Characterization of a transport and detoxification pathway for the antitumour drug bleomycin in Saccharomyces cerevisiae.
BLM (bleomycin) is effective in combination therapy against various cancers including testicular cancer. However, several other cancers such as colon cancer are refractory to BLM treatment. The exact mechanism for this differential response of cancer cells to the drug is not known. In the present study, we created fluorescently labelled BLM-A5, which retained nearly full genotoxic potential, and used this molecule to conduct the first study to understand the transport pathway of the drug in Saccharomyces cerevisiae. Uptake studies revealed that fluoro-BLM-A5 is transported into the cell in a concentration-dependent manner. Transport of a non-saturating concentration of fluoro-BLM-A5 was modest for the first 90 min, but thereafter it was sharply induced until 300 min. The inducible transport was completely abolished by the addition of cycloheximide, suggesting that BLM-A5 uptake into the cell is dependent on new protein synthesis. Interestingly, transport of fluoro-BLM-A5 was blocked if the cells were preincubated with increasing concentrations of spermine. Moreover, a mutant lacking the Ptk2 kinase, necessary for positively regulating polyamine transport, was defective in fluoro-BLM-A5 uptake and exhibited extreme resistance to the drug. A simple interpretation of these results is that BLM-A5 may enter the cell through the polyamine transport system. We showed further that after the uptake, fluoro-BLM-A5 accumulated into the vacuole of the parent, but localized to the cytoplasm of mutants disrupted for the END3 gene required for an early step of the endocytotic pathway. In general, mutants with a defect in the endocytic pathway to the vacuole were hypersensitive to BLM-A5. We suggest that BLM-A5 is transported across the yeast plasma membrane and sequestered into the vacuole for detoxification.
Aouida
Mustapha
M
Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, University of Montreal, 5415, Boul. de l'Assomption, Montreal, Quebec, Canada H1T 2M4.
Leduc
Anick
A
Wang
Huijie
H
Ramotar
Dindial
D
D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D001761:Bleomycin / Q000031:analogs & derivatives / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000172:deficiency / Q000502:physiology; D004249:DNA Damage / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008658:Inactivation, Metabolic / Q000502:physiology*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D013096:Spermine / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D014617:Vacuoles / Q000737:chemistry
D003598:Cytoskeletal Proteins; C091908:END3 protein, S cerevisiae; D005456:Fluorescent Dyes; D029701:Saccharomyces cerevisiae Proteins; D001761:Bleomycin; D013096:Spermine; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016650:Fluorescein-5-isothiocyanate