RESUMEN
In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.
Asunto(s)
Quimiocina CCL20/metabolismo , Quimiotaxis de Leucocito/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/biosíntesis , Enfermedad de Hodgkin/virología , Linfocitos T Reguladores/inmunología , Adulto , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Femenino , Citometría de Flujo , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica , Masculino , Microdisección , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Células de Reed-Sternberg , Escape del Tumor/inmunología , Regulación hacia ArribaRESUMEN
Classical Hodgkin lymphoma (cHL) is characterized histologically by a minority of malignant Hodgkin Reed-Sternberg cells surrounded by abundant inflammatory cells, generally believed to be of major importance in the pathophysiology of the disease. Here, we present data that link inflammatory cell-derived arachidonic acid metabolites, the cysteinyl leukotrienes (CysLT), to the pathogenesis of cHL. Two HL cell lines, L1236 and KMH2, were shown to express functional CysLT(1) receptors, responding with a robust calcium signal upon leukotriene (LT) D(4) challenge. LTD(4) stimulated protein release of tumor necrosis factor-alpha, interleukin-6 and -8 by L1236 cells and interleukin-8 by KMH2 cells. Importantly, all these LTD(4)-induced effects were blocked by the CysLT(1) receptor-specific antagonist zafirlukast. Immunohistochemical studies of cHL biopsies and microarray analysis of microdissected cells revealed that the CysLT(1) receptor is expressed also by primary Hodgkin Reed-Sternberg cells. As these cells are surrounded by CysLT-producing eosinophils, macrophages and mast cells, our results suggest the CysLTs as mediators in the pathogenesis of cHL, contributing to the aberrant cytokine network of this lymphoma.
Asunto(s)
Cisteína/química , Enfermedad de Hodgkin/fisiopatología , Leucotrienos/fisiología , Adolescente , Adulto , Anciano , Señalización del Calcio , Línea Celular Tumoral , Niño , Preescolar , Femenino , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica , Leucotrieno D4/farmacología , Leucotrienos/química , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismoRESUMEN
BACKGROUND: Invasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells. METHODS: We examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR. RESULTS: Using GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC. CONCLUSION: IDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Perfilación de la Expresión Génica , Microdisección/métodos , Análisis de Matrices Tisulares/métodos , Biomarcadores , Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/genética , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/patología , Colágeno Tipo III/genética , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Rayos LáserRESUMEN
To overcome some limitations of in vitro established cell-line tumor models for Hodgkin lymphoma (HL), we explored whether culturing in a three-dimensional (3D) matrix could improve the quality of the model. We used a novel designer peptide based self-organizing matrix. The gene expression profile of the 3D-cultured HL derived cell-line L1236 was compared with that of suspension-cultured (2D) L1236, as well as to the gene expression profile found in HL tumor samples. To validate our results we also included a gene expression data set of laser captured Hodgkin-Reed - Sternberg (H-RS) cells. The gene expression profiles were analyzed using Affymetrix technology. We found that the 3D culture affected gene expression of a HL derived cell-line inducing a more tumor-related expression profile. 3D culture affected the expression of 500 genes in L1236, upregulating genes involved in immune response and apoptosis and downregulating genes involved in cell division. It also affected genes involved in actin filament polymerization.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/patología , Células de Reed-Sternberg/patología , Apoptosis , Biopsia , Línea Celular Tumoral , Biología Computacional , Humanos , Ganglios Linfáticos/patología , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/química , ARN Neoplásico/metabolismo , Células de Reed-Sternberg/citologíaRESUMEN
Bicalutamide is a non-steroidal anti-androgen commonly used in the treatment of prostate carcinoma. We analysed the transcriptional response to bicalutamide treatment with the aim of explaining the inhibition of telomerase in the androgen-sensitive cell line LNCaP and the effects of bicalutamide on the androgen-insensitive cell line DU145. Cells treated with 80 muM bicalutamide in steroid-depleted medium for 1 day were analysed in duplicate by Affymetrix Human Genome Focus Arrays. Response to bicalutamide in LNCaP cells was represented by downregulation of androgen-regulated genes, activation of the p53 pathway and inhibition of telomerase, which was associated with downregulation of v-myc avian myelocytomatosis viral oncogene homologue (MYC) and telomerase reverse transcriptase subunit. In DU145 cells we observed the influence of cell density on bicalutamide effectivity such that highly confluent cells showed lesser sensitivity than low confluent ones. In conclusion, we provide an explanation for telomerase inhibition after androgen receptor blockade in LNCaP cells and we also report activation of the p53 pathway in LNCaP cells and in-vitro sensitivity to bicalutamide of low confluent androgen-insensitive DU145 cells. These findings might have implications for both experimental and clinical research into prostate cancer. In particular, activation of the p53 pathway after treatment with 80 microM bicalutamide could justify usage of bicalutamide dosages higher than 150 mg daily in androgen-sensitive carcinoma therapy.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Anilidas/farmacología , Genes p53/efectos de los fármacos , Neoplasias de la Próstata/genética , Telomerasa/metabolismo , Andrógenos/farmacología , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Nitrilos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/biosíntesis , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales de Tetrazolio , Tiazoles , Compuestos de TosiloRESUMEN
The characterisation of monoclonal antibodies (MAbs) and their epitopes is important prior to their application as molecular probes. In this study, Western blotting using IgG1 Fc and pFc' fragments was employed to screen seven MAbs before pepscan analysis to determine their reactivity to potentially linear epitopes. MAbs PNF69C, PNF110A, X1A11 and MAbs WC2, G7C, JD312, 1A1 detected epitopes within the C(H)3 and C(H)2 domains, respectively. However, only four MAbs showed pepscan profiles that highlighted likely target residues. In particular, MAbs PNF69C and PNF110A that have previously been characterised with pan-IgG and anti-G3m(u) specificity, detected the peptide motif 338-KAKGQPR-344 which was located within the N-terminal region of the C(H)3 domain. Furthermore the majority of residues were present in all four IgG subclasses. Consequently the peptide identified was consistent with the pan-IgG nature of these antibodies. By using PCImdad, a molecular display programme, this sequence was visualised as surface accessible, located in the C(H)2/C(H)3 inter-domain region and proximal to the residue arginine(435). It is speculated that this residue may be important for phenotypic expression of G3m(u) and specificity of these reagents. Pepscan analysis of MAbs G7C and JD312 (both pan-IgG) highlighted the core peptide sequence 290-KPREE-294, which was present in the C(H)2 domain and was common to all four IgG subclasses. PCImdad also showed this region to be highly accessible and was consistent with previous bioinformatic and autoimmune analysis of IgG. Overall these MAbs may serve as useful anti-IgG or anti-G3m(u) reagents and probes of immunoglobulin structure.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mapeo Epitopo , Epítopos/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Animales , Anticuerpos Antiidiotipos/clasificación , Anticuerpos Antiidiotipos/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Pruebas de Inhibición de Hemaglutinación , Humanos , Fragmentos Fc de Inmunoglobulinas/clasificación , Inmunoglobulina G/clasificación , Cadenas Pesadas de Inmunoglobulina/inmunología , Técnicas In VitroRESUMEN
Connective tissue growth factor (CTGF) has a major role in development of fibrosis and in the wound-healing process. Microarray analysis of 44 classical Hodgkin lymphoma (cHL) samples showed higher CTGF messenger RNA expression in the nodular sclerosis (NS) than in the mixed cellularity (MC) subtype. When analyzed by immunohistochemical analysis, Hodgkin-Reed-Sternberg (H-RS) cells and macrophages in 23 cHLs and "popcorn" cells in 2 nodular lymphocyte predominant Hodgkin lymphomas showed expression of CTGF protein correlating with the extent of fibrosis. In NS, CTGF was also expressed in fibroblasts and occasional lymphocytes. Malignant cells in 32 samples of various non-Hodgkin lymphomas were negative for CTGF. A staining pattern of stromal cells similar to that of NS cHL was seen in anaplastic large cell lymphoma. Macrophages stained positively in Burkitt lymphomas and in some mantle cell lymphomas. The high occurrence of fibrosis in cHL may be related to CTGF expression by malignant H-RS cells.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Enfermedad de Hodgkin/metabolismo , Linfoma de Células B/metabolismo , Biomarcadores de Tumor/análisis , Factor de Crecimiento del Tejido Conjuntivo/genética , Perfilación de la Expresión Génica , Humanos , ARN Mensajero/metabolismo , Células de Reed-Sternberg/metabolismoRESUMEN
The Epstein-Barr virus (EBV) contributes to the growth and survival of Hodgkin lymphoma (HL) cells. Here we report that down-regulation of the transforming growth factor-beta (TGF-beta) target gene, protein tyrosine phosphatase receptor kappa (PTPRK), followed EBV infection of HL cells and was also more frequently observed in the Hodgkin and Reed-Sternberg (HRS) cells of EBV-positive compared with EBV-negative primary HL. The viability and proliferation of EBV-positive HL cells was decreased by overexpression of PTPRK, but increased following the knockdown of PTPRK expression in EBV-negative HL cells, demonstrating that PTPRK is a functional tumor suppressor in HL. EBV suppressed the TGF-beta-mediated activation of PTPRK expression, suggesting disruption of TGF-beta signaling upstream of PTPRK. This was confirmed when we showed that the Epstein-Barr nuclear antigen-1 (EBNA1) decreased Smad2 protein levels and that this was responsible for PTPRK down-regulation. EBNA1 decreased the half-life of Smad2 but did not interact with Smad2. By down-regulating Smad2 protein expression, EBNA1 apparently disables TGF-beta signaling, which subsequently decreases transcription of the PTPRK tumor suppressor. We speculate that loss of the phosphatase function of PTPRK may activate as-yet-unidentified growth-promoting protein tyrosine kinases, which in turn contribute to the pathogenesis of EBV-positive HL.
Asunto(s)
Infecciones por Virus de Epstein-Barr/complicaciones , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/virología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , División Celular/fisiología , Línea Celular , Supervivencia Celular/fisiología , Regulación hacia Abajo/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Genes Supresores de Tumor/fisiología , Enfermedad de Hodgkin/metabolismo , Humanos , Masculino , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteína Smad2/metabolismoRESUMEN
Polycomb group (PcG) proteins are chromatin modifiers that are necessary for the maintenance and renewal of embryonic and adult stem cells. However, overexpression of the PcG protein, Bmi-1, causes lymphoma in transgenic mice. We show that Bmi-1 is up-regulated in Hodgkin lymphoma (HL) cells by the Epstein-Barr virus (EBV) oncogene latent membrane protein-1 (LMP1) and that this up-regulation is mediated by NF-kappaB signaling. We also show that Bmi-1 is up-regulated by NF-kappaB in EBV-negative HL cells. Down-regulation of LMP1 and Bmi-1 decreased the survival of HL cells, suggesting that Bmi-1 may mediate the prosurvival effects of LMP1-induced NF-kappaB signaling in HL cells. Transcriptional targets of Bmi-1 were identified after its knockdown in an HL cell line. We show here that Bmi-1 and LMP1 down-regulate the ataxia telangiectasia-mutated (ATM) tumor suppressor and conclude that Bmi-1 contributes to LMP1-induced oncogenesis in HL.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Enfermedad de Hodgkin/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Supervivencia Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Perfilación de la Expresión Génica , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Humanos , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba , Proteínas de la Matriz Viral/genéticaRESUMEN
A proportion of patients with Hodgkin lymphoma carry Epstein-Barr virus (EBV), an oncogenic herpesvirus, in their tumor cells. Although it is generally assumed that EBV contributes to the malignant phenotype of Hodgkin lymphoma cells, direct evidence in support of this is lacking. Here we show that EBV infection of Hodgkin lymphoma cells results in the induction of autotaxin, a secreted tumor-associated factor with lysophospholipase-D activity. Up-regulation of autotaxin increased the generation of lysophosphatidic acid (LPA) and led to the enhanced growth and survival of Hodgkin lymphoma cells, whereas specific down-regulation of autotaxin decreased LPA levels and reduced cell growth and viability. In lymphoma tissues, autotaxin expression was mainly restricted to CD30+ anaplastic large-cell lymphomas and Hodgkin lymphoma; in the latter, high levels of autotaxin were strongly associated with EBV positivity (P = .006). Our results identify the induction of autotaxin and the subsequent generation of LPA as key molecular events that mediate the EBV-induced growth and survival of Hodgkin lymphoma cells and suggest that this pathway may provide opportunities for novel therapeutic intervention.