THE ROLE OF PROTEIN CHAPERONES IN THE SURVIVAL FROM ANTHRACYCLINE-INDUCED OXIDATIVE STRESS IN SACCHAROMYCES CEREVISIAE

Abstract

Several S. cerevisiae deletion strains involving heat-shock response factors were among the most sensitive mutants identified in a previous genetic screen for doxorubicin hypersensitivity. These strains included ydj1Δ, ssz1Δ and zuo1Δ mutants. In addition, new1Δ, whose function was unknown, also displayed significant sensitivity to anthracyclines. We further investigated the basis for the sensitivity of these mutants. We determined that heat-shock could partially rescue the sensitivity of the strains to doxorubicin, including the homologous recombination mutant rad52Δ, which is sensitive to doxorubicin-mediated DNA double strand breaks (DSBs). However, none of the heat-shock response mutants were sensitive to DSBs, but were highly sensitive to reactive oxygen species (ROS) generated by quinone-ring-containing agents, such as anthracyclines and menadione. A fluorescent-based assay indicates that doxorubicin causes protein aggregation. Interestingly, the disaggregase mutant hsp104Δ is not sensitive to anthracyclines or menadione suggesting that Hsp104p does not play a role in disaggregating doxorubicin-induced protein aggregates. However New1p, which has been recently shown to be a novel disaggregase, is essential for cell viability after exposure to anthracyclines and menadione and it is not involved in thermotolerance. Our data suggest that in S. cerevisiae, doxorubicin produces protein aggregation through ROS and requires Ydj1p and New1p for resolution.

Keywords: Anthracyclines; Cancer; Heat-Shock Response; Molecular Chaperones; Oxidative Stress.

Figure 1

Heat-shock rescues strains sensitivity to doxorubicin. Serial dilutions of selected strains were plated onto YPD with or without doxorubicin, as described in the Materials and Methods. The plates were incubated at either 30°C (normal temperature) or 37°C (heat-shock). Growth was scored after 3 days of incubation. The result of a representative experiment is presented.

Figure 2

Doxorubicin exposure leads to protein aggregation. The evaluation of doxorubicin-induced protein aggregation was performed in wild type strain using a model substrate of firefly luciferase (FFL) fused to green fluorescent protein (GFP) as described in the Materials and Methods. a) Serial dilutions of the control (− Doxo) and doxorubicin-exposed cells (+ Doxo) were plated onto Leu-drop out plates and incubated at 30°C to determine growth. b) Protein aggregation as determined by aggregation of the FFL-GFP fusion reporter. GFP and DIC images are presented for doxorubicin treated (− Doxo) and control (− Doxo) wild type cells.

Figure 3

HSP104 is not essential for protection of cells to doxorubicin. Cells were grown to exponential phase in YPD at 30°C and switched to 39°C to induce the heat-shock response. Aliquots were transferred to 50°C and incubated for the indicated time, then placed on ice before spotting. Serial dilutions (5-fold) were spotted onto YPD plates and incubated at 30°C. Cell growth was monitored daily and colonies were counted at day 3.

Figure 4

Heat-shock defective strains are not sensitive to DNA double-strand breaks generated by doxorubicin. Serial dilutions of heat-shock response mutants were plated onto etoposide-containing plates as described in the Materials and Methods. The rad52Δ sensitive strain was used as the control. Growth was scored after 3 days of incubation at 30°C. Sensitivity was assessed by comparison to growth of the strains on plates without etoposide.

Figure 5

Strains defective in the heat-shock response are sensitive to agents that generate oxidative stress. Serial dilutions of heat-shock response mutants exposed to menadione were plated onto non-selective plates as described in the Materials and Methods. The sod1Δ sensitive strain was used as the control. Growth was scored after 3 days of incubation at 30°C, and sensitivity was assessed by comparison to the growth of the untreated cells (No Drug) and to cells grown on plates containing doxorubicin (+ Doxo).