A FUNCTIONAL GENOMIC APPROACH TO IDENTIFY GENES THAT MODULATE DOXORUBICIN CYTOTOXICITY IN YEAST

T.J.Westmoreland, J.R. Marks, J.A. Olson, Jr., K.L. Blackwell, S.A. Snyder,
C.B. Bennett* Duke University Medical Center Box 3873 Durham, NC 27710
*benne048@mc.duke.edu

INTRODUCTION Doxorubicin (DX) and ionizing radiation (IR) are two anticancer agents effective in the treatment of breast cancer.  These agents are thought to promote lethality by inducing DNA double strand breaks (DSB) through free radical production (1).  DX also induces DSBs by the inhibition of topoisomerase II and is an S phase specific inhibitor of replication (1,2).  Resistance to DX has been found to occur in petite (respiratory deficient) yeast strains (2). In order to identify novel genetic targets required for resistance to the lethal effects of these agents, we have screened the Saccharomyces cerevisiae diploid deletion strain collection for IR and DX sensitive mutants.

METHODS We exposed isogenic diploid yeast strains individually deleted for 1076 nonessential genes to IR and DX. Sensitivity was determined by comparing deletion strains to wildtype (WT).  Strain sensitivity was confirmed by survival curve analysis.  Briefly, log phase cells in liquid medium were exposed to DX. Hourly aliquots were plated, and relative survival was determined.  Cell cycle progression of DX sensitive strains was determined for single (G1) cells exposed to DX.  Each strain was grown to late log phase, and individual G1 cells were placed into a grid pattern on rich nutrient plates containing DX.  The cells were scored hourly for 72 hours to determine the onset of S phase and the time required for microcolony formation. 

RESULTS We identified 167 yeast gene deletions that sensitized cells to the lethal effects of DX. Of these, 23% were in mitochondria functions.  Also, 65 deletions were found to be sensitive to IR of which 43 (66%) were cross sensitive to DX.  Surprisingly, 22 gene deletions showed enhanced resistance to DX.  Among previously identified IR sensitive deletion mutants (4), 42 were cross sensitive to DX.  We identified 5 members of the CCR4-NOT transcriptional complex that were cross sensitive to DX and IR. CCR4 and DHH1, two members of the complex, are highly conserved checkpoint genes that interact genetically with BRCA1 in yeast and when deleted show prolonged G1-S phase cell cycle transition following IR damage (3).  An increase in the production of petite colonies that survived DX was observed following treatment of these mutant cells. These cells were found to have increased DX resistance confirming that functional mitochondria play an important role in DX toxicity in yeast (1). We examined the cell cycle progression of CCR4-NOT mutants (ccr4D, dhh1D, and pop2D) vs WT along with their isogenic petite counterparts during continual exposure to DX.  We found that single unbudded cells (G1) progressed slowly through the cell cycle eventually forming viable microcolonies for both WT and WT petite cells.  For ccr4D and pop2D strains, G1 cells either arrested as budded cells or progressed into small microcolonies that lysed. For ccr4D and pop2D petite strains, most G1 cells arrested as budded cells that lysed.  DHH1D G1 cells progressed into small microcolonies that lysed.

DISCUSSION We identified 167 genes required for resistance to DX in yeast.  Among these, we identified 5 members of the CCR4-NOT complex. Petite strains deleted for members of this complex are more resistant to DX.  Therefore, functional mitochondria are required for the cytotoxic effects of DX.  However, both respiratory proficient and petite ccr4D and pop2D cells that transit S phase in the presence of DX arrested as budded cells that lysed. This indicates that DX also mediates inhibition of DNA synthesis independently of mitochondrial function.  Therefore, in yeast CCR4-NOT mutants, inhibition of DNA synthesis plays a critical role in DX-induced cytotoxicity.  Some viable gene deletions confer enhanced resistance to DX yet retain functional mitochondria.  Therefore, cellular targets other than replication and mitochondria are required to mediate DX cytotoxicity in yeast.  Since DX treatment in humans can lead to significant cardiomyopathy (1), our approach may help elucidate new genes and pathways that modulate DX cytotoxicity.

ACKNOWLEDGMENT We would like to thank Cheryl Morgan with the Duke University Medical Center Morris Pharmacy for her thoughtful gift of doxorubicin.

REFERENCES

1. Wallace, K.B. (2003) Pharm. & Tox. 93, 105-115
2. Patel, S., Sprung, A.U., Keller, B.A., Heaton, V.J., and Fisher, L.M. (1997) Mol. Pharm. 52, 658-666
3. Westmoreland, T.J., Olson, J.A., Jr., Saito, W.Y., Huper, G., Marks, J.R., and Bennett, C.B. (2003) J. Surg Res. 113, 62-73
4. Westmoreland, T.J., Marks, J.R., Olson, J.A., Jr., Thompson, E.M., Resnick, M.A., and Bennett, C.B. Submitted Eukaryotic Cell