RESUMO
AIMS: Exercise training (ET) has a cardioprotective effect and can alter the molecular response to myocardial infarction (MI). The Neuregulin 1 (NRG1)/ErbB signaling plays a critical role in cardiac repair and regeneration in the failing heart. We sought to investigate whether ET following MI could activate the NRG1/ErbB signaling and promote cardiac repair and regeneration. MAIN METHODS: Male Sprague-Dawley rats were used to establish the MI model. Exercise-trained animals were subjected to four weeks of exercise (16m/min, 50min/d, 5d/wk) following the surgery. AG1478 was used as an inhibitor of ErbB (1mg/kg body weight, administered i.v. every other day during the process of training). NRG1/ErbB signaling activation, cardiomyocyte (CM) proliferation and apoptosis were evaluated. KEY FINDINGS: In the exercise-trained rats, NRG1 expression was up-regulated and ErbB/PI3K/Akt signaling was activated compared with the MI group. In addition, ET preserved heart function accompanied with increased numbers of BrdU(+) CMs, PCNA(+) CMs and c-kit(+) cells, and reduced apoptosis level in the MI rats. In contrast, blocking ErbB signaling by AG1478 attenuated the ET-induced cardiac repair and regeneration. SIGNIFICANCE: ET up-regulates NRG1 expression and activates ErbB2, ErbB4 and PI3K/Akt signal transduction to promote cardiac repair through endogenous regeneration. Activation of ErbB may be an underlying mechanism for the ET-induced cardiac repair and regeneration following MI.
Assuntos
Modelos Animais de Doenças , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Neuregulina-1/biossíntese , Proteínas Oncogênicas v-erbB/biossíntese , Condicionamento Físico Animal/métodos , Animais , Masculino , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Quinazolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Tirfostinas/farmacologiaRESUMO
Neuregulin1, a protein involved in signaling through the ErbB receptors, is required for the proper development of multiple organ systems. A complete understanding of the expression profile of Neuregulin1 is complicated by the presence of multiple isoform variants that result from extensive alternative splicing. Remarkably, these numerous protein products display a wide range of divergent functional roles, making the characterization of tissue-specific isoforms critical to understanding signaling. Recent evidence suggests an important role for Neuregulin1 signaling during olfactory epithelium development and regeneration. In order to understand the physiological consequences of this signaling, we sought to identify the isoform-specific and cell type-specific expression pattern of Neuregulin1 in the adult olfactory mucosa using a combination of RT-qPCR, FACS, and immunohistochemistry. To complement this information, we also analyzed the cell-type specific expression patterns of the ErbB receptors using immunohistochemistry. We found that multiple Neuregulin1 isoforms, containing predominantly the Type I and Type III N-termini, are expressed in the uninjured olfactory mucosa. Specifically, we found that Type III Neuregulin1 is highly expressed in mature olfactory sensory neurons and Type I Neuregulin1 is highly expressed in duct gland cells. Surprisingly, the divergent localization of these Neuregulin isoforms and their corresponding ErbB receptors does not support a role for active signaling during normal turnover and maintenance of the olfactory mucosa. Conversely, we found that injury to the olfactory epithelium specifically upregulates the Neuregulin1 Type I isoform bringing the expression pattern adjacent to cells expressing both ErbB2 and ErbB3 which is compatible with active signaling, supporting a functional role for Neuregulin1 specifically during regeneration.
Assuntos
Neuregulina-1/metabolismo , Mucosa Olfatória/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Regeneração/fisiologia , Animais , Éxons , Regulação da Expressão Gênica , Genes erbB , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuregulina-1/biossíntese , Neuregulina-1/genética , Mucosa Olfatória/lesões , Neurônios Receptores Olfatórios/metabolismo , Proteínas Oncogênicas v-erbB/biossíntese , Proteínas Oncogênicas v-erbB/genética , Isoformas de Proteínas , Regeneração/genética , Transdução de SinaisRESUMO
Overexpression of members of the ErbB receptor family is common in oral squamous cell carcinomas (OSCC); however, their prognostic value for aggressive OSCC has been debated. Extranodal spread to cervical lymph nodes is the most significant prognostic indicator in OSCC. In the present study, we investigated the clinical significance of single versus paired overexpression of members of the ErbB receptor family in 82 OSCC patients with lymph nodes metastasis, with or without capsular rupture (CR) followed by at least 10 years. Immunohistochemistry analysis revealed a common overexpression of ErbB1 (P = 0.021), ErbB2 (P = 0.001), ErbB4 (P = 0.048), as well as MMP-2 (P = 0.043) in OSCC cases with CR+. Increased expression of ErbB1 was associated with MMP-2 in tumors with advanced clinical stages, including poorly differentiated (grade III) tumors (P < 0.050). Vascular embolization was associated with MMP-2 (P = 0.021) and MMP-13 (P = 0.010) overexpression. Survival analysis revealed a lower survival probability in tumors overexpressing ErbB1 (P = 0.038), ErbB4 (P = 0.043), and MMP-12 (P = 0.050). As well a strong association was observed in cases with high risk of recurrence and strong immunostaining for ErbB1 (P = 0.017), ErbB4 (P = 0.008), MMP-1 (P = 0.003), MMP-2 (P = 0.016), MMP-10 (P = 0.041), and MMP-13 (P = 0.005). Stratified multivariate survival analysis revealed a strong prognostic interdependence of ErbB1 and ErbB4 cooverexpression in predicting the worst overall and disease-free survivals (P = 0.0013 and P = 0.0004, respectively). Taken together, these results support a cooperation of ErbB1, ErbB4, and members of the MMP family in predicting OSCC invasion and poor clinical outcomes.
Assuntos
Carcinoma de Células Escamosas/genética , Receptores ErbB/genética , Neoplasias Bucais/genética , Proteínas Oncogênicas v-erbB/genética , Adulto , Idoso , Carcinoma de Células Escamosas/patologia , Receptores ErbB/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Metaloproteinase 13 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/biossíntese , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Estadiamento de Neoplasias , Proteínas Oncogênicas v-erbB/biossíntese , Prognóstico , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptor ErbB-4 , Resultado do TratamentoRESUMO
Glucagon-like peptide 2 (GLP-2) is an enteroendocrine hormone trophic for intestinal mucosa; it has been shown to increase enteric neuronal expression of vasoactive intestinal polypeptide (VIP) in vivo. We hypothesized that GLP-2 would regulate VIP expression in enteric neurons via a phosphatidylinositol-3 kinase-γ (PI3Kγ) pathway. The mechanism of action of GLP-2 was investigated using primary cultures derived from the submucosal plexus (SMP) of the rat and mouse colon. GLP-2 (10(-8) M) stimulation for 24 h increased the proportion of enteric neurons expressing VIP (GLP-2: 40 ± 6% vs. control: 22 ± 5%). GLP-2 receptor expression was identified by immunohistochemistry on neurons (HuC/D+) and glial cells (GFAP+) but not on smooth muscle or fibroblasts in culture. Over 1-4 h, GLP-2 stimulation of SMP increased phosphorylated Akt/Akt ratios 6.1-fold, phosphorylated ERK/ERK 2.5-fold, and p70S6K 2.2-fold but did not affect intracellular cAMP. PI3Kγ gene deletion or pharmacological blockade of PI3Kγ, mammalian target of rapamycin (mTOR), and MEK/ERK pathways blocked the increase in VIP expression by GLP-2. GLP-2 increased the expression of growth factors and their receptors in SMP cells in culture [IGF-1r (3.2-fold increase), EGFr (5-fold), and ErbB-2-4r (6- to 7-fold)] and ligands [IGF-I (1.5-fold), amphiregulin (2.5-fold), epiregulin (3.2-fold), EGF (7.5-fold), heparin-bound EGF (2.0-fold), ß-cellulin (50-fold increase), and neuregulins 2-4 (300-fold increase) (by qRT-PCR)]. We conclude that GLP-2 acts on enteric neurons and glial cells in culture via a PI3Kγ/Akt pathway, stimulating neuronal differentiation via mTOR and ERK pathways, and expression of receptors and ligands for the IGF-I and ErbB pathways.
Assuntos
Sistema Nervoso Entérico/metabolismo , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Neurônios/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Peptídeo Intestinal Vasoativo/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Proteínas ELAV/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Receptores ErbB/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Camundongos , Neurregulinas/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Oncogênicas v-erbB/biossíntese , Proteínas Oncogênicas v-erbB/genética , Fosfatidilinositol 3-Quinase/fisiologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
The role of ErbB family in discreet pituitary functions is reviewed. Several ErbB receptor ligands, EGF, TGFα, and heregulin are differentially expressed in normal gonadotroph and lacto-somatotroph lineages, and other elements of the anterior pituitary. ErbB receptors, i.e. EGFR and ErbB2, are also localized to the anterior pituitary with preferential EGFR lactosomatotroph expression. EGF regulates CRH and ACTH secretion and corticotroph proliferation as well as exhibiting autocrine and paracrine effects on gonadotrophs and on lactosomatotroph proliferation, gene and protein expression, and hormonal secretion. EGF and EGFR are expressed in both functioning and non-functioning pituitary adenomas, with higher expression in more aggressive tumor subtypes. ErbB2 receptor is detected in all tumor subtypes, particularly in invasive tumors. ErbB tyrosine kinase inhibitors regulate hormonal secretion, cell morphology, and proliferation in lacto-somatotroph tumors, reflecting the emerging application of targeted pituitary therapeutics.
Assuntos
Proteínas Oncogênicas v-erbB/biossíntese , Hipófise/metabolismo , Receptor ErbB-2/biossíntese , Animais , Fator de Crescimento Epidérmico/metabolismo , Humanos , Neuregulina-1/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Neoplasias Hipofisárias/metabolismo , Receptor ErbB-2/metabolismo , Fator de Crescimento Transformador alfa/metabolismoRESUMO
BACKGROUND: ErbB receptors and their cognate ligands are implicated in cancer progression. Their expression in gastrointestinal cancer, however, has not been systemically studied. MATERIALS AND METHODS: The expression of four ErbB receptors and a panel of ErbB ligands were determined by reverse transcription-PCR in two gastric (TMK1, MKN-45) and two colon (SW1116, HT-29) cancer cell lines. Cell proliferation was measured by MTT assay while gene knockdown was achieved by RNA interference. RESULTS: ErbB1, ErbB2 and ErbB3 receptors and five known or putative ErbB ligands, namely, epiregulin, epidermal growth factor (EGF), heparin-binding EGF, transforming growth factor alpha (TGFalpha) and neuroglycan-C were expressed in all four cell lines. Knockdown of neuroglycan-C, however, did not affect cell proliferation. CONCLUSION: This study profiles the expression of ErbB receptors and their cognate ligands in gastric and colon cancer cells. These findings might lay the basis for the development of ErbB pathway-directed therapeutics for gastrointestinal cancer.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Proteínas Oncogênicas v-erbB/biossíntese , Neoplasias Gástricas/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Neoplasias do Colo/patologia , Fator de Crescimento Epidérmico/biossíntese , Epirregulina , Células HT29 , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Ligantes , Neurregulinas/biossíntese , Neoplasias Gástricas/patologiaRESUMO
PURPOSE: To correlate p53 and ErbB receptors status with disease-free survival (DFS) and overall survival (OS) in locally advanced breast cancer. PATIENTS AND METHODS: Sixty patients were included in a single-center, open-label, phase II trial (1998-2003). Analysis of Erb receptors and p53 status and estrogen receptor/progesterone receptor data were available for 33 patients. Neoadjuvant epirubicin 75 mg/m2 and paclitaxel 175-200 mg/m2 were administered every 21 days. The patients underwent surgery and radiation therapy and adjuvant chemo/hormonotherapy. RESULTS: Approximately two thirds of the patients demonstrated overexpression of ErbB receptors and had mutant p53 overexpression. The disease recurred in 11/33 patients and 7 died (median follow-up 56 months). Detrimental effects on OS were established in cases of combined defective p53 expression and ErbB1-ErbB3 heterodimeric receptor overexpression. In contrast, normal p53 together with the same overexpressed heterodimeric combination of ErbB receptors showed no statistically significant effect. CONCLUSION: In terms of the clinical impact of combinations of ErbB receptors with or without mutant p53, only the overexpressed various ErbB1-ErbB3 dimeric combinations and the ErbB1/ErbB2/ErbB3 triplet combination with mutated p53 were related to a significantly poorer outcome. This observation may help in the development of new strategies required for blocking these molecular pathways and improving the outcome of patients with locally advanced breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Proteínas Oncogênicas v-erbB/biossíntese , Receptor Cross-Talk , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
PURPOSE: To assess ErbB-4 expression in advanced human prostate cancer (PC) cell lines, the role of ErbB-4 in motility, migration, and proliferative/tumorigenic potential of PC cells, and efficacy of anti-ErbB-4 monoclonal antibody (Mab) treatment on PC cells in vitro and tumor growth in vivo. MATERIALS AND METHODS: Established advanced human PC cell lines (PC-3, Cl-1, and Du-145) were evaluated for ErbB-4 expression. Several Cl-1 cell line clones expressing various levels of ErbB-4 were isolated, their motility, migration capacity, and in vitro proliferation as well as survival following Mab treatment were evaluated. Tumorigenicity and proliferation capacity of these clones in vivo and efficacy of Mab treatment on tumor growth were estimated by measurements of subcutaneous tumors developed in nude mice. RESULTS: PC cell lines studied express ErbB-4. Both PC-3 and Du-145 cell lines express high ErbB-4 levels; only 50% of Cl-1 cells express ErbB-4 with large heterogeneity. Cl-1 sub-clones highly expressing ErbB-4 showed increased cell motility, migration, and proliferation rate in vitro and enhanced growth in vivo, compared to clones with low ErbB-4 expression. Mab treatment inhibited the growth of cells expressing high but not low ErbB-4 levels in vitro and decreased the growth of subcutaneous tumors in nude mice generated by ErbB-4 highly expressing cells. CONCLUSIONS: High expression of ErbB-4 in prostate cancer Cl-1 cell clones correlated with high proliferative and migration capacity and high tumorigenic potential. The inhibitory effect of Mab on cell proliferation and on subcutaneous tumor growth suggests ErbB-4's potential as a target for molecular anticancer therapy.
Assuntos
Proteínas Oncogênicas v-erbB/biossíntese , Neoplasias da Próstata/patologia , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Feminino , Formazans/química , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Organismos Livres de Patógenos EspecíficosRESUMO
OBJECTIVE: The purpose of this study is to verify the hypothesis that ErbB-2 protein is overexpressed in human middle ear cholesteatomas and to elucidate the relationship between overexpression of ErbB-2 protein, cell proliferation, and apoptosis. STUDY DESIGN: Prospective review of 20 patients between 2001 and 2003 with middle ear cholesteatoma. METHODS: Middle ear cholesteatoma matrix and retroauricular skin were immunostained with anti-ErbB-2, Ki-67, and single-stranded DNA (ssDNA) antibody. The distribution of immunoreactivity to these antibodies and labeling indices were compared between cholesteatoma and retroauricular skin. RESULTS: In matrix of middle ear cholesteatoma, ErbB-2 and ssDNA were expressed in the keratinocytes of all layers and Ki-67 was expressed in the keratinocytes of the basal, lower spinous, and occasionally granular layer. In retroauricular skin, ErbB-2 and Ki-67 were expressed in the keratinocytes of the basal and occasionally lower spinous layer and ssDNA was expressed in the keratinocytes of all layers. Labeling indices against anti-ErbB-2, Ki-67, and ssDNA antibody were significantly greater in cholesteatoma as compared with retroauricular skin. CONCLUSIONS: In cases of cholesteatoma, ErbB-2 protein was overexpressed and cell proliferation and apoptosis of keratinocytes were accelerated. ErbB-2 protein could modulate terminal differentiation and apoptosis in the keratinocytes of all layers in cholesteatoma matrix and cell proliferation in the keratinocytes of the basal and lower spinous layer in normal skin.
Assuntos
Colesteatoma da Orelha Média/metabolismo , Regulação da Expressão Gênica , Proteínas Oncogênicas v-erbB/biossíntese , Adolescente , Adulto , Idoso , Colesteatoma da Orelha Média/genética , Colesteatoma da Orelha Média/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas v-erbB/genética , Estudos ProspectivosRESUMO
Oligodendrogliomas of all grades overexpress epidermal growth factor receptor (EGFR), whereas deletion of ink4a/arf is found only in high-grade tumors. We used the S100 beta promoter to generate transgenic mice expressing v-erbB, a transforming allele of EGFR. These mice developed low-grade oligodendroglioma. Transgenic animals heterozygous for ink4a/arf or p53 developed high-grade tumors. Comparative genomic hybridization revealed loss of distal mouse chromosome 4, a region orthologous with human chromosome 1p, which is commonly lost in oligodendroglioma. Our results demonstrate that overexpression of EGFR, an epigenetic observation of uncertain significance in human oligodendroglioma, can initiate oligodendroglioma in the mouse. Furthermore, p53 pathway mutations can mediate the transition from low to high grade. These models hold promise for studying tumor lineage, identifying contributing genetic alterations and evaluating preclinical therapies in this important neoplasm.
Assuntos
Neoplasias Encefálicas/genética , Receptores ErbB/biossíntese , Genes erbB-1/genética , Oligodendroglioma/genética , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Aberrações Cromossômicas , Cromossomos de Mamíferos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Modelos Animais de Doenças , Receptores ErbB/genética , Genes p53 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Mutação , Fatores de Crescimento Neural/genética , Oligodendroglioma/metabolismo , Oligodendroglioma/patologia , Proteínas Oncogênicas v-erbB/biossíntese , Proteínas Oncogênicas v-erbB/genética , Regiões Promotoras Genéticas , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/genética , Células Tumorais CultivadasAssuntos
Adenocarcinoma/metabolismo , Receptores ErbB/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas Oncogênicas v-erbB/biossíntese , Adenocarcinoma/química , Adulto , Idoso , Receptores ErbB/análise , Feminino , Humanos , Neoplasias Pulmonares/química , Masculino , Pessoa de Meia-IdadeRESUMO
ErbB subfamily genes, known as proto-oncogenes, encode receptor tyrosine kinases, and are expressed in relation to tumorigenesis of the mammary gland in humans. In this study, we examined the expression of erbB subfamily mRNAs in two canine normal mammary glands and 12 mammary tumor samples by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Each primer set was designed from the nucleotide sequence of the region conserved in erbB subfamily cDNA among other species. No erbB subfamily mRNAs were expressed in the normal mammary gland. In contrast, all of the subfamily mRNAs were expressed in a benign mammary tumor, and more than one type of the subfamily mRNA were observed in 11 malignant mammary tumors. The length of RT-PCR products were 380 bp for erbB1, 500 bp for erbB2, 644 bp for erbB3, and 416 bp for erbB4. These sequences were highly homologous to the cDNA sequences of other species. Therefore, these results suggest that the expression of erbB subfamily mRNAs in canine mammary tumors plays an important role in tumorigenesis of the mammary gland.
Assuntos
Doenças do Cão/genética , Genes erbB/genética , Neoplasias Mamárias Animais/genética , Animais , Sequência de Bases , Doenças do Cão/metabolismo , Cães , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Mamárias Animais/metabolismo , Dados de Sequência Molecular , Proteínas Oncogênicas v-erbB/biossíntese , Proteínas Oncogênicas v-erbB/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Homologia de Sequência do Ácido NucleicoRESUMO
Most cervical carcinomas appear to arise from cervical intraepithelial neoplasia (CIN) lesions. In addition to infection with high-risk human papilloma viruses, which is indicative of an increased risk of progression, alterations of oncogenes and tumor suppressor genes play a role. Genetic studies of CIN lesions, primary cervical carcinoma, and metastases may shed light on the relative importance of various genetic alterations involved in the progression of CIN to invasive carcinoma. We examined tumor material from 10 patients with squamous cell carcinoma of the uterine cervix and synchronous CIN lesions and lymph node metastases. The CIN component, invasive carcinoma, and lymph node metastases were analyzed separately for loss of heterozygosity (LOH) on the following loci: VHL (3p21), HLA region (6p22-23), PGL (11q 22-24), E6 associated protein (15q11-13), TP53 (17p13), DCC (18q21.1), and chromosomes 1, 2, 4, 9, 20, and X. Using immunohistochemistry, the expression of the EGF receptor, ERBB2, and TP53 was determined. In CIN lesions, frequent LOH was found at chromosome arms 3p, 6p, and 11q. Primary invasive carcinoma showed additional LOH at chromosome arms 6q, 17p, and 18q. In lymph node metastases, an additional locus on the X chromosome displayed LOH. All carcinomas and synchronous lesions but one showed high expression levels of the EGF receptor. TP53 staining, when present, was found in all synchronous lesions. Focal staining of ERBB2 was found in one CIN lesion, two invasive carcinomas, and four metastases. The molecular alterations accumulated in a fashion that paralleled the progression of the tumors. These results indicate that cervical tumorigenesis occurs in a stepwise fashion, including infection and integration of oncogenic HPV and several specific genetic alterations. Genes Chromosomes Cancer 26:346-354, 1999.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias do Colo do Útero/genética , Adulto , Idoso , Biomarcadores Tumorais , Carcinoma de Células Escamosas/patologia , DNA de Neoplasias , Progressão da Doença , Receptores ErbB/análise , Receptores ErbB/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Pessoa de Meia-Idade , Proteínas Oncogênicas v-erbB/análise , Proteínas Oncogênicas v-erbB/biossíntese , Papillomaviridae/genética , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/biossíntese , Neoplasias do Colo do Útero/patologiaRESUMO
The avian erythroblastosis viral oncogene (v-erbB) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v-erbB in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v-erbB-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc-Grb2-Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação a Calmodulina/isolamento & purificação , Linhagem Celular Transformada , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Embrião de Galinha , Colágeno/metabolismo , Receptores ErbB/biossíntese , Fibroblastos , Proteína Adaptadora GRB2 , Genes erbB , Proteínas Oncogênicas v-erbB/biossíntese , Proteínas Oncogênicas v-erbB/isolamento & purificação , Biossíntese de Proteínas , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Domínios de Homologia de srcRESUMO
OBJECTIVES: To investigate protein expression and genetic aberrations of p53 and HER-2 in renal cell carcinoma (RCC) as well as possible association of p53 and HER-2 abnormalities with the tumor behavior of RCC. METHODS: Formalin-fixed and paraffin-embedded tissue blocks from 70 patients with RCC were studied. Protein expression of p53 and HER-2 was assessed immunohistochemically. Following deoxyribonucleic acid extraction from the paraffin sections using a microdissection technique, p53 gene mutations within exons 5 to 8 were examined by polymerase chain reaction (PCR) single-strand conformation polymorphism and HER-2 gene amplification by a differential PCR. RESULTS: Of the 70 RCCs, 16 (22.9%) showed protein expression of p53, with 7 (10%) demonstrating mutations within exons 5 to 7. Additionally, 28 RCCs (40%) displayed protein expression of HER-2, with 12 (17.1%) demonstrating gene amplification. All cases showing p53 mutations or HER-2 amplification represented protein expression of p53 or HER-2, respectively. No cases with negative immunostaining for p53 or HER-2 protein displayed positive results at gene level. Statistical analysis revealed a close relationship between p53 protein expression and the tumor grade, as well as a significant association of HER-2 protein expression and gene amplification with the tumor stage of RCC. CONCLUSIONS: Our observations suggest that the abnormalities of p53 and HER-2 may be involved in the pathogenesis of RCC and that other mechanisms leading to protein expression of p53 and HER-2 may coexist within a single tumor in addition to the genetic aberrations.
Assuntos
Carcinoma de Células Renais/genética , Genes erbB-2/genética , Genes p53/genética , Neoplasias Renais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/metabolismo , Feminino , Humanos , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Oncogênicas v-erbB/biossíntese , Proteína Supressora de Tumor p53/biossínteseRESUMO
We propose that chronically denervated Schwann cells may be less able to respond to axonal signals than their acutely denervated counterparts, and that this lack of sensitivity may be one reason why axons fail to regenerate into chronically denervated nerve stumps. To test this proposal we have used in situ hybridization, and quantitative and qualitative immunohistochemistry to compare the expression of c-erbB2 and c-erbB4 receptors in Schwann cells denervated for up to 6 months in vivo, with that seen in Schwann cells denervated for similar periods of time but then exposed to regenerating axons. The results were correlated with the extent of axonal regeneration in each experimental group as assessed from transverse sections which had been double-immunolabelled using anti S-100 and anti-beta tubulin III antibodies. Since c-erbBs are receptors for neuronally derived neuregulins we probed the appropriate axotomised DRG neurons for expression of GGF2 mRNA. When the denervated distal stumps were anastomosed to acutely transected proximal stumps, GGF expression in DRGs increased transiently during the first week: we assume that secreted GGF2 derived from regrowing axon sprouts would have been available to Schwann cells in all distal stumps. Endoneurial cell proliferation (predominantly Schwann cell proliferation); levels of expression of c-erbB receptors by Schwann cells, and the degree to which axons regenerated into the distal stumps all decreased as the period of prior denervation increased: the longer the time of denervation, the lower the expression of c-erbBs in Schwann cells, and the smaller the percentage of bands of Bungner which were re-innervated.
Assuntos
Regeneração Nervosa/fisiologia , Proteínas Oncogênicas v-erbB/biossíntese , Células de Schwann/fisiologia , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Western Blotting , Denervação , Feminino , Proteína GAP-43 , Fator de Maturação da Glia , Imuno-Histoquímica , Hibridização In Situ , Glicoproteínas de Membrana/biossíntese , Microscopia Eletrônica , Modelos Neurológicos , Compressão Nervosa , Fatores de Crescimento Neural/biossíntese , Proteínas do Tecido Nervoso/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Células de Schwann/ultraestruturaRESUMO
Mucin production, when heavily sialylated, can promote cancer cell invasion and metastasis, and modulate the immune recognition system of the host. To explore the prognostic implication of sialomucin expression in lung cancer, we studied 116 patients with non-small-cell lung cancer (NSCLC). Tumor specimens were stained immunohistochemically with monoclonal antibodies (mAbs) against mucin glycoprotein (17Q2, HMFG2, SM3), and histochemically with periodic acid-Schiff/alcian blue to differentiate neutral mucin from acid mucin, and with high-iron diamine/alcian blue to differentiate sialomucin from sulfomucin. The expression status of two established molecular prognostic factors, the p53 and erbB-2 oncoproteins, were evaluated immunohistochemically. The staining was performed on two separately archived, paraffin-embedded tumor blocks for each patient, with normal lung as a control. Correlations were subsequently made among stains and various clinicopathologic factors. All analyses were blinded, and included Kaplan-Meier survival estimates with Cox proportional hazards regression modeling. Associations were established among adenocarcinoma histotype and erbB-2 overexpression, sialomucin expression, and 17Q2 and HMFG2 immunohistochemical positivity (p < 0.05). Sialomucin expression was closely linked to erbB-2 overexpression (p = 0.01). Significant univariate predictors (p < 0.05) of recurrence and cancer death were surgical stage, p53 expression, erbB-2 overexpression, and sialomucin expression. These four factors remained as independent predictors of early recurrence (p < 0.05) after multivariate analysis. For cancer death prediction, p53 and sialomucin expression had a marginal effect. We concluded that sialomucin expression is also a poor indicator of prognosis, which is associated with erbB-2 oncoprotein overexpression, early postoperative recurrence, and cancer death in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Mucinas/biossíntese , Recidiva Local de Neoplasia/metabolismo , Proteínas Oncogênicas v-erbB/biossíntese , Análise Atuarial , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Sialomucinas , Proteína Supressora de Tumor p53/biossínteseAssuntos
Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultura Livres de Soro , Fator de Crescimento Epidérmico/farmacologia , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Macrófagos/citologia , Camundongos , Proteínas Oncogênicas v-erbB/biossíntese , Células Estromais/citologiaRESUMO
The human alpha2-integrin gene is transcriptionally downregulated in a nontumorigenic human mammary epithelial cell line, MTSV1-7, and its clonal variant HB2, overexpressing the Erb-B2 oncogene. In this study, we have used deletion mutations within the alpha2-integrin promoter inserted 5' of the chloramphenicol acetyltransferase or luciferase reporter genes to identify the element that is responsible for the Erb-B2-mediated downregulation. The results of the transient-transfection assay showed that the Sp1 binding element located in the core region (positions --64 to +1) of the alpha2-integrin promoter plays an essential role in the alpha2-integrin promoter activity and its downregulation by Erb-B2. By gel shift assay, we have demonstrated that this element binds with a high degree of affinity not only to Sp1, but also to Sp3. The downregulation of the alpha2-integrin promoter activity could also be achieved by overexpression of v-Hras (v-ras), suggesting that the signals generated by Erb-B2, which lead to downregulation of the alpha2-integrin gene expression, may proceed through the ras pathway. Both the Erb-B2- and the v-ras-overexpressing cells exhibited a Sp1 DNA binding activity lower than that of the parental line, while the relative levels of Sp1 protein in these cells were not altered. The Erb-B2- and v-ras-mediated downregulation could be reversed by the overexpression of Sp1 and by a dominant negative variant of ras (rasN17), confirming the importance of Sp1 and the ras pathway. The inhibitory effects of Erb-B2 on transcriptional activity of the alpha2-integrin promoter were observed in transient-cotransfection assays using alpha2-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha2-integrin reporter gene construct was transfected into MTSV1-7 or HB2 cells permanently overexpressing Erb-B2 or v-ras. The effects of Erb-B2 or v-ras on the transcriptional activity of the alpha2-integrin promoter were observed in nontumorigenic luminal epithelial cell lines (MTSV1-7 and HB2) as well as in the breast cancer cell line T47D. These data suggest that in luminal epithelial cells and the breast cancers which develop from them, the Erb-B2 proto-oncogene signaling leads to inhibition of (alpha)2(beta)1-integrin gene expression and could contribute to the disruption of tissue architecture seen in breast cancers.
Assuntos
Antígenos CD/biossíntese , Regulação para Baixo , Genes erbB-2 , Genes ras , Fator de Transcrição Sp1/metabolismo , Sequência de Bases , Sítios de Ligação , Mama , Linhagem Celular , Núcleo Celular/metabolismo , Epitélio/imunologia , Epitélio/metabolismo , Feminino , Genes Reporter , Vetores Genéticos , Humanos , Integrina alfa2 , Proteínas Nucleares/metabolismo , Proteína Oncogênica p21(ras)/biossíntese , Proteínas Oncogênicas v-erbB/biossíntese , Regiões Promotoras Genéticas , Proto-Oncogene Mas , TransfecçãoRESUMO
PURPOSE: To study the c-erbB-2 oncogene in primary transitional cell bladder cancer. MATERIALS AND METHODS: Ninety-five patients with known clinical follow-up and epidermal growth factor receptor (EGFr) status were studied for expression of c-erbB-2 by immunostaining. Possible mechanisms underlying increased staining for c-erbB-2 protein were investigated by analyzing DNA and RNA encoding c-erbB-2. RESULTS: Strong positive staining for c-erbB-2 was detected in 20 (21%) tumors, with weaker staining in a further 13 (14%). There was no correlation between increased staining for c-erbB-2 and tumor stage, grade, or EGFr status. There was a low rate of amplification of the c-erbB-2 gene (1 of 24) on Southern blotting with a higher rate of elevated c-erbB-2 mRNA (4 of 44) with dot blot hybridization. For pT1 tumors, the rate of recurrence was higher for those tumors which were positive for c-erbB-2. CONCLUSIONS: c-erbB-2 oncoprotein is expressed by a significant proportion of transitional cell tumors of the bladder. In this study, the prognostic significance of c-erbB-2 expression appears limited.