Mechanism of selectivity of an angiogenesis inhibitor from screening a genome-wide set of Saccharomyces cerevisiae deletion strains.
BACKGROUND
The synthetic tripeptide arsenical 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO) is an angiogenesis inhibitor that targets the mitochondria of actively dividing but not quiescent endothelial cells, arresting their proliferation and causing apoptosis. Normal endothelial cells are much more sensitive to GSAO than tumor cells. To elucidate the mechanism of tumor cell resistance, we identified yeast genes that are necessary for resistance to GSAO.


METHODS
We screened a genome-wide set of 4546 Saccharomyces cerevisiae deletion strains to identify GSAO-sensitive strains. We then examined GSAO accumulation in and proliferation activity of endothelial cells (BAECs) and tumor cells treated with GSAO and modulators of pathways and proteins identified in the yeast screen. We also examined GSAO effects on proliferation of mammalian cells transfected with transporter protein constructs.


RESULTS
Eighty-eight deletion strains were sensitive to GSAO. The most sensitive strains had deletions of genes whose products are involved in vacuolar function (corresponding to drug transport in mammalian cells) and glutathione synthesis. BAECs were more sensitive to GSAO than tumor cells, and cell sensitivity to GSAO was approximately proportional to cellular glutathione levels. Treatment of BAECs and tumor cells with MK-571, an inhibitor of multidrug resistance-associated protein (MRP), or with buthionine sulfoximine, an inhibitor of glutathione synthesis, increased their sensitivity to GSAO. Mammalian cells transfected with MRP1 or MRP2 were resistant to GSAO, whereas cells transfected with MRP3, MRP4, MRP5, P-glypoprotein, or breast cancer resistance protein were not.


CONCLUSIONS
Differences in MRP activity and cellular glutathione levels contribute to the selectivity of GSAO for endothelial versus tumor cells. MRP1 and/or MRP2 may transport GSAO from resistant cells, with glutathione acting as a cotransporter. Genetic screening in yeast is a powerful tool for understanding drug action in mammalian cells.
Dilda
Pierre J
PJ
Center for Vascular Research, University of New South Wales, Department of Haematology, Prince of Wales Hospital, Sydney, Australia.
Don
Anthony S
AS
Tanabe
Kara M
KM
Higgins
Vincent J
VJ
Allen
John D
JD
Dawes
Ian W
IW
Hogg
Philip J
PJ
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001152:Arsenicals / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D018432:Drug Resistance, Multiple / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016681:Genome, Fungal; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D015151:Immunoblotting; D024101:Mitochondrial Proteins / Q000235:genetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D012269:Ribosomal Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
D020533:Angiogenesis Inhibitors; D001152:Arsenicals; D005978:Glutathione; D024101:Mitochondrial Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C426645:4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide; C093947:MRP2 protein, S cerevisiae; C489541:MRP4 protein, S cerevisiae; C112002:MRP51 protein, S cerevisiae; C114462:multidrug resistance-associated protein 3; C421109:multidrug resistance-associated protein 1