Mft1, identified from a genome-wide screen of the yeast haploid mutants, mediates cell cycle arrest to counteract quinoxaline-induced toxicity

Abstract

Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest.

Keywords: Saccharomyces cerevisiae; and antitumor activities; antibacterial; antifungal; cell cycle arrest; drug resistance; genome-wide screening; quinoxaline sensitive mutants.

FIGURE 1

Identification of the QXN-sensitive yeast mutants from the haploid mutant collection. Briefly, frozen stocks of the haploid collection were allowed to grow overnight in YPD media and subjected to the robotic screen as outlined in the illustration shown in Supplementary Figure S3. Each spot represents a mutant from the collection that was spotted on solid YPD plates without and with 5 mM of QXN. Following 2 days of growth at 30°C, the plates were photographed. The opened square highlighted the mutants (e.g., ddc1Δ and mft1Δ) that are sensitive to QXN and the arrows point to the assigned gene name from the Saccharomyces Genome Database. The data are representative of three independent High-Throughput screens.

FIGURE 2

Spot test analysis confirms the sensitivity of the mutants to QXN. Cells were grown overnight, serially diluted and spotted onto plates with the indicated concentrations of QXN. The plates shown in panels (A–C) were photographed after 48 h incubation at 30°C. The QXN-sensitive strains are compared to the parent strain BY4741. The experiment was independently repeated at least three times.

FIGURE 3

Comparison of the sensitivity of the parent, the single mutants mft1Δ and ddc1Δ and the double mutant ddc1Δ mft1Δ towards QXN and HU. (A,B), The indicated strains were grown overnight, serially diluted and spotted onto plates containing QXN and HU, respectively. The data is representative of two independent experiments. The plates were photographed after 48 h incubation at 30°C. BY4741 is the parent. (C–E), Cells from overnight cultures were adjusted to OD₆₀₀ of ∼0.15 and allowed to grow in the absence (panel C) and presence (panels D and E) of the indicated QXN concentrations. The OD₆₀₀ was taken at the indicated time and plotted. The results shown are the averages of three independent experiments and the error bars indicate the standard deviation.

FIGURE 4

Cell cycle analysis of the parent and the indicated mutants in response to QXN and following recovery. The exponentially growing parent and mutant strains were untreated and treated with QXN (9 mM for 4 h), and the cells were washed and allowed to grow in fresh media without QXN. Samples were taken at 0, 30, 60 and 120 min as recovery time for FACS analysis as described in the Materials and Methods. (A–D), The percentage of cells in the indicated phases for the untreated, treated, and post-treatment (recovery) for the parent and the mutants. Error bars represent mean ± SD. *p < 0.05, **p < 0.01. See Supplementary Figure S4 for the graphic representation of the cell cycle profiles for the indicated strains.