RESUMO
DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.
Assuntos
Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA , Estresse Oxidativo , Transporte Ativo do Núcleo Celular , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Inibidor de Quinase Dependente de Ciclina p19/genética , Reparo do DNA , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal , Ligação ProteicaRESUMO
The elucidation of the molecular mechanisms underlying the effects of traditional Chinese medicines in clinical practice is a key step toward their worldwide application, and this topic is currently a subject of intense research interest. Rg1, a component of ginsenoside, has recently been shown to perform several pharmacological functions; however, the underlying mechanisms of these effects remain unclear. In the present study, we investigated whether Rg1 has an anti-senescence effect on hematopoietic stem cells (HSCs) and the possible molecular mechanisms driving any effects. The results showed that Rg1 could effectively delay tert-butyl hydroperoxide (t-BHP)-induced senescence and inhibit gene expression in the p16(INK4a)-Rb and p19(Arf)-p53-p21(Cip/Waf1) signaling pathways in HSCs. Our study suggested that these two signaling pathways might be potential targets for elucidating the molecular mechanisms of the Rg1 anti-senescence effect.
Assuntos
Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p19/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ginsenosídeos/farmacologia , Células-Tronco Hematopoéticas/citologia , Proteína do Retinoblastoma/genética , Proteína Supressora de Tumor p53/genética , Animais , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/química , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismo , terc-Butil Hidroperóxido/farmacologiaRESUMO
DNA damage, which perturbs genomic stability, has been linked to cognitive decline in the aging human brain, and mutations in DNA repair genes have neurological implications. Several studies have suggested that DNA damage is also increased in brain disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise mechanisms connecting DNA damage with neurodegeneration remain poorly understood. CDK5, a critical enzyme in the development of the central nervous system, phosphorylates a number of synaptic proteins and regulates dendritic spine morphogenesis, synaptic plasticity and learning. In addition to these physiological roles, CDK5 has been involved in the neuronal death initiated by DNA damage. We hypothesized that p19INK4d, a member of the cell cycle inhibitor family INK4, is involved in a neuroprotective mechanism activated in response to DNA damage. We found that in response to genotoxic injury or increased levels of intracellular calcium, p19INK4d is transcriptionally induced and phosphorylated by CDK5 which provides it with greater stability in postmitotic neurons. p19INK4d expression improves DNA repair, decreases apoptosis and increases neuronal survival under conditions of genotoxic stress. Our in vivo experiments showed that decreased levels of p19INK4d rendered hippocampal neurons more sensitive to genotoxic insult resulting in the loss of cognitive abilities that rely on the integrity of this brain structure. We propose a feedback mechanism by which the neurotoxic effects of CDK5-p25 activated by genotoxic stress or abnormal intracellular calcium levels are counteracted by the induction and stabilization of p19INK4d protein reducing the adverse consequences on brain functions.
Assuntos
Cálcio/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Reparo do DNA/genética , Hipocampo/metabolismo , Neurônios/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Cognição/fisiologia , Quinase 5 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p19/genética , Citotoxinas/farmacologia , Dano ao DNA , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais , Transcrição Gênica , Zinostatina/farmacologiaRESUMO
BACKGROUND: During evolution, organisms with renewable tissues have developed mechanisms to prevent tumorigenesis, including cellular senescence and apoptosis. Cellular senescence is characterized by a permanent cell cycle arrest triggered by both endogenous stress and exogenous stress. The p19INK4d, a member of the family of cyclin-dependent kinase inhibitors (INK4), plays an important role on cell cycle regulation and in the cellular DNA damage response. We hypothesize that p19INK4d is a potential factor involved in the onset and/or maintenance of the senescent state. METHODS: Senescence was confirmed by measuring the cell cycle arrest and the senescence-associated ß-galactosidase activity. Changes in p19INK4d expression and localization during senescence were determined by Western blot and immunofluorescence assays. Chromatin condensation was measured by microccocal nuclease digestion and histone salt extraction. RESULTS: The data presented here show for the first time that p19INK4d expression is up-regulated by different types of senescence. Changes in senescence-associated hallmarks were driven by modulation of p19 expression indicating a direct link between p19INK4d induction and the establishment of cellular senescence. Following a senescence stimulus, p19INK4d translocates to the nucleus and tightly associates with chromatin. Moreover, reduced levels of p19INK4d impair senescence-related global genomic heterochromatinization. Analysis of p19INK4d mRNA and protein levels in tissues from differently aged mice revealed an up-regulation of p19INK4d that correlates with age. CONCLUSION: We propose that p19INK4d participates in the cellular mechanisms that trigger senescence by contributing to chromatin compaction. GENERAL SIGNIFICANCE: This study provides novel insights into the dynamics process of cellular senescence, a central tumor suppressive mechanism.
Assuntos
Envelhecimento/genética , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p19/genética , Heterocromatina/genética , Animais , Carcinogênese , Pontos de Checagem do Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA/genética , Regulação da Expressão Gênica , Camundongos , beta-Galactosidase/biossínteseRESUMO
The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.
Assuntos
Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Cloroquina/farmacologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/metabolismo , Humanos , Modelos Biológicos , Mutagênicos/toxicidade , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Proteínas Supressoras de Tumor/metabolismo , Raios UltravioletaRESUMO
DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, ß-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with (32)P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/genética , Reparo do DNA/genética , DNA/genética , Peptídeos beta-Amiloides/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cisplatino/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células HEK293 , Humanos , Mutação , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Raios UltravioletaRESUMO
Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.
Assuntos
Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA , Fator de Transcrição E2F1/metabolismo , Ativação Transcricional/efeitos da radiação , Raios Ultravioleta , Animais , Ciclo Celular , Cricetinae , Inibidor de Quinase Dependente de Ciclina p19/genética , DNA/farmacologia , Reparo do DNA , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/fisiologia , Células HEK293 , Humanos , Elementos de Resposta , Transcrição Gênica/efeitos da radiaçãoRESUMO
BACKGROUND: A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. CONCLUSIONS/SIGNIFICANCE: The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.
Assuntos
Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p19/genética , Fator de Transcrição E2F1/metabolismo , Periodicidade , Regulação para Cima/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proliferação de Células , Sequência Conservada/genética , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Retroalimentação Fisiológica , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transcrição GênicaRESUMO
p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.
Assuntos
Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA , Peptídeos beta-Amiloides/farmacologia , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Northern Blotting , Linhagem Celular , Sobrevivência Celular , Cisplatino/farmacologia , Cricetinae , Reparo do DNA , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Regulação para CimaRESUMO
Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.