RESUMEN
Oncolytic poxviruses have demonstrated initial promising results in patients with cancer in clinical trials, yet further improvements are needed. It has been shown that a single point mutation in the A34R gene resulted in the production of more total progeny virus and more extracellular enveloped virus (EEV), a form that can be immune-evasive and with enhanced spread. We have genetically engineered a new oncolytic poxvirus (designated vA34R) by incorporating this mutated A34R gene into a viral backbone (vvDD) which was designed for tumor-selective replication. This rationally designed virus can evade neutralization from antipoxvirus antibodies and is highly cytotoxic to cancer cells. It demonstrates improved spread and increased replication within the peritoneal cavity resulting in improved antitumor effects in a peritoneal carcinomatosis (PC) model of MC38 colon cancer. Impressively, after carrier cell-mediated delivery in the preimmunized host, vA34R displayed high replication in tumor nodules yet low accumulation in normal tissues thus enhancing the therapeutic index leading to 70% long-term cures. These results demonstrate that vA34R gains an enhanced therapeutic index for PC via immune evasion, increased spread, and production of more progeny virus. Thus, vA34R may be a potent oncolytic virus (OV) for patients with PC, even after prior exposure to vaccinia virus (VV).
Asunto(s)
Vectores Genéticos/fisiología , Mutación , Virus Oncolíticos/fisiología , Poxviridae/fisiología , Proteínas Virales/genética , Animales , Carcinoma/inmunología , Carcinoma/mortalidad , Carcinoma/terapia , Línea Celular Tumoral , Efecto Citopatogénico Viral , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Humanos , Ratones , Viroterapia Oncolítica , Neoplasias Peritoneales/inmunología , Neoplasias Peritoneales/mortalidad , Neoplasias Peritoneales/terapia , Infecciones por Poxviridae/inmunología , Infecciones por Poxviridae/virología , Virus Vaccinia/fisiología , Replicación ViralRESUMEN
Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2'-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.
Asunto(s)
Epigénesis Genética/fisiología , Genes Homeobox/fisiología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Regulación hacia Arriba/fisiología , Animales , Trióxido de Arsénico , Arsenicales/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Epigénesis Genética/efectos de los fármacos , Genes Homeobox/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Inhibidores de Histona Desacetilasas , Humanos , Masculino , Ratones , Óxidos/farmacología , Piridinas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Uromodulin (Tamm Horsfall glycoprotein) is the most abundant protein found in normal human urine. Its function has yet to be determined. Identifying mutations in the uromodulin gene may be helpful in understanding the function of uromodulin. There has been 1 report of 4 families suffering from mutations in the uromodulin gene, resulting in the autosomal dominant transmission of hypouricosuric hyperuricemia and chronic renal failure. This case report describes another family with similar clinical manifestations. METHODS: A family was identified with clinical characteristics of hypouricosuric hyperuricemia and renal failure occurring in a mother and daughter. Clinical characteristics were identified, and laboratory studies were obtained in the proband and the proband's daughter. A genetic analysis was performed to evaluate for mutations in the uromodulin gene. RESULTS: The proband suffered from hyperuricemia at an early age and progressive renal failure with end-stage renal disease developing at age 49 years. The proband's daughter suffered from hyperuricemia, a reduced fractional excretion of uric acid, and mild renal insufficiency. A g.2105G > A mutation in exon 4 of the uromodulin gene resulting in a substitution of tyrosine for cysteine was identified in both the proband and the proband's daughter. The clinical characteristics were similar to those of other patients suffering from uromodulin mutations and to those of patients suffering from medullary cystic kidney disease type 2 and familial juvenile hyperuricemic nephropathy. CONCLUSION: Uromodulin associated kidney disease results in hyperuricemia and renal failure. The specific uromodulin mutation found in this family is consistent with the hypothesis that mutations disrupt highly conserved cysteine residues in the uromodulin protein. Potential mechanisms for these pathologic changes are discussed. The authors would appreciate referral of other families for screening for mutations.
Asunto(s)
Hiperuricemia/genética , Fallo Renal Crónico/etiología , Mucoproteínas/genética , Sustitución de Aminoácidos , Niño , Cisteína/química , Exones/genética , Femenino , Genes Dominantes , Gota/etiología , Humanos , Hiperuricemia/complicaciones , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Mucoproteínas/química , Alineación de Secuencia , Relación Estructura-Actividad , UromodulinaRESUMEN
Medullary cystic kidney disease type 2 is an uncommon autosomal dominant condition characterized by juvenile onset hyperuricemia, precocious gout and chronic renal failure progressing to end-stage renal disease in the 4th through 7th decades of life. A family suffering from this condition is described. The patient in the index case presented with renal insufficiency as a child. A renal biopsy revealed tubular atrophy, and immunohistochemical staining of the tissue for uromodulin (Tamm Horsfall protein) revealed dense deposits in renal tubular cells. Genetic testing revealed a single nucleotide mutation (c.899G>A) resulting in an exchange of a cysteine residue for tyrosine (C300Y). Medullary cystic kidney disease type 2 (also known as uromodulin-associated kidney disease) likely represents a form of endoplasmic reticulum storage disease, with deposition of the abnormal uromodulin protein in the endoplasmic reticulum, leading to tubular cell atrophy and death.
Asunto(s)
Riñón/metabolismo , Riñón/patología , Mucoproteínas/genética , Mucoproteínas/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Adenina , Niño , Guanina , Humanos , Inmunohistoquímica/métodos , Masculino , Riñón Poliquístico Autosómico Dominante/metabolismo , Polimorfismo de Nucleótido Simple , Coloración y Etiquetado , UromodulinaRESUMEN
The amelogenesis imperfectas (AI) are a group of hereditary enamel defects characterized by clinical and genetic diversity. The most common AI types are inherited as autosomal traits. Three mutations of the enamelin (ENAM) gene have been found in cases of autosomal dominant hypoplastic AI. The gene(s) responsible for hypocalcified forms of AI have not been identified, although a number of autosomal genes have been proposed as candidates for AI based on their expression by ameloblasts, including ameloblastin and enamelin (chromosome 4q13.3), tuftelin (chromosome 1q21), enamelysin (chromosome 11q22.3-q23) and kallikrein 4 (chromosome 19q13.3-q13.4). To localize the gene(s) responsible for autosomal dominant hypocalcified AI, we evaluated support for/against linkage of AI to genetic markers spanning five AI candidate genes in two extended families. Our data excluded all proposed candidate gene regions as causal for autosomal dominant hypocalcified AI in these families. These linkage findings provide further evidence for genetic heterogeneity among families with autosomal dominant AI and indicate that, at least, some forms of autosomal dominant hypocalcified AI are not caused by a gene in the five most commonly reported AI candidate genes.
Asunto(s)
Amelogénesis Imperfecta/genética , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 4/genética , ADN/genética , Proteínas del Esmalte Dental/genética , Femenino , Genes Dominantes/genética , Ligamiento Genético/genética , Marcadores Genéticos/genética , Humanos , Calicreínas/genética , Escala de Lod , Masculino , Metaloproteinasa 20 de la Matriz , Metaloproteinasas de la Matriz/genética , Mutación/genética , Calcificación de Dientes/genéticaRESUMEN
Hereditary gingival fibromatosis (HGF) is a rare, autosomal dominant form of gingival overgrowth. Affected individuals have a benign, slowly progressive, nonhemorrhagic, fibrous enlargement of the oral masticatory mucosa. Genetic loci for autosomal dominant forms of HGF have been localized to chromosome 2p21-p22 (HGF1) and chromosome 5q13-q22 (HGF2). To identify the gene responsible for HGF1, we extended genetic linkage studies to refine the chromosome 2p21-p22 candidate interval to approximately 2.3 Mb. Development of an integrated physical and genetic map of the interval identified 16 genes. Sequencing of these genes, in affected and unaffected HGF1 family members, identified a mutation in the Son of sevenless-1 (SOS1) gene in affected individuals. In this report, we describe the genomic structure of the SOS1 gene and present evidence that insertion of a cytosine between nucleotides 126,142 and 126,143 in codon 1083 of the SOS1 gene is responsible for HGF1. This insertion mutation, which segregates in a dominant manner over four generations, introduces a frameshift and creates a premature stop codon, abolishing four functionally important proline-rich SH3 binding domains normally present in the carboxyl-terminal region of the SOS1 protein. The resultant protein chimera contains the wild-type SOS1 protein for the N-terminal amino acids 1-1083 fused to a novel 22-amino acid carboxyl terminus. Similar SOS1 deletion constructs are functional in animal models, and a transgenic mouse construct with a comparable SOS1 chimera produces a phenotype with skin hypertrophy. Clarification of the functional role of this SOS1 mutant has implications for understanding other forms of gingival fibromatosis and corrective gingival-tissue management.
Asunto(s)
Fibromatosis Gingival/clasificación , Fibromatosis Gingival/genética , Ligamiento Genético/genética , Mutación/genética , Proteína SOS1/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Brasil , Cromosomas Humanos Par 2/genética , Análisis Mutacional de ADN , Exones/genética , Femenino , Genes Dominantes/genética , Humanos , Intrones/genética , Escala de Lod , Masculino , Datos de Secuencia Molecular , Linaje , Mapeo Físico de Cromosoma , Estructura Terciaria de Proteína , ARN Mensajero/análisis , ARN Mensajero/genética , Proteína SOS1/químicaRESUMEN
BACKGROUND: We have recently identified a mutation in the uromodulin gene in a large family affected with hyperuricemia, gout, and renal failure. The purpose of this investigation is to provide a comprehensive characterization of the clinical findings of this syndrome in family members who had a mutation in the uromodulin gene. METHODS: An extended family suffering from hyperuricemia and gout was identified by a local practitioner. After consent was obtained, patients provided a directed clinical history and blood and urine specimens for chemical and genetic testing. All family members were tested for the presence of uromodulin gene mutations by direct DNA sequence analysis. The clinical and biochemical characteristics of family members carrying the affected mutation were then investigated. RESULTS: Thirty-nine family members were found to have an exon 5 uromodulin gene mutation (g.1966 1922 del), and 29 unaffected family members were identified. The cardinal clinical features in individuals with the uromodulin mutation included hyperuricemia, decreased fractional excretion of uric acid, and chronic interstitial renal disease leading to end-stage renal disease (ESRD) in the fifth through seventh decade. Women did not always develop hyperuricemia or gout, but still developed progressive chronic renal failure. CONCLUSION: Mutation of the uromodulin gene resulted in hyperuricemia, reduced fractional excretion of uric acid, and renal failure. Genetic testing will be required to definitively identify individuals suffering from this condition. We are interested in studying other families that may suffer from this condition and would appreciate any such referrals.