Interaction of Estramustine with Tubulin Isotypes

Laing, Naomi; Dahllöf, Björn; Hartley-Asp, Beryl; Ranganathan, Sulabha; Tew, Kenneth D.

doi:10.1021/bi961445w

Cited by 66 publications

(38 citation statements)

References 32 publications

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“…pacitaxel binding to Ptubulin and estramustine binding to microtubule-associated proteins and tubulin (Dahllof et al, 1993;Speicher et al, 1994;Laing et al, 1997). The present results and our previous findings in estramustineresistant prostate cell lines suggest that resistance to paclitaxel and estramustine may share a common basis despite the differing binding sites on the microtubule.…”

Section: Discussionsupporting

confidence: 67%

Altered β-tubulin isotype expression in paclitaxel-resistant human prostate carcinoma cells

Ranganathan

Benetatos²,

Colarusso³

et al. 1998

Br J Cancer

195

133

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Summary To investigate the role of ,B-tubulin isotype composition in resistance to paclitaxel, an anti-microtubule agent, human prostate carcinoma (DU-145) cells were intermittently exposed to increasing concentrations of paclitaxel. Cells that were selected and maintained at 10 nm paclitaxel (Pac-1 0) were fivefold resistant to the drug. Pac-1 0 cells accumulated radiolabelled paclitaxel to the same extent as DU-1 45 cells and were negative for MDR-1. Analysis of Pac-10 and DU-145 cells by flow cytometry showed similar cell cycle patterns. Immunofluorescent staining revealed an overall increase of a-and 1-tubulin levels in Pac-10 cells compared with DU-145 cells. Examination of 3-tubulin isotype composition revealed a significant increase in pill isotype in the resistant cells, both by immunofluorescence and by western blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the isotypes confirmed the increase observed for the ,,, by exhibiting ninefold higher ,,, mRNA levels and also showed fivefold increase of the Plv. transcript. In addition, analysis of paclitaxel-resistant cells that were selected at increasing levels of the drug (Pac 2, 4, 6, 8 and 10) exhibited a positive correlation between increasing P,,, levels and increasing resistance to paclitaxel. Increased expression of specific ,B-tubulin isotypes and subsequent incorporation into microtubules may alter cellular microtubule dynamics, providing a defence against the anti-microtubule effects of paclitaxel and other tubulin-binding drugs.Paclitaxel has gained considerable attention in cancer therapy in recent years and is successfully used in treating a variety of tumours, including those of the breast, ovary and lung. In treatment of prostate cancer, paclitaxel is inactive when used as a single agent (Roth et al, 1993). However, in combination with estramustine, another anti-microtubule agent, paclitaxel has significant activity against hormone refractory prostate cancer (Hudes et al, 1995).Despite its preclinical and clinical success, the exact mechanism of action of paclitaxel is not known. At low concentrations, paclitaxel blocks mitosis by kinetic stabilization of spindle microtubules (Jordan et al, 1993). Paclitaxel differs from the other anti-microtubule agents such as vinblastine and colchicine by causing microtubule polymerization instead of depolymerization.The c4p-tubulin heterodimer is the major component of microtubules. Most of the anti-microtubule agents, including paclitaxel, vinblastine, colchicine and estramustine bind to 3-tubulin. Paclitaxel binding sites on 3-tubulin were identified at the N-terminal 31 amino acids and at residues 217-231 of the protein . These two binding sites are part of the colchicine binding site and are highly conserved among species.Both a-and ,-tubulins are encoded by multigene families and exist as several isotypes in cells. 3-Tubulin exists as six isotypes that are evolutionarily conserved across species and differ from each other predominantly at the carboxy terminus. Sever...

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Section: Discussionsupporting

confidence: 67%

Altered β-tubulin isotype expression in paclitaxel-resistant human prostate carcinoma cells

Ranganathan

Benetatos²,

Colarusso³

et al. 1998

Br J Cancer

195

133

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“…reduced ability of the drug to induce the conformational change required to suppress microtubule dynamics). Other microtubule-targeted drugs, including estramustine and colchicine, interact more weakly with ␤III-tubulin than with several other isotypes in vitro (34,35). In addition, colchicine binding studies and cysteine cross-linking studies suggest that ␤III-tubulin has a more rigid conformation than the other ␤-isotypes examined (18,36).…”

Section: Overexpression Of ␤Iii-tubulin Does Not Cause An Inherent Inmentioning

confidence: 99%

βIII-Tubulin Induces Paclitaxel Resistance in Association with Reduced Effects on Microtubule Dynamic Instability

Kamath

Wilson

Cabral

et al. 2005

Journal of Biological Chemistry

235

175

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The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the ␤III-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the ␤-subunit of tubulin in microtubules and suppressing microtubule dynamic instability, leading to mitotic arrest and cell death. We hypothesized that ␤III-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule dynamic instability in resistant cells or by rendering the microtubules less sensitive to the suppression of dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either ␤I-or ␤III-tubulin, we analyzed microtubule dynamic instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of dynamic instability between the two ␤-tubulin-overexpressing cell types. However, in the presence of 150 nM paclitaxel, dynamic instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing ␤III-tubulin than in cells overexpressing similar levels of ␤I-tubulin (suppressed 47%). The results suggest that overexpression of ␤III-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule dynamics. The results also suggest that endogenous regulators of microtubule dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.

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“…EM has been found to bind weakly to microtubule-associated proteins (MAP) and to inhibit microtubule assembly in vitro (13,14). EM has been shown to bind to tubulin dimers (15,16) and weakly inhibits the polymerization of MAPsfree tubulin into microtubules (16). Furthermore, EM has been shown to suppress the dynamic instability of individual MAP-free microtubules in vitro (16).…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, EM has been shown to suppress the dynamic instability of individual MAP-free microtubules in vitro (16). The EM binding site on tubulin has been suggested to be distinct from the colchicine and vinblastine sites (16) and may partially overlap with the Taxol-binding site in tubulin (15). Interestingly, EMP has been shown to bind to brain MAPs and to depolymerize MAP-rich microtubules in vitro (17).…”

Section: Introductionmentioning

confidence: 99%

Kinetic Stabilization of Microtubule Dynamics by Estramustine Is Associated with Tubulin Acetylation, Spindle Abnormalities, and Mitotic Arrest

2008

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Estramustine (EM) alone or in combination with other anticancer agents is clinically used for the treatment of hormone refractory prostate cancer. Furthermore, EM has been shown to potently inhibit the proliferation of different types of cancer cells in culture apparently by targeting microtubules; however, the antiproliferative mechanism of action of EM is not clear. In this work, we have shown that EM strongly suppressed the dynamic instability of individual microtubules in MCF-7 cells by reducing the rates of growing and shortening excursions and increasing the time microtubule spent in the pause state. At its half maximal proliferation inhibitory concentration (IC 50 ), EM exerted strong suppressive effects on the dynamics of microtubules in MCF-7 cells without detectably affecting either the organization or the polymerized mass of microtubules. At relatively high concentrations (5 Â IC 50 ), EM significantly depolymerized microtubules in the cells. Furthermore, the microtubules were found highly acetylated, supporting the conclusion that they were stabilized by the drug. EM treatment induced spindle abnormalities in MCF-7 cells, and a major population of the arrested mitotic cells was multipolar. EM also perturbed the microtubule-kinetochore interaction, thereby activating the spindle assembly checkpoint and leading to apoptotic cell death. [Cancer Res 2008; 68(15):6181-9]

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Interaction of Estramustine with Tubulin Isotypes

Cited by 66 publications

References 32 publications

Altered β-tubulin isotype expression in paclitaxel-resistant human prostate carcinoma cells

Altered β-tubulin isotype expression in paclitaxel-resistant human prostate carcinoma cells

βIII-Tubulin Induces Paclitaxel Resistance in Association with Reduced Effects on Microtubule Dynamic Instability

Kinetic Stabilization of Microtubule Dynamics by Estramustine Is Associated with Tubulin Acetylation, Spindle Abnormalities, and Mitotic Arrest

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