RESUMEN
Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.
Asunto(s)
Inestabilidad Genómica , ARN Interferente Pequeño/fisiología , Transducción de Señal , Enfermedad de Charcot-Marie-Tooth/genética , Biología Computacional , Daño del ADN , Reparación del ADN/genética , Replicación del ADN/genética , Regulación hacia Abajo , Genes cdc , Biblioteca Genómica , Genómica , Células HeLa , Histonas/metabolismo , Humanos , Fosforilación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Cyclin B1 should have some rate limiting function for cell cycle progression. To test this, we measured the effect of siRNA-mediated depletion of cyclin B1 on mitotic entry and timing. We depleted cyclin B1 in HeLa and hTert-RPE1 cells to levels equivalent or below those achieved in the telophase-to-G(1) window. Average cyclin B1/Cdk1 activity was measured in HeLa cells and depleted by approximately 99%. In both cell lines, this caused approximately 20% increase in the G(2) and approximately 20% increase the M traverse time. However, co-depletion of cyclin B1 and B2 induced a profound increase in G(2) cells, a dramatic reduction in mitotic cells, and an increase in a 4C cycling population. We conclude that any residual levels of cyclin B1 were not sufficient to promote stable mitotic entry and transition in absence of normal levels of cyclin B2. Therefore, we conclude that B cyclin is necessary for mitosis but cyclin B1 is not. Nocodazole treated, cyclin B1-depleted HeLa cells arrested but exited that arrest at higher rates than controls, suggesting that the duration of the spindle checkpoint was affected. In B1 depleted cells, population growth was delayed but evidence of cell death was not consistently observed. A strong phenotype of mitotic chromosomal aberration was observed in HeLa cells depleted for either cyclin but not in RPE cells. In B1 or B2 depleted cells, maloriented chromosomes at metaphase were increased 10 fold and one third of affected metaphase cells entered anaphase without congression. Lagging chromosomes at anaphase were dramatically increased. The aggregate evidence from our study and others suggests that the common effect of cyclin B1 depletion is mild cell cycle perturbation. Lack of uniformity in other phenotypes suggest that these are low penetrance effects that are exacerbated or compensated in some systems by other mechanisms.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Genes cdc/fisiología , Mitosis/genética , Anafase/genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proliferación Celular , Aberraciones Cromosómicas , Segregación Cromosómica/genética , Ciclina B/genética , Ciclina B1 , Ciclina B2 , Regulación hacia Abajo/genética , Células HeLa , Humanos , Metafase/genética , Nocodazol/farmacología , Interferencia de ARN/fisiología , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Factores de TiempoRESUMEN
Many epitopes are phosphorylated during mitosis. These epitopes are useful biomarkers for mitotic cells. The most commonly used are MPM-2 and serine 10 of histone H3. Here we investigated the use of an antibody generated against a phospho peptide matching residues 774-788 of the human retinoblastoma protein 1 (Rb) to detect mitotic cells. Human cell lines were stained with DNA dyes and antibodies reactive with epitopes defined by antibody MPM-2, phospho-S10-histone-H3, and the phospho-serine peptide, TRPPTLSPIPHIPRC (phospho-S780-Rb). Immunoreactivity and DNA content were measured by flow and image cytometry. Correlation and pattern recognition analyses were performed on list mode data. Western blots and immunoprecipitation were used to investigate the number of peptides reactive with phospho-S780-Rb and the relationship between reactivity with this antibody and MPM-2. Costaining for bromodeoxyuridine (BrdU) was used to determine acid resistance of the phospho-S780-Rb epitope. Cell cycle related phospho-S780-Rb immunofluorescence correlated strongly with that of MPM-2. Laser scanning cytometry showed that phospho-S780-Rb immunofluorescence is expressed at high levels on all stages of mitotic cells. Western blotting and immunoprecipitation showed that the epitope is expressed on several peptides including Rb protein. Costaining of BrdU showed that the epitope is stable to acid. Kinetic experiments showed utility in complex cell cycle analysis aimed at measuring cell cycle transition state timing. The phospho-S780-Rb epitope is a robust marker of mitosis that allows cytometric detection of mitotic cells beginning with chromatin condensation and ending after cytokinesis. Costaining of cells with DNA dyes allows discrimination and counting of mitotic cells and post-cytokinetic ("newborn") cells. To facilitate use without confusion about specificity, we suggest the trivial name, pS780 for this mitotic epitope.
Asunto(s)
Biomarcadores de Tumor , Biología Celular , Citometría de Flujo/métodos , Mitosis , Ciclo Celular , Línea Celular Tumoral , Técnicas Citológicas/métodos , Células HeLa , Humanos , Células K562 , Microscopía Confocal/métodos , Fosforilación , Proteína de Retinoblastoma/metabolismoRESUMEN
An essential component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV-induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/efectos de la radiación , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN/metabolismo , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN , ADN Polimerasa Dirigida por ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Humanos , Ratones , Oocitos/fisiología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Rayos Ultravioleta , Proteínas de Xenopus , Xenopus laevis , Proteína de la Xerodermia Pigmentosa del Grupo A/genéticaRESUMEN
Current models suggest that cyclin B1/cdk1 regulates the G2 to M transition and that its activity is maximal during the period from prophase to metaphase in mammalian cells. Although data are lacking, the idea that cyclin B1/cdk1 regulates the transit time from prophase to metaphase is reasonable. Development of small molecule inhibitors of cyclin dependent kinases (cdk's) as cancer therapeutics presents an opportunity to evaluate the effects of inhibiting cdk's in asynchronous cell populations. Analysis of cdk1 inhibitors is complicated by their ability to inhibit other cdk's in vitro at higher concentrations. In this study we measured the effects of two cdk1 inhibitors on S, G2, and M transit for Hela cells and correlated these effects on cyclin B1/cdk1 and cyclin A/cdk2 activities. Dose responses demonstrate that low concentrations of both compounds inhibited the activity of cdk1 but not cdk2 in HeLa cells. The partial loss of cdk1 activity at low doses induced a prophase accumulation during a 3 h period and an increased transit time through mitosis. In addition, both inhibitors lengthened the G2 transit time with progressively greater effect on mid and late G2. High doses of both inhibitors increased the S phase time, which correlated with the inhibition of cdk2 activity. These results suggest that cdk1-cyclin activity is rate limiting for cell cycle progression during a period from mid G2 through prophase.