Antitumour activity of the silybin-phosphatidylcholine complex, IdB 1016, against human ovarian cancer.

This study aimed to assess, in an in vivo experimental model, the growth inhibitory effects of IdB 1016 (Silipide, a complex of silybin/phosphatidylcholine) when used as a single agent against human ovarian cancer. We also wanted to investigate the mechanism of the antiangiogenic action by assessing Vascular Endothelial Growth Factor (VEGF) levels and by using macroarray technology to evaluate the regulation of a panel of genes involved in angiogenesis. We also aimed to establish the plasma and tumour bioavailability of silybin after repeated administration of IdB 1016. Female nude mice bearing human ovarian cancer xenografts (A2780) received 450 mg/kg/day IdB 1016 daily by oral gavage until the end of the study. At sacrifice, blood and tumour specimens were collected and subsequently processed for the determination of silybin levels, VEGF levels or a gene expression profile. IdB 1016 was significantly active in inhibiting ovarian tumour growth. Treatment with 450 mg/kg/day for a total of 20 administrations produced a tumour weight inhibition (TWI%) of 78% and a Log10 Cell Kill (LCK) of 1.1. Free silybin levels were found to be 7.0+/-5.3 microg/ml and 183.5+/-85.9 ng/g tissue (mean+/-standard deviation (S.D.)) in the plasma and tumour samples, respectively. No significant differences were found in the concentration of human VEGF in xenografts from control and IdB 1016-treated mice. The array analysis suggested the downregulation of the VEGR receptor 3 and the upregulation of angiopoietin-2 as potential mechanisms for the antiangiogenic activity. In conclusion, these findings suggest IdB 1016 is a good candidate, with a relevant clinical potential, for use in the management of recurrent ovarian cancer. A phase II, non-randomised clinical study is now ongoing in our Institute aimed at evaluating the efficacy of daily administrations of IdB 1016 in the serological recurrence of ovarian cancer.

Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly.

The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.

Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIV ribozymes in a human T lymphoblastoid cell line.

Human immunodeficiency virus (HIV) infection represents one of the most challenging systems for gene therapy. Thanks to the extended knowledge of the molecular biology of the HIV life cycle, many different strategies have been developed including transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes, and intracellular antibody fragments. In this paper, we have tested in a human T lymphoblastoid cell line the antiviral activity of ribozymes specifically designed to co-localize inside the nucleus with the Rev pre-mRNA before it is spliced and transported to the cytoplasm. This result was obtained by inserting the ribozyme in the spliceosomal U1 small nuclear RNA (snRNA) and in a derivative that has perfect complementarity with the 5' splice site of the Rev pre-mRNA. These ribozymes were tested in human T cell clones and were shown to be very efficient in inhibiting viral replication. Not only were the p24 levels in the culture medium drastically reduced but so were the intracellular HIV transcripts. Control disabled ribozymes enabled us to show the specificity of the ribozyme activity. Therefore, these constructs have potential utility for gene therapy of HIV-1 infection.

Use of adenoviral VAI small RNA as a carrier for cytoplasmic delivery of ribozymes.

The in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several features: RNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of "natural" small RNA coding genes as expressing cassettes gives high chances to fulfill these requirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1. The conserved 5' leader sequence of HIV was chosen as a target, because it is present in all the viral transcripts and is highly conserved. Hammerhead ribozymes were substituted to different portions of the VAI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compartmentalization, and assembly in specific ribonucleoparticles containing the La antigen. Interesting differences in the activity of the different chimera were found in both in vitro cleavage assays and S100 extracts of injected oocytes where the catalytic activity of the ribozymes in the RNP context can be analyzed.

U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus.

The in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector.

Processing of the intron-encoded U16 and U18 snoRNAs: the conserved C and D boxes control both the processing reaction and the stability of the mature snoRNA.

A novel class of small nucleolar RNAs (snoRNAs), encoded in introns of protein coding genes and originating from processing of their precursor molecules, has recently been described. The L1 ribosomal protein (r-protein) gene of Xenopus laevis and its human homologue contain two snoRNAs, U16 and U18. It has been shown that these snoRNAs are excised from their intron precursors by endonucleolytic cleavage and that their processing is alternative to splicing. Two sequences, internal to the snoRNA coding region, have been identified as indispensable for processing the conserved boxes C and D. Competition experiments have shown that these sequences interact with diffusible factors which can bind both the pre-mRNA and the mature U16 snoRNA. Fibrillarin, which is known to associate with complexes formed on C and D boxes of other snoRNAs, is found in association with mature U16 RNA, as well as with its precursor molecules. This fact suggests that the complex formed on the pre-mRNA remains bound to U16 throughout all the processing steps. We also show that the complex formed on the C and D boxes is necessary to stabilize mature snoRNA.

Self-cleaving motifs are found in close proximity to the sites utilized for U16 snoRNA processing.

A class of small nucleolar RNAs (snoRNAs) is encoded in introns of protein-coding genes. The U16 snoRNA belongs to this class; it is encoded in the third intron of the Xenopus laevis (Xl) L1 ribosomal protein encoding gene and is released from the pre-mRNA by processing both in vivo and in vitro systems. In this paper, we show that in close proximity to the U16 snoRNA processing sites, sequences displaying self-cleaving activity are present. These elements are conserved in the two copies of the Xl L1 and in the single copy of the X. tropicalis L1. The catalytic activity corresponds to that already described for the minimal hairpin ribozyme [Dange et al., Science 242 (1990) 585-588]; it is Mn(2+)-dependent, produces 2'-3' cyclic phosphate and 5'-OH termini and comprises an essential GAAA element. Here we show that the 2'-OH group of the G residue is essential for catalysis.

Two different snoRNAs are encoded in introns of amphibian and human L1 ribosomal protein genes.

We previously reported that the third intron of the X.laevis L1 ribosomal protein gene encodes for a snoRNA called U16. Here we show that four different introns of the same gene contain another previously uncharacterized snoRNA (U18) which is associated with fibrillarin in the nucleolus and which originates by processing of the pre-mRNA. The pathway of U18 RNA release from the pre-mRNA is the same as the one described for U16: primary endonucleolytic cleavages upstream and downstream of the U18 coding region produce a pre-U18 RNA which is subsequently trimmed to the mature form. Both the gene organization and processing of U18 are conserved in the corresponding genes of X.tropicalis and H.sapiens. The L1 gene thus has a composite structure, highly conserved in evolution, in which sequences coding for a ribosomal protein are intermingled with sequences coding for two different snoRNAs. The nucleolar localization of these different components suggests some common function on ribosome biosynthesis.

A novel small nucleolar RNA (U16) is encoded inside a ribosomal protein intron and originates by processing of the pre-mRNA.

We report that the third intron of the L1 ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre-mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human L1 ribosomal protein gene a highly homologous region is found which can be released from the pre-mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the L1 ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components.

The mechanisms controlling ribosomal protein L1 pre-mRNA splicing are maintained in evolution and rely on conserved intron sequences.

Sequences corresponding to the third intron of the X.laevis L1 ribosomal protein gene were isolated from the second copy of the X.laevis gene and from the single copy of X.tropicalis. Sequence comparison revealed that the three introns share an unusual sequence conservation which spans a region of 110 nucleotides. In addition, they have the same suboptimal 5' splice sites. The three introns show similar features upon oocyte microinjection: they have very low splicing efficiency and undergo the same site specific cleavages which lead to the accumulation of truncated molecules. Computer analysis and RNAse digestions have allowed to assign to the conserved region a specific secondary structure. Mutational analysis has shown that this structure is important for conferring the cleavage phenotype to these three introns. Competition experiments show that the cleavage phenotype can be prevented by coinjection of excess amounts of homologous sequences.

Identification of the sequences responsible for the splicing phenotype of the regulatory intron of the L1 ribosomal protein gene of Xenopus laevis.

Splicing of the regulated third intron of the L1 ribosomal protein gene of Xenopus laevis has been studied in vivo by oocyte microinjection of wild-type and mutant SP6 precursor RNAs and in vitro in the heterologous HeLa nuclear extract. We show that two different phenomena combine to produce the peculiar splicing phenotype of this intron. One, which can be defined constitutive, shows the same features in the two systems and leads to the accumulation of spliced mRNA, but in very small amounts. The low efficiency of splicing is due to the presence of a noncanonical 5' splice site which acts in conjunction with sequences present in the 3' portion of the intron. The second leads to the massive conversion of the pre-mRNA into site specific truncated molecules. This has the effect of decreasing the concentration of the pre-mRNA available for splicing. We show that this aberrant cleavage activity occurs only in the in vivo oocyte system and depends on the presence of an intact U1 RNA.