Canstatin gene electrotransfer combined with radiotherapy: preclinical trials for cancer treatment.

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.

Dimerization of the Epstein-Barr virus ZEBRA protein in the yeast two-hybrid system. Comparison Of a ZEBRA variant with the B95-8 form.

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.

Amino-acid change in the Epstein-Barr-virus ZEBRA protein in undifferentiated nasopharyngeal carcinomas from Europe and North Africa.

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).

Qualitative analysis of the expression of Epstein-Barr virus lytic genes in nasopharyngeal carcinoma biopsies.

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.

Chicken tyrosine hydroxylase gene: isolation and functional characterization of the 5' flanking region.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines. We describe here the isolation of the chicken TH gene and the analysis of 3 kb of its 5' flanking region. The chicken TH transcription unit spans 19 kb. The 60-bp proximal promoter contains a TATA box and a cyclic AMP response element (CRE) sequence. The 5' flanking region contains several AP1-, AP2-, and octamer-like sequences as well as a glucocorticoid response element at position -1.4 kb. A construct containing the 3-kb 5' flanking DNA fused to the chloramphenicol acetyltransferase (CAT) gene was transiently transfected into PC12 cells, and the effect of various effectors was tested. Only forskolin increased the CAT activity, likely owing to the presence of the CRE sequence. Constructs prepared by progressively deleting the 5' flanking DNA were transfected into PC12 and QT6 (quail transformed fibroblasts) cells. In both cell types, the transcriptional activity increased with deletion of the 5' flanking region. These results show that the 60-bp region containing the TATA box and the CRE is sufficient to act as a constitutive promoter for the chicken TH gene and that this region appears to be negatively controlled by upstream sequences.

Expression of the Epstein-Barr virus immediate early gene, BZLF1, in nasopharyngeal carcinoma tumor cells.

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.

Chicken major histocompatibility complex class II B genes: analysis of interallelic and interlocus sequence variance.

Five different chicken B-LB genes were cloned and sequenced. The comparison of these sequences shows that they can be classified as members of two different families, the B-LBII family (containing the B-LBI and B-LBII genes) and the B-LBIII family (containing the B-LBIII, B-LBIV, and B-LBV genes). The extent of polymorphism within each of these families was assessed by in vitro amplification of DNA fragments encompassing exon 2 in several haplotypes. The nucleotide sequences were determined, and pairwise relationships were evaluated. In the course of this work, a sixth gene termed B-LBVI was identified, defining a third family (B-LBVI family). Polymorphism of the B-LBIII or B-LBVI families is far less extensive than that of the B-LBII family. In this latter, the distribution of conserved and polymorphic residues is similar to what has been described in mammals. These families seem to have been generated by gene duplication events giving rise to several isotypes, as observed in mammals. However, phylogenetic analyses indicate that these families are not homologous to their mammalian counterparts. Evaluation of the level of transcription of these different genes showed that genes from the B-LBII family are predominantly transcribed over those of the other families.