Heightened sensitivity to paclitaxel in class IVa β-tubulin-transfected cells is lost as expression increases

Stably transfected Chinese hamster ovary cell lines expressing increasing levels of beta 4a, a class IV neuronal-specific beta-tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that beta 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 beta 4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that beta 4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of beta 4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. beta 4a-Tubulin differs from the more ubiquitous beta 4b isotype at relatively few amino acid residues, yet beta 4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of beta 4a expression, beta 4a and beta 4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into beta 4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala(115) to serine in beta 4atubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into beta 4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.