Random Mutagenesis of β-Tubulin Defines a Set of Dispersed Mutations That Confer Paclitaxel Resistance
- 359 Downloads
Previous research showed that mutations in β1-tubulin are frequently involved in paclitaxel resistance but the question of whether the mutations are restricted by cell-type specific differences remains obscure.
To circumvent cellular constraints, we randomly mutagenized β-tubulin cDNA, transfected it into CHO cells, and selected for paclitaxel resistance.
A total of 26 β1-tubulin mutations scattered throughout the sequence were identified and a randomly chosen subset were confirmed to confer paclitaxel resistance using site-directed mutagenesis of β-tubulin cDNA and transfection into wild-type cells. Immunofluorescence microscopy and biochemical fractionation studies indicated that cells expressing mutant tubulin had decreased microtubule polymer and frequently suffered mitotic defects that led to the formation of large multinucleated cells, suggesting a resistance mechanism that involves destabilization of the microtubule network. Consistent with this conclusion, the mutations were predominantly located in regions that are likely to be involved in lateral or longitudinal subunit interactions. Notably, fourteen of the new mutations overlapped previously reported mutations in drug resistant cells or in patients with developmental brain abnormalities.
A random mutagenesis approach allowed isolation of a wider array of drug resistance mutations and demonstrated that similar mutations can cause paclitaxel resistance and human neuronal abnormalities.
KEY WORDScell division drug resistance microtubule assembly mutant tubulin neuronal disorders
Chinese hamster ovary
ACKNOWLEDGMENTS AND DISCLOSURES
These studies were supported by grant CA85935 from the National Institutes of Health to FC.
- 1.Leandro-Garcia LJ, Leskela S, Landa I, Montero-Conde C, Lopez-Jimenez E, Leton R, et al. Tumoral and tissue-specific expression of the major human beta-tubulin isotypes. Cytoskeleton (Hoboken). 2010;67:214–23.Google Scholar
- 11.Begaye A, Trostel S, Zhao Z, Taylor RE, Schriemer DC, Sackett DL. Mutations in the beta-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding. Cell Cycle. 2011;10Google Scholar
- 19.Cabral F. Factors determining cellular mechanisms of resistance to antimitotic drugs. Drug Resistance Updates. 2001;3:1–6.Google Scholar
- 55.DeLano WL. MacPyMOL: A PyMOL-based molecular graphics application for MacOS X. South San Francisco: DeLano Scientific LLC; 2005.Google Scholar
- 59.Poruchynsky MS, Kim JH, Nogales E, Annable T, Loganzo F, Greenberger LM, et al. Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analogue, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability. Biochemistry. 2004;43:13944–54.PubMedCrossRefGoogle Scholar