RESUMO
The phosphoinositide-3-kinase like kinases (PIKK) such as ATM and ATR play a key role in initiating the cellular DNA damage response (DDR). One key ATM target is the cyclin-dependent kinase inhibitor p27Kip1 that promotes G1 arrest. ATM activates p27Kip1-induced arrest in part through phosphorylation of p27Kip1 at Serine 140. Here we show that this site is dephosphorylated by the type 2C serine/threonine phosphatase, WIP1 (Wildtype p53-Induced Phosphatase-1), encoded by the PPM1D gene. WIP1 has been shown to dephosphorylate numerous ATM target sites in DDR proteins, and its overexpression and/or mutation has often been associated with oncogenesis. We demonstrate that wildtype, but not phosphatase-dead WIP1, efficiently dephosphorylates p27Kip1 Ser140 both in vitro and in cells and that this dephosphorylation is sensitive to the WIP1-specific inhibitor GSK 2830371. Increased expression of wildtype WIP1 reduces stability of p27Kip1 while increased expression of similar amounts of phosphatase-dead WIP1 has no effect on p27Kip1 protein stability. Overexpression of wildtype p27Kip1 reduces cell proliferation and colony forming capability relative to the S140A (constitutively non-phosphorylated) form of p27. Thus, WIP1 plays a significant role in homeostatic modulation of p27Kip1 activity following activation by ATM.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína Fosfatase 2C/metabolismo , Serina/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Células HEK293 , Humanos , Células MCF-7 , Mutação/genética , Fosfopeptídeos/metabolismo , Fosforilação , Estabilidade Proteica , Reprodutibilidade dos Testes , Ensaio Tumoral de Célula-TroncoRESUMO
Clonal hematopoiesis (CH), in which stem cell clones dominate blood production, becomes increasingly common with age and can presage malignancy development. The conditions that promote ascendancy of particular clones are unclear. We found that mutations in PPM1D (protein phosphatase Mn2+/Mg2+-dependent 1D), a DNA damage response regulator that is frequently mutated in CH, were present in one-fifth of patients with therapy-related acute myeloid leukemia or myelodysplastic syndrome and strongly correlated with cisplatin exposure. Cell lines with hyperactive PPM1D mutations expand to outcompete normal cells after exposure to cytotoxic DNA damaging agents including cisplatin, and this effect was predominantly mediated by increased resistance to apoptosis. Moreover, heterozygous mutant Ppm1d hematopoietic cells outcompeted their wild-type counterparts in vivo after exposure to cisplatin and doxorubicin, but not during recovery from bone marrow transplantation. These findings establish the clinical relevance of PPM1D mutations in CH and the importance of studying mutation-treatment interactions. VIDEO ABSTRACT.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Células Clonais/efeitos dos fármacos , Doxorrubicina/farmacologia , Hematopoese/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Proteína Fosfatase 2C/genética , Idoso , Animais , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Doxorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HEK293 , Hematopoese/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteína Fosfatase 2C/metabolismoRESUMO
Scientific progress depends on formulating testable hypotheses informed by the literature. In many domains, however, this model is strained because the number of research papers exceeds human readability. Here, we developed computational assistance to analyze the biomedical literature by reading PubMed abstracts to suggest new hypotheses. The approach was tested experimentally on the tumor suppressor p53 by ranking its most likely kinases, based on all available abstracts. Many of the best-ranked kinases were found to bind and phosphorylate p53 (P value = 0.005), suggesting six likely p53 kinases so far. One of these, NEK2, was studied in detail. A known mitosis promoter, NEK2 was shown to phosphorylate p53 at Ser315 in vitro and in vivo and to functionally inhibit p53. These bona fide validations of text-based predictions of p53 phosphorylation, and the discovery of an inhibitory p53 kinase of pharmaceutical interest, suggest that automated reasoning using a large body of literature can generate valuable molecular hypotheses and has the potential to accelerate scientific discovery.
Assuntos
Indexação e Redação de Resumos , Quinases Relacionadas a NIMA/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Células HCT116 , Células HEK293 , Humanos , Quinases Relacionadas a NIMA/genética , Processamento de Linguagem Natural , Fosforilação , PubMed , Proteína Supressora de Tumor p53/genéticaRESUMO
The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with current chemotherapeutic approaches.
Assuntos
Aminopiridinas/farmacologia , Dipeptídeos/farmacologia , Mutação , Neuroblastoma/tratamento farmacológico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Alelos , Linhagem Celular Tumoral , Feminino , Loci Gênicos , Humanos , Masculino , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2C , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutations in the TP53 tumour suppressor gene occur in half of all human cancers, indicating its critical importance in inhibiting cancer development. Despite extensive studies, the mechanisms by which mutant p53 enhances tumour progression remain only partially understood. Here, using data from the Cancer Genome Atlas (TCGA), genomic and transcriptomic analyses were performed on 2256 tumours from 10 human cancer types. We show that tumours with TP53 mutations have altered gene expression profiles compared to tumours retaining two wild-type TP53 alleles. Among 113 known p53-up-regulated target genes identified from cell culture assays, 10 were consistently up-regulated in at least eight of 10 cancer types that retain both copies of wild-type TP53. RPS27L, CDKN1A (p21(CIP1)) and ZMAT3 were significantly up-regulated in all 10 cancer types retaining wild-type TP53. Using this p53-based expression analysis as a discovery tool, we used cell-based assays to identify five novel p53 target genes from genes consistently up-regulated in wild-type p53 cancers. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2 and FOXM1 were disproportionately up-regulated in many TP53 mutant cancer types. Finally, > 93% of tumours with a TP53 mutation exhibited greatly reduced wild-type p53 messenger expression, due to loss of heterozygosity or copy neutral loss of heterozygosity, supporting the concept of p53 as a recessive tumour suppressor. The data indicate that tumours with wild-type TP53 retain some aspects of p53-mediated growth inhibitory signalling through activation of p53 target genes and suppression of cell cycle regulatory genes.
Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Mutação , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Perda de Heterozigosidade , Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G(1) phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G(1)/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G(1) exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome.
Assuntos
Dano ao DNA/fisiologia , Fase G1/fisiologia , Produtos do Gene tax/genética , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fase S/fisiologia , Adulto , Animais , Western Blotting , Células Cultivadas , Dano ao DNA/efeitos da radiação , Reparo do DNA/fisiologia , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Imunofluorescência , Fase G1/efeitos da radiação , Histonas/genética , Histonas/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Imunoprecipitação , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteína Fosfatase 2C , Dímeros de Pirimidina , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase S/efeitos da radiação , Raios UltravioletaRESUMO
PURPOSE: We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC). METHODS: A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay. RESULTS: Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p = 0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p < 0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173, 55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p = 0.122). CONCLUSIONS: Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação para Baixo , Epigênese Genética , Fator 3-beta Nuclear de Hepatócito/genética , Neoplasias Pulmonares/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Metilação de DNA , Análise Mutacional de DNA , Feminino , Dosagem de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Regiões Promotoras GenéticasRESUMO
The HTLV-I oncoprotein Tax interferes with DNA double strand break repair. Since non-homologous end joining (NHEJ) is a major pathway used to repair DNA double strand breaks we examined the effect of Tax on this pathway, with particular interest in the expression and function of Ku80, a critical component of the NHEJ pathway. Tax expression decreased Ku80 mRNA and protein levels, and repressed transcription from the Ku80 promoter. Conversely, Ku80 mRNA increased following siRNA knockdown of Tax in HTLV-I infected cells. Tax expression was associated with an elevated number of micronuclei and nucleoplasmic bridges, hallmarks of improper DNA double strand break repair. Our studies identified Tax as a transcriptional repressor of Ku80 that correlates with decreased DNA repair function. The reduction of Ku80 transcription by Tax may deplete the cell of an essential DNA break binding protein, resulting in reduced repair of DNA double strand breaks and accumulation genomic mutations.
Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Antígenos Nucleares/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Viral/química , Proteínas de Ligação a DNA/genética , Produtos do Gene tax/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Autoantígeno Ku , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Osteosarcoma is the primary malignant cancer of bone and particularly affects adolescents and young adults, causing debilitation and sometimes death. As a model for human osteosarcoma, we have been studying p53(+/-) mice, which develop osteosarcoma at high frequency. To discover genes that cooperate with p53 deficiency in osteosarcoma formation, we have integrated array comparative genomic hybridization, microarray expression analyses in mouse and human osteosarcomas, and functional assays. In this study, we found seven frequent regions of copy number gain and loss in the mouse p53(+/-) osteosarcomas but have focused on a recurrent amplification event on mouse chromosome 9A1. This amplicon is syntenic with a similar chromosome 11q22 amplicon identified in several human tumor types. Three genes on this amplicon, the matrix metalloproteinase gene MMP13 and the antiapoptotic genes Birc2 (cIAP1) and Birc3 (cIAP2), show elevated expression in mouse and human osteosarcomas. We developed a functional assay using clonal osteosarcoma cell lines transduced with lentiviral short hairpin RNA vectors to show that down-regulation of MMP13, Birc2, or Birc3 resulted in reduced tumor growth when transplanted into immunodeficient recipient mice. These experiments revealed that high MMP13 expression enhances osteosarcoma cell survival and that Birc2 and Birc3 also enhance cell survival but only in osteosarcoma cells with the chromosome 9A1 amplicon. We conclude that the antiapoptotic genes Birc2 and Birc3 are potential oncogenic drivers in the chromosome 9A1 amplicon.
Assuntos
Neoplasias Ósseas/genética , Proteínas Inibidoras de Apoptose/genética , Metaloproteinase 13 da Matriz/genética , Osteossarcoma/genética , Proteína Supressora de Tumor p53/deficiência , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína 3 com Repetições IAP de Baculovírus , Neoplasias Ósseas/enzimologia , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/genética , Hibridização Genômica Comparativa , Amplificação de Genes , Instabilidade Genômica , Humanos , Proteínas Inibidoras de Apoptose/deficiência , Metaloproteinase 13 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Osteossarcoma/enzimologia , Fosfoproteínas/biossíntese , Fosfoproteínas/deficiência , Fosfoproteínas/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases , Proteínas de Sinalização YAPRESUMO
The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.
Assuntos
Expressão Gênica , Proteínas Proto-Oncogênicas/genética , RNA Antissenso/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Animais , Linhagem Celular , Eletroporação , Granulócitos/citologia , Granulócitos/metabolismo , Células HL-60 , Humanos , Separação Imunomagnética , Células Jurkat , Macrófagos/metabolismo , Camundongos , Modelos Genéticos , Biossíntese de Proteínas , Interferência de RNA , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/metabolismo , Transcrição Gênica , Células U937RESUMO
Viruses have been linked to approximately 20% of all human tumors worldwide. These transforming viruses encode viral oncoproteins that interact with cellular proteins to enhance viral replication. The transcriptional and post-transcriptional effects of these viral oncoproteins ultimately result in cellular transformation. Historically, viral research has been vital to the discovery of oncogenes and tumor suppressors with more current research aiding in unraveling some mechanisms of carcinogenesis. Interestingly, since transforming viruses affect some of the same pathways that are dysregulated in human cancers, their study enhances our understanding of the multistep process of tumorigenesis. This review will examine the cellular mechanisms targeted by oncogenic human viruses and the processes by which these effects contribute to transformation. In particular, we will focus on three transforming viruses, human T-cell leukemia virus type-I, hepatitis B virus and human papillomavirus. These viruses all encode specific oncogenes that promote cell cycle progression, inhibit DNA damage checkpoint responses and prevent programmed cell death in an effort to promote viral propagation. While the transforming properties of these viruses are probably unintended consequences of replication strategies, they provide excellent systems in which to study cancer development.
Assuntos
Transformação Celular Viral , Neoplasias , Vírus Oncogênicos , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Reparo do DNA , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Oncogenes , Vírus Oncogênicos/genética , Vírus Oncogênicos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Receptor fas/metabolismoRESUMO
Both PU.1 (also called SFPI1), an Ets-family transcription factor, and AML1 (also called RUNX1), a DNA-binding subunit of the CBF transcription factor family, are crucial for the generation of all hematopoietic lineages, and both act as tumor suppressors in leukemia. An upstream regulatory element (URE) of PU.1 has both enhancer and repressor activity and tightly regulates PU.1 expression. Here we show that AML1 binds to functionally important sites within the PU.1 upstream regulatory element and regulates PU.1 expression at both embryonic and adult stages of development. Analysis of mice carrying conditional AML1 knockout alleles and knock-in mice carrying mutations in all three AML1 sites of the URE proximal region demonstrated that AML1 regulates PU.1 both positively and negatively in a lineage dependent manner. Dysregulation of PU.1 expression contributed to each of the phenotypes observed in these mice, and restoration of proper PU.1 expression rescued or partially rescued each phenotype. Thus, our data demonstrate that PU.1 is a major downstream target gene of AML1.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Animais , Plaquetas/metabolismo , Camundongos , Camundongos Knockout , Elementos Reguladores de Transcrição , Linfócitos T/metabolismoRESUMO
BACKGROUND: The HTLV-I oncoprotein, Tax, is a pleiotropic protein whose activity is partially regulated by its ability to interact with, and perturb the functions of, numerous cellular proteins. Tax is predominantly a nuclear protein that localizes to nuclear foci known as Tax Speckled Structures (TSS). We recently reported that the localization of Tax and its interactions with cellular proteins are altered in response to various forms of genotoxic and cellular stress. The level of cytoplasmic Tax increases in response to stress and this relocalization depends upon the interaction of Tax with CRM1. Cellular pathways and signals that regulate the subcellular localization of Tax remain to be determined. However, post-translational modifications including sumoylation and ubiquitination are known to influence the subcellular localization of Tax and its interactions with cellular proteins. The sumoylated form of Tax exists predominantly in the nucleus while ubiquitinated Tax exists predominantly in the cytoplasm. Therefore, we hypothesized that post-translational modifications of Tax that occur in response to DNA damage regulate the localization of Tax and its interactions with cellular proteins. RESULTS: We found a significant increase in mono-ubiquitination of Tax in response to UV irradiation. Mutation of specific lysine residues (K280 and K284) within Tax inhibited DNA damage-induced ubiquitination. In contrast to wild-type Tax, which undergoes transient nucleocytoplasmic shuttling in response to DNA damage, the K280 and K284 mutants were retained in nuclear foci following UV irradiation and remained co-localized with the cellular TSS protein, sc35. CONCLUSION: This study demonstrates that the localization of Tax, and its interactions with cellular proteins, are dynamic following DNA damage and depend on the post-translational modification status of Tax. Specifically, DNA damage induces the ubiquitination of Tax at K280 and K284. Ubiquitination of these residues facilitates the dissociation of Tax from sc35-containing nuclear foci, and stimulates nuclear export of Tax through the CRM1 pathway.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Dano ao DNA/fisiologia , Produtos do Gene tax/fisiologia , Infecções por HTLV-I/fisiopatologia , Linhagem Celular Transformada , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/química , Humanos , Processamento de Proteína Pós-Traducional , Ubiquitinação/fisiologiaRESUMO
Serum response factor (SRF) was recently shown to bind and activate the human T-cell leukemia virus type 1 (HTLV-1) promoter at bases -116 to -125 relative to the transcription start site. In addition to the SRF binding site (CArG box), serum response elements (SRE) also typically contain a binding site for a member of the ternary complex factor (TCF) family. Here we demonstrate the presence of two TCF binding sites upstream of the viral CArG box. Binding of the TCF family member Elk-1 to these sites was shown to activate transcription of the promoter. Based on these results, the position of the previously described viral SRE (vSRE) within the HTLV-1 promoter can be extended from -116 to -157 to include the two newly identified TCF sites. Purified Elk-1 bound to a probe containing the vSRE, and this complex formed a ternary complex with SRF. In addition, the complex formed by nuclear extract on this probe contained Elk-1, as shown by electrophoretic mobility shift assay supershift. Both of the predicted TCF sites independently bound Elk-1. Elk-1 activated transcription of the HTLV-1 long terminal repeat (LTR), and mutations within either of the TCF sites or the CArG box reduced responsiveness of the LTR to Elk-1. Chromatin immunoprecipitation demonstrated that Elk-1 associates with the HTLV-1 LTR in vivo. These results identify a functional SRE within the HTLV-1 LTR and suggest that both Elk-1 and SRF play important roles in regulating basal HTLV-1 gene expression.
Assuntos
Regulação Viral da Expressão Gênica , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , RNA Viral/biossíntese , Sequências Repetidas Terminais , Transcrição Gênica/fisiologia , Proteínas Elk-1 do Domínio ets/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/biossínteseRESUMO
During 'emergency' situations such as infections, host defense requires rapid mobilization of bone marrow granulocyte progenitors. 'Steady-state' granulopoiesis is absolutely dependent on the C/EBPalpha transcription factor, but the transcriptional mechanisms underlying emergency granulopoiesis remain unclear. Here we show that large numbers of granulocytes were generated from C/EBPalpha-deficient progenitors after cytokine stimulation in vivo. Cytokine treatment or fungal infection induced upregulation of C/EBPbeta but not C/EBPalpha or C/EBPepsilon transcripts in granulocyte progenitors, and C/EBPbeta-deficient progenitors showed decreased emergency-induced granulopoiesis in vitro and in vivo. C/EBPbeta inhibited proliferation less severely than did C/EBPalpha. These data suggest a critical function for C/EBPbeta in emergency granulopoiesis, which demands both differentiation and proliferation of granulocyte precursors.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Granulócitos/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Infecções/imunologia , Animais , Animais Congênicos , Proteína alfa Estimuladora de Ligação a CCAAT/deficiência , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Candidíase/imunologia , Candidíase/fisiopatologia , Ciclo Celular , Diferenciação Celular , Células Cultivadas/metabolismo , Ensaio de Unidades Formadoras de Colônias , Estradiol/farmacologia , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-3/genética , Interleucina-3/fisiologia , Células K562/citologia , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/fisiologia , Transdução Genética , Regulação para CimaAssuntos
Adenocarcinoma Bronquioloalveolar/genética , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Mutação , Doença Aguda , Diferenciação Celular/genética , Regulação para Baixo , Células Epiteliais , Predisposição Genética para Doença , Humanos , Leucemia Mieloide/genéticaRESUMO
Mutations constitutively activating FLT3 kinase are detected in approximately 30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal-regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPalpha mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPalpha. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPalpha may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação Puntual , Tirosina Quinase 3 Semelhante a fms/genética , Idoso , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/genética , Feminino , Granulócitos/patologia , Humanos , Células K562 , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Masculino , Células Mieloides/patologia , Fosforilação , Piperazinas/farmacologia , Quinazolinas/farmacologia , Serina/metabolismo , Células Tumorais Cultivadas , Células U937 , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The leucine zipper family transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) inhibits proliferation and promotes differentiation in various cell types. In this study, we show, using a lung-specific conditional mouse model of C/EBPalpha deletion, that loss of C/EBPalpha in the respiratory epithelium leads to respiratory failure at birth due to an arrest in the type II alveolar cell differentiation program. This differentiation arrest results in the lack of type I alveolar cells and differentiated surfactant-secreting type II alveolar cells. In addition to showing a block in type II cell differentiation, the neonatal lungs display increased numbers of proliferating cells and decreased numbers of apoptotic cells, leading to epithelial expansion and loss of airspace. Consistent with the phenotype observed, genes associated with alveolar maturation, survival, and proliferation were differentially expressed. Taken together, these results identify C/EBPalpha as a master regulator of airway epithelial maturation and suggest that the loss of C/EBPalpha could also be an important event in the multistep process of lung tumorigenesis. Furthermore, this study indicates that exploring the C/EBPalpha pathway might have therapeutic benefits for patients with respiratory distress syndromes.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/deficiência , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Alvéolos Pulmonares/patologia , Insuficiência Respiratória/genética , Insuficiência Respiratória/patologia , Animais , Apoptose , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular , Proliferação de Células , Epitélio/metabolismo , Epitélio/patologia , Deleção de Genes , Perfilação da Expressão Gênica , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/crescimento & desenvolvimento , Deleção de SequênciaRESUMO
The PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent "priming" of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Animais , Medula Óssea/metabolismo , Células Cultivadas , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transativadores/deficiência , Transativadores/genéticaRESUMO
Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in specimens from patients with non-small-cell lung cancer who have a response to anilinoquinazoline EGFR inhibitors. Despite the dramatic responses to such inhibitors, most patients ultimately have a relapse. The mechanism of the drug resistance is unknown. Here we report the case of a patient with EGFR-mutant, gefitinib-responsive, advanced non-small-cell lung cancer who had a relapse after two years of complete remission during treatment with gefitinib. The DNA sequence of the EGFR gene in his tumor biopsy specimen at relapse revealed the presence of a second point mutation, resulting in threonine-to-methionine amino acid change at position 790 of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance.