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J. Biol. Chem., Vol. 282, Issue 37, 27058-27066, September 14, 2007

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Heightened Sensitivity to Paclitaxel in Class IVa -Tubulin-transfected Cells Is Lost as Expression Increases^*

From the Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77030

Stably transfected Chinese hamster ovary cell lines expressing increasing levels of 4a, a class IV neuronal-specific -tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that 4a reduced microtubule assembly in proportion to its abundance and thereby caused supersensitivity to microtubule disruptive drugs such as colcemid, vinblastine, and nocodazole. However, the response to paclitaxel was more complex. Low expression of 4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that 4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of 4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. 4a-Tubulin differs from the more ubiquitous 4b isotype at relatively few amino acid residues, yet 4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of 4a expression, 4a and 4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into 4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala¹¹⁵ to serine in 4a-tubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into 4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.

Received for publication, May 17, 2007 , and in revised form, July 5, 2007.

^* This work was supported by National Institutes of Health Grant CA85935 (to F. C.). 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.

¹ To whom correspondence should be addressed: Dept. of Integrative Biology and Pharmacology, The University of Texas Medical School, 6431 Fannin St., Houston, TX 77030. Tel.: 713-500-7485; Fax: 713-500-7455; E-mail: fernando.r.cabral{at}uth.tmc.edu.

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