Heightened sensitivity to paclitaxel in Class IVa beta-tubulin-transfected cells is lost as expression increases.
Stably transfected Chinese hamster ovary cell lines expressing increasing levels of beta4a, a class IV neuronal-specific beta-tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that beta4a 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 beta4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that beta4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of beta4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. beta4a-Tubulin differs from the more ubiquitous beta4b isotype at relatively few amino acid residues, yet beta4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of beta4a expression, beta4a and beta4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into beta4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala(115) to serine in beta4a-tubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into beta4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.
Yang
Hailing
H
Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77030.
Cabral
Fernando
F
Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77030. Electronic address: fernando.r.cabral@uth.tmc.edu.
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017239:Paclitaxel / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014162:Transfection; D014404:Tubulin / Q000737:chemistry / Q000145:classification*; D050257:Tubulin Modulators / Q000494:pharmacology*
D014404:Tubulin; D050257:Tubulin Modulators; D017239:Paclitaxel