RESUMEN
Lamin A is a main constituent of the nuclear lamina and contributes to nuclear shaping, mechano-signaling transduction and gene regulation, thus affecting major cellular processes such as cell cycle progression and entry into senescence, cellular differentiation and stress response. The role of lamin A in stress response is particularly intriguing, yet not fully elucidated, and involves prelamin A post-translational processing. Here, we propose prelamin A as the tool that allows lamin A plasticity during oxidative stress response and permits timely 53BP1 recruitment to DNA damage foci. We show that while PCNA ubiquitination, p21 decrease and H2AX phosphorylation occur soon after stress induction in the absence of prelamin A, accumulation of non-farnesylated prelamin A follows and triggers recruitment of 53BP1 to lamin A/C complexes. Then, the following prelamin A processing steps causing transient accumulation of farnesylated prelamin A and maturation to lamin A reduce lamin A affinity for 53BP1 and favor its release and localization to DNA damage sites. Consistent with these observations, accumulation of prelamin A forms in cells under basal conditions impairs histone H2AX phosphorylation, PCNA ubiquitination and p21 degradation, thus affecting the early stages of stress response. As a whole, our results are consistent with a physiological function of prelamin A modulation during stress response aimed at timely recruitment/release of 53BP1 and other molecules required for DNA damage repair. In this context, it becomes more obvious how farnesylated prelamin A accumulation to toxic levels alters timing of DNA damage signaling and 53BP1 recruitment, thus contributing to cellular senescence and accelerated organismal aging as observed in progeroid laminopathies.
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Heat shock activates the transcription of arrays of Satellite III (SatIII) DNA repeats in the pericentromeric heterochromatic domains of specific human chromosomes, the longest of which is on chromosome 9. Long non-coding SatIII RNAs remain associated with transcription sites where they form nuclear stress bodies or nSBs. The biology of SatIII RNAs is still poorly understood. Here, we show that SatIII RNAs and nSBs are detectable up to four days after thermal stress and are linked to defects in chromosome behavior during mitosis. Heat shock perturbs the execution of mitosis. Cells reaching mitosis during the first 3 h of recovery accumulate in pro-metaphase. During the ensuing 48 h, this block is no longer detectable; however, a significant fraction of mitoses shows chromosome segregation defects. Notably, most of lagging chromosomes and chromosomal bridges are bound to nSBs and contain arrays of SatIII DNA. Disappearance of mitotic defects at the end of day 2 coincides with the processing of long non-coding SatIII RNAs into a ladder of small RNAs associated with chromatin and ranging in size from 25 to 75 nt. The production of these molecules does not rely on DICER and Argonaute 2 components of the RNA interference apparatus. Thus, massive transcription of SatIII DNA may contribute to chromosomal instability.
Asunto(s)
Cromosomas Humanos/metabolismo , ADN Satélite/metabolismo , Factores de Transcripción del Choque Térmico/genética , ARN Largo no Codificante/metabolismo , Segregación Cromosómica , Células HeLa , Humanos , Mitosis , ARN Pequeño no Traducido/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Protein acetylation and deacetylation events are finely regulated by lysine-acetyl-transferases and lysine-deacetylases and constitute an important tool for the activation or inhibition of specific cellular pathways. One of the most important lysine-acetyl-transferases is p300, which is involved in the regulation of gene expression, cell growth, DNA repair, differentiation, apoptosis, and tumorigenesis. A well-known target of p300 is constituted by the tumor suppressor protein p53, which plays a critical role in the maintenance of genomic stability and whose activity is known to be controlled by post-translational modifications, among which acetylation. p300 activity toward p53 is negatively regulated by the NAD-dependent deacetylase SIRT1, which deacetylates p53 preventing its transcriptional activation and the induction of p53-dependent apoptosis. However, the mechanisms responsible for p53 regulation by p300 and SIRT1 are still poorly understood. Here we identify the nucleosome assembly protein TSPY-Like 2 (TSPYL2, also known as TSPX, DENTT, and CDA1) as a novel regulator of SIRT1 and p300 function. We demonstrate that, upon DNA damage, TSPYL2 inhibits SIRT1, disrupting its association with target proteins, and promotes p300 acetylation and activation, finally stimulating p53 acetylation and p53-dependent cell death. Indeed, in response to DNA damage, cells silenced for TSPYL2 were found to be defective in p53 activation and apoptosis induction and these events were shown to be dependent on SIRT1 and p300 function. Collectively, our results shed new light on the regulation of p53 acetylation and activation and reveal a novel TSPYL2 function with important implications in cancerogenesis.
Asunto(s)
Proteína p300 Asociada a E1A/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Sirtuina 1/genética , Proteína p53 Supresora de Tumor/genética , Células A549 , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Proliferación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Procesamiento Proteico-Postraduccional/genética , Activación Transcripcional/efectos de los fármacos , GemcitabinaRESUMEN
Alternative splicing is a pervasive mechanism that molds the transcriptome to meet cell and organism needs. However, how this layer of gene expression regulation is coordinated with other aspects of the cell metabolism is still largely undefined. Glucose is the main energy and carbon source of the cell. Not surprisingly, its metabolism is finely tuned to satisfy growth requirements and in response to nutrient availability. A number of studies have begun to unveil the connections between glucose metabolism and splicing programs. Alternative splicing modulates the ratio between M1 and M2 isoforms of pyruvate kinase in this way determining the choice between aerobic glycolysis and complete glucose oxidation in the Krebs cycle. Reciprocally, intermediates in the Krebs cycle may impact splicing programs at different levels by modulating the activity of 2-oxoglutarate-dependent oxidases. In this review we discuss the molecular mechanisms that coordinate alternative splicing programs with glucose metabolism, two aspects with profound implications in human diseases.
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Genome integrity is continuously threatened by endogenous sources of DNA damage including reactive oxygen species (ROS) produced by cell metabolism. Factors of the RNA interference (RNAi) machinery have been recently involved in the cellular response to DNA damage (DDR) in proliferating cells. To investigate the impact of component of RNAi machinery on DDR activation in terminally differentiated cells, we exploited cytoplasmic hybrid (cybrid) cell lines in which mitochondria of sporadic Parkinson's disease patients repopulate neuroblastoma SH-SY5Y-Rho(0) cells. Upon differentiation into dopaminergic neuron-like cells, PD63 cybrid showed increased intracellular level of ROS and chronic DDR activation, compared to other cybrids with the same nuclear background. Importantly, DDR activation in these cells can be prevented by ROS scavenging treatment suggesting that ROS production is indeed causative of nuclear DNA damage. Sequence analysis of the mitogenomes identified a rare and heteroplasmic missense mutation affecting a highly conserved residue of the ND5-subunit of respiratory complex I, which accounts for ROS increase. We demonstrated that the assembly of nuclear DDR foci elicited by oxidative stress in these cells relies on DROSHA, providing the first evidence that components of RNAi machinery play a crucial role also in the mounting of ROS-induced DDR in non-replicating neuronal cells.
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Daño del ADN , Mutación Missense , NADH Deshidrogenasa/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ribonucleasa III/metabolismo , Alelos , Secuencia de Aminoácidos , Diferenciación Celular , Línea Celular , Citoplasma/metabolismo , Histonas/metabolismo , Humanos , NADH Deshidrogenasa/química , FosforilaciónRESUMEN
Etoposide derives from podophyllotoxin, a toxin found in the American Mayapple. It was first synthesized in 1966 and approved for cancer therapy in 1983 by the U.S. Food and Drug Administration (Hande, 1998[25]). Starting from 1980s several studies demonstrated that etoposide targets DNA topoisomerase II activities thus leading to the production of DNA breaks and eliciting a response that affects several aspects of cell metabolisms. In this review we will focus on molecular mechanisms that account for the biological effect of etoposide.
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Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.
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Forma de la Célula/genética , ADN Ligasas/genética , Replicación del ADN/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Western Blotting , Adhesión Celular/genética , Ciclo Celular/genética , Línea Celular , Línea Celular Transformada , Movimiento Celular/genética , Daño del ADN , ADN Ligasa (ATP) , ADN Ligasas/deficiencia , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Microscopía Fluorescente , Mutación , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de Tiempo/métodosRESUMEN
Alternative splicing emerges as a potent and pervasive mechanism of gene expression regulation that expands the coding capacity of the genome and forms an intermediate layer of regulation between transcriptional and post-translational networks. Indeed, alternative splicing occupies a pivotal position in developmental programs and in the cell response to external and internal stimuli. Not surprisingly, therefore, its deregulation frequently leads to human disease. In this review we provide an updated overview of the impact of alternative splicing on tumorigenesis. Moreover, we discuss the intricacy of the reciprocal interactions between alternative splicing programs and signal transduction pathways, which appear to be crucially linked to cancer progression in response to the tumor microenvironment. Finally, we focus on the recently described interplay between splicing and chromatin organization which is expected to shed new lights into gene expression regulation in normal and cancer cells.
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Empalme Alternativo , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Transducción de Señal/genética , Transición Epitelial-Mesenquimal/genética , Humanos , Modelos Genéticos , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina , Microambiente Tumoral/genéticaRESUMEN
BACKGROUND: The amount of gene expression data available in public repositories has grown exponentially in the last years, now requiring new data mining tools to transform them in information easily accessible to biologists. RESULTS: By exploiting expression data publicly available in the Gene Expression Omnibus (GEO) database, we developed a new bioinformatics tool aimed at the identification of genes whose expression appeared simultaneously altered in different experimental conditions, thus suggesting co-regulation or coordinated action in the same biological process. To accomplish this task, we used the 978 human GEO Curated DataSets and we manually performed the selection of 2,109 pair-wise comparisons based on their biological rationale. The lists of differentially expressed genes, obtained from the selected comparisons, were stored in a PostgreSQL database and used as data source for the CorrelaGenes tool. Our application uses a customized Association Rule Mining (ARM) algorithm to identify sets of genes showing expression profiles correlated with a gene of interest. The significance of the correlation is measured coupling the Lift, a well-known standard ARM index, and the χ(2) p value. The manually curated selection of the comparisons and the developed algorithm constitute a new approach in the field of gene expression profiling studies. Simulation performed on 100 randomly selected target genes allowed us to evaluate the efficiency of the procedure and to obtain preliminary data demonstrating the consistency of the results. CONCLUSIONS: The preliminary results of the simulation showed how CorrelaGenes could contribute to the characterization of molecular pathways and biological processes integrating data obtained from other applications and available in public repositories.
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Perfilación de la Expresión Génica/métodos , Transcriptoma , Algoritmos , Minería de Datos , Regulación hacia Abajo , Humanos , Internet , Regulación hacia ArribaRESUMEN
It is well-known that DNA-damaging agents induce genome instability, but only recently have we begun to appreciate that chromosomes are fragile per se and frequently subject to DNA breakage. DNA replication further magnifies such fragility, because it leads to accumulation of single-stranded DNA. Recent findings suggest that chromosome fragility is similarly increased during transcription. Transcripts produced by RNA polymerase II (RNAPII) are subject to multiple processing steps, including maturation of 5' and 3' ends and splicing, followed by transport to the cytoplasm. RNA maturation starts on nascent transcripts and is mediated by a number of diverse proteins and ribonucleoprotein particles some of which are recruited cotranscriptionally through interactions with the carboxy-terminal domain of RNAPII. This coupling is thought to maximize efficiency of pre-mRNA maturation and directly impacts the choice of alternative splice sites. Mounting evidence suggests that lack of coordination among different RNA maturation steps, by perturbing the interaction of nascent transcripts with the DNA template, has deleterious effects on genome stability. Thus, in the absence of proper surveillance mechanisms, transcription could be a major source of DNA damage in cancer. Recent high-throughput screenings in human cells and budding yeast have identified several factors implicated in RNA metabolism that are targets of DNA damage checkpoint kinases: ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3 related) (Tel1 and Mec1 in budding yeast, respectively). Moreover, inactivation of various RNA processing factors induces accumulation of γH2AX foci, an early sign of DNA damage. Thus, a complex network is emerging that links DNA repair and RNA metabolism. In this review we provide a comprehensive overview of the role played by pre-mRNA processing factors in the cell response to DNA damage and in the maintenance of genome stability.
RESUMEN
In this work, we show that vertical, high aspect-ratio (HAR) photonic crystals (PhCs), consisting of periodic arrays of 5 µm wide gaps with depth of 50 µm separated by 3 µm thick silicon walls, fabricated by electrochemical micromachining, can be used as three-dimensional microincubators, allowing cell lines to be selectively grown into the gaps. Silicon micromachined dice incorporating regions with different surface profiles, namely flat silicon and deeply etched PhC, were used as microincubators for culturing adherent cell lines with different morphology and adhesion properties. We extensively investigated and compared the proliferative behavior on HAR PhCs of eight human cell models, with different origins, such as the epithelial (SW613-B3; HeLa; SW480; HCT116; HT29) and the mesenchymal (MRC-5V1; CF; HT1080). We also verified the contribution of cell sedimentation into the silicon gaps. Fluorescence microscopy analysis highlights that only cell lines that exhibit, in the tested culture condition, the behavior typical of the mesenchymal phenotype are able to penetrate into the gaps of the PhC, extending their body deeply in the narrow gaps between adjacent silicon walls, and to grow adherent to the vertical surfaces of silicon. Results reported in this work, confirmed in various experiments, strongly support our statement that such three-dimensional microstructures have selection capabilities with regard to the cell lines that can actively populate the narrow gaps. Cells with a mesenchymal phenotype could be exploited in the next future as bioreceptors, in combination with HAR PhC optical transducers, e.g., for label-free optical detection of cellular activities involving changes in cell adhesion and/or morphology (e.g., apoptosis) in a three-dimensional microenvironment.
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Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Microtecnología/instrumentación , Microtecnología/métodos , Fotones , Silicio/química , Línea Celular , Cristalización , Células Epiteliales/citología , Humanos , Microscopía FluorescenteRESUMEN
This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.
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ADN Ligasas/deficiencia , Fibroblastos/metabolismo , Proteoma/análisis , Línea Celular Transformada , Cromatografía Liquida , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/química , ADN Ligasa (ATP) , Electroforesis en Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogéneas/análisis , Ribonucleoproteínas Nucleares Heterogéneas/química , Humanos , Concentración de Iones de Hidrógeno , Pliegue de Proteína , Proteínas/análisis , Proteínas/química , Proteínas/clasificación , Proteoma/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.
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Daño del ADN , Replicación del ADN , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Línea Celular Transformada , ADN Ligasa (ATP) , ADN Ligasas/genética , Humanos , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Fosforilación , Proteómica , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina , Estrés Fisiológico/genéticaRESUMEN
BACKGROUND: Terminally differentiated (TD) cells permanently exit the mitotic cycle while acquiring specialized characteristics. Although TD cells can be forced to reenter the cell cycle by different means, they cannot be made to stably proliferate, as attempts to induce their replication constantly result in cell death or indefinite growth arrest. There is currently no biological explanation for this failure. PRINCIPAL FINDINGS: Here we show that TD mouse myotubes, reactivated by depletion of the p21 and p27 cell cycle inhibitors, are unable to complete DNA replication and sustain heavy DNA damage, which triggers apoptosis or results in mitotic catastrophe. In striking contrast, quiescent, non-TD fibroblasts and myoblasts, reactivated in the same way, fully replicate their DNA, do not suffer DNA damage, and proliferate even in the absence of growth factors. Similar results are obtained when myotubes and fibroblasts are reactivated by forced expression of E1A or cyclin D1 and cdk4. CONCLUSIONS: We conclude that the inability of myotubes to complete DNA replication must be ascribed to peculiar features inherent in their TD state, rather than to the reactivation method. On reviewing the literature concerning reactivation of other TD cell types, we propose that similar mechanisms underlie the general inability of all kinds of TD cells to proliferate in response to otherwise mitogenic stimuli. These results define an unexpected basis for the well known incompetence of mammalian postmitotic cells to proliferate. Furthermore, this trait might contribute to explain the inability of these cells to play a role in tissue repair, unlike their counterparts in extensively regenerating species.
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Diferenciación Celular/fisiología , Replicación del ADN/fisiología , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiología , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Ensayo Cometa , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Replicación del ADN/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Mitosis/genética , Mitosis/fisiología , Interferencia de ARNRESUMEN
Fragile sites are hot spots for sister chromatid exchanges, translocations, deletions, complex rearrangements, and gene amplification. It has been hypothesized that rearrangements at fragile sites derive from unreplicated regions resulting from stalled forks that escape the ATR replication checkpoint. In the present study, we investigated the role of the Claspin (CLSPN) gene, which codes for an adaptor protein in the ATR pathway, during DNA replication stress in human cells. We show that the inhibition of the CLSPN gene leads to both genome instability and fragile site expression. Following aphidicolin treatment, we found a transient increase of Claspin synthesis due to its requirement to checkpoint activation. However, Claspin synthesis decreased after a prolonged aphidicolin treatment. We propose that CLSPN modulation, following an extreme replication block, allows rare cells to escape checkpoint mechanisms and enter mitosis with a defect in genome assembly. Our observations provide the basis for a better understanding of cell cycle checkpoints deregulation in cancer.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Aberraciones Cromosómicas , Sitios Frágiles del Cromosoma/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Afidicolina/farmacología , Western Blotting , Células Cultivadas , Replicación del ADN , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Hibridación Fluorescente in Situ , MitosisRESUMEN
DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.
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Proteínas de Ciclo Celular/metabolismo , ADN Ligasas/metabolismo , Replicación del ADN/fisiología , Proteínas Nucleares/metabolismo , Anticuerpos/análisis , Línea Celular , Daño del ADN/genética , Daño del ADN/fisiología , ADN Ligasa (ATP) , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunoprecipitación , Modelos Biológicos , Unión Proteica , Proteómica/métodosRESUMEN
46BR.1G1 cells derive from a patient with a genetic syndrome characterized by drastically reduced replicative DNA ligase I (LigI) activity and delayed joining of Okazaki fragments. Here we show that the replication defect in 46BR.1G1 cells results in the accumulation of both single-stranded and double-stranded DNA breaks. This is accompanied by phosphorylation of the H2AX histone variant and the formation of gammaH2AX foci that mark damaged DNA. Single-cell analysis demonstrates that the number of gammaH2AX foci in LigI-defective cells fluctuates during the cell cycle: they form in S phase, persist in mitosis, and eventually diminish in G(1) phase. Notably, replication-dependent DNA damage in 46BR.1G1 cells only moderately delays cell cycle progression and does not activate the S-phase-specific ATR/Chk1 checkpoint pathway that also monitors the execution of mitosis. In contrast, the ATM/Chk2 pathway is activated. The phenotype of 46BR.1G1 cells is efficiently corrected by the wild-type LigI but is worsened by a LigI mutant that mimics the hyperphosphorylated enzyme in M phase. Notably, the expression of the phosphomimetic mutant drastically affects cell morphology and the organization of the cytoskeleton, unveiling an unexpected link between endogenous DNA damage and the structural organization of the cell.
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Ciclo Celular , Daño del ADN , ADN Ligasas/deficiencia , Replicación del ADN , Línea Celular , Forma de la Célula , Citoesqueleto/patología , Roturas del ADN de Doble Cadena , ADN Ligasa (ATP) , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Mutantes , Fosforilación , Piel/citologíaRESUMEN
Ribosomal S6 kinase 2 (S6K2) acts downstream of the mammalian target of rapamycin (mTOR). Here, we show that some S6K2 localize at the centrosome throughout the cell cycle. S6K2 is found in the pericentriolar area of the centrosome. S6K2 centrosomal localization is unaffected by serum withdrawal or treatment with rapamycin, wortmannin, U0126, or phorbol-12-myristate-13-acetate (PMA). Unlike S6K2, S6 kinase 1 (S6K1) does not localize at the centrosome, suggesting the two kinases may also have nonoverlapping functions. Our data suggest that centrosomal S6K2 may have a role in the phosphoinositide-3-kinase (PI3K)/Akt/mTOR signaling pathway that has also been detected in the centrosome.
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Ciclo Celular/fisiología , Centrosoma/enzimología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/fisiología , Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TORRESUMEN
Etoposide is a potent anti-tumor drug that belongs to the class of topoisomerase poisons. Although its molecular target, i.e. DNA topoisomerase II, has been identified more than 20 years ago, the cellular response to etoposide is still poorly understood. The cytotoxicity of the drug stems from its ability to stabilize a covalent complex between DNA topoisomerase II and DNA that results in a high level of DNA damage. Here, we review the present knowledge about the strategy used by the cells to deal with the etoposide-induced DNA damage. New and unanticipated effects of topoisomerase II poisoning on cell metabolism are recently emerging, among which the ability to activate cell cycle checkpoint pathways and to affect gene expression at different levels, including chromatin remodeling and alternative splicing of gene transcripts. The elucidation of the effects of etoposide on cell metabolism will increase our ability to exploit this drug in cancer therapy and will expand our comprehension of the cancerous cell.