Evidence for distinct DNA‐ and RNA‐based mechanisms of 5‐fluorouracil cytotoxicity in Saccharomyces cerevisiae

Abstract

5-Fluorouracil (5FU) is an effective chemotherapeutic drug developed as an inhibitor of thymidylate synthetase (TS). Inhibition of TS leads to 'thymine-less death', a condition resulting from depletion of dTTP pools and misincorporation of dUTP into newly synthesized or repaired DNA. 5FU is also incorporated into RNA and a growing body of evidence suggests that RNA-based effects play a significant role in its cytotoxicity. Indeed, recent experiments in yeast showed that defects in the nuclear RNA exosome subunit Rrp6p cause hypersensitivity to 5FU. The present study asked whether the 5FU hypersensitivity of an rrp6-yeast strain reflects the DNA-or RNA-based effects of 5FU. Genetic analyses suggest that while a DNA repair mutation, apn1-, causes sensitivity to 5FU-induced DNA damage, an rrp6-mutation causes hypersensitivity, due to the RNA-based effects of 5FU. Analysis of a strain with normal DNA and RNA metabolism grown in the presence of 5FU shows that UMP suppresses the 5FU-induced defect more than dTMP, suggesting that the RNA-based toxicity of 5FU predominates in these cells. These findings underscore the importance of understanding the RNA-based mechanism of 5FU cytotoxicity and highlight the use of yeast as a model system for elucidating its details.

RNA-Based 5-Fluorouracil Toxicity Requires the Pseudouridylation Activity of Cbf5p

“…Accordingly, we asked if depletion of Cbf5p affects the RNA-based 5FU hypersensitivity of an rrp6-D mutant (Hoskins and Butler 2007). We used strains containing CBF5 under the control of the tetO 7 repressible promoter, which allows repression of CBF5 transcription by the addition of doxycycline to the media (Gari et al 1997).…”

“…Several experiments have shown that uridine, not thymidine, relieves the cytotoxic and apoptotic effects of 5FU (Engelbrecht et al 1984;Linke et al 1996;Pritchard et al 1997;Bunz et al 1999;Hoskins and Butler 2007). Furthermore, a good correlation exists between the incorporation of 5FU into RNA and the cytotoxicity of the drug, and the co-therapeutic methotrexate enhances incorporation of 5FU into RNA (Greenhalgh and Parish 1990;Parker and Cheng 1990;Longley et al 2003).…”

Fungal Homoserine Kinase ( thr1 Δ) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation

“…An additional virtue for targeting Thr1p and Thr4p is the demonstration that thr1⌬ and thr4⌬ mutants are hypersensitive to the clinically relevant drug 5-fluorocytosine, even under conditions of high threonine. Since 5-fluorocytosine is converted into 5-fluorouracil, which inhibits RNA and DNA metabolism (25,28,29,53), and thr1⌬ mutants are hypersensitive to DNA-damaging agents (2, 3), 5-fluorocytosine-induced DNA damage may explain the observed hypersensitivity. Thus, novel Thr1p or Thr4p inhibitors would have great potential as sole (C. neoformans) and combination (C. albicans) therapies.…”

Rrp6, Rrp47 and Cofactors of the Nuclear Exosome

“…[31][32][33] This effect; (i) results from incorporation of the base analogue into RNA, (ii) is abolished by a mutation in Rrp6 that inhibits its degradation, but not by one that inhibits its 3' end maturation function, (iii) correlates with the accumulation of poly(A)+ RNA degradation intermediates and (iv) is independent of the transcription-coupled DNA repair pathway. 32,34 Moreover, hypersensitivity of rrp6-∆ strains to 5FU requires a catalytically active pseudouridine synthetase, Cbf5. 35 Some pseudouridine synthetases cannot convert 5FU…”