Human brain and testis specific betaIII-tubulin was amplified from a cDNA library, modified to encode a C-terminal hemagglutinin antigen epitope tag, and cloned into a vector that allows tetracycline regulated expression in mammalian cells. Immunofluorescence analysis of transfected Chinese hamster ovary cells demonstrated that expressed HA-tagged betaIII-tubulin is able to assemble with endogenous tubulin into microtubules even though betaIII-tubulin is not a normal constituent of these cells. A stable G418-resistant clone with moderate HAbetaIII-tubulin expression displayed weak (1.5-2-fold) resistance to paclitaxel. A second clone with higher HAbetaIII-tubulin expression could not grow unless tetracycline was present to repress transcription of the transfected cDNA. Analysis of cellular microtubules in each of these clones indicated that incorporation of HAbetaIII-tubulin led to a significant expression-dependent decrease in assembled tubulin. Paclitaxel resistant cells were also directly selected from the transfected cell population using a paclitaxel concentration 4 times higher than the minimum toxic dose. Few cells were able to survive the selection and they grew very slowly. Western blot analysis of these resistant cells revealed very high HAbetaIII-tubulin expression that led to almost complete replacement of endogenous beta-tubulin at steady state. Transfected betaIII-tubulin with no epitope tag behaved in a very similar fashion indicating that presence of the HA tag had no discernible functional effect. The results demonstrate that betaIII-tubulin diminishes microtubule assembly, is toxic when present at high levels, but is able to confer weak resistance to paclitaxel when expressed at moderate levels in mammalian cells.
Copyright 2003 Wiley-Liss, Inc.