Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae.
Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for beta-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated beta-tubulin gene into a S. cerevisiae strain with diminished transporter activity and developed the first paclitaxel-sensitive budding yeast strain. In the presence of paclitaxel, cytoplasmic microtubules were stable to cold depolymerization. Paclitaxel-treated cells showed evidence of a mitotic block, with an increase in large-budded cells and cells with a 2N DNA content and DNA fragmentation, identified by FACS analysis and the TUNEL assay. In the presence of paclitaxel, the number of dead cells in cultures increased three-fold and cells containing reactive oxygen species were present. We conclude that paclitaxel blocks mitosis in this strain, leading to an apoptotic-like cell death. This strain will also be useful in further studies of the effect of microtubule dynamics on various cellular processes in S. cerevisiae.
Foland
Travis B
TB
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045-7534, USA.
Dentler
William L
WL
Suprenant
Kathy A
KA
Gupta
Mohan L
ML
Himes
Richard H
RH
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics; D005800:Genes, Fungal; D008870:Microtubules / Q000187:drug effects*; D008938:Mitosis / Q000187:drug effects*; D009154:Mutation; D017239:Paclitaxel / Q000378:metabolism / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D017239:Paclitaxel