RESUMEN
Accumulation of methionine sulfoxide (Met(O)) is a significant feature of human cataract and previous studies have shown that methionine sulfoxide reductase A (MsrA), which acts to repair Met(O), can defend human lens cells against oxidative stress induced cell death. A key feature of oxidative stress is increased reactive oxygen species (ROS) in association with loss of mitochondrial function. Here, we sought to establish a potential role for MsrA in the accumulation of ROS in lens cells and the corresponding mitochondrial membrane potential in these cells. Targeted gene silencing was used to establish populations of lens cells expressing different levels of MsrA, and the mitochondrial membrane potential and ROS levels of these cell populations were monitored. Decreased MsrA levels were found to be associated with loss of cell viability, decreased mitochondrial membrane potential, and increased ROS levels in the absence of oxidative stress. These effects were augmented upon oxidative stress treatment. These results provide evidence that MsrA is a major determinant for accumulation of ROS in lens cells and that increased ROS levels in lens cells are associated with a corresponding decrease in mitochondrial membrane potential that is likely related to the requirement for MsrA in lens cell viability.
Asunto(s)
Silenciador del Gen/fisiología , Cristalino/metabolismo , Mitocondrias/fisiología , Oxidorreductasas/genética , Especies Reactivas de Oxígeno/metabolismo , Supervivencia Celular/genética , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Potenciales de la Membrana/fisiología , Metionina Sulfóxido Reductasas , Estrés Oxidativo/fisiología , Oxidorreductasas/análisis , ARN Interferente Pequeño/genéticaRESUMEN
Previous studies have indicated that replication stress can trigger apoptosis-like cell death, accompanied (where tested) by production of reactive oxygen species (ROS), in mammalian cells and budding yeast (Saccharomyces cerevisiae). In mammalian cells, inappropriate entry into mitosis also leads to cell death. Here, we report similar responses in fission yeast (Schizosaccharomyces pombe). We used ROS- and death-specific fluorescent stains to measure the effects of mutations in replication initiation and checkpoint genes in fission yeast on the frequencies of ROS production and cell death. We found that certain mutant alleles of each of the four tested replication initiation genes caused elevated ROS and cell death. Where tested, these effects were not enhanced by checkpoint-gene mutations. Instead, when cells competent for replication but defective in both the replication and damage checkpoints were treated with hydroxyurea, which slows replication fork movement, the frequencies of ROS production and cell death were greatly increased. This was a consequence of elevated CDK activity, which permitted inappropriate entry into mitosis. Thus, studies in fission yeast are likely to prove helpful in understanding the pathways that lead from replication stress and inappropriate mitosis to cell death in mammalian cells.
Asunto(s)
División Celular/fisiología , Replicación del ADN , Mitosis/fisiología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Schizosaccharomyces/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/fisiología , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Genes cdc , Humanos , Hidroxiurea/metabolismo , Mutación , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Schizosaccharomyces/citología , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
PURPOSE: Methionine-sulfoxide reductases are unique, in that their ability to repair oxidized proteins and MsrA, which reduces S-methionine sulfoxide, can protect lens cells against oxidative stress damage. To date, the roles of MsrB1, -B2 and -B3 which reduce R-methionine sulfoxide have not been established for any mammalian system. The present study was undertaken to identify those MsrBs expressed by the lens and to evaluate the enzyme activities, expression patterns, and abilities of the identified genes to defend lens cells against oxidative stress damage. METHODS: Enzyme activities were determined with bovine lens extracts. The identities and spatial expression patterns of MsrB1, -B2, and -B3 transcripts were examined by RT-PCR in human lens and 21 other tissues. Oxidative stress resistance was measured using short interfering (si)RNA-mediated gene-silencing in conjunction with exposure to tert-butyl hydroperoxide (tBHP) and MTS viability measurements in SRA04/01 human lens epithelial cells. RESULTS: Forty percent of the Msr enzyme activity present in the lens was MsrB, whereas the remaining enzyme activity was MsrA. MsrB1 (selenoprotein R, localized in the cytosol and nucleus), MsrB2 (CBS-1, localized in the mitochondria), and MsrB3 (localized in the endoplasmic reticulum and mitochondria) were all expressed by the lens. These genes exhibit asymmetric expression patterns between different human tissues and different lens sublocations, including lens fibers. All three genes are required for lens cell viability, and their silencing in lens cells results in increased oxidative-stress-induced cell death. CONCLUSIONS: The present data suggest important roles for both MsrA and -Bs in lens cell viability and oxidative stress protection. The differential tissue distribution and lens expression patterns of these genes, coupled with increased oxidative-stress-induced cell death on their deletion provides evidence that they are important for lens cell function, resistance to oxidative stress, and, potentially, cataractogenesis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Cristalino/enzimología , Estrés Oxidativo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Supervivencia Celular , Células Cultivadas , Células Epiteliales/enzimología , Silenciador del Gen/fisiología , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cristalino/citología , Metionina Sulfóxido Reductasas , Proteínas de Microfilamentos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Factores de Transcripción , terc-Butilhidroperóxido/farmacologíaRESUMEN
In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.
Asunto(s)
Apoptosis , Ciclo Celular/fisiología , Daño del ADN/efectos de los fármacos , Replicación del ADN , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Alquilantes/farmacología , Animales , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Saccharomyces cerevisiae/citología , Schizosaccharomyces/citologíaRESUMEN
The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase epsilon) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.