RESUMEN
Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alpha v beta3 and alpha v beta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.
Asunto(s)
Neoplasias de la Mama/terapia , Colágeno Tipo IV/genética , Terapia Genética/métodos , Fragmentos de Péptidos/genética , Neoplasias de la Próstata/terapia , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Apoptosis , Neoplasias de la Mama/radioterapia , Línea Celular Tumoral , Colágeno Tipo IV/sangre , Terapia Combinada , Electroporación , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Músculo Esquelético/metabolismo , Fragmentos de Péptidos/sangre , Neoplasias de la Próstata/radioterapia , Dosificación Radioterapéutica , Proteínas Recombinantes de Fusión/genética , Albúmina Sérica/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epstein-Barr virus (EBV) is a herpes virus associated with several human tumors. The EBV protein, ZEBRA, is a transactivator of the basic leucine zipper family (bZip). It binds to specific sequences on DNA and is able to interact with cellular proteins such as p53. The interaction of the ZEBRA protein with its cognate DNA sequences is stable as long as the dimerization domain is functional. Recent work from this laboratory identified a ZEBRA variant (Z206) with a single amino acid change at residue 206. An alanine is substituted for a serine, and this replacement is present in 72% of nasopharyngeal carcinoma from Europe and North Africa. As amino acid 206 lies within the dimerization domain it could be instrumental in interactions with other proteins. The yeast two-hybrid system was used to study ZEBRA-protein interactions. As ZEBRA by itself is a transactivator in yeast, it cannot be used directly in this assay. This paper describes modifications in ZEBRA amino acid sequences, rendering it usable in the yeast two-hybrid assay. We compared the dimerization capacity of the Z206 variant to that of ZEBRA from B95-8 (Z95) and observed that reporter gene activity with Z206 was consistently lower than that of Z95 (P < 0.05). Furthermore, no interaction was found to occur between either form of ZEBRA (Z206 or Z95) and the tumor suppressor, p53 in the yeast two-hybrid system.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/metabolismo , Leucina Zippers , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Virales/metabolismo , Sustitución de Aminoácidos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/genética , Variación Genética , Herpesvirus Humano 4/genética , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Different Epstein-Barr-virus(EBV) variants were found to be associated with nasopharyngeal carcinoma (NPC). The type-C variant lacks the BamHI site between the BamHI W1* and I* regions and the type-f variant has an extra BamHI site in the BamHI F fragment. The BNLF1 gene (which encodes the LMP1 protein) from a nude-mouse-passaged CAO strain and from NPC biopsies from Taiwanese patients also exhibits variations resulting in structural and functional differences in the protein. The BZLF1 gene encodes the ZEBRA protein which triggers the EBV lytic cycle. A difference has been observed in 8 amino acids in the ZEBRA sequence in B95-8 (Z95) and P3HR1 (ZP3) cell lines. EBV found in NPC biopsies and peripheral-blood cells from Asians was predominantly of the ZP3 type (72%), while 81% of samples from different EBV-associated diseases and peripheral-blood cells from North Africa or Europe were of the Z95 type. We found that an alanine 206 had been replaced by a serine in the Z95 sequence in 72% of the NPC biopsies from European and North African patients. The Zser206 variant is found in a significantly lower percentage (p < 0.001) of other EBV-positive tissues from individuals in the same region (10-33%). In contrast, a 30-bp deletion is observed near the 3' end of the LMP1 gene in the majority of EBV (86%) from NPC and peripheral-blood cells from Asians, whereas a significantly lower percentage (p < 0.001) of NPC biopsies from European and North African patients (56%) have this deletion, as do lymphocytes from control individuals from the same region (36 and 55% respectively).
Asunto(s)
Carcinoma/virología , Proteínas de Unión al ADN/genética , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virología , Transactivadores/genética , Proteínas Virales , África del Norte , Secuencia de Aminoácidos , Carcinoma/genética , Europa (Continente) , Genes Virales , Humanos , Huésped Inmunocomprometido , Datos de Secuencia Molecular , Neoplasias Nasofaríngeas/genética , Mutación Puntual , Polimorfismo de Longitud del Fragmento de Restricción , Eliminación de Secuencia , Proteínas de la Matriz Viral/genética , Proteínas Estructurales Virales/genéticaRESUMEN
We recently showed that BZLF1, the gene encoding the Epstein-Barr virus (EBV) ZEBRA protein, was expressed in all eight nasopharyngeal carcinoma (NPC) specimens studied. We present here studies on the expression of EBV lytic cycle genes in the same eight NPC biopsies to determine if production of the ZEBRA transactivator could lead to a complete productive cycle. The tumour lesions exhibit a number of different patterns of limited lytic gene expression. In three out of eight tumours neither BRLF1 nor BMLF1 expression could be detected. Otherwise BMLF1 mRNA was expressed in all the other specimens. Three specimens also expressed BRLF1. Two specimens not only exhibited BZLF1, BMLF1 and BRLF1 transcripts, but also expressed the late gene BLLF1 which encodes the membrane protein gp220. The early gene product BBLF2 could not be detected in any of the eight NPC. However, expression of the late gene encoding the lytic truncated form of LMP1 (D1LMP) was found in seven of the eight NPC biopsies. Thus, it could be suggested that the EBV abortive lytic cycle occurred in most of the NPC studied.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Expresión Génica , Genes Virales , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Neoplasias Nasofaríngeas/virología , Reacción en Cadena de la Polimerasa/métodos , Transactivadores/biosíntesis , Proteínas Virales/biosíntesis , Secuencia de Bases , Biopsia , Cartilla de ADN , ADN Complementario , Proteínas de Unión al ADN/análisis , Genes Inmediatos-Precoces , Herpesvirus Humano 4/metabolismo , Humanos , Datos de Secuencia Molecular , Neoplasias Nasofaríngeas/patología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Viral/análisis , ARN Viral/biosíntesis , Transactivadores/análisis , Proteínas Virales/análisisRESUMEN
The Epstein-Barr virus has been shown to be associated with nasopharyngeal carcinoma (NPC). We have shown that anti-ZEBRA transactivator antibodies were present in sera of most NPC patients. We investigated the expression of the BZLF1 gene in fresh NPC tumor biopsies. RNA transcripts of this gene could be detected by in situ hybridization. This expression is restricted to tumor cells and is not present in infiltrating lymphocytes. ZEBRA protein could also be visualized in a fraction of these cells. A cDNA library made from polyadenylated mRNA of an NPC tumor biopsy was constructed and analyzed by PCR. Transcripts of the BZLF1 gene were unspliced, incompletely spliced, or fully processed. A transcript lacking the middle exon was also observed. Fully processed BZLF1 mRNA was also detected in six more NPC tumor biopsies.