RESUMO
DNA damage in eukaryotic cells induces signaling pathways mediated by the ATM, p53 and ERK proteins, but the interactions between these pathways are not completely known. To address this issue, we performed a time course analysis in human embryonic fibroblast cells treated with DNA-damaging agents. DNA damage induced the phosphorylation of p53 at Ser 15 (p-p53) and the phosphorylation of ERK (p-ERK). Inhibition of p53 by a dominant negative mutant or in p53(-/-) fibroblast cells abolished ERK phosphorylation. ERK inhibitor prevented p53 phosphorylation, indicating that phosphorylations of p53 and p-ERK are interdependent each other. A time course analysis showed that ATM interacted with p-p53 and p-ERK in early time (0.5 h) and interaction between ATM-bound p-p53 and p-ERK or ATM-bound p-ERK and p-p53 occurred in late time (3 h) of DNA damage. These results indicate that ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex between p-p53 and p-ERK in response to DNA damage to cause growth arrest.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ativação Enzimática , Fibroblastos/metabolismo , Humanos , Camundongos , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/metabolismoRESUMO
Since the detailed comparison of DNA repair activities among mammalian embryonic fibroblast cells with different replicative life spans has not been investigated, we tested DNA repair activities in embryonic fibroblast cells derived from mammals including human, dog, rat, and mouse. The cell viability after treatment of four DNA damage agents appeared to be decreased in the order of human embryonic fibroblasts (HEFs) > dog embryonic fibroblasts (DEFs) > rat embryonic fibroblasts (REFs) > mouse embryonic fibroblasts (MEFs) although statistical significance was lacking. The amounts of strand breaks and AP (apurinic/apyrimidinic) sites also appear to be decreased in the order of HEFs > DEFs > REFs ≥ MEFs after treatment of DNA damage agents. The DNA repair activities and rates including base excision repair (BER), nucleotide excision repair (NER) and double-strand break repair (DSBR) including non-homologous end-joining (NHEJ) decreased again in the order of HEFs > DEFs > REFs ≥ MEFs. BER and NHEJ activities in 3% O(2) also decreased in the order of HEFs > DEFs > REFs > MEFs. This order in DNA repair activity appears to be coincident with that of replicative life span of fibroblasts and that of life span of mammals. These results indicate that higher DNA repair activity is related with longer replicative life span in embryonic fibroblast cells.
Assuntos
Proliferação de Células , Senescência Celular , Dano ao DNA , Reparo do DNA , Fibroblastos/metabolismo , Animais , Camptotecina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Etoposídeo/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Sprague-DawleyRESUMO
Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells. PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1/Cip1/Sdi level. Depletion of p21Waf1/Cip1/Sdi induced cell death and PD98059 induced additional cell death. DNA-damage-induced increase in cytoplasmic localization and phosphorylation of threonine residues of p21Waf1/Cip1/Sdi was reversed by PD98059. Thus, the results suggest that ERK pathway mediates anti-apoptotic effects through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to mild DNA damage.
Assuntos
Apoptose/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoplasma/metabolismo , Dano ao DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Western Blotting , Caspase 3/metabolismo , Fracionamento Celular , Linhagem Celular , Citometria de Fluxo , Humanos , Imunoprecipitação , Fosforilação , Sais de Tetrazólio , TiazóisRESUMO
In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon gamma (IFNgamma). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor alpha (TNFalpha) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFkappaB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/fisiologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Proteína Amiloide A Sérica/genética , Animais , Apoptose , Diferenciação Celular , Divisão Celular , Células Cultivadas , Feminino , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Amiloide A Sérica/metabolismo , TransfecçãoRESUMO
In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFalpha treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFalpha induced WDNM1 expression showed the NFkappaB-dependent mechanism since it's expression was abrogated in IkappaBalphaM (super-repressor of NFkappaB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFalpha-NFkappaB signal pathway, is associated with HC11 cell apoptosis.
Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Glândulas Mamárias Animais/fisiologia , Proteínas de Neoplasias/biossíntese , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Diferenciação Celular/efeitos dos fármacos , Clonagem Molecular , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Camundongos , Microscopia de Fluorescência , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Protein farnesylation is one of the most common lipid modifications and has an important role in the regulation of various cellular functions. We have recently developed a novel proteomics strategy, designated the tagging-via-substrate (TAS) approach, for the detection and proteomic analysis of farnesylated proteins. This chapter describes the principle of TAS technology and details the method for detection and enrichment of farnesylated proteins.
Assuntos
Prenilação de Proteína , Proteômica/métodos , Animais , Azidas/química , Células COS , Chlorocebus aethiops , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Choque Térmico HSP40/química , Humanos , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Proteínas ras/químicaRESUMO
Progesterone has a potential protective effect against ovarian carcinoma induced by estrogen. Progesterone is also known to cause apoptosis while tamoxifen induces growth arrest. Therefore, we attempted to determine whether combined treatment with progesterone and tamoxifen has a synergistic effect on anti-cancer activity. Although progesterone is known to cause apoptosis while tamoxifen induces growth arrest in many cancer cells, the detailed action of progesterone and tamoxifen and the anticancer effect of combined treatment have not been tested in ovarian cancer cells. Therefore, we tested the growth and apoptosis activity of progesterone and tamoxifen and the anticancer effect of combined treatment of progesterone and tamoxifen in ovarian cancer cells. Ovarian cancer cells, PA-1, were treated with progesterone, tamoxifen, or a combination of progesterone and tamoxifen. The anti-cancer effects were investigated by use of flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, DNA fragmentation analysis, and Western blot analysis. We found that 100 µM progesterone induced typical apoptosis in PA-1 cells. Treatment of PA-1 cells with 10 µM tamoxifen resulted in an increase in the levels of p21, p27, p16 and phospho-pRb, indicating typical G1 arrest. Co-treatment of PA-1 cells with 100 µM progesterone and 10 µM tamoxifen resulted in typical apoptosis, similar to that induced by treatment with 100 µM progesterone alone. These results indicate that progesterone caused apoptosis and tamoxifen induced G1 arrest. Combined treatment with tamoxifen and progesterone caused apoptosis similar to that induced by treatment with progesterone alone and had no additional anti-cancer effect in ovarian cancer cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Progesterona/farmacologia , Tamoxifeno/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Even though CR has shown to enhance base excision repair (BER) and nucleotide excision repair (NER) capacities, it has not been reported whether CR can enhance non-homologous end joining (NHEJ) activity. To examine the effect of CR on NHEJ activity, ad libitum (AL)- and calorie restricted (CR)-dieted rats were used. Age-dependent decline of NHEJ activity was apparent in the lung, liver, and kidney and appeared to be slightly decreased in spleen. CR reduced age-dependent decline of NHEJ activity in all tissues, even though the extent of recovery was variable among tissues. Moreover, CR appeared to reduce age-dependent decline of XRCC4 protein level. These results suggest that CR could reduce age-dependent decline of NHEJ activity in various tissues of rats possibly through up-regulation of XRCC4.
Assuntos
Envelhecimento/metabolismo , Restrição Calórica , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Baço/metabolismo , Envelhecimento/genética , Animais , Western Blotting , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , Masculino , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio , Regulação para CimaRESUMO
Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.
RESUMO
Like phosphorylation, protein sumoylation likely represents a dynamic PTM to alter protein function in support of cell regulatory systems. The broad-spectrum impact of transient or chronic engagement of signal transduction cascades on protein sumoylation has not been explored. Here, we find that epidermal growth factor (EGF) stimulation evokes a rapid alteration in small ubiquitin modifier (SUMO) target selection, while oncogene expression alters steady-state SUMO-protein profiles. A proteomic SUMO target analysis in melanoma cells identified proteins involved in cellular signaling, growth control, and neural differentiation.
Assuntos
Proteínas/metabolismo , Proteoma/metabolismo , Proteína SUMO-1/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Proteínas/genética , Proteoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Interferente Pequeno/genética , Proteína SUMO-1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.
Assuntos
Lisina/metabolismo , Proteômica , Acetilação , Sequência de Aminoácidos , Animais , Células Cultivadas , Biologia Computacional , Fibroblastos/citologia , Células HeLa , Humanos , Lisina/química , Espectrometria de Massas , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Peptídeos/química , Proteínas/química , Proteínas/isolamento & purificação , Especificidade por SubstratoRESUMO
Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.
Assuntos
Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Proteínas de Membrana/análise , Proteômica/métodos , Linhagem Celular Tumoral , Membrana Celular/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificaçãoRESUMO
A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.