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J. Biol. Chem., Vol. 279, Issue 48, 49940-49947, November 26, 2004

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In Vivo Measurement of Microtubule Dynamics Using Stable Isotope Labeling with Heavy Water

EFFECT OF TAXANES^*

Patrizia Fanara, Scott Turner, Robert Busch, Salena Killion, Mohamad Awada, Holly Turner, Ablatt Mahsut, Kristen L. LaPrade, Julie M. Stark, and Marc K. Hellerstein¶||

From the KineMed, Inc., Emeryville, California 94608, the ^¶Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720, and the ^||Department of Medicine, University of California, San Francisco General Hospital, San Francisco, California 94110

Microtubules are dynamic polymers with central roles in the mitotic checkpoint, mitotic spindle assembly, and chromosome segregation. Agents that block mitotic progression and cell proliferation by interfering with microtubule dynamics (microtubule-targeted tubulin-polymerizing agents (MTPAs)) are powerful antitumor agents. Effects of MTPAs (e.g. paclitaxel) on microtubule dynamics have not yet been directly demonstrated in intact animals, however. Here we describe a method that measures microtubule dynamics as an exchange of tubulin dimers into microtubules in vivo. The incorporation of deuterium (²H₂) from heavy water (²H O) into tubulin dimers and polymers is measured by gas chromatography/mass spectrometry. In cultured human lung and breast cancer cell lines, or in tumors implanted into nude mice, tubulin dimers and polymerized microtubules exhibited nearly identical label incorporation rates, reflecting their rapid exchange. Administration of paclitaxel during 24 h of ²H₂O labeling in vivo reduced ²H labeling in polymers while increasing ²H in dimers, indicating diminished flux of dimers into polymers (i.e. inhibition of microtubule dynamic equilibrium). In vivo inhibition of microtubule dynamics was dose-dependent and correlated with inhibition of DNA replication, a stable isotopic measure of tumor cell growth. In contrast, microtubule polymers from sciatic nerve of untreated mice were not in dynamic equilibrium with tubulin dimers, and paclitaxel increased label incorporation into polymers. Our results directly demonstrate altered microtubule dynamics as an important action of MTPAs in vivo. This sensitive and quantitative in vivo assay of microtubule dynamics may prove useful for pre-clinical and clinical development of the next generation of MTPAs as anticancer drugs.

Received for publication, August 23, 2004

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: KineMed, Inc., 5980 Horton St., Ste. 470, Emeryville, CA 94608. Tel.: 510-655-6525; Fax: 510-655-6506; E-mail: pfanara{at}kinemed.com.

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