RESUMEN
Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and in situ hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.
Asunto(s)
Coinfección/complicaciones , Infecciones por Herpesviridae/complicaciones , Neoplasias/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/complicaciones , Infecciones Tumorales por Virus/complicaciones , Animales , Gammaherpesvirinae , Macaca mulatta , Virus de la Inmunodeficiencia de los SimiosRESUMEN
Background: Despite advances in immunotherapeutic strategies for neuroblastoma (NBL), relapse remains a significant cause of mortality for high risk patients. The discovery of novel tumor associated antigens to improve efficacy and minimize the toxicities of immunotherapy is therefore warranted. Receptor Tyrosine Kinase-like Orphan Receptor-1 and 2 (ROR1 and ROR2) have been found to be expressed in several malignancies with limited expression in healthy tissues. Objectives: Given their role in tumor migration and proliferation and the fact that they were originally cloned from a NBL cell line, we hypothesized that ROR1 and ROR2 could serve as potential targets for anti-ROR1 and anti-ROR2 based immunotherapies in NBL. Methods: We characterized the mRNA and protein expression of ROR1 and ROR2 in NBL cell lines and tissue microarrays of patient samples. To explore the potential of ROR1 targeting, we performed in vitro cytotoxicity assays against NBL using NK92 cells as effector cells. Results: Both ROR1 and ROR2 are expressed across all stages of NBL. In patients with non-MYC amplified tumors, expression of ROR1/ROR2 correlated with survival and prognosis. Moreover, in a proof-of-concept experiment, pretreatment of NBL cell line with anti-ROR1 antibody showed additive cytotoxicity with NK92 cells. Conclusions: ROR1 and ROR2 could serve as novel targets for immunotherapy in NBL. The additive effect of anti-ROR1 antibodies with NK cells needs to be explored further to evaluate the possibility of combining anti-ROR1 antibodies with immune effectors such as NK92 cells as a potential off-the shelf immunotherapy for NBL and other ROR1 expressing malignancies.
Asunto(s)
Inmunoterapia/métodos , Neuroblastoma/inmunología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/inmunología , Línea Celular Tumoral , Humanos , PronósticoRESUMEN
Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic ß-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, ß-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rß (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/etiología , Modelos Animales de Enfermedad , Células Secretoras de Insulina/metabolismo , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Interleucina-15/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Humanos , Interleucina-15/antagonistas & inhibidores , Interleucina-15/sangre , Subunidad alfa del Receptor de Interleucina-15/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacologíaRESUMEN
Melanocortin 1 receptor (MC1R) signaling stimulates black eumelanin production through a cAMP-dependent pathway. MC1R polymorphisms can impair this process, resulting in a predominance of red phaeomelanin. The red hair, fair skin and UV sensitive phenotype is a well-described melanoma risk factor. MC1R polymorphisms also confer melanoma risk independent of pigment. We investigated the effect of Mc1r deficiency in a mouse model of UV-induced melanoma. C57BL/6-Mc1r+/+-HGF transgenic mice have a characteristic hyperpigmented black phenotype with extra-follicular dermal melanocytes located at the dermal/epidermal junction. UVB induces melanoma, independent of melanin pigmentation, but UVA-induced and spontaneous melanomas are dependent on black eumelanin. We crossed these mice with yellow C57BL/6-Mc1re/e animals which have a non-functional Mc1r and produce predominantly yellow phaeomelanin. Yellow C57BL/6-Mc1re/e-HGF mice produced no melanoma in response to UVR or spontaneously even though the HGF transgene and its receptor Met were expressed. Total melanin was less than in C57BL/6-Mc1r+/+-HGF mice, hyperpigmentation was not observed and there were few extra-follicular melanocytes. Thus, functional Mc1r was required for expression of the transgenic HGF phenotype. Heterozygous C57BL/6-Mc1re/+-HGF mice were black and hyperpigmented and, although extra-follicular melanocytes and skin melanin content were similar to C57BL/6-Mc1r+/+-HGF animals, they developed UV-induced and spontaneous melanomas with significantly less efficiency by all criteria. Thus, heterozygosity for Mc1r was sufficient to restore the transgenic HGF phenotype but insufficient to fully restore melanoma. We conclude that a previously unsuspected melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma and postulate that this pathway is involved in human melanoma.
Asunto(s)
Factor de Crecimiento de Hepatocito/fisiología , Melanoma Experimental/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptor de Melanocortina Tipo 1/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Femenino , Humanos , Masculino , Melaninas/fisiología , Melanoma Experimental/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND & AIMS: Treatment of inflammatory bowel disease would benefit from specific targeting of therapeutics to the intestine. We developed a strategy for localized delivery of the immunosuppressive cytokine interleukin (IL)-27, which is synthesized actively in situ by the food-grade bacterium Lactococcus lactis (LL-IL-27), and tested its ability to reduce colitis in mice. METHODS: The 2 genes encoding mouse IL-27 were synthesized with optimal codon use for L lactis and joined with a linker; a signal sequence was added to allow for product secretion. The construct was introduced into L lactis. Colitis was induced via transfer of CD4(+)CD45RB(hi) T cells into Rag(-/-) mice to induce colitis; 7.5 weeks later, LL-IL-27 was administered to mice via gavage. Intestinal tissues were collected and analyzed. RESULTS: LL-IL-27 administration protected mice from T-cell transfer-induced enterocolitis and death. LL-IL-27 reduced disease activity scores, pathology features of large and small bowel, and levels of inflammatory cytokines in colonic tissue. LL-IL-27 also reduced the numbers of CD4(+) and IL-17(+) T cells in gut-associated lymphoid tissue. The effects of LL-IL-27 required production of IL-10 by the transferred T cells. LL-IL-27 was more effective than either LL-IL-10 or systemic administration of recombinant IL-27 in reducing colitis in mice. LL-IL-27 also reduced colitis in mice after administration of dextran sodium sulfate. CONCLUSIONS: LL-IL-27 reduces colitis in mice by increasing the production of IL-10. Mucosal delivery of LL-IL-27 could be a more effective and safer therapy for inflammatory bowel disease.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Enterocolitis/inmunología , Factores Inmunológicos/administración & dosificación , Enfermedades Inflamatorias del Intestino , Interleucina-10/inmunología , Interleucinas/administración & dosificación , Mucosa Intestinal/inmunología , Lactococcus lactis , Administración Oral , Animales , Modelos Animales de Enfermedad , Factores Inmunológicos/farmacología , Interleucinas/inmunología , Mucosa Intestinal/efectos de los fármacos , Ratones , Linfocitos T , Transformación BacterianaRESUMEN
Inflammation and hypoxia are known to promote the metastatic progression of tumours. The CCAAT/enhancer-binding protein-δ (C/EBPδ, CEBPD) is an inflammatory response gene and candidate tumour suppressor, but its physiological role in tumourigenesis in vivo is unknown. Here, we demonstrate a tumour suppressor function of C/EBPδ using transgenic mice overexpressing the Neu/Her2/ERBB2 proto-oncogene in the mammary gland. Unexpectedly, this study also revealed that C/EBPδ is necessary for efficient tumour metastasis. We show that C/EBPδ is induced by hypoxia in tumours in vivo and in breast tumour cells in vitro, and that C/EBPδ-deficient cells exhibit reduced glycolytic metabolism and cell viability under hypoxia. C/EBPδ supports CXCR4 expression. On the other hand, C/EBPδ directly inhibits expression of the tumour suppressor F-box and WD repeat-domain containing 7 gene (FBXW7, FBW7, AGO, Cdc4), encoding an F-box protein that promotes degradation of the mammalian target of rapamycin (mTOR). Consequently, C/EBPδ enhances mTOR/AKT/S6K1 signalling and augments translation and activity of hypoxia-inducible factor-1α (HIF-1α), which is necessary for hypoxia adaptation. This work provides new insight into the mechanisms by which metastasis-promoting signals are induced specifically under hypoxia.
Asunto(s)
Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteínas F-Box/biosíntesis , Regulación de la Expresión Génica , Hipoxia , Neoplasias Mamarias Animales/secundario , Metástasis de la Neoplasia/patología , Ubiquitina-Proteína Ligasas/biosíntesis , Animales , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Proteína 7 que Contiene Repeticiones F-Box-WD , Glucólisis , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Animales/fisiopatología , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia/fisiopatologíaRESUMEN
We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato/genética , Secuencia de Bases , Interferón gamma , Nefritis Lúpica/inmunología , Eliminación de Secuencia , Animales , Anticuerpos Antinucleares/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Nefritis Lúpica/genética , Macrófagos/inmunología , Macrófagos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones NoqueadosRESUMEN
We employed Francisella tularensis live vaccine strain (LVS) to study mechanisms of protective immunity against intracellular pathogens and, specifically, to understand protective correlates. One potential molecular correlate identified previously was interleukin-6 (IL-6), a cytokine with pleotropic roles in immunity, including influences on T and B cell functions. Given its role as an immune modulator and the correlation with successful anti-LVS vaccination, we examined the role IL-6 plays in the host response to LVS. IL-6-deficient (IL-6 knockout [KO]) mice infected with LVS intradermally or intranasally or anti-IL-6-treated mice, showed greatly reduced 50% lethal doses compared to wild-type (WT) mice. Increased susceptibility was not due to altered splenic immune cell populations during infection or decreased serum antibody production, as IL-6 KO mice had similar compositions of each compared to WT mice. Although LVS-infected IL-6 KO mice produced much less serum amyloid A and haptoglobin (two acute-phase proteins) than WT mice, there were no other obvious pathophysiological differences between LVS-infected WT and IL-6 KO mice. IL-6 KO or WT mice that survived primary LVS infection also survived a high-dose LVS secondary challenge. Using an in vitro overlay assay that measured T cell activation, cytokine production, and abilities of primed splenocytes to control intracellular LVS growth, we found that IL-6 KO total splenocytes or purified T cells were slightly defective in controlling intracellular LVS growth but were equivalent in cytokine production. Taken together, IL-6 is an integral part of a successful immune response to primary LVS infection, but its exact role in precipitating adaptive immunity remains elusive.
Asunto(s)
Vacunas Bacterianas/inmunología , Francisella tularensis/inmunología , Interleucina-6/inmunología , Tularemia/inmunología , Animales , Vacunas Bacterianas/metabolismo , Vacunas Bacterianas/farmacología , Francisella tularensis/metabolismo , Haptoglobinas/inmunología , Haptoglobinas/metabolismo , Interleucina-6/metabolismo , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Amiloide A Sérica/inmunología , Proteína Amiloide A Sérica/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tularemia/metabolismo , Tularemia/microbiología , Tularemia/prevención & control , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/metabolismo , Vacunas Atenuadas/farmacologíaRESUMEN
SMAD4 (MAD homologue 4 (Drosophila)), also known as DPC4 (deleted in pancreatic cancer), is a tumour suppressor gene that encodes a central mediator of transforming growth factor-beta signalling. Germline mutations in SMAD4 are found in over 50% of patients with familial juvenile polyposis, an autosomal dominant disorder characterized by predisposition to hamartomatous polyps and gastrointestinal cancer. Dense inflammatory cell infiltrates underlay grossly normal appearing, non-polypoid colonic and gastric mucosa of patients with familial juvenile polyposis. This prominent stromal component suggests that loss of SMAD4-dependent signalling in cells within the epithelial microenvironment has an important role in the evolution of intestinal tumorigenesis in this syndrome. Here we show that selective loss of Smad4-dependent signalling in T cells leads to spontaneous epithelial cancers throughout the gastrointestinal tract in mice, whereas epithelial-specific deletion of the Smad4 gene does not. Tumours arising within the colon, rectum, duodenum, stomach and oral cavity are stroma-rich with dense plasma cell infiltrates. Smad4(-/-) T cells produce abundant T(H)2-type cytokines including interleukin (IL)-5, IL-6 and IL-13, known mediators of plasma cell and stromal expansion. The results support the concept that cancer, as an outcome, reflects the loss of the normal communication between the cellular constituents of a given organ, and indicate that Smad4-deficient T cells ultimately send the wrong message to their stromal and epithelial neighbours.
Asunto(s)
Neoplasias Gastrointestinales/inmunología , Transducción de Señal , Proteína Smad4/metabolismo , Linfocitos T/metabolismo , Poliposis Adenomatosa del Colon/etiología , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Animales , Comunicación Celular , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Neoplasias Gastrointestinales/metabolismo , Neoplasias Gastrointestinales/patología , Eliminación de Gen , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Proteína Smad4/genéticaRESUMEN
Vaccine-based expansion of T cells is one approach to enhance the graft-versus-tumor effect of allogeneic bone marrow transplantation (BMT), but the complex immunobiology of the allogeneic environment on responses to tumor vaccines has not been well characterized. We hypothesized that subclinical graft-versus-host disease (GVHD) impairs immunity, but modulation of gamma interferon (IFN-gamma) signaling could reverse this effect. Dendritic cell vaccines and donor lymphocyte infusions (DLIs) were incorporated into a minor histocompatibility antigen-mismatched, T cell-depleted, allogeneic BMT mouse model. Animals were then challenged with H-Y expressing tumors. CD4(+) and CD8(+) responses to H-Y were diminished in vaccinated allogeneic versus syngeneic BMT recipients with DLI doses below the threshold for clinical GVHD, especially in thymectomized hosts. IFN-gamma receptor 1-deficient (IFN-gammaR1(-/-)) T cells cannot cause GVHD but also have diminished vaccine responses. Remarkably, IFN-gammaR1(-/-) bone marrow abrogates GVHD, allowing higher DLI doses to be tolerated, but improves vaccine responses and tumor protection. We conclude that tumor vaccines administered after allogeneic BMT can augment graft-versus-tumor if GVHD is avoided and that prevention of IFN-gamma signaling on donor bone marrow is an effective approach to preventing GVHD while preserving immunocompetence.
Asunto(s)
Trasplante de Médula Ósea/fisiología , Enfermedad Injerto contra Huésped/prevención & control , Efecto Injerto vs Tumor/genética , Interferón gamma/metabolismo , Receptores de Interferón/genética , Animales , Trasplante de Médula Ósea/métodos , Femenino , Enfermedad Injerto contra Huésped/complicaciones , Enfermedad Injerto contra Huésped/genética , Efecto Injerto vs Tumor/inmunología , Terapia de Inmunosupresión/efectos adversos , Interferón gamma/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Receptores de Interferón/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Especificidad por Sustrato/genética , Especificidad por Sustrato/inmunología , Acondicionamiento Pretrasplante/métodos , Células Tumorales Cultivadas , Escape del Tumor/genética , Escape del Tumor/inmunología , Receptor de Interferón gammaRESUMEN
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, yet molecular events associated with the genesis and progression of this potentially fatal disease are largely unknown. For the molecules and pathways that have been implicated, genetic validation has been impeded by lack of a mouse model of RMS. Here we show that simultaneous loss of Ink4a/Arf function and disruption of c-Met signaling in Ink4a/Arf(-/-) mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF) induces RMS with extremely high penetrance and short latency. In cultured myoblasts, c-Met activation and Ink4a/Arf loss suppress myogenesis in an additive fashion. Our data indicate that human c-MET and INK4a/ARF, situated at the nexus of pathways regulating myogenic growth and differentiation, represent critical targets in RMS pathogenesis. The marked synergism in mice between aberrant c-Met signaling and Ink4a/Arf inactivation, lesions individually implicated in human RMS, suggests a therapeutic combination to combat this devastating childhood cancer.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Factor de Crecimiento de Hepatocito/metabolismo , Rabdomiosarcoma/metabolismo , Transducción de Señal , Neoplasias de los Tejidos Blandos/metabolismo , Proteína p14ARF Supresora de Tumor/genética , Animales , Células Cultivadas , Humanos , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-met/metabolismo , Rabdomiosarcoma/genética , Neoplasias de los Tejidos Blandos/genéticaRESUMEN
Obesity is a major health hazard that is caused by a combination of genetic and behavioral factors. Several models of obesity have been described in mice that have defects in the production of peptide hormones, in the function of cell membrane receptors, or in a transcription factor required for neuronal cell development. We have been investigating the function of a family of genes (POTE and ANKRD26) that encode proteins that are associated with the inner aspect of the cell membrane and that contain both ankyrin repeats and spectrin helices, motifs known to interact with signaling proteins in the cell. To assess the function of ANKRD26, we prepared a mutant mouse with partial inactivation of the Ankrd26 gene. We find that the homozygous mutant mice develop extreme obesity, insulin resistance, and an increase in body size. The obesity is associated with hyperphagia with no reduction in energy expenditure and activity. The Ankrd26 protein is expressed in the arcuate and ventromedial nuclei within the hypothalamus and in the ependyma and the circumventricular organs that act as an interface between the peripheral circulation and the brain. In the enlarged hearts of the mutant mice, the levels of both phospho-Akt and mTOR were elevated. These results show that alterations in an unidentified gene can lead to obesity and identify a molecular target for the treatment of obesity.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Gigantismo/genética , Obesidad/genética , Factores de Transcripción/fisiología , Animales , Tamaño Corporal , Mapeo Cromosómico , ADN Complementario/metabolismo , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Exones , Etiquetas de Secuencia Expresada , Genotipo , Homocigoto , Humanos , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Mutación , Factores de Transcripción/genéticaRESUMEN
Cancer cells undergo significant changes in carbohydrate expression, and these alterations can be useful as biomarkers and therapeutic targets. In this study, we investigated the expression of carbohydrate antigens containing a terminal GalNAcalpha1-3Gal or GalNAcalpha1-6Gal on human cervix and cervical carcinoma. Monoclonal antibodies to each of these carbohydrates were generated by immunizing rabbits with the corresponding antigen conjugated to KLH followed by hybridoma production. Antibodies were screened and evaluated using a combination of carbohydrate microarray profiling, ELISA, dot blot and immunohistochemical staining to verify specificity. Antibody 132-3 was found to selectively recognize GalNAcalpha1-3Gal with little cross-reactivity to other structurally similar antigens such as GalNAcalpha1-6Gal, blood group A, Forssman antigen and the Tn antigen on both solution assays and human tissue. Although GalNAcalpha1-6Gal expression was not detected, GalNAcalpha1-3Gal expression was found on 55% of squamous cell carcinomas. Expression in normal tissue was observed but was restricted to the suprabasal epithelial layer. Importantly, we found expression of the antigen on cervical cancer had a statistically significant correlation with the 5-year survival rate of the patients (48 vs. 85% for antigen negative vs. positive, p = 0.017). Expression of GalNAcalpha1-3Gal did not correlate with other clinical factors including tumor stage, size and lymph node metastasis, indicating the antigen is a new, independent biomarker for the prognosis of cervical cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Disacáridos/análisis , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/metabolismo , Adulto , Anticuerpos Monoclonales/inmunología , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Cuello del Útero/química , Cuello del Útero/patología , Distribución de Chi-Cuadrado , Disacáridos/química , Disacáridos/inmunología , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Metástasis Linfática , Persona de Mediana Edad , Estructura Molecular , Pronóstico , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The lamin B receptor (LBR) is an integral nuclear envelope protein that interacts with chromatin and has homology to sterol reductases. Mutations in LBR result in Pelger-Huët anomaly and HEM-Greenberg skeletal dysplasia, whereas in mice Lbr mutations result in ichthyosis. To further understand the function of the LBR and its role in disease, we derived a novel mouse model with a gene-trap insertion into the Lbr locus (Lbr(GT/GT)). Phenotypically, the Lbr(GT/GT) mice are similar to ichthyosis mice. The Lbr(GT/GT) granulocytes lack a mature segmented nucleus and have a block in late maturation. Despite these changes in nuclear morphology, the innate granulocyte immune function in the killing of Staphylococcus aureus bacteria appears to be intact. Granulocyte differentiation requires the transcription factor C/EBPepsilon. We identified C/EBPepsilon binding sites within the Lbr promoter and used EMSAs and luciferase assays to show that Lbr is transcriptionally regulated by C/EBPepsilon. Our findings indicate that the Lbr(GT/GT) mice are a model for Pelger-Huët anomaly and that Lbr, under transcriptional regulation of C/EBPepsilon, is necessary for morphological but not necessarily functional granulocyte maturation.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Neutrófilos/citología , Anomalía de Pelger-Huët/genética , Anomalía de Pelger-Huët/fisiopatología , Receptores Citoplasmáticos y Nucleares/genética , Transcripción Genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Diferenciación Celular , Forma del Núcleo Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Insercional , Activación Neutrófila , Neutrófilos/fisiología , Anomalía de Pelger-Huët/embriología , Anomalía de Pelger-Huët/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Staphylococcus aureus/fisiología , Receptor de Lamina BRESUMEN
Inactivation of the p53 pathway represents the most common molecular defect of human cancer. But in the setting of melanoma, a highly aggressive and invariably fatal malignancy in its advanced disseminated form, mutation/deletion of p53 is relatively rare, whereas its positive regulator ARF is often lost. Here, we show that genetic deficiency in Arf but not p53 facilitates rapid development of melanoma in a genetically engineered mouse model. This difference is accounted for, at least in part, by the unanticipated observation that, unlike fibroblasts, senescence control in melanocytes is strongly regulated by Arf and not p53. Moreover, oncogenic NRAS collaborates with deficiency in Arf, but not p53, to fully transform melanocytes. Our data demonstrate that ARF and p53, although linked in a common pathway, suppress tumorigenesis through distinct, lineage-dependent mechanisms and suggest that ARF helps restrict melanoma progression by executing the oncogene-induced senescence program in benign nevi. Thus, therapeutics designed to restore wild-type p53 function may be insufficient to counter melanoma and other malignancies in which ARF holds p53-independent tumor suppressor activity.
Asunto(s)
Senescencia Celular , Melanoma/patología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Proteína p14ARF Supresora de Tumor/fisiología , Proteína p53 Supresora de Tumor , Animales , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Melanocitos/patología , Melanoma/etiología , Ratones , Proteína p14ARF Supresora de Tumor/deficiencia , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
The tumor-associated Tn antigen has been investigated extensively as a biomarker and therapeutic target. Cancer vaccines containing the Tn antigen as a single tumor antigen or as a component of a polyvalent vaccine have progressed into phase I and II clinical trials. One major focus of Tn-based vaccines is the treatment of prostate cancer patients. Although expression of the antigen on prostate tumors is a critical prerequisite, previous reports investigating Tn expression in prostate tumors have produced conflicting results. Using a combination of immunohistochemistry and carbohydrate microarray profiling, we show that only 4% to 26% of prostate tumors express the Tn antigen. Based on our results, the majority of prostate cancer patients do not express the appropriate antigen. Therefore, efforts to preselect the subset of prostate cancer patients with Tn-positive tumors or apply Tn vaccines to other cancers with higher rates of antigen expression could significantly improve clinical response rates. Because conflicting information on carbohydrate expression is a general problem for the field, the approach described in this article of analyzing antigen expression with multiple antibodies and using carbohydrate microarray profiles to interpret the results will be useful for the development of other carbohydrate-based cancer vaccines and diagnostics.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Biomarcadores de Tumor/metabolismo , Vacunas contra el Cáncer/uso terapéutico , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Anciano , Animales , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Biomarcadores de Tumor/inmunología , Carbohidratos/inmunología , Carcinoma de Células Transicionales/inmunología , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/terapia , Ensayos Clínicos como Asunto , Humanos , Hiperplasia/inmunología , Hiperplasia/metabolismo , Hiperplasia/terapia , Masculino , Neoplasias de la Próstata/metabolismo , ConejosRESUMEN
The transforming growth factor-beta (TGF-beta) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-beta is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-beta has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-beta reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-beta receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-beta involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-beta. These data suggest new roles for the TGF-beta pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation.
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Neoplasias de la Mama/patología , Células Madre Neoplásicas/patología , Factor de Crecimiento Transformador beta/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Diferenciación Celular/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Humanos , Proteína 1 Inhibidora de la Diferenciación/biosíntesis , Proteína 1 Inhibidora de la Diferenciación/genética , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/deficiencia , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Factor de Crecimiento Transformador beta/deficiencia , Trasplante HeterólogoRESUMEN
BACKGROUND: Development of therapies for patients with BRCA1 mutations has been hampered by lack of readily available in vitro and in vivo models. We recently showed that transplantation of transgenic mammary tumors as cell suspensions into naïve recipients generates reproducible tumors with remarkable stability of gene expression profile. We examined the expression profiles of original and serially transplanted mammary tumors from Brca1 deficient mice, and tumor derived cell lines to validate their use for preclinical testing and studies of tumor biology. METHODS: Original tumors, serially transplanted and multiple cell lines derived from Brca1 mammary tumors were characterized by morphology, gene and protein expression, and cell surface markers. RESULTS: Gene expression among Brca1 tumors showed more heterogeneity than among previously characterized tumors from MMTV-PyMT and -Wnt1 models. Gene expression data segregated Brca1 tumors into 3 distinct types: basal, mixed luminal, and tumors with epithelial-to-mesenchymal transition (EMT). Serial transplantation of individual tumors and multiple cell lines derived from the original tumors recapitulated the molecular characteristics of each tumor of origin. One tumor had distinct features of EMT and gave rise to cell lines that contained a distinct CD44+/CD24-/low population that may correlate with human breast cancer stem cells. CONCLUSION: Although individual tumors expanded by transplantation maintain the genomic profile of the original tumors, the heterogeneity among Brca1 tumors limits the extent of their use for preclinical testing. However, cell lines offer a robust material for understanding tumor biology and response to therapies driven by BRCA1 deficiency.
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Proteína BRCA1/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Mutación/genética , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Mamarias Experimentales/patología , Ratones , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , FenotipoRESUMEN
The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.
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Ancirinas/metabolismo , Perfilación de la Expresión Génica/métodos , Espermatocitos/metabolismo , Testículo/metabolismo , Animales , Ancirinas/inmunología , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Humanos , Inmunohistoquímica/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Espermatocitos/inmunología , Testículo/inmunologíaRESUMEN
BACKGROUND: Although severe hepatitis and liver tumors occur in a high percentage of A/J male mice naturally infected with Helicobacter hepaticus, these effects have not been observed after injection of adult mice with the bacteria. OBJECTIVES: We tested the hypothesis that perinatal exposure to the bacteria is required for liver tumorigenesis. METHODS: A/J female mice were infected by intragastric (ig) or intraperitoneal (ip) treatment with 1.5 x 10(8) H. hepaticus before pregnancy. We examined offspring at progressive time intervals, including some kept until natural death in old age. A/J, BALB/c, and C57BL/6 weanling male mice were similarly treated ig with the bacteria and observed for up to 2 years. RESULTS: After ip bacterial infection of A/J females, 41% of their male offspring developed hepatitis and 33% had hepatocellular tumors, including 18% with hepatocellular carcinoma. Treatment by the ig route resulted in a similar incidence of hepatitis in offspring (35%) but fewer total liver tumors (8%) and carcinomas (4%). By contrast, ig instillation of H. hepaticus in weanling A/J, C57BL/6, or BALB/c mice resulted in low incidence of hepatitis (0-20%) and few liver tumors, despite presence of bacteria confirmed in feces. CONCLUSIONS: Results indicate that a high incidence of liver tumors in mice infected with H. hepaticus requires perinatal exposure. Contributing perinatal factors could include known high sensitivity of neonatal liver to tumor initiation, and/or modulation of immune response to the bacterium or its toxins. Mechanisms of human perinatal sensitivity to such phenomena can be studied with this model.