RESUMEN
Differential HLA-C levels influence several human diseases, but the mechanisms responsible are incompletely characterized. Using a validated prediction algorithm, we imputed HLA-C cell surface levels in 228 individuals from the 1000 Genomes dataset. We tested 68,726 SNPs within the MHC for association with HLA-C level. The HLA-C promoter region variant, rs2395471, 800 bp upstream of the transcription start site, gave the most significant association with HLA-C levels (p = 4.2 × 10-66). This imputed expression quantitative trait locus, termed impeQTL, was also shown to associate with HLA-C expression in a genome-wide association study of 273 donors in which HLA-C mRNA expression levels were determined by quantitative PCR (qPCR) (p = 1.8 × 10-20) and in two cohorts where HLA-C cell surface levels were determined directly by flow cytometry (n = 369 combined, p < 10-15). rs2395471 is located in an Oct1 transcription factor consensus binding site motif where the A allele is predicted to have higher affinity for Oct1 than the G allele. Mobility shift electrophoresis demonstrated that Oct1 binds to both alleles in vitro, but decreased HLA-C promoter activity was observed in a luciferase reporter assay for rs2395471_G relative to rs2395471_A on a fixed promoter background. The rs2395471 variant accounts for up to 36% of the explained variation of HLA-C level. These data strengthen our understanding of HLA-C transcriptional regulation and provide a basis for understanding the potential consequences of manipulating HLA-C levels therapeutically.
Asunto(s)
Antígenos HLA-C/biosíntesis , Antígenos HLA-C/genética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas/genética , Algoritmos , Alelos , Sitios de Unión/genética , Conjuntos de Datos como Asunto , Genoma Humano/genética , Estudio de Asociación del Genoma Completo , Células HeLa , Humanos , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Aplastic anemia (AA) is characterized by hypocellular marrow and peripheral pancytopenia. Because interferon gamma (IFN-γ) can be detected in peripheral blood mononuclear cells of AA patients, it has been hypothesized that autoreactive T lymphocytes may be involved in destroying the hematopoietic stem cells. We have observed AA-like symptoms in our IFN-γ adenylate-uridylate-rich element (ARE)-deleted (del) mice, which constitutively express a low level of IFN-γ under normal physiologic conditions. Because no T-cell autoimmunity was observed, we hypothesized that IFN-γ may be directly involved in the pathophysiology of AA. In these mice, we did not detect infiltration of T cells in bone marrow (BM), and the existing T cells seemed to be hyporesponsive. We observed inhibition in myeloid progenitor differentiation despite an increase in serum levels of cytokines involved in hematopoietic differentiation and maturation. Furthermore, there was a disruption in erythropoiesis and B-cell differentiation. The same phenomena were also observed in wild-type recipients of IFN-γ ARE-del BM. The data suggest that AA occurs when IFN-γ inhibits the generation of myeloid progenitors and prevents lineage differentiation, as opposed to infiltration of activated T cells. These results may be useful in improving treatment as well as maintaining a disease-free status.
Asunto(s)
Anemia Aplásica/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Madre Hematopoyéticas/inmunología , Interferón gamma/inmunología , Anemia Aplásica/genética , Anemia Aplásica/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea/métodos , Diferenciación Celular/genética , Linaje de la Célula/genética , Eritropoyesis/efectos de los fármacos , Eritropoyesis/genética , Eritropoyesis/inmunología , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Ratones Endogámicos BALB C , Ratones Noqueados , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND & AIMS: Liver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector GLuc, to assess de novo liver tumor progression, as well as the detection of innate immune responses or responses induced by therapeutic intervention. METHODS: Tracking de novo liver tumor progression with GLuc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. RESULTS: Rising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8(+) T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL(+) hepatocytes following liver MET transfection demonstrating that these cells provide protection from MET-induced hepatocyte apoptosis. Treatment with IL-18+IL-12 of mice displaying established HCC decreased tumor burden which was associated with decreased levels of serum luciferase. CONCLUSIONS: Hydrodynamic delivery of the SBT vector GLuc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer. This revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.
Asunto(s)
Adenoma de Células Hepáticas/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/inmunología , Inmunidad Celular , Neoplasias Hepáticas Experimentales/inmunología , Luciferasas/sangre , Proteínas Recombinantes/uso terapéutico , Adenoma de Células Hepáticas/tratamiento farmacológico , Adenoma de Células Hepáticas/patología , Animales , Apoptosis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Progresión de la Enfermedad , Hepatocitos/patología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Interleucina-12/uso terapéutico , Interleucina-18/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/patología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
(1) Background: The hedgehog (HH) signaling pathway is a key regulator of embryonic patterning, tissue regeneration, stem cell renewal, and cancer growth. The smoothened (SMO) protein regulates the HH signaling pathway and has demonstrated oncogenic activity. (2) Methods: To clarify the role of the HH signaling pathway in tumorigenesis, the expression profile of key HH signaling molecules, including SMO, PTCH1, GLI1, GLI2, and GLI3, were determined in 33 cancer cell lines and normal prostate cells and tissues. We performed a computational analysis of the upstream region of the SMO gene to identify the regulatory elements. (3) Results: Three potential CpG islands and several putative SMO promoter elements were identified. Luciferase reporter assays mapped key SMO promoter elements, and functional binding sites for SP1, AP1, CREB, and AP-2α transcription factors in the core SMO promoter region were confirmed. A hypermethylated SMO promoter was identified in several cancer cell lines suggesting an important role for epigenetic silencing of SMO expression in certain cancer cells. (4) Discussion: These results have important implications for our understanding of regulatory mechanisms controlling HH pathway activity and the molecular basis of SMO gene function. Moreover, this study may prove valuable for future research aimed at producing therapeutic downregulation of SMO expression in cancer cells.