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1.
Cancer Sci ; 107(6): 734-45, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26998741

RESUMO

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Proteína Supressora de Tumor p53/metabolismo , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/metabolismo , Morte Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fucose/metabolismo , Humanos , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , alfa-L-Fucosidase/biossíntese
2.
Sci Rep ; 6: 19174, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26754925

RESUMO

The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53-dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Nucleares/genética , Fatores de Transcrição/metabolismo , Proliferação de Células , Dano ao DNA , Elementos Facilitadores Genéticos , Expressão Gênica , Regulação da Expressão Gênica , Fatores de Transcrição de Choque Térmico , Humanos , Proteínas Imediatamente Precoces/metabolismo , Família Multigênica , Complexos Multiproteicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Fosforilação , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Transdução de Sinais , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo
3.
World J Biol Chem ; 6(3): 139-47, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26322172

RESUMO

Deregulated c-Myc expression is a hallmark of many human cancers. We have recently identified a role of mammalian homolog of yeast SPT-ADA-GCN5-acetyltransferas (SAGA) complex component, SAGA-associated factor 29 (SGF29), in regulating the c-Myc overexpression. Here, we discuss the molecular nature of SFG29 in SPT3-TAF9-GCN5-acetyltransferase complex, a counterpart of yeast SAGA complex, and the mechanism through which the elevated SGF29 expression contribute to oncogenic potential of c-Myc in hepatocellularcarcinoma (HCC). We propose that the upstream regulation of SGF29 elicited by sex-determining region Y (Sry) is also augmented in HCC. We hypothesize that c-Myc elevation driven by the deregulated Sry and SGF29 pathway is implicated in the male specific acquisition of human HCCs.

4.
Stem Cells ; 33(9): 2652-63, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26013162

RESUMO

The acquisition of stemness is a hallmark of aggressive human hepatocellular carcinoma (hHCC). The stem cell marker OCT4 is frequently expressed in HCCs, and its expression correlates with those of putative cancer stem cell (CSC) markers and CSC properties. Here, we describe a novel mechanism of CSC maintenance by SRY through OCT4. We previously reported that Sry is involved in tumor malignancy in rodent HCCs. However, the oncogenic function of SRY in hHCCs is poorly understood. Ectopic expression of SRY increased multiple stem cell factors, including OCT4 and CD13. The OCT4 promoter contained SRY-binding sites that were directly activated by SRY. In HCC-derived cells, SRY knockdown decreased OCT4 expression and cancer stem-like phenotypes such as self-renewal, chemoresistance, and tumorigenicity. Conversely, OCT4 and SRY overexpression promoted cancer stem-like phenotypes. OCT4 knockdown in SRY clones downregulated the self-renewal capacity and chemoresistance. These data suggest that SRY is involved in the maintenance of cancer stem-like characteristics through OCT4. Moreover, CSCs of HCC-derived cells differentiated into Tuj1-positive neuron-like cells by retinoic acid. Noteworthily, SRY was highly expressed in some hHCC patients. Taken together, our findings imply a novel therapeutic strategy against CSCs of hHCCs.


Assuntos
Carcinoma Hepatocelular/metabolismo , Sistemas de Liberação de Medicamentos , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/biossíntese , Proteína da Região Y Determinante do Sexo/biossíntese , Animais , Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Silenciamento de Genes/métodos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Proteína da Região Y Determinante do Sexo/genética
5.
Proc Natl Acad Sci U S A ; 111(52): 18691-6, 2014 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-25512506

RESUMO

Communication between cancer cells and their microenvironment controls cancer progression. Although the tumor suppressor p53 functions in a cell-autonomous manner, it has also recently been shown to function in a non-cell-autonomous fashion. Although functional defects have been reported in p53 in stromal cells surrounding cancer, including mutations in the p53 gene and decreased p53 expression, the role of p53 in stromal cells during cancer progression remains unclear. We herein show that the expression of α-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts (CAFs), was increased by the ablation of p53 in lung fibroblasts. CAFs enhanced the invasion and proliferation of lung cancer cells when cocultured with p53-depleted fibroblasts and required contact between cancer and stromal cells. A comprehensive analysis using a DNA chip revealed that tetraspanin 12 (TSPAN12), which belongs to the tetraspanin protein family, was derepressed by p53 knockdown. TSPAN12 knockdown in p53-depleted fibroblasts inhibited cancer cell proliferation and invasion elicited by coculturing with p53-depleted fibroblasts in vitro, and inhibited tumor growth in vivo. It also decreased CXC chemokine ligand 6 (CXCL6) secretion through the ß-catenin signaling pathway, suggesting that cancer cell contact with TSPAN12 in fibroblasts transduced ß-catenin signaling into fibroblasts, leading to the secretion of CXCL6 to efficiently promote invasion. These results suggest that stroma-derived p53 plays a pivotal role in epithelial cancer progression and that TSPAN12 and CXCL6 are potential targets for lung cancer therapy.


Assuntos
Fibroblastos/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Transdução de Sinais , Tetraspaninas/metabolismo , Animais , Linhagem Celular Tumoral , Quimiocina CXCL6/genética , Quimiocina CXCL6/metabolismo , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Tetraspaninas/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
6.
Biochem Biophys Res Commun ; 453(1): 117-23, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25261720

RESUMO

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the 110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas de Ligação a RNA/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2/farmacologia , Administração Oral , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Aralia/química , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HeLa , Células Hep G2 , Humanos , Lipoproteínas HDL/antagonistas & inibidores , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos Nus , Peso Molecular , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Receptores de Lipoproteínas/antagonistas & inibidores , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2/química , Proteínas Inativadoras de Ribossomos Tipo 2/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Cell Rep ; 7(2): 527-538, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24726368

RESUMO

In lung cancer progression, p53 mutations are more often observed in invasive tumors than in noninvasive tumors, suggesting that p53 is involved in tumor invasion and metastasis. To understand the nature of p53 function as a tumor suppressor, it is crucial to elucidate the detailed mechanism of the alteration in epithelial cells that follow oncogenic KRAS activation and p53 inactivation. Here, we report that KRAS activation induces epithelial-mesenchymal transition and that p53 inactivation is required for cell motility and invasiveness. Furthermore, TSPAN2, a transmembrane protein, is responsible for cell motility and invasiveness elicited by p53 inactivation. TSPAN2 is highly expressed in p53-mutated lung cancer cells, and high expression of TSPAN2 is associated with the poor prognosis of lung adenocarinomas. TSPAN2 knockdown suppresses metastasis to the lungs and liver, enabling prolonged survival. TSPAN2 enhances cell motility and invasiveness by assisting CD44 in scavenging intracellular reactive oxygen species.


Assuntos
Movimento Celular , Neoplasias Pulmonares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tetraspaninas/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Nus , Mutação , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Tetraspaninas/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
8.
Biochem Biophys Res Commun ; 444(3): 382-6, 2014 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-24468084

RESUMO

The 14-3-3 family of proteins regulates various signaling pathways involved in cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the ß isoform of the 14-3-3 protein promotes cell growth and tumorigenicity of rat K2 hepatocellular carcinoma cells. We identified fourteen-three-three beta interactant 1 (FBI1)/Akirin2 as a binding partner of 14-3-3ß and showed that the complex of these proteins promotes tumorigenicity and metastasis of K2 cells. In addition, we demonstrated that FBI1/Akirin2 downregulation shortened the duration of MAPK activity. Because 14-3-3ß and FBI1/Akirin2 overexpression is observed in various cancer cell lines, 14-3-3ß-FBI1/Akirin2 oncogenic function should be elucidated in different types of cancer. In this study, we used LLC1 Lewis lung carcinoma cells as a model. We established FBI1/Akirin2 knockdown cell clones through transfection of an antisense FBI1/Akirin2 expression vector and assessed the capacity for cell growth in vitro and tumorigenicity and metastasis in vivo. FBI1/Akirin2 downregulation decreased anchorage-independent growth, whereas the growth rate in monolayer culture was not affected. Moreover, an in vivo assay in nude mice showed that FBI1/Akirin2 overexpression is required for LLC1 tumor growth and metastasis. These results suggest that FBI1/Akirin2 plays an important role in oncogenesis of LLC1 lung carcinoma cells, and this protein may also serve as an oncogene in other cancers.


Assuntos
Proteínas 14-3-3/fisiologia , Carcinoma Pulmonar de Lewis/patologia , Metástase Neoplásica , Animais , Linhagem Celular Tumoral , Ratos
9.
Acta Histochem Cytochem ; 47(6): 303-10, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25859064

RESUMO

Excretory organs contain epithelial cells that form a filtration membrane specialized for ultrafiltration to produce primary urine. In vertebrates, the filtration membrane is made up of slit diaphragm (SD) formed by glomerular podocytes. Basal metazoans such as flatworms are also known have filtration epithelial cells, called flame cells, which exhibit SD-like structures. The molecular components of podocyte SD have been studied in detail, while those of the SD-like structures in basal metazoans including flatworms remain to be clarified. To determine whether the SD-like structures in flatworms have molecular components common to the SD in vertebrate podocytes, we examined the expression of gene homologue for mammalian nephrin, which encodes an essential transmembrane protein that participates in the formation of the SD, in a species of flatworms, planarian (Dugesia japonica). Flame cells were distributed throughout the entire body of the planarian, but the nephrin-expressing cells identified by in situ hybridization were mainly detected at body periphery excluding head region. The distribution pattern of nephrin-expressing cells was similar to that of proliferating cell nuclear antigen-expressing neoblasts, which are pluripotent stem cells characteristic to planarians. These findings indicated that the SD-like structures can be formed without the Nephrin protein in planarian flame cells.

10.
PLoS One ; 8(11): e78716, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223164

RESUMO

Basal cell adhesion molecule (BCAM), known to be a splicing variant of Lutheran glycoprotein (LU), is an immunoglobulin superfamily membrane protein that acts as a laminin α5 receptor. The high affinity of BCAM/LU for laminin α5 is thought to contribute to the pathogenesis of sickle red blood cells and to various developmental processes. However, the function of BCAM in carcinogenesis is poorly understood. Based on microarray expression analysis, we found that BCAM was one of the target genes of the oncogenic 14-3-3ß-FBI1/Akirin2 complex, which acts as a transcriptional repressor and suppresses MAPK phosphatase-1 gene expression. To elucidate the detailed function of BCAM in malignant tumors, we established BCAM-expressing hepatoma K2 cells. These cells lost the malignant characteristics of parental cells, such as anchorage-independent growth, migration, invasion, and tumorigenicity. Moreover, luciferase reporter assays and chromatin immunoprecipitation analysis revealed that the 14-3-3ß-FBI1/Akirin2 complex bound to the BCAM promoter and repressed transcription. Thus, these data indicate that BCAM is a suppressive oncoprotein, and that FBI1/Akirin2 is involved in tumorigenicity and metastasis of hepatoma through the downregulation of suppressive oncogenes.


Assuntos
Proteínas 14-3-3/genética , Sistema do Grupo Sanguíneo Lutheran/genética , Proteínas Oncogênicas/genética , Proteínas Repressoras/genética , Proteínas 14-3-3/metabolismo , Animais , Northern Blotting , Western Blotting , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação Neoplásica da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Sistema do Grupo Sanguíneo Lutheran/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Transplante Heterólogo
11.
Brain Res ; 1524: 12-25, 2013 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-23769735

RESUMO

U-box protein PRP19ß, a splicing variant of PRP19α, suppresses neuronal differentiation and conversely promotes astrocyte differentiation as a neuron/glia switch molecule. However, the mechanistic basis of PRP19ß in astrocyte differentiation is not well understood. Here, we demonstrated that PRP19ß regulates the stability of protein tyrosine phosphatase 1B (PTP1B) via ubiquitination during N(6),2'-O-dibutyryl cyclic AMP (cAMP)-induced astrocyte differentiation of C6 cells. Only overexpression of PRP19ß conferred astrocyte properties at a certain level, and induced more astrocyte markers, glial fibrillary acidic protein (GFAP) and S100ß, in the presence of cAMP, whereas its down-regulation by antisense RNA showed a suppressive effect. In addition, ectopic expression of PRP19ß led to robust phosphorylation of signal transducer and activator of transcription 3 (STAT3) accompanying the reduction in PTP1B stability during astrocyte differentiation. Immunological analysis revealed that PRP19ß interacted with PTP1B and ubiquitinated PTP1B via its U-box region. Forced expression of the U-box deletion mutant of PRP19ß resulted in inhibition of astrocyte differentiation. Moreover, down-regulation of PTP1B by short hairpin (sh)RNA enhanced astrocyte differentiation, while forced expression of PTP1B showed an inhibitory effect. Thus, these results indicate that PRP19ß activates the gp130/Janus kinase (JAK)/STAT signaling pathway during astrocyte differentiation of C6 cells via PTP1B ubiquitination.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Imuno-Histoquímica , Imunoprecipitação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
PLoS One ; 8(2): e56997, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23451132

RESUMO

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6-12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.


Assuntos
Linhagem da Célula , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Primers do DNA , Camundongos , Proteína Meis1 , Neurônios/citologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligação Proteica
13.
J Biol Chem ; 288(19): 13269-77, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23536184

RESUMO

BACKGROUND: It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. RESULTS: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. CONCLUSION: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. SIGNIFICANCE: Cellular transformation renders cells more susceptible to some DNA-damaging agents. Anti-cancer drugs generally target cancer cells rather than normal somatic cells. However, the factors that determine this differential sensitivity are poorly understood. Here we show that Arf/p53-dependent down-regulation of H2AX induced the selective survival of normal cells after drug treatment, resulting in the preferential targeting of cancer cells. Treatment with camptothecin, a topoisomerase I inhibitor, caused normal cells to down-regulate H2AX and become quiescent, a process mediated by both Arf and p53. In contrast, transformed cells that harbor mutations in either Arf or p53 do not down-regulate H2AX and are more sensitive to drugs unless they have developed drug resistance. Such transformation-associated changes in H2AX expression rendered cancer cells more susceptible to drug-induced damage (by two orders of magnitude). Thus, the expression of H2AX and γH2AX (phosphorylated form of H2AX at Ser-139) is a critical factor that determines drug sensitivity and should be considered when administering chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação para Baixo , Histonas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Camptotecina/farmacologia , Forma Celular , Células Cultivadas , Senescência Celular , Cisplatino/farmacologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Histonas/genética , Humanos , Hidroxiureia/farmacologia , Camundongos , Camundongos Knockout , Mutação , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína Supressora de Tumor p53/genética
14.
Biochem Biophys Res Commun ; 432(1): 34-9, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23376716

RESUMO

Normal cells undergo a growth-arrested status that is produced by p53-dependent down-regulation of histone H2AX. Immortality is developed after abrogation of the H2AX-diminished state, which is associated with genomic instability (often with tetraploidy) and the induction of mutations in either the Arf or p53 gene. However, the role of Arf in control of H2AX expression and genome stability is still unclear. Here, we show that both Arf and p53 are required for the down-regulation of H2AX and formation of the growth-arrested state. Wild-type (WT) mouse embryonic fibroblasts (MEFs) subjected to tetraploidization with DNA lesions did not undergo mitotic catastrophe-associated cell death and stayed in a growth-arrested state, until immortality was attained with mutations in the Arf/p53 module and recovery of H2AX expression. Whereas tetraploidization was essential for immortalization of WT MEFs, this event was not required for immortalization of MEFs containing mutations in Arf/p53 and these cells still underwent mitotic catastrophe-associated cell death. Thus, WT MEFs are protected from immortalization with genome stability, which is abrogated with tetraploidization and mutation of either Arf or p53.


Assuntos
Pontos de Checagem do Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Diploide , Instabilidade Genômica , Tetraploidia , Proteína Supressora de Tumor p53/fisiologia , Células 3T3 , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Histonas/metabolismo , Camundongos , Camundongos Knockout , Mitose , Proteína Supressora de Tumor p53/genética
15.
J Biol Chem ; 286(20): 18251-60, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21454683

RESUMO

The common polymorphism of p53 at codon 72, either encoding proline or arginine, has drawn attention as a genetic factor associated with clinical outcome or cancer risk for the last 2 decades. We now show that these two polymorphic variants differ in protein structure, especially within the N-terminal region and, as a consequence, differ in post-translational modification at the N terminus. The arginine form (p53-72R) shows significantly enhanced phosphorylation at Ser-6 and Ser-20 compared with the proline form (p53-72P). We also show diminished Mdm2-mediated degradation of p53-72R compared with p53-72P, which is at least partly brought about by higher levels of phosphorylation at Ser-20 in p53-72R. Furthermore, enhanced p21 expression in p53-72R-expressing cells, which is dependent on phosphorylation at Ser-6, was demonstrated. Differential p21 expression between the variants was also observed upon activation of TGF-ß signaling. Collectively, we demonstrate a novel molecular difference and simultaneously suggest a difference in the tumor-suppressing function of the variants.


Assuntos
Códon , Predisposição Genética para Doença , Neoplasias , Polimorfismo Genético , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53 , Linhagem Celular , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
16.
Biochem Biophys Res Commun ; 408(1): 38-44, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21453680

RESUMO

Transcription factors play a crucial role in the development of various tissues. In particular, the transcription factors of the basic helix-loop-helix (bHLH) family are crucial regulators of neurodifferentiation. Previous studies suggested that the bHLH transcription factor Hand2 is essential for sympathetic nervous system neuron differentiation in vivo, but the molecular mechanisms involved have not been well elucidated. It is important for understanding their mode of action in cellular differentiation to clarify how these bHLH factors regulate distinct transcriptional targets in a temporally and spatially controlled manner. Recent reports on ES cell differentiation suggested that its molecular mechanism mimics that of in vivo neurogenesis. However, the diverse nature of ES cell populations has prevented efficient analysis. To address this issue, we previously established a cell line in P19 embryonal carcinoma (EC) cells. Efficient sympathetic nervous system (SNS) neuron differentiation is induced in the cell line. Using this cell line, we succeeded in showing that the interaction of bHLH transcription factor Hand2 with E2a is required for transcription of Phox2b, which is essential for autonomic nervous system neuron development, and this binding activates this expression in SNS differentiation. Moreover, we also demonstrated that Hes5 regulated the transcription of Phox2b as a negative regulator and it inhibited the SNS differentiation. These findings have enabled us to determine the novel regulatory mechanism of Phox2b in SNS differentiation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Neurogênese/genética , Neurônios/citologia , Sistema Nervoso Simpático/citologia , Fatores de Transcrição/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Imunoprecipitação , Camundongos , RNA Interferente Pequeno/genética , Transcrição Gênica
17.
J Neurosci Res ; 88(13): 2787-97, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20629186

RESUMO

PRP19alpha and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19 alpha/14-3-3beta/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Analysis of PRP19 alpha mutants revealed that the phosphorylation of PRP19 alpha at Thr 193 by Akt was critical for its binding with 14-3-3beta to translocate into the nuclei and for PRP19 alpha/14-3-3beta/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19 alpha or sense 14-3-3beta RNAs promoted NGF-induced neuronal differentiation, whereas down-regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19 alpha T193A lost its binding ability with 14-3-3beta and acted as a dominant-negative mutant in neuronal differentiation. These results imply that Akt-dependent phosphorylation of PRP19 alpha at Thr193 triggers PRP19 alpha/14-3-3beta/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre-mRNAs.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteína Oncogênica v-akt/metabolismo , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Chlorocebus aethiops , Eletroforese em Gel Bidimensional/métodos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoprecipitação/métodos , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Células PC12 , Fosforilação , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Tetraciclina/farmacologia , Treonina/metabolismo
18.
Biochem Biophys Res Commun ; 390(2): 223-9, 2009 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-19785992

RESUMO

The basic helix-loop-helix transcription factor Hand2 is induced by bone morphogenetic proteins (BMPs) in neural crest-derived precursor cells during the early stage of development of the autonomic nervous system (ANS). Previous studies showed that Hand2 was essential for the ANS differentiation. However, regulatory mechanism of pluripotent genes has not been elucidated in ANS differentiation. Here, we show that Hand2 regulated nanog expression in ANS differentiation. Our studies demonstrated that the forced expression of Hand2 promoted the ANS differentiation program in P19 embryonal carcinoma (EC) cells without aggregation. Furthermore, our results suggested that Hand2 bound to the promoter of nanog, a gene required for embryonic stem cells self-renewal, and suppressed nanog expression after Hand2 induction. The rapid downregulation of nanog mRNA during ANS differentiation correlated with the Hand2 transcriptional activity and nanog promoter methylation. These findings are evidence for a presence of the novel regulatory mechanism of nanog in ANS differentiation.


Assuntos
Sistema Nervoso Autônomo/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Neurogênese/genética , Animais , Sistema Nervoso Autônomo/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Células-Tronco Embrionárias/metabolismo , Camundongos , Proteína Homeobox Nanog
19.
Cell ; 136(3): 535-50, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19203586

RESUMO

p53 And Akt are critical players regulating tumorigenesis with opposite effects: whereas p53 transactivates target genes to exert its function as a tumor suppressor, Akt phosphorylates its substrates and transduces downstream survival signals. In addition, p53 and Akt negatively regulate each other to balance survival and death signals within a cell. We now identify PHLDA3 as a p53 target gene that encodes a PH domain-only protein. We find that PHLDA3 competes with the PH domain of Akt for binding of membrane lipids, thereby inhibiting Akt translocation to the cellular membrane and activation. Ablation of endogenous PHLDA3 results in enhanced Akt activity and decrease of p53-dependent apoptosis. We also demonstrate the suppression of anchorage-independent cell growth by PHLDA3. Loss of the PHLDA3 genomic locus was frequently observed in primary lung cancers, suggesting a role of PHLDA3 in tumor suppression. Our results reveal a new mode of coordination between the p53 and Akt pathways.


Assuntos
Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Proteína Oncogênica v-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais
20.
J Biol Chem ; 284(8): 5050-5, 2009 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-19106089

RESUMO

Cell cycle regulation and biochemical responses upon nutrients and growth factors are the major regulatory mechanisms for cell sizing in mammals. Recently, we identified that the death effector domain-containing DEDD impedes mitotic progression by inhibiting Cdk1 (cyclin-dependent kinase 1) and thus maintains an increase of cell size during the mitotic phase. Here we found that DEDD also associates with S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase, and supports its activity by preventing inhibitory phosphorylation of S6K1 brought about by Cdk1 during the mitotic phase. DEDD(-/-) cells showed reduced S6K1 activity, consistently demonstrating decreased levels in activating phosphorylation at the Thr-389 site. In addition, levels of Cdk1-dependent inhibitory phosphorylation at the C terminus of S6K1 were enhanced in DEDD(-/-) cells and tissues. Consequently, as in S6K1(-/-) mice, the insulin mass within pancreatic islets was reduced in DEDD(-/-) mice, resulting in glucose intolerance. These findings suggest a novel cell sizing mechanism achieved by DEDD through the maintenance of S6K1 activity prior to cell division. Our results also suggest that DEDD may harbor important roles in glucose homeostasis and that its deficiency might be involved in the pathogenesis of type 2 diabetes mellitus.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Mitose , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Proteína Quinase CDC2/genética , Tamanho Celular , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Diabetes Mellitus Tipo 2/genética , Ativação Enzimática/genética , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Homeostase/genética , Insulina/genética , Insulina/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Estrutura Terciária de Proteína/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética
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