Yeast Assay Highlights the Intrinsic Genomic Instability of Human PML Intron 6 over Intron 3 and the Role of Replication Fork Proteins.

Human acute promyelocytic leukemia (APL) is characterized by a specific balanced translocation t(15;17)(q22;q21) involving the PML and RARA genes. In both de novo and therapy-related APL, the most frequent PML breakpoints are located within intron 6, and less frequently in intron 3; the precise mechanisms by which these breakpoints arise and preferentially in PML intron 6 remain unsolved. To investigate the intrinsic properties of the PML intron sequences in vivo, we designed Saccharomyces cerevisiae strains containing human PML intron 6 or intron 3 sequences inserted in yeast chromosome V and measured gross chromosomal rearrangements (GCR). This approach provided evidence that intron 6 had a superior instability over intron 3 due to an intrinsic property of the sequence and identified the 3' end of intron 6 as the most susceptible to break. Using yeast strains invalidated for genes that control DNA replication, we show that this differential instability depended at least upon Rrm3, a DNA helicase, and Mrc1, the human claspin homolog. GCR induction by hydrogen peroxide, a general genotoxic agent, was also dependent on genetic context. We conclude that: 1) this yeast system provides an alternative approach to study in detail the properties of human sequences in a genetically controlled situation and 2) the different susceptibility to produce DNA breaks in intron 6 versus intron 3 of the human PML gene is likely due to an intrinsic property of the sequence and is under replication fork genetic control.

Loss of the thioredoxin reductase Trr1 suppresses the genomic instability of peroxiredoxin tsa1 mutants.

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a Can(R) mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the Can(R) mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H2O2 bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.

Selective cytotoxicity of Aniba rosaeodora essential oil towards epidermoid cancer cells through induction of apoptosis.

Essential oils are complex mixtures of odorous and volatile compounds derived from secondary plant metabolism. They can be isolated from many plants by mechanical pressing or hydro- and steam-distillation and are known to induce a wide range of biological effects through their antibacterial, antifungal, cytotoxic, antioxidant and antimutagenic activities. In order to explore their beneficial properties on human skin cells, we investigated the effects of an essential oil from rosewood Aniba rosaeodora (REO) on the human epidermoid carcinoma cell line A431, on immortal HaCaT cells thought to represent an early stage of skin carcinogenesis, on transformed normal HEK001 keratinocytes and on primary normal NHEK keratinocytes. In a defined range of concentrations, REO selectively killed A431 and HaCaT cells. The same treatments had only a minor cytotoxic effect on HEK001 and NHEK cells. Preferentially in A431 and HaCaT cells, REO triggered the production of reactive oxygen species, induced depolarization of the mitochondrial membrane and caused caspase-dependent cell death characterized by phosphatidylserine externalization, an early marker of apoptosis. Both intrinsic and extrinsic apoptotic pathways were implicated in REO-induced cell death. The identification of selective induction of apoptosis in precancerous and cancerous skin cells by REO highlights the potential anticancer activity of this essential oil.

Peroxiredoxin Tsa1 is the key peroxidase suppressing genome instability and protecting against cell death in Saccharomyces cerevisiae.

Peroxiredoxins (Prxs) constitute a family of thiol-specific peroxidases that utilize cysteine (Cys) as the primary site of oxidation during the reduction of peroxides. To gain more insight into the physiological role of the five Prxs in budding yeast Saccharomyces cerevisiae, we performed a comparative study and found that Tsa1 was distinguished from the other Prxs in that by itself it played a key role in maintaining genome stability and in sustaining aerobic viability of rad51 mutants that are deficient in recombinational repair. Tsa2 and Dot5 played minor but distinct roles in suppressing the accumulation of mutations in cooperation with Tsa1. Tsa2 was capable of largely complementing the absence of Tsa1 when expressed under the control of the Tsa1 promoter. The presence of peroxidatic cysteine (Cys(47)) was essential for Tsa1 activity, while Tsa1(C170S) lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or tsa1(CCS) lacking the peroxidatic and resolving Cys) and recombinational repair (rad51), dying cells displayed irregular cell size/shape, abnormal cell cycle progression, and significant increase of phosphatidylserine externalization, an early marker of apoptosis-like cell death. The tsa1(CCS) rad51- or tsa1 rad51-induced cell death did not depend on the caspase Yca1 and Ste20 kinase, while the absence of the checkpoint protein Rad9 accelerated the cell death processes. These results indicate that the peroxiredoxin Tsa1, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect S. cerevisiae cells against toxic levels of DNA damage that occur during aerobic growth.