RESUMO
BACKGROUND: We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals. RESULTS: Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera. CONCLUSION: This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.
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The transcription coactivator, Yes-associated protein (YAP), which is a nuclear effector of the Hippo signaling pathway, has been shown to be a mechano-transducer. By using mutant fish and human 3D spheroids, we have recently demonstrated that YAP is also a mechano-effector. YAP functions in three-dimensional (3D) morphogenesis of organ and global body shape by controlling actomyosin-mediated tissue tension. In this chapter, we present a platform that links the findings in fish embryos with human cells. The protocols for analyzing tissue tension-mediated global body shape/organ morphogenesis in vivo and ex vivo using medaka fish embryos and in vitro using human cell spheroids represent useful tools for unraveling the molecular mechanisms by which YAP functions in regulating global body/organ morphogenesis.
Assuntos
Desenvolvimento Embrionário/genética , Morfogênese/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mutação , Proteínas Nucleares/metabolismo , Oryzias , Esferoides Celulares , Fatores de Transcrição/metabolismoRESUMO
Thyroid-stimulating hormone (TSH)-secreting tumors (TSH-omas) are pituitary tumors that constitutively secrete TSH. The molecular genetics underlying this abnormality are not known. We discovered that a knock-in mouse harboring a mutated thyroid hormone receptor (TR) beta (PV; TRbeta(PV/PV) mouse) spontaneously developed TSH-omas. TRbeta(PV/PV) mice lost the negative feedback regulation with highly elevated TSH levels associated with increased thyroid hormone levels (3,3',5-triiodo-l-thyronine [T3]). Remarkably, we found that mice deficient in all TRs (TRalpha1(-/-) TRbeta(-/-)) had similarly increased T3 and TSH levels, but no discernible TSH-omas, indicating that the dysregulation of the pituitary-thyroid axis alone is not sufficient to induce TSH-omas. Comparison of gene expression profiles by cDNA microarrays identified overexpression of cyclin D1 mRNA in TRbeta(PV/PV) but not in TRalpha1(-/-) TRbeta(-/-) mice. Overexpression of cyclin D1 protein led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway only in TRbeta(PV/PV) mice. The liganded TRbeta repressed cyclin D1 expression via tethering to the cyclin D1 promoter through binding to the cyclic AMP response element-binding protein. That repression effect was lost in mutant PV, thereby resulting in constitutive activation of cyclin D1 in TRbeta(PV/PV) mice. The present study revealed a novel molecular mechanism by which an unliganded TRbeta mutant acts to contribute to pituitary tumorigenesis in vivo and provided mechanistic insights into the understanding of pathogenesis of TSH-omas in patients.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hipofisárias/etiologia , Receptores dos Hormônios Tireóideos/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Proliferação de Células , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Fatores de Transcrição E2F , Deleção de Genes , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Imunoprecipitação , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Hipófise/metabolismo , Hormônios Hipofisários/metabolismo , Neoplasias Hipofisárias/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores beta dos Hormônios Tireóideos , Fatores de Tempo , Ativação Transcricional , Transfecção , Tri-Iodotironina/metabolismoRESUMO
Oral squamous cell carcinoma (OSCC) has an absolute majority of all oral cancer. We used proteomic technology to analyze the protein expression profile in OSCC tissues and accompanying surrounding normal tissues in four oral locations (buccal mucosa, gingival mucosa, oral floor, and tongue). Ten protein spots were overexpressed more strongly in cancer tissues than normal ones, and were identified as proliferating cell nuclear antigen, 14-3-3 ε, 14-3-3 σ, proteasome subunit α type 5, translationally controlled tumor protein, eukaryotic translation initiation factor 3 subunit, macrophage capping protein, and mitochondrial isocitrate dehydrogenase subunit α. Macrophage capping protein and mitochondrial isocitrate dehydrogenase subunit α had two spots. Especially, we focused on 14-3-3 σ protein, one of the eight identified proteins, and assessed its expression level in four oral locations of OSCC by using differential display methods. The expression level of 14-3-3 σ protein was upregulated in four locations of oral cavity. Eight proteins which we identified in this study may play an important role in OSCC carcinogenesis and progression and could be used as diagnostic biomarkers of OSCC.
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Heat stress causes severe constraints on numerous physiological functions of cells, such as the repression of splicing of mRNA precursors. In this study, we performed proteomic profiling of a nuclear fraction of Jurkat cells during heat stress using 2-DE and LC-MS/MS. We found 10 protein spots whose expression had changed after heat stress at 43 degrees C for 30 min. Seven of those protein spots, periodic tryptophan protein 1 homolog (PWP1), importin beta-1 subunit, sumoylated protein, splicing factor 3a subunit 3 (SF3a3), TAR DNA-binding protein 43, U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit (U2AF35) and small ubiquitin-related modifier-1 (SUMO-1) were downregulated, while three other protein spots, Protein SET, 40S ribosomal protein SA and 60S acidic ribosomal protein P0 were upregulated by the heat stress. We focused on the downregulation of two splicing factors, which might participate in the repression of pre-mRNA processing by heat stress, leading to cell apoptosis.
Assuntos
Apoptose , Eletroforese em Gel Bidimensional/métodos , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/análise , Proteínas Nucleares/análise , Espectrometria de Massas em Tandem , Cromatografia Líquida , Regulação para Baixo , Transtornos de Estresse por Calor , Humanos , Células Jurkat , Ribonucleoproteína Nuclear Pequena U2/metabolismoRESUMO
In the central nervous system, stressful conditions can easily cause the oxidation of lipoprotein particles, followed by the oxidative modification of apolipoproteins such as apolipoprotein E (apoE) and the production of free radicals and aldehydes. We have confirmed that oxidized very-low-density lipoprotein (VLDL) inhibits the proliferation, viability and differentiation of neuronal PC12 cells leading to cell death. The cells internalized intact apoE, but did not internalize oxidized apoE. The phosphorylation of stathmin and various mitogen-activated protein (MAP) kinases including extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) was examined in PC12 cells exposed to native and oxidized VLDL, H(2)O(2) (which generates free radicals), and 4-hydroxy-2-nonenal (HNE) (an aldehyde). Oxidized VLDL and H(2)O(2) reduced stathmin phosphorylation while HNE increased it, suggesting that oxidized VLDL and H(2)O(2) stimulated similar signal transduction pathways. Based on the results, free radicals, but not aldehydes may play a major role in the neuronal cell death induced by lipoprotein oxidation. Furthermore, the phosphorylation status of MAP kinases indicated that the activation of the JNK cascade might be required for neuronal cell death.
Assuntos
Apolipoproteínas E/metabolismo , Morte Celular , Lipoproteínas VLDL/metabolismo , Proteínas dos Microtúbulos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Fosfoproteínas/metabolismo , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Neurônios/enzimologia , Células PC12 , Fosforilação , Ratos , EstatminaRESUMO
The effects of various lipoproteins on the growth and the differentiation of cultured normal human keratinocytes were investigated. Primary cultures of human epidermal keratinocytes were obtained from neonatal foreskin, and then added with lipoproteins, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Cell growth potential was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VLDL and LDL enhanced keratinocytes growth and LDL receptor expression at the plasma membrane level. These effects were more remarkably observed in cells cultured with VLDL than in cells cultured with LDL. Apolipoprotein E (ApoE) was highly increased in VLDL treated cells. These results suggest that VLDL binds with high affinity to cell surface receptors and induces cell proliferation.
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Queratinócitos/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/farmacologia , Lipoproteínas VLDL/farmacologia , Apolipoproteínas E/biossíntese , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Epidérmicas , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Receptores de LDL/biossínteseRESUMO
The oxidative modification of apolipoprotein (apo) E and lipid peroxidation in human very-low-density lipoprotein (VLDL) induced by peroxynitrite and cupric ions in vitro were strongly suppressed by enrichment with alpha-tocopherol (alpha-Toc; 170 microM). Alpha-Toc also suppressed the decrease in the heparin-binding activity of apoE in the VLDL oxidation. These results suggest that alpha-Toc protected apoE in VLDL from oxidative stress.
Assuntos
Apolipoproteínas E/metabolismo , Lipoproteínas VLDL/metabolismo , Molsidomina/análogos & derivados , alfa-Tocoferol/farmacologia , Sulfato de Cobre/farmacologia , Heparina/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Molsidomina/farmacologia , OxirreduçãoRESUMO
The molecular genetics underlying thyroid carcinogenesis is not well understood. We have recently created a mutant mouse by targeting a mutation (PV) into the thyroid hormone receptor beta gene (TRbetaPV mouse). TRbetaPV/PV mice spontaneously develop follicular thyroid carcinoma through pathological progression of hyperplasia, capsular and vascular invasion, anaplasia and eventually metastasis to distant organs. TRbetaPV/PV mice provide an unusual opportunity to study the alterations in gene regulation that occur during thyroid carcinogenesis. To this end, we profiled the genomic changes in the thyroids of TRbetaPV/PV mice at 6 months of age, at which time metastasis had begun. From arrays of 20 000 mouse cDNAs, 185 genes were up-regulated (2-17-fold) and 92 were down-regulated (2-20-fold). Functional clustering of named genes with reported functions (100 genes) indicated that approximately 39% of these genes were tumor-, metastasis/invasion- and cell-cycle-related. Among the activated tumor-related genes identified, cyclin D1, pituitary tumor transforming gene-1, cathespin D and transforming growth factor alpha were also found to over-express in human thyroid cancers. Analyses of the gene profiles suggested that the signaling pathways mediated by thyrotropin, peptide growth factors, transforming growth factor-beta, tumor necrosis factor-alpha and nuclear factor-kappaB were activated, whereas pathways mediated by peroxisome proliferation activated receptor gamma were repressed. These results indicate that complex alterations of multiple signaling pathways contribute to thyroid carcinogenesis. The critical genes associated with thyroid follicular carcinogenesis uncovered in the present study could serve as signature genes for diagnostic purposes, as well as for possible therapeutic targets.