Identification of genes required for protection from doxorubicin by a genome-wide screen in Saccharomyces cerevisiae

Abstract

Anthracyclines are chemotherapeutic agents commonly used to treat a broad range of malignancies. Although effective, these drugs present serious complications, most notably cardiotoxicity. To determine the mechanisms that mediate cytoprotection from doxorubicin, we have screened the collection of Saccharomyces cerevisiae haploid gene deletion mutants. We have identified 71 deletion strains that display varying degrees of hypersensitivity to doxorubicin at a concentration that does not significantly reduce the viability of wild-type cells. Complementation of the doxorubicin-sensitive phenotype of the deletion strains with the wild-type genes proves that the sensitivity of the strain to doxorubicin is due to the gene deletion. The genes that mediate cytoprotection from doxorubicin belong to multiple pathways including DNA repair, RNA metabolism, chromatin remodeling, amino acid metabolism, and heat shock response. In addition, proteins with mitochondrial, osmosensing, vacuolar, and ribosomal functions are also required for protection from doxorubicin. We tested the sensitivity of the deletion strains to other cytotoxic agents, which resulted in different drug-specific sensitive groups. Most of the identified genes have mammalian homologues that participate in conserved pathways. Our data may prove useful to develop strategies aimed at sensitizing tumor cells to doxorubicin as well as protecting cardiac cells from its cytotoxic effects.

Figure 1

Determination of the concentration of doxorubicin for the screen. A, wild-type parental strain (BY4741) and rad52 and msh2 mutants were tested for growth on media containing different concentrations of doxorubicin (Doxo) as indicated in Materials and Methods. Serial dilutions (1:10–1:10⁵) of saturated stationary cultures (cell density, ~2 × 10⁷) were spotted in the plates containing 0, 5, 20, and 100 µmol/L doxorubicin, respectively. Growth was scored after 3 d of incubation at 30°C. The 1:10 dilution of the different concentration plates is shown. B, quantification of the survival of the tested strains. Survival was determined by counting the number of colonies in the respective dilutions and calculated based on the growth in plates lacking doxorubicin.

Figure 2

Confirmation of the sensitivity of individual deletion mutant strains to doxorubicin. Candidate doxorubicin-sensitive strains identified in the screen were retested by plating serial dilutions of overnight cultures from the original library onto plates containing 20 µmol/L doxorubicin. To account for growth rate differences between the strains, growth in a control plate lacking doxorubicin was also tested. Sensitivity was determined by growth in doxorubicin-containing plates relative to the growth in plates with no drug. The wild-type control shows no significant growth defect (>83% survival) in doxorubicin at the concentration used, consistent with the data in Fig. 1. The sensitive strains displayed varying degrees of sensitivity in doxorubicin plates and were classified as slightly sensitive strains (2–10-fold sensitivity; e.g., tat1), sensitive strains (10–100-fold sensitivity; e.g., ssz1), or hypersensitive strains (>100-fold sensitivity; e.g., snf2, ydj1, etc). The strains were retested twice, displaying consistent phenotype. Selected strains are shown.

Figure 3

Complementation of the doxorubicin sensitivity of selected deletion strains. Strains were transformed with expression plasmids containing their specific deleted genes or the empty vector and tested for complementation. Serial dilutions of stationary cultures were spotted in selective media and selective media plus doxorubicin. Growth was determined after 3 d of incubation at 30°C.