubule assembly in vitro at their physiological concentrations. Of the ones that have been examined, thioredoxin and thioredoxin reductase are much better candidates for being physiological regulators than are either cystamine or glutathione.
- Published
- 1991
- Full Text
- View/download PDF
31. Contrasting effects of maytansine and vinblastine on the alkylation of tubulin sulfhydryls.
- Author
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Luduena RF and Roach MC
- Subjects
- Alkylation, Animals, Cattle, Dimethyl Sulfoxide pharmacology, Iodoacetamide metabolism, Kinetics, Microtubules metabolism, Podophyllotoxin pharmacology, Sulfhydryl Compounds, Brain metabolism, Maytansine pharmacology, Oxazines pharmacology, Tubulin metabolism, Vinblastine pharmacology
- Published
- 1981
- Full Text
- View/download PDF
32. Detection of energy transfer between tryptophan residues in the tubulin molecule and bound bis(8-anilinonaphthalene-1-sulfonate), an inhibitor of microtubule assembly, that binds to a flexible region on tubulin.
- Author
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Prasad AR, Luduena RF, and Horowitz PM
- Subjects
- Anilino Naphthalenesulfonates pharmacology, Animals, Brain metabolism, Cattle, Energy Transfer, Fluorescent Dyes, Kinetics, Spectrometry, Fluorescence, Anilino Naphthalenesulfonates metabolism, Microtubules drug effects, Tryptophan, Tubulin metabolism
- Abstract
The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 microM, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37 degrees C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin-Weber plots were obtained for bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated for Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.
- Published
- 1986
- Full Text
- View/download PDF
33. Alpha- and beta-tubulin: separation and partial sequence analysis.
- Author
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Luduena RF and Woodward DO
- Subjects
- Amino Acid Sequence, Animals, Brain ultrastructure, Chick Embryo, Cyanogen Bromide, Electrophoresis, Polyacrylamide Gel, Macromolecular Substances, Male, Microtubules analysis, Peptides analysis, Peptides isolation & purification, Sea Urchins ultrastructure, Sperm Tail ultrastructure, Brain Chemistry, Nerve Tissue Proteins, Sea Urchins analysis, Sperm Tail analysis, Spermatozoa analysis, Tubulin analysis, Tubulin isolation & purification
- Published
- 1975
- Full Text
- View/download PDF
34. The interaction of cystamine with bovine brain tubulin.
- Author
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Banerjee A, Jordan MA, Little M, and Luduena RF
- Subjects
- Animals, Biopolymers, Cattle, Cystamine pharmacology, Mitosis drug effects, Sulfhydryl Compounds pharmacology, Sulfhydryl Reagents pharmacology, Cystamine metabolism, Tubulin metabolism
- Abstract
Microtubule assembly in vitro is sensitive to a variety of non-physiological sulfhydryl-oxidizing agents, but the physiological significance of this phenomenon is unknown, since no physiological sulfhydryl-oxidizing agent has been shown to affect microtubule assembly in vitro. We have accordingly investigated the interaction of tubulin with cystamine. We have found that millimolar concentrations of cystamine inhibit microtubule assembly and induce an abnormal form of tubulin polymerization. Cystamine-induced polymerization does not occur at cold temperature. Formation of the polymer requires reaction of cystamine with two sulfhydryls which become available at 37 degrees C. In addition, cystamine reacts with about three sulfhydryls at 0 degrees C without inducing polymerization. This latter set of sulfhydryls appear to include one or both of the previously defined beta s sulfhydryls whose reaction with N, N'-ethylene-bis(iodoacetamide) is markedly inhibited by GTP, maytansine and vinblastine [Roach, M. C. & Luduena, R. F. (1984) J. Biol. Chem. 259, 12063-12071]. Cystamine's specific manner of interacting with tubulin suggests that it may mimic an endogenous sulfhydryl-directed regulator of microtubule assembly.
- Published
- 1987
- Full Text
- View/download PDF
35. Interaction of reduced glutathione with bovine brain tubulin.
- Author
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Banerjee A, Jordan MA, and Luduena RF
- Subjects
- Animals, Cattle, Colchicine metabolism, Glutathione analogs & derivatives, Glutathione pharmacology, Glutathione Disulfide, Maytansine pharmacology, Microscopy, Electron, Osmolar Concentration, Podophyllotoxin pharmacology, Polymers metabolism, Temperature, Brain Chemistry, Glutathione metabolism, Tubulin metabolism
- Abstract
Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.
- Published
- 1985
- Full Text
- View/download PDF
36. Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures.
- Author
-
Palomares R, Prasad V, Luduena RF, and Manso-Martínez R
- Subjects
- Adenosine Diphosphate metabolism, Adenosine Diphosphate pharmacology, Adenosine Triphosphate metabolism, Adenosine Triphosphate pharmacology, Animals, Cattle, Fluorides pharmacology, Guanosine Triphosphate metabolism, Kinetics, Microtubules drug effects, Phosphates metabolism, Phosphorylation, Cerebral Cortex analysis, Guanosine Triphosphate pharmacology, Microtubule Proteins metabolism, Microtubules physiology
- Abstract
Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [gamma-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [gamma-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [gamma-32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [gamma-32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [gamma-32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [gamma-32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.
- Published
- 1987
- Full Text
- View/download PDF
37. Kinetics of association and dissociation of colchicine-tubulin complex from brain and renal tubulin. Evidence for the existence of multiple isotypes of tubulin in brain with differential affinity to colchicine.
- Author
-
Banerjee A and Luduena RF
- Subjects
- Animals, Binding Sites, Cattle, Kinetics, Protein Binding, Spectrometry, Fluorescence, Brain metabolism, Colchicine metabolism, Kidney metabolism, Tubulin metabolism
- Abstract
The kinetics of colchicine binding to bovine brain tubulin have been reported to be biphasic under pseudo first order conditions [(1978) Biochemistry 17, 4466-4472]. Unlike brain tubulin, the kinetics of colchicine binding to bovine renal tubulin are monophasic. The apparent on-rate constant for the binding of colchicine to renal tubulin is found to be very close to that of the faster binding component in brain tubulin. Similarly, the dissociation of colchicine-tubulin complex in the presence of iodide is biphasic for brain tubulin but monophasic for renal tubulin. Since brain and renal tubulin apparently differ in beta-tubulin, our results suggest that the biphasic nature of the kinetics for bovine brain tubulin could possibly originate from the existence of multiple isotypes of tubulin differing in drug binding affinity.
- Published
- 1987
- Full Text
- View/download PDF
38. The role of the B-ring of colchicine in the stability of the colchicine-tubulin complex.
- Author
-
Banerjee A, Barnes LD, and Luduena RF
- Subjects
- Colchicine analogs & derivatives, Kinetics, Molecular Conformation, Podophyllotoxin pharmacology, Protein Binding drug effects, Structure-Activity Relationship, Temperature, Colchicine metabolism, Tubulin metabolism
- Abstract
The binding of colchicine to tubulin is a slow, temperature-dependent and a poorly reversible process. Colchicine analogues modified in the B-ring of colchicine have been reported to bind to tubulin fairly rapidly (Ray, K., Bhattacharyya, B. and Biswas, B.B. (1981) J. Biol. Chem. 256, 6241-6244). In an effort to test the role of the B-ring in the reversibility of the colchicine-tubulin binding reaction, we have studied the kinetics of dissociation of the drug-tubulin complex for two B-ring-modified colchicine analogues under conditions in which the association reaction was blocked with a 40-fold excess of podophyllotoxin. In both cases, the dissociation was biphasic. The off-rate constants were determined and the results strongly suggest that the B-ring part of colchicine is responsible for the stability of the drug-tubulin complex. The dissociation data have been explained in terms of a binding model in which the binding of colchicine to tubulin involves a three-subdomain interaction rather than the previously suggested two-subdomain model (Andreu, J.M. and Timasheff, S.N. (1982) Biochemistry 21, 534-543).
- Published
- 1987
- Full Text
- View/download PDF
39. Interactions of vinblastine and maytansine with tubulin.
- Author
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Luduena RF, Anderson WH, Prasad V, Jordan MA, Ferrigni KC, Roach MC, Horowitz PM, Murphy DB, and Fellous A
- Subjects
- Animals, Brain drug effects, Brain metabolism, Erythrocytes drug effects, Kinetics, Macromolecular Substances, Microtubule-Associated Proteins metabolism, Microtubules drug effects, Microtubules ultrastructure, Protein Binding, Structure-Activity Relationship, Maytansine metabolism, Microtubules metabolism, Oxazines metabolism, Tubulin metabolism, Vinblastine metabolism
- Published
- 1986
- Full Text
- View/download PDF
40. Effect of estramustine phosphate on the assembly of trypsin-treated microtubules and microtubules reconstituted from purified tubulin with either tau, MAP2, or the tubulin-binding fragment of MAP2.
- Author
-
Fridén B, Wallin M, Deinum J, Prasad V, and Luduena R
- Subjects
- Binding Sites, Estramustine metabolism, Microtubules metabolism, Octoxynol, Polyethylene Glycols pharmacology, tau Proteins, Estramustine pharmacology, Microtubule-Associated Proteins metabolism, Microtubules drug effects, Nitrogen Mustard Compounds pharmacology, Peptide Fragments metabolism, Trypsin pharmacology, Tubulin metabolism
- Abstract
Estramustine phosphate, an estradiol nitrogen-mustard derivative is a microtubule-associated protein (MAP)-binding microtubule inhibitor, used in the therapy of prostatic carcinoma. It was found to inhibit assembly and to induce disassembly of microtubules reconstituted from phosphocellulose-purified tubulin with either tau, microtubule-associated protein 2, or chymotrypsin-digested microtubule-associated protein 2. Estramustine phosphate also inhibited assembly of trypsin-treated microtubules, completely depleted of high-molecular-weight microtubule-associated proteins, but with their microtubule-binding fragment present. In all cases estramustine phosphate induced disassembly to about 50%, at a concentration of approximately 100 microM, at similar protein concentrations. However, estramustine phosphate did not affect dimethyl sulfoxide-induced assembly of phosphocellulose-purified tubulin. Estramustine phosphate is a reversible inhibitor, as the nonionic detergent Triton X-100 was found to counteract the inhibition in a concentration-dependent manner. The reversibility was nondisruptive, as Triton X-100 itself did not affect microtubule assembly, microtubule protein composition, or morphology. This new reversible MAPs-dependent inhibitor estramustine phosphate affects the tubulin assembly, induced by tau, as well as by the small tubulin-binding part of MAP2 with the same concentration dependency. This indicates that tau and the tubulin-binding part of MAP2, in addition to their assembly promoting functions also have binding site(s) for estramustine phosphate in common.
- Published
- 1987
- Full Text
- View/download PDF
41. Comparative structure and chemistry of tubulins from different eukaryotes.
- Author
-
Luduena RF and Little M
- Subjects
- Animals, Macromolecular Substances, Peptide Fragments analysis, Species Specificity, Biological Evolution, Tubulin
- Abstract
Electrophoretic and peptide mapping have been used to examine alpha- and beta-tubulins from chordates, tunicates, echinoderms, mollusks, brachiopods, ferns, fungi, green algae and heliozoans. Cytoplasmic, ciliary, flagellar, and axopodial tubulins were examined. The results show that beta-tubulin is more conserved than alpha-tubulin. The large differences seen between axonemal and cytoplasmic tubulins and the similarity of all axonemal tubulins examined indicate that the genes for these two tubulin classes diverged prior to the appearance of metazoa and metaphyta. Comparisons of alpha-tubulins appear useful for tracing phyletic relationship within kingdoms whereas comparisons of beta-tubulins may be better for relating the kingdoms to each other.
- Published
- 1981
- Full Text
- View/download PDF
42. Bis(8-anilinonaphthalene-1-sulfonate) as a probe for tubulin decay.
- Author
-
Prasad AR, Luduena RF, and Horowitz PM
- Subjects
- Animals, Brain metabolism, Cattle, Circular Dichroism, Kinetics, Protein Binding, Protein Conformation, Quantum Theory, Spectrometry, Fluorescence, Thermodynamics, Tubulin isolation & purification, Anilino Naphthalenesulfonates metabolism, Fluorescent Dyes metabolism, Tubulin metabolism
- Abstract
The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) has been used to detect structural correlates of the well-known but poorly understood decay of tubulin function, by which tubulin loses its ability to polymerize and bind drugs in a complex, time-dependent way. The present results indicate that the decay of tubulin is accompanied by the appearance of hydrophobic areas, which bind a total of six Bis-ANS molecules with a dissociation constant of 19 microM. This binding seems to be a result of localized structural changes that are taking place in the tubulin molecule and can be used as a probe for these changes. In particular, circular dichroism measurements revealed no significant changes in the average secondary structure of the protein during the time required for complete binding of the Bis-ANS molecules. Preincubation of tubulin with the antimitotic drugs colchicine, podophyllotoxin, and vinblastine slows the rate of appearance of the hydrophobic region. Vinblastine has the maximal effect followed by colchicine and podophyllotoxin. In contrast, preincubation with maytansine has no effect. In addition, lowering the temperature decreases the rate of appearance of this region. These results correlate with the effect of drugs on the alkylation of tubulin sulfhydryl groups by iodoacetamide [Luduena, R.F., & Roach, M.C. (1981) Biochemistry 20, 4444-4450] and with the ability of inhibitors of microtubule assembly to permit the polymerization of tubulin into nonmicrotubule structures.
- Published
- 1986
- Full Text
- View/download PDF
43. Synthesis and antitumor activity of tropolone derivatives. 6. Structure-activity relationships of antitumor-active tropolone and 8-hydroxyquinoline derivatives.
- Author
-
Yamato M, Hashigaki K, Yasumoto Y, Sakai J, Luduena RF, Banerjee A, Tsukagoshi S, Tashiro T, and Tsuruo T
- Subjects
- Animals, Antineoplastic Agents therapeutic use, Colchicine metabolism, Leukemia P388 drug therapy, Mice, Structure-Activity Relationship, Tropolone chemical synthesis, Tropolone therapeutic use, Antineoplastic Agents chemical synthesis, Cycloheptanes, Hydroxyquinolines, Oxyquinoline, Tropolone analogs & derivatives
- Abstract
The bis derivative 6 of 8-hydroxyquinoline, which, like tropolones, readily forms a chelate, was synthesized and found to have high potency (dose = 12.5 mg/kg, T/C % = 164) against leukemia P388 in mice approximately equivalent to that of the bistropolone 1b. 8-Hydroxyquinoline analogues with broad structural variation were synthesized and their structure-activity relationships followed the same pattern as in the tropolone series. In addition, the bistropolones 1a-e were tested for their ability to bind to tubulin and found to have no such property. The results of this study suggested that bistropolone and bis(8-hydroxyquinoline) derivatives must form a chelate with the metal necessary for the enzyme, such as ribonucleotide reductase, which catalyzes the DNA biosynthetic pathways.
- Published
- 1987
- Full Text
- View/download PDF
44. The effect of 2-(4-methyl-1-piperazinylmethyl) acrylophenone dihydrochloride on the alkylation of tubulin.
- Author
-
Luduena RF, Roach MC, Trcka PP, Mallevais ML, and MacRae T
- Subjects
- Alkylation, Colchicine pharmacology, Fructose-Bisphosphate Aldolase metabolism, Iodoacetamide metabolism, Kinetics, Urea pharmacology, Vincristine pharmacology, Microtubules drug effects, Piperazines pharmacology, Tubulin metabolism
- Abstract
2-(4-Methyl-1-piperazinylmethyl) acrylophenone dihydrochloride (MPMAP) is a novel inhibitor of microtubule assembly in vitro and in vivo whose molecular mechanism of action has not been investigated (M. L. Mallevais, A. Delacourte, I. Lesieur, D. Lesieur, M. Cazin, C. Brunet, and M. Luyckx (1984) Biochimie 66, 477-482). We have examined the effect of MPMAP on the alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide) (EBI). MPMAP is a very potent inhibitor of tubulin alkylation by iodo[14C]acetamide. MPMAP gives half-maximal inhibition at a concentration of 15 microM. MPMAP also inhibits the alkylation of denatured tubulin and of aldolase, implying that it reacts strongly with sulfhydryl groups. MPMAP does not, however, interfere with formation by EBI of a crosslink between cysteines 239 and 354 in the beta subunit of tubulin, suggesting that these sulfhydryls are located in a cleft in the tubulin molecule.
- Published
- 1987
- Full Text
- View/download PDF
45. Isolation and partial characterization of alpha and beta-tubulin from outer doublets of sea-urchin sperm and microtubules of chick-embryo brain.
- Author
-
Luduena RF and Woodward DO
- Subjects
- Amino Acid Sequence, Amino Acids analysis, Animals, Brain cytology, Chick Embryo, Cyanogen Bromide, Electrophoresis, Electrophoresis, Polyacrylamide Gel, Flagella analysis, Hydrolysis, Male, Proteins analysis, Sea Urchins, Sodium Dodecyl Sulfate, Spermatozoa cytology, Urea, Brain Chemistry, Microtubules analysis, Nerve Tissue Proteins isolation & purification, Proteins isolation & purification, Spermatozoa analysis
- Abstract
Two kinds of tubulin (alpha and beta) have been described in microtubules from many different systems. In this study a discontinuous acrylamide-gel system containing sodium dodecyl sulfate was used to separate milligram quantities of alpha- and beta-tubulin from microtubules of chick-embryo brain and from outer doublets of sea-urchin sperm. The isolated tubulins were characterized by peptide mapping and automated sequencing of the first 25 NH(2)-terminal amino acids. Our results show that alpha- and beta-tubulin are related but distinctly different proteins and that each one has been highly conserved in the course of evolution.
- Published
- 1973
- Full Text
- View/download PDF
46. Evolutionary aspects of tubulin structure.
- Author
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Little M, Krämmer G, Singhofer-Wowra M, and Luduena RF
- Subjects
- Amino Acid Sequence, Animals, Brain Chemistry, Chickens, Humans, Peptide Fragments analysis, Physarum genetics, Rats, Schizosaccharomyces genetics, Sea Urchins, Sequence Homology, Nucleic Acid, Species Specificity, Swine, Biological Evolution, Tubulin genetics
- Published
- 1986
- Full Text
- View/download PDF
47. N,N'-Ethylene-bis(iodoacetamide) as a probe for structural and functional characteristics of brine shrimp, squid, and bovine tubulins.
- Author
-
Luduena RF, Roach MC, MacRae TH, and Langford GM
- Subjects
- Alkylation, Animals, Benzimidazoles pharmacology, Cattle, Cell Fractionation, Colchicine pharmacology, Maytansine pharmacology, Microtubules ultrastructure, Molecular Weight, Nocodazole, Podophyllotoxin pharmacology, Species Specificity, Tubulin isolation & purification, Vinblastine pharmacology, Artemia metabolism, Cross-Linking Reagents pharmacology, Decapodiformes metabolism, Ethylenediamines pharmacology, Tubulin metabolism
- Abstract
We have developed a simple probe for certain functionally significant features of the tubulin molecule. When bovine brain tubulin is treated with N,N'-ethylene-bis(iodoacetamide) (EBI), two intrachain cross-links, designated beta s and beta *, are formed in beta-tubulin, each one with a unique effect on the electrophoretic mobility of beta on gels containing sodium dodecyl sulfate. Formation of the beta * cross-link, which involves at least one assembly-critical sulfhydryl, is completely inhibited by colchicine and its congeners, while that of beta s is inhibited completely by maytansine and GTP and partly by vinblastine. To see how conserved this complex pattern is in evolution we examined tubulins from the brine shrimp Artemia and the squid Loligo. In both tubulins EBI forms the beta * cross-link in a reaction inhibitable by colchicine, podophyllotoxin, and nocodazole. In each tubulin, EBI appears to form a second intrachain cross-link in a reaction that can be inhibited completely by maytansine and GTP and partly by vinblastine. In Artemia, this cross-link alters the electrophoretic mobility to a slightly smaller extent than is the case for beta s in bovine brain, but in Loligo the alteration is much greater. It seems that the ligand-binding sites, the critical sulfhydryls, and their spatial interrelationships are strongly conserved and that the beta s sulfhydryls or the sequence between them are less strongly conserved in evolution.
- Published
- 1985
- Full Text
- View/download PDF
48. The effect of TN-16 on the alkylation of tubulin.
- Author
-
Roach MC and Luduena RF
- Subjects
- Alkylation, Animals, Colchicine metabolism, Dose-Response Relationship, Drug, Ethylenediamines metabolism, Iodoacetamide pharmacology, Antineoplastic Agents pharmacology, Pyrrolidinones pharmacology, Tubulin metabolism
- Abstract
The synthetic anti-tumor drug 3-(1-anilinoethylidene)-5-benzylpyrrolidine-2,4-dione (TN-16) is known to block microtubule assembly and colchicine binding to tubulin, although its structure does not resemble those of either colchicine, podophyllotoxin, or nocodazole (Arai, FEBS Lett. 155:273-276 (1983]. We have found that TN-16 affects the intra-chain cross-linking of beta-tubulin by N,N'-ethylene-bis(iodoacetamide) in a manner identical to that of colchicine, podophyllotoxin, and nocodazole, but different from that of vinblastine or maytansine. TN-16 also inhibits alkylation of tubulin by iodo[14C]acetamide, as do colchicine and its congeners. TN-16 appears to bind to tubulin at the colchicine binding site and one of its phenyl groups is likely to bind at the site on tubulin where colchicine's A ring binds.
- Published
- 1985
- Full Text
- View/download PDF
49. Tau microheterogeneity: an immunological approach with monoclonal antibodies.
- Author
-
Fellous A, Ohayon R, Mazie JC, Rosa F, Luduena RF, and Prasad V
- Subjects
- Aging, Animals, Antigen-Antibody Complex, Brain Chemistry, Cerebellum growth & development, Genetic Variation, Microscopy, Electron, Rats, Tubulin analysis, tau Proteins, Antibodies, Monoclonal, Brain growth & development, Microtubule-Associated Proteins analysis, Microtubules ultrastructure, Nerve Tissue Proteins analysis
- Abstract
The family of tau polypeptides purified from mammalian brain exhibit both extensive heterogeneity and large similarities in their chemical, physical, and functional properties. All the tau isoforms generated at a transcriptional or posttranscriptional level share the property of interacting with tubulin dimers in a specific manner. They strengthen longitudinal interactions between tubulin dimers and thus may stabilize microtubules once they are formed. Mild proteolysis or phosphorylation does not remove but only modulates the tau specific function that is probably related to the conserved sequences of the molecules. Monoclonal antibodies raised against tau were found to recognize epitopes conserved not only between species but also in different tissues. Using indirect immunofluorescence, a specific staining pattern was observed on rat neuronal cells and also on human skin fibroblasts. The same antibodies did not recognize glial cells, suggesting that these cells either do not contain detectable levels of tau or contain tau molecules different from the neuronal ones. These data suggest that tau protein is widely distributed, highly conserved, and may be preferentially associated with special subclasses of microtubules.
- Published
- 1986
- Full Text
- View/download PDF
50. Chemical modification of bovine brain tubulin with the guanine nucleotide affinity analogue 5'-p-fluorosulfonylbenzoylguanosine.
- Author
-
Prasad AR and Luduena RF
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Brain Chemistry, Cattle, Guanosine metabolism, Guanosine Triphosphate pharmacology, Microtubules metabolism, Sulfhydryl Compounds analysis, Affinity Labels metabolism, Guanosine analogs & derivatives, Tubulin metabolism
- Abstract
Bovine brain tubulin purified in the absence of GTP and MgCl2, reacts with 5'-p-fluorosulfonylbenzoylguanosine (5'-FSBG)2, an affinity analog of GTP and two moles of the reagent are incorporated per mole of tubulin at 0 degree C. 5'-FSBG is unable to promote the polymerization of tubulin into microtubules. 2 mM GTP, podophyllotoxin and vinblastine provide almost 50% protection against the modification, when added individually. Combination of these ligands gives maximal protection. Tubulin modified with 5'-FSBG lost two sulfhydryl groups per mole of tubulin and reduction with beta-mercaptoethanol led to the loss of the 2 moles of FSBG that had been incorporated. These data are interpreted on the basis that the modification of tubulin by 5'-FSBG proceeds via a thiosulfonate intermediate between the analogue and a reactive thiol group at or near that portion of the GTP binding site of tubulin where the phosphate moiety of GTP binds.
- Published
- 1987
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