These results indicate that sphingosine/ceramide biosynthesis is required to prevent mitochondria from becoming toxic to cells. In support of this conclusion, it has Cell Cycle inhibitor recently been shown that ceramide-depleted
mitochondria were more sensitive to hydrogen peroxide and ethidium bromide [40] and that ceramide depletion in yeast mitochondria is associated with programmed cell death and oxidative stress [41]. A previous study from our laboratory explored drug-induced haploinsufficiency as a genome-wide approach to study the mechanism of action of drugs [6]. This work identified sphingosine/ceramide biosynthesis as the vital pathway inhibited by dhMotC. Interestingly, none of the 21 heterozygous mutants showing increased sensitivity to dhMotC was deleted of a gene involved in mitochondrial function. Therefore, the drug-induced haploinsufficiency screen, despite its genome-wide
coverage, only partially revealed the mechanism of action of dhMotC, concealing genes of mitochondrial function involved in the mechanism by which dhMotC kills cells. A second screen carried out in the present study, to identify suppressors of drug sensitivity, clearly showed that increasing the find more expression of genes encoding mitochondrial proteins can substantially BKM120 clinical trial increase resistance to dhMotC, further strengthening the link between mitochondria and the mechanism of action of the compound. Interestingly, comparing the results from the drug-induced haploinsufficiency screen [6] and the suppressor screen showed only 1 common gene, SUI2, a subunit of the translation Montelukast Sodium initiation factor eIF2 involved in amino acid starvation [42]. This seemed surprising since the screens are conceptually
similar in that they both rely on gene dose to identify drug-gene interactions. Differences between screens may be related to 1) stoichiometry, e.g. knockdown of 1 subunit of a protein complex is sufficient to reduce its activity and increase drug sensitivity while overexpression of 1 subunit of a protein complex is not sufficient to increase its activity and confer resistance, 2) redundancy, i.e. overexpression of a single gene is sufficient to confer resistance while knockout of redundant genes is necessary to detect sensitivity, and 3) unanticipated technical differences. Alternatively, the results may indicate a more complex relationship between gene dosage and drug sensitivity than has been generally considered. The third screen carried out in this study was a chemical-genetic synthetic lethality screen to identify nonessential genes that increase sensitivity to dhMotC when completely deleted in haploid strains.