RESUMO
Spatial estimates of crop evapotranspiration with high accuracy from the field to watershed scale have become increasingly important for water management, particularly over irrigated agriculture in semiarid regions. Here, we provide a comprehensive assessment on patterns of annual agricultural water use over California's Central Valley, using 30-m daily evapotranspiration estimates based on Landsat satellite data. A semiempirical Priestley-Taylor approach was locally optimized and cross-validated with available field measurements for major crops including alfalfa, almond, citrus, corn, pasture, and rice. The evapotranspiration estimates explained >70% variance in daily measurements from independent sites with an RMSE of 0.88 mm day-1. When aggregated over the Valley, we estimated an average evapotranspiration of 820 ± 290 mm yr-1 in 2014. Agricultural water use varied significantly across and within crop types, with a coefficient of variation ranging from 8% for Rice (1,110 ± 85 mm yr-1) to 59% for Pistachio (592 ± 352 mm yr-1). Total water uses in 2016 increased by 9.6%, as compared to 2014, mostly because of land-use conversion from fallow/idle land to cropland. Analysis across 134 Groundwater Sustainability Agencies (GSAs) further showed a large variation of agricultural evapotranspiration among and within GSAs, especially for tree crops, e.g., almond evapotranspiration ranging from 339 ± 80 mm yr-1 in Tracy to 1,240 ± 136 mm yr-1 in Tri-County Water Authority. Continuous monitoring and assessment of the dynamics and spatial heterogeneity of agricultural evapotranspiration provide data-driven guidance for more effective land use and water planning across scales.
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Dual-energy computed tomography (DECT) combines the advantages of conventional CT with the ability to detect bone marrow oedema (BMO), which was previously limited to magnetic resonance imaging (MRI). By analysing DECT virtual non-calcium (VNCa) maps, radiologists can improve the detection of subtle and occult fractures and approximate the acuity/healing of fractures of indeterminate age. This review highlights the role of DECT in the assessment of musculoskeletal trauma, particularly among elderly, post-menopausal women and those at risk for osteoporosis. DECT is especially useful in investigating trabecular bone predominant regions (e.g., vertebral bodies, pelvis, hip, and long bone metaphyses) for stress (i.e., fatigue or insufficiency) and fragility fractures. CT is often performed first due to its increased availability, especially in the emergency setting, shorter imaging duration, and possible patient contraindications to magnetic resonance imaging (MRI). By enabling BMO detection, DECT may have a role in triaging patients for definitive MRI assessment. Understanding the role of anatomical, pathological, and patient factors in image interpretation can improve radiologist adoption of DECT, increase diagnostic confidence, and improve patient management.
Assuntos
Doenças da Medula Óssea/diagnóstico por imagem , Edema/diagnóstico por imagem , Fraturas Ósseas/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Diagnóstico Diferencial , Humanos , Interpretação de Imagem Radiográfica Assistida por ComputadorRESUMO
Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.
Assuntos
Transformação Celular Neoplásica , Epigênese Genética , Receptores ErbB , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Glioblastoma , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Receptores ErbB/biossíntese , Receptores ErbB/genética , Amplificação de Genes , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/patologia , HumanosRESUMO
BACKGROUND: The human epidermal growth factor receptor (EGFR) is an important therapeutic target in oncology, and three different types of EGFR inhibitors have been approved for the treatment of cancer patients. However, there has been no clear association between the expression levels of EGFR protein in the tumours determined by the FDA-approved EGFR PharmDx kit (Dako) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors. METHOD: In this study, we investigated the potential of our anti-EGFR monoclonal antibodies (mAbs; ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild-type EGFR and/or the type-III deletion mutant form of EGFR (EGFRvIII) in formalin-fixed, paraffin-embedded human tumour specimens. RESULTS: We found that the anti-EGFR mAb in the EGFR PharmDx kit stained both wild-type and EGFRvIII-expressing cells in formalin-fixed, paraffin-embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR10 and ICR9 were specific for the wild-type EGFR. CONCLUSION: We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild-type EGFR protein in tumour specimens using immunohistochemistry, and to determine their prognostic significance, as well as predictive value for response to therapy with EGFR antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores ErbB/análise , Neoplasias/diagnóstico , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/imunologia , Humanos , Imuno-Histoquímica , Proteínas Mutantes/análise , Proteínas Mutantes/imunologia , Neoplasias/química , Inclusão em Parafina , Valor Preditivo dos TestesRESUMO
The epidermal growth factor receptor (EGFR) is essential to multiple physiological and neoplastic processes via signaling by its tyrosine kinase domain and subsequent activation of transcription factors. EGFR overexpression and alteration, including point mutations and structural variants, contribute to oncogenesis in many tumor types. In this study, we identified an in-frame splice variant of the EGFR called mini-LEEK (mLEEK) that is more broadly expressed than the EGFR and is overexpressed in several cancers. Unlike previously characterized EGFR variants, mLEEK lacks the extracytoplasmic, transmembrane and tyrosine kinase domains. mLEEK localizes in the nucleus and functions as a transcription factor to regulate target genes involved in the cellular response to endoplasmic reticulum (ER) stress, including the master regulator of the unfolded protein response (UPR) pathways, molecular chaperone GRP78/Bip. We demonstrated that mLEEK regulates GRP78 transcription through direct interaction with a cis-regulatory element within the gene promoter. Several UPR pathways were interrogated and mLEEK expression was found to attenuate the induction of all pathways upon ER stress. Conversely, knockdown of mLEEK resulted in caspase-mediated cell death and sensitization to ER stress. These findings indicate that mLEEK levels determine cellular responses to unfavorable conditions that cause ER stress. This information, along with the overexpression of mLEEK in tumors, suggests unique strategies for therapeutic intervention. Furthermore, the identification of mLEEK expands the known mechanisms by which the EGFR gene contributes to oncogenesis and represents the first link between two previously disparate areas in cancer cell biology: EGFR signaling and the UPR.
Assuntos
Receptores ErbB/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Transcrição Gênica , Ativação Transcricional , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Receptores ErbB/genética , Feminino , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/genética , Melanoma/metabolismo , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Pele/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Resposta a Proteínas não DobradasRESUMO
The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2α, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2α isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2α mRNA expression than normal lung control tissue. To determine the importance of JNK2α in NSCLC progression, we reduced JNK2α expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2α had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2α induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2α can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2α2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2α is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2α.
Assuntos
Adenocarcinoma Bronquioloalveolar/enzimologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma de Células Escamosas/enzimologia , Transformação Celular Neoplásica/metabolismo , Neoplasias Pulmonares/enzimologia , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Isoenzimas/metabolismo , Pulmão/enzimologia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Proteína Quinase 9 Ativada por Mitógeno/análise , RNA Interferente Pequeno/farmacologia , Fator de Transcrição STAT3/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Anti-epidermal growth factor receptor strategies are now established in cancer treatment We have recently described the presence of EGFRvIII (a variant EGFR) in prostatic tumours from UK white men and this is now a target for anti-prostate cancer treatments. However, there has been no report on the expression of this abnormal protein in black men. MATERIALS AND METHODS: We determined EGFRvIII expression in sections of normal, benign hyperplastic (BPH) and carcinomatous (CaP) prostatic archival tissues from Nigerian men and UK white men using streptavidin immunohistochemical techniques. The EGFRvIII immunoreactivity was scored visually using a semi-quantitative method and the results compared statistically. RESULTS: EGFRvIII expression increased with increasing malignancy in both study populations (CaP > BPH > Normal p, <0.0001). Furthermore, EGFRvIII expression was similar in both BPH and CaP tissues in black and white men (p, 0.86 and 0.31 respectively). CONCLUSION: These results demonstrate that EGFRvIII immunoreactivity in prostatic tumours in black men is similar to that in white men. Anti-cancer treatments directed at the EGFRvIII should be equally effective in men from both subpopulations.
Assuntos
População Negra , Receptores ErbB/fisiologia , Hiperplasia Prostática/fisiopatologia , Neoplasias da Próstata/fisiopatologia , População Branca , Estudos de Casos e Controles , Receptores ErbB/genética , Receptores ErbB/imunologia , Humanos , Imuno-Histoquímica , Masculino , Nigéria/epidemiologia , Hiperplasia Prostática/genética , Hiperplasia Prostática/imunologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Estados Unidos/epidemiologiaRESUMO
Prostatic carcinogenesis has been associated with alterations in the expression of the androgen receptor (AR) and the epidermal growth factor receptor (WT-EGFR), and over-expression of the constitutively active variant epidermal growth factor receptor (EGFRvIII). Changes in the expression of AR, WT-EGFR and EGFRvIII were evaluated in serial sections from 26 normal and 26 benign hyperplastic and 50 prostate cancer tissues using specific immunostaining techniques. The loss of AR expression in peri-epithelial stroma as prostatic tissues de-differentiated correlated strongly with the depletion of WT-EGFR and with increasing expression of the EGFRvIII in the adjacent epithelium. In contrast, changes in epithelial AR immunopositivity in these tissues correlated weakly with the changes in normal and variant EGFR levels. This is the first report correlating the changes in the expression of these three proteins in archival material from the different human prostatic tissue histotypes. The loss of expression of proteins that contribute to the regulation of prostatic homeostasis (AR and WT-EGFR) correlates strongly with the expression of a constitutively active variant EGF receptor (EGFRvIII) in human prostate cancer. These changes occur at an early stage of neoplastic transformation and may contribute to the progression of the disease to hormone independence.
Assuntos
Receptores ErbB/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Endotélio Vascular/metabolismo , Humanos , Imunoquímica , MasculinoRESUMO
CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate-responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.
Assuntos
Receptores de Hialuronatos/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Fracionamento Celular , Linhagem Celular , Genes Reporter , Humanos , Ionomicina/farmacologia , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Ativação TranscricionalRESUMO
Storage of unstained paraffin slides may lead to the deterioration of specimens and failure to detect cellular proteins immunohistochemically. Although the implication of age-induced alterations on multicenter immunohistochemical studies would be considerable, they have not been investigated previously. The current study was undertaken to examine the effect of this factor further and to explore new ways of overcoming the resultant shortcomings. The authors now report on the immunodetection of a host of antigens in similarly preserved unstained serial paraffin slides obtained from three centers using a panel of eight antibodies. Staining of recently prepared sections from the authors' centers resulted in similar strong patterns in seven of eight antibodies, with one antibody demonstrating variable immunoreactivity. However, storage of unstained paraffin sections at room temperature resulted in a variable but progressive decrease in expression of several tissue antigens. Although the loss in antigenicity was proportional to the length of storage, the effect was reversible if super antibody concentrations were used. The authors conclude that recently prepared paraffin sections from centers with similar fixation protocols have similar immunoreactivity and are suitable for use in comparative multicenter studies. However, in view of the delays that may attend tissue transportation during these projects, the authors suggest that test systems should be checked for age-induced antigen degradation by incubating sections with higher antibody concentrations.
Assuntos
Inclusão em Parafina , Manejo de Espécimes , Anticorpos/imunologia , Humanos , Imuno-Histoquímica , Estudos Multicêntricos como AssuntoRESUMO
The Grb2-associated binder-1 (Gab1) is one of the major adapter molecules downstream of growth factor receptor signaling. Even though insulin causes tyrosine phosphorylation of Gab1, its role in insulin signaling has not been identified yet. We have demonstrated that insulin increased expression of early growth response gene-1 (egr-1), which is one of the most important transcription factors involved in cell proliferation and differentiation. In the present study, the possible role of Gab1 in insulin-induced egr-1 expression was studied using Rat1 fibroblasts expressing human insulin receptors and wildtype Gab1 (HIRc/Gab1(WT)), Gab1 with three tyrosines in the phosphatidylinositol (PI) 3'-kinase binding domain mutated to phenylalanine (HIRc/Gab1(DeltaPI3K)), or histidinol resistance only (HIRc/HIS). Insulin-induced egr-1 expression in HIRc/Gab1(DeltaPI3K) cells was much lower than in the other cells, as determined by Northern blot analysis. These results suggest that Gab1 is involved in the signaling pathway for insulin-induced egr-1 expression through increasing PI3'-kinase activity. The MAP kinase activity increased less with insulin treatment in HIRc/Gab1(DeltaPI3K) cells than in other cells. Inhibition of MAP kinase by the MEK inhibitor completely abolished insulin-induced egr-1 expression. These results suggest that Gab1 increases MAP kinase activity through its PI3'-kinase binding site, which then leads to egr-1 expression. Our results indicate that Gab1 is involved in the control of egr-1 expression regulated by insulin.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/fisiologia , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Androstadienos/farmacologia , Animais , Sítios de Ligação , Butadienos/farmacologia , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Proteína 1 de Resposta de Crescimento Precoce , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Flavonoides/farmacologia , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/química , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Transcrição/biossíntese , Transfecção , WortmaninaRESUMO
B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Linfócitos B/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Ligação Proteica , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Proto-Oncogênicas c-aktRESUMO
Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.
Assuntos
Células Epiteliais/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/ultraestrutura , Cães , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Microtúbulos/metabolismo , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
Epidermal growth factor receptor vIII (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the EGFR. Its expression has been detected in gliomas and various other human malignancies. To more fully characterize the function and potential biological role of EGFRvIII in regulating cell proliferation and in tumorigenesis, we transfected EGFRvIII cDNA into a nontumorigenic, interleukin 3 (IL-3)-dependent murine hematopoietic cell line (32D cells). We observed 32D cells expressing high levels of EGFRvIII (32D/EGFRvIII P5) to be capable of abrogating the IL-3-dependent pathway in the absence of ligands. In contrast, the parental cells, 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells, all depended on IL-3 or EGF-like ligands for growth. 32D/EGFRvIII P5 cells subjected to long-term culture conditions in the absence of IL-3 revealed further elevation of EGFRvIII expression levels. These results suggested that the IL-3-independent phenotype is mediated by EGFRvIII. The level of expression is a critical driving force for the IL-3-independent phenotype. Dose-response analysis revealed 32D/EGFRvIII cells to require 500-fold higher concentrations (50 ng/ml) of EGF to further stimulate the EGF-mediated proliferation than in the 32D/EGFR cells (100 pg/ml). Similar effects were also observed in beta-cellulin-mediated proliferation. Moreover, 32D cells expressing high levels of EGFRvIII formed large tumors in nude mice, even when no exogenous EGF ligand was administered. In contrast, no tumors grew in mice injected with 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells or low-expressing clone 32D/EGFRvIII C2 cells or the parental 32D cells. The changes of the ligand specificity support the notion for an altered conformation of EGFRvIII to reveal an activated ligand-independent oncoprotein with tumorigenic activity analogous to v-erbB. These studies clearly demonstrate that EGFRvIII is capable of transforming a nontumorigenic, IL-3-dependent murine hematopoietic cell line (32D cells) into an IL-3-independent and ligand-independent malignant phenotype in vitro and in vivo. To delineate the biological significance of EGFRvIII in human breast cancer, we expressed EGFRvIII in the MCF-7 human breast cancer cell line. Expression of EGFRvIII in MCF-7 cells produced a constitutively activated EGFRvIII receptor. Expression of EGFRvIII in MCF-7 cells also elevated ErbB-2 phosphorylation, presumably through heterodimerization and cross-talk. These MCF-7/EGFRvIII transfectants exhibited an approximately 3-fold increase in colony formation in 1% serum with no significant effect observed at higher percentages of serum. A similar result was also seen in anchorage-dependent assays. Furthermore, EGFRvIII expression significantly enhanced tumorigenicity of MCF-7 cells in athymic nude mice with P < 0.001. Collectively, these results provide the first evidence that EGFRvIII could play a pivotal role in human breast cancer progression.
Assuntos
Neoplasias da Mama/metabolismo , Receptores ErbB/metabolismo , Animais , Divisão Celular , DNA Complementar/metabolismo , Receptores ErbB/genética , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Receptor ErbB-2/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Earlier studies have demonstrated an unexplained depletion of the epidermal growth factor receptor (EGFR) protein expression in prostatic cancer. We now attribute this phenomenon to the presence of a variant EGFR (EGFRvIII) that is highly expressed in malignant prostatic neoplasms. In a retrospective study, normal, benign hyperplastic and malignant prostatic tissues were examined at the mRNA and protein levels for the presence of this mutant receptor. The results demonstrated that whilst EGFRvIII was not present in normal prostatic glands, the level of expression of this variant protein increased progressively with the gradual transformation of the tissues to the malignant phenotype. The selective association of high EGFRvIII levels with the cancer phenotype underlines the role that this mutant receptor may maintain in the initiation and progression of malignant prostatic growth, and opens the way for new approaches in the management of this disease including gene therapy.
Assuntos
Receptores ErbB/análise , Proteínas de Neoplasias/análise , Neoplasias da Próstata/química , Análise de Variância , Western Blotting , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Fenótipo , RNA Mensageiro/análise , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.
Assuntos
DNA/biossíntese , Neuritos/fisiologia , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Apoptose , Sobrevivência Celular , Dados de Sequência Molecular , Fator de Crescimento Neural/farmacologia , Células PC12 , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Coelhos , Ratos , Proteínas Quinases S6 Ribossômicas/metabolismo , Tirosina/metabolismoRESUMO
Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4, 5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.
Assuntos
Proteínas Sanguíneas , Sequência Conservada , Células Epiteliais/citologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Sequência de Aminoácidos , Arginina/genética , Sítios de Ligação/genética , Morfogênese , Mutagênese Sítio-Dirigida , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Homologia de Sequência de Aminoácidos , Triptofano/genéticaRESUMO
Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.