Resistance to Peloruside A and Laulimalide: Functional Significance of Acquired βI-tubulin Mutations at Sites Important for Drug-Tubulin Binding
Peter T. Northcote,
John H. Miller.
Cancer cell lines selected for resistance to microtubule targeting agents (MTA) often have acquired mutations
in the β-tubulin binding sites for these agents. Despite strong correlational evidence, the functional and quantitative
significance of such mutations in the resistance to MTA remains unknown. We recently showed that peloruside A (PLA)
and laulimalide (LAU)-resistant cancer cell lines, 1A9-R1 (R1) and 1A9-L4 (L4), generated through multi-step selection
of human 1A9 ovarian cancer cells with high concentrations of either PLA (for R1) or LAU (for L4) have single distinct
mutations in their βI-tubulin gene. The R1 cells have a mutation at amino acid position 296 (A296T), and the L4 cells
have a mutation at position 306 (R306H/C), both of which lie at the putative binding sites of PLA and LAU. To gain
insights on the functional role of these mutations in the resistance phenotype, R1 and L4 cells were transfected with wild
type βI-tubulin. MTT cell proliferation assays revealed that restoration of wild type βI-tubulin expression partially
sensitized the R1 and L4 cells to PLA and LAU. Cell cycle analysis and intracellular tubulin polymerization assays
demonstrated that the increased sensitivity was correlated with an increased ability of PLA and LAU to induce G2-M
arrest and tubulin polymerization in the cells. Unlike paclitaxel-selected clones of 1A9 cells, both R1 and L4 cells
exhibited a functional p53 gene, and the abundance of the mismatch repair gene hMSH2 (human mutS homolog 2) was
comparable to the parental 1A9 cells. This study provides the first direct evidence that A296 and R306 of βI-tubulin are
important determinants of the PLA and LAU response in cancer cells.
Keywords: Binding site, β-tubulin mutations, drug resistance, hMSH2, laulimalide, microtubule, p53, paclitaxel, peloruside A.
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