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Yeast response to the DNA damaging beta-lapachone

ABSTRACT: β-lapachone (b-lap) is an anticancer agent that selectively induces cell death in human cancer cells. A mechanism for b-lap cytotoxicity was shown to be mediated by the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation in several tumour cells. To further characterise the molecular effects of b-lap action, we compared the gene expression profile of yeast cells treated or not with b-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were enriched in the set of differentially expressed genes. Accordingly, b-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, and inhibitor of NADH dehydrogenases (NADH-DH). In addition, a mutant defective in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to b-lap treatment, thus resembling tumour cell responses to b-lap exposure. However, b-lap treatment elicited similar ROS production in WT and nde2 cells, and dicoumarol did not affect cell viability in b-lap treated yeasts. Most interestingly, DNA damage responses triggered by b-lap were abolished in the nde2 mutant. Furthermore, the nde2 mutant was sensitive to DNA damaging agents other than b-lap. These data indicated that ROS production did not mediate b-lap cytotoxicity and highlighted a role of Nde2p in DNA damage checkpoints. Amino acid biosynthesis genes were also differentially expressed in b-lap treated cells, suggesting that b-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Supporting this, b-lap treatment increased phosphorylation of eIF2 alpha in a Gcn2p-dependent manner. eIF2alpha phosphorylation required Gcn1p and Gcn20p and surprisingly Nde2p. Gcn2p was also shown to be required for the G1 checkpoint triggered by b-lap and for cell survival. Remarkably, b-lap treatment also increased phosphorylation of eIF2 alpha in breast tumour cells, which was partially dependent on the Nde2p orthologue AMID. These findings i) suggest that Gcn2p and Nde2p are key determinants of b-lap toxicity in yeast and human cells, ii) uncover a new functional connection between Nde2p, Gcn2p and DNA damage checkpoints conserved throughout evolution and iii) suggest that pharmacological modulation of Gcn2p could provide an effective strategy for antitumour drug discovery.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE15412 | GEO | 2011/12/31

SECONDARY ACCESSION(S): PRJNA117087

REPOSITORIES: GEO

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Project description:β-lapachone (b-lap) is an anticancer agent that selectively induces cell death in human cancer cells. A mechanism for b-lap cytotoxicity was shown to be mediated by the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation in several tumour cells. To further characterise the molecular effects of b-lap action, we compared the gene expression profile of yeast cells treated or not with b-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were enriched in the set of differentially expressed genes. Accordingly, b-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, and inhibitor of NADH dehydrogenases (NADH-DH). In addition, a mutant defective in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to b-lap treatment, thus resembling tumour cell responses to b-lap exposure. However, b-lap treatment elicited similar ROS production in WT and nde2 cells, and dicoumarol did not affect cell viability in b-lap treated yeasts. Most interestingly, DNA damage responses triggered by b-lap were abolished in the nde2 mutant. Furthermore, the nde2 mutant was sensitive to DNA damaging agents other than b-lap. These data indicated that ROS production did not mediate b-lap cytotoxicity and highlighted a role of Nde2p in DNA damage checkpoints. Amino acid biosynthesis genes were also differentially expressed in b-lap treated cells, suggesting that b-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Supporting this, b-lap treatment increased phosphorylation of eIF2 alpha in a Gcn2p-dependent manner. eIF2alpha phosphorylation required Gcn1p and Gcn20p and surprisingly Nde2p. Gcn2p was also shown to be required for the G1 checkpoint triggered by b-lap and for cell survival. Remarkably, b-lap treatment also increased phosphorylation of eIF2 alpha in breast tumour cells, which was partially dependent on the Nde2p orthologue AMID. These findings i) suggest that Gcn2p and Nde2p are key determinants of b-lap toxicity in yeast and human cells, ii) uncover a new functional connection between Nde2p, Gcn2p and DNA damage checkpoints conserved throughout evolution and iii) suggest that pharmacological modulation of Gcn2p could provide an effective strategy for antitumour drug discovery. Direct comparison between wt samples and b-lapachone treated-wt samples. Four biological replicates. Dye was swapped in two of them

Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.

Project description:Two nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), control yeast growth and metabolism in response to changes in nutrient availability. Starvation for amino acids activates the GAAC pathway, involving Gcn2p phosphorylation of eIF2 and preferential translation of GCN4, a transcription activator of genes involved in amino acid metabolism. TOR senses nitrogen availability and regulates gene expression through transcription factors, such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome in response to amino acid starvation and rapamycin treatment. Of the ~2500 genes whose expression was changed by 2-fold or greater, Gcn4p and Gln3p were required for 542 and 657 genes, respectively. While Gcn4p activates a common core of 57 genes in response to amino acid starvation or rapamycin treatment, the different stress arrangements allow for variations in Gcn4p-directed transcription. With few exceptions, genes requiring Gcn2p eIF2 kinase for induced expression also required Gcn4p, emphasizing the role of Gcn2p as an upstream activator of Gcn4p-directed transcription. There is also significant coordination between the GAAC and TOR pathways, with Gcn4p being required for activation of more genes during rapamycin treatment than Gln3p. Importantly, TOR regulates the GAAC-directed transcription of genes required for assimilation of nitrogen sources, such as Î³-amino-butyric acid. Therefore, yeast has integrated gene expression responses to amino acid abundance and nitrogen source quality through the control of Gcn2p phosphorylation of eIF2 and GCN4 translation. Keywords: gene expression In this study, we carried out microarray analyses in a collection of yeast strains deleted for GCN2, GCN4, and GLN3, individually or in combinations, to explore the importance of the TOR and GAAC pathways in directing the transcriptome in response to amino acid starvation and rapamycin treatment.

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Yeast response to the DNA damaging beta-lapachone

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