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New insights into the RNA-based mechanism of action of the anticancer drug 5'-fluorouracil in eukaryotic cells.

Mojardín L, Botet J, Quintales L, Moreno S, Salas M - PLoS ONE (2013)

Bottom Line: This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment.The transcription of several tRNA genes was also significantly induced after drug exposure.Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Cantoblanco, Madrid, Spain.

ABSTRACT
5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

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Phenotypic effects of 5FU in S. pombe cells.(A) Survival time course after 500 µM 5FU (EC50) treatment. The values represent the means ± s.d. of three independent experiments. (B) FACS analysis of the time course experiment indicating heterogeneity in DNA content at 240 min in 5FU-treated cells compared to untreated cells. (C) Microscopy analysis and DAPI staining show cells with elongated phenotype after 5FU exposure for 240 min suggesting a cell cycle delay in the presence of the drug.
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pone-0078172-g001: Phenotypic effects of 5FU in S. pombe cells.(A) Survival time course after 500 µM 5FU (EC50) treatment. The values represent the means ± s.d. of three independent experiments. (B) FACS analysis of the time course experiment indicating heterogeneity in DNA content at 240 min in 5FU-treated cells compared to untreated cells. (C) Microscopy analysis and DAPI staining show cells with elongated phenotype after 5FU exposure for 240 min suggesting a cell cycle delay in the presence of the drug.

Mentions: We performed a time course analysis of S. pombe viability after incubating cells with 500 µM 5FU (the half maximal effective concentration dose, EC50, at 240 min) in order to evaluate the phenotypic effects associated to drug treatment (Figure 1A). Flow cytometry experiments revealed that 5FU-treated cells showed an abnormal DNA content at 240 min when compared to untreated cells (Figure 1B), probably due to a delayed entry into S phase. Consistent with this result, 5FU has been described as a G1/S phase blocker in human cell lines [19]. In addition, microscopy analysis and nuclear DAPI staining showed that a proportion of the cells treated with 5FU for 240 min became elongated with a characteristic cell cycle delay phenotype (Figure 1C).


New insights into the RNA-based mechanism of action of the anticancer drug 5'-fluorouracil in eukaryotic cells.

Mojardín L, Botet J, Quintales L, Moreno S, Salas M - PLoS ONE (2013)

Phenotypic effects of 5FU in S. pombe cells.(A) Survival time course after 500 µM 5FU (EC50) treatment. The values represent the means ± s.d. of three independent experiments. (B) FACS analysis of the time course experiment indicating heterogeneity in DNA content at 240 min in 5FU-treated cells compared to untreated cells. (C) Microscopy analysis and DAPI staining show cells with elongated phenotype after 5FU exposure for 240 min suggesting a cell cycle delay in the presence of the drug.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815194&req=5

pone-0078172-g001: Phenotypic effects of 5FU in S. pombe cells.(A) Survival time course after 500 µM 5FU (EC50) treatment. The values represent the means ± s.d. of three independent experiments. (B) FACS analysis of the time course experiment indicating heterogeneity in DNA content at 240 min in 5FU-treated cells compared to untreated cells. (C) Microscopy analysis and DAPI staining show cells with elongated phenotype after 5FU exposure for 240 min suggesting a cell cycle delay in the presence of the drug.
Mentions: We performed a time course analysis of S. pombe viability after incubating cells with 500 µM 5FU (the half maximal effective concentration dose, EC50, at 240 min) in order to evaluate the phenotypic effects associated to drug treatment (Figure 1A). Flow cytometry experiments revealed that 5FU-treated cells showed an abnormal DNA content at 240 min when compared to untreated cells (Figure 1B), probably due to a delayed entry into S phase. Consistent with this result, 5FU has been described as a G1/S phase blocker in human cell lines [19]. In addition, microscopy analysis and nuclear DAPI staining showed that a proportion of the cells treated with 5FU for 240 min became elongated with a characteristic cell cycle delay phenotype (Figure 1C).

Bottom Line: This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment.The transcription of several tRNA genes was also significantly induced after drug exposure.Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Cantoblanco, Madrid, Spain.

ABSTRACT
5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

Show MeSH
Related in: MedlinePlus