Cellular senescence bypass screen identifies new putative tumor suppressor genes.

Senescence is a mechanism that limits cellular lifespan and constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a role in tumorigenesis, we have designed and performed a large-scale antisense-based genetic screen in primary mouse embryo fibroblasts (MEFs). Out of this screen, we have identified five different genes through which loss of function partially bypasses senescence. These genes belong to very different biochemical families: csn2 (component of the Cop9 signalosome), aldose reductase (a metabolic enzyme) and brf1 (subunit of the RNA polymerase II complex), S-adenosyl homocysteine hydrolase and Bub1. Inactivation, at least partial, of these genes confers resistance to both p53- and p16INK4a-induced proliferation arrest. Furthermore, such inactivation inhibits p53 but not E2F1 transcriptional activity and impairs DNA-damage-induced transcription of p21. Since the aim of the screen was to identify new regulators of tumorigenesis, we have tested their inactivation in human tumors. We have found, either by northern blot or quantitative reverse transcriptase-PCR analysis, that the expression of three genes, Csn2, Aldose reductase and Brf1, is lost at different ratios in tumors of different origins. These genes are located at common positions of loss of heterogeneity (15q21.2, 7q35 and 14q32.33); therefore,we have measured genomic losses of these specific genes in different tumors. We have found that Csn2 and Brf1 also show genomic losses of one allele in different tumors. Our data suggest that the three genes identified in the genome-wide loss-of-function genetic screen are putative tumor suppressors located at 15q21.2; 7q35 and 14q32.33.

Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles.

We have demonstrated earlier the usefulness of recombinant porcine parvovirus (PPV) virus-like particles (VLPs) as an efficient recombinant vaccine for PPV. Here, we have demonstrated that preparations of PPV VLPs could be contaminated by recombinant baculoviruses. Since these baculoviruses can be a problem for the registration and safety requirements of the recombinant vaccine, we have tested different baculovirus inactivation strategies, studying simultaneously the integrity and immunogenicity of the VLPs. These methods were pasteurization, treatment with detergents and alkylation with binary ethylenimine (BEI). The structural and functional integrity of the PPV VLPs after the inactivation treatments were analyzed by electron microscopy, hemagglutination, double antibody sandwich (DAS)-ELISA and immunogenicity studies. Binary ethylenimine and Triton X-100 inactivated particles maintained all the original structural and antigenic properties. In addition, PPV VLPs were subjected to size-exclusion chromatography to analyze the presence of VP2 monomers or any other contaminant. The resulting highly purified material was used as the standard of reference to quantify PPV VLPs in order to determine the dose of vaccine by DAS-ELISA. After immunization experiments in guinea pigs, the antibody titers obtained with all the inactivation procedures were very similar. Triton X-100 treatment was selected for further testing in animals because of the speed, simplicity and safety of the overall procedure.

An optimized amphiphilic cationic peptide as an efficient non-viral gene delivery vector.

BACKGROUND: Due to their chemical definition and reduced size, the use of peptides as gene delivery systems is gaining interest as compared to the more common polymeric non-viral vectors. To achieve gene transfer efficiencies that would make peptides a realistic alternative to existing methods, we have evaluated and attempted to concert those properties with a direct impact on the activity of the system. These considerations have led to the design, synthesis and characterization of a 23-residue cationic peptide which we term RAWA. METHODS: We have characterized RAWA biophysically and functionally. Biophysical studies include evaluation of DNA condensation and membrane perturbing activities. DNA transfer activity has been evaluated in cell culture at controlled DNA-to-peptide stoichiometries, using a luciferase gene as reporter. Requirements for additional effectors such as chloroquine and peptide cofactors have also been considered. RESULTS: RAWA displays in vitro DNA condensing activity similar to that of protamines, reaching maximum effect at a peptide-to-DNA molar charge ratio (CR) of 4 (+/-). The reduced membrane perturbing activity diminishes its cytotoxic potential. In COS-7 cells, transfection efficiency with RAWA peptiplexes, compares favorably with well-recognized systems, including Lipofectamine Plus, Superfect, GenePorter and FuGene. The peptide-associated activity between free and DNA-bound species has been mapped by analyzing dependency on chloroquine treatment. The lack of significant serum inhibition and low toxicity make this system advantageous for potential in vivo application. A ternary complex including the acid-triggered fusogenic JTS-1 peptide is presented as a potential strategy for further in vivo studies. CONCLUSIONS: We have developed a gene delivery system based on an amphipathic cationic peptide with improved DNA condensation ability and reduced cytotoxicity, which maintains membrane binding and perturbing activities. Observed efficiency with this molecule is very high and compares favorably with currently available transfection systems.

A chimeric fusion protein containing transforming growth factor-alpha mediates gene transfer via binding to the EGF receptor.

Fusion proteins engineered to incorporate distinct functions which co-operate in mediating the cell-type specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vector systems for targeted gene transfer. Here we have chosen the EGF receptor overexpressed in many human tumors of epithelial origin as a target for a novel modular fusion protein. We have fused a cDNA fragment of the human EGF receptor ligand TGF-alpha to sequences encoding the translocation domain of Pseudomonas exotoxin A as an endosome escape activity, and the DNA-binding domain of the yeast GAL4 transcription factor. Upon bacterial expression, this TEG fusion protein displayed specific binding to EGF receptors. Complexes of the chimeric protein and plasmid DNA carrying a luciferase reporter gene, after condensation with poly-L-lysine resulted in an up to 150-fold increase in reporter gene expression in EGF receptor expressing cells in comparison to poly-L-lysine-DNA complexes alone. While in COS-1 cells no additional endosome escape activity was required, in A431 cells gene delivery was dependent on the simultaneous presence of the endosome destabilizing reagent chloroquine indicating that cell-type specific factors such as different intracellular routing of protein-DNA complexes greatly influence transfection efficiency.

A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery.

Modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vectors for targeted gene delivery. Here, we describe a novel DNA carrier protein termed GD5 that mimics the structure of the bacterial diphtheria toxin (DT) and facilitates target cell-specific gene transfer via receptor-mediated endocytosis. GD5 carries at the N terminus the DNA-binding domain of the yeast transcription factor Gal4, which is connected to a C-terminal antibody fragment specific for the tumor-associated ErbB2 antigen via an internal DT translocation domain as an endosome escape activity. Bacterially expressed GD5 protein specifically bound to ErbB2-expressing cells and formed protein-DNA complexes with a luciferase reporter gene construct. These complexes, after compensation of excess negative charge with poly-L-lysine, served as a specific transfection vector for ErbB2-expressing cells. Inhibitors of endosomal acidification drastically reduced GD5-mediated transfection, indicating that the DT translocation domain of GD5, similar to the parental toxin, is strictly dependent on the transit through an acidic environment. Our results suggest that fusion proteins that employ the natural endosome escape mechanism of bacterial toxins might aid in the development of efficient nonviral vectors for applications in gene therapy.

Oxidation of sulfhydryl groups of ribonuclease inhibitor in epithelial cells is sufficient for its intracellular degradation.

Ribonuclease inhibitor (RI) is a cytoplasmic protein (50 kDa) that inhibits a variety of pancreatic type RNases. The porcine inhibitor contains 30 cysteine residues, all of which occur in the reduced state. It is well known that in vitro modification of the thiol groups inactivates the protein and greatly increases its susceptibility to proteolysis. Here we show that oxidation of thiol groups in RI can also occur within the cell. Induction of an oxidative insult in cultured LLC-PK1 cells, either with a general oxidant, H2O2, or with a thiol-specific oxidant, diamide, led to the loss of RI activity. By using specific antibodies it was demonstrated that the decrease correlated with a decline in the amount of RI protein in the cells. Furthermore, analysis of RI mRNA levels and half-life of the protein excluded inhibition of the synthesis of RI as the cause of its depletion. The results indicate that oxidation of thiol groups in RI is sufficient to cause its rapid inactivation and disappearance from the cell. Most likely this results from intracellular degradation of the protein.

Target cell-specific DNA transfer mediated by a chimeric multidomain protein. Novel non-viral gene delivery system.

Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains: 1) an ErbB-2 -specific single chain antibody confers target cell specificity, 2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enable sequence-specific high affinity binding to DNA. Carrier protein purified from bacterial lysates displayed both ErbB-2-specific and DNA sequence-specific binding in vitro. Complexes which formed spontaneously by the interaction of the fusion protein with a luciferase reporter gene construct carrying a GAL4-specific recognition sequence, after condensation of the DNA and compensation of excess negative charge with poly-L-lysine were able to transfect ErbB-2-expressing cells in vitro in a cell-specific manner. Transient expression of the luciferase gene driven by the SV40 early promoter was observed and correlates with the amount of carrier protein in the complex. Truncated forms of the carrier protein lacking either the cell recognition domain or the translocation domain failed to facilitate efficient DNA transfer.

Residues 287-301 of human ribonuclease inhibitor do not affect ribonuclease activity and inhibitor binding; a reply.

Residues 287-301 of human placental ribonuclease inhibitor have been reported to inhibit pancreatic ribonuclease A in a similar way as the entire inhibitor (Crevel-Thieffry, I., Cotterill, S. and Schuller, E. (1992) Biochim. Biophys. Acta 1122, 107-112). Using three different assays, we were unable to observe inhibition by the synthetic peptide. Moreover, the peptide did not compete with the entire inhibitor for binding to RNAase A.

Inactivation of ribonuclease inhibitor by thiol-disulfide exchange.

Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.

Theoretical treatment of tight-binding inhibition of an enzyme. Ribonuclease inhibitor as special case.

A general treatment of very tight-binding inhibition is described. It was applied to purified endogenous RNAase inhibitor from rat testis. This treatment discriminates among the different types of inhibition and allows for calculation of the inhibition parameters. When very tight-binding inhibitions are studied at similar molar concentrations of both enzyme and inhibitor, a further approach is required. This is also described and applied to the RNAase inhibitor. A Ki value of 3.2 x 10(-12) M was found for this inhibitor protein. On the basis of this result, it was considered inappropriate to classify this type of inhibitor in terms of competitive or non-competitive, as has been done for such inhibitors so far. Functional consequences of this analysis are discussed for the RNAase-RNAase inhibitor system.

Ribonuclease-RNAase inhibitor complex from rat testis. Purification of the RNAase inhibitor.

The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.

Purification, molecular and enzymic characterization of an acid RNase from the insect Ceratitis capitata.

An acid ribonuclease has been purified from the insect Ceratitis capitata. The specific activity of the purified enzyme is 580 units/mg. This enzyme is a single polypeptide chain of about 35.5 kDa, containing only one disulfide bridge and no free -SH groups. The A0.1%1cm at 280 nm is 1.90. The hydrodynamic radius of the native enzyme is 2.5 nm. The secondary structure of this RNase is composed of 10% alpha-helix, 31% beta-structure and 59% aperiodic conformation with an average number of residues per helical segment of 10, based on circular dichroic measurements. Optimum parameters for the enzyme activity are pH 5.5, 0.15 M ionic strength and 40 degrees C. Divalent cations are not required for the enzymic catalysis. This enzyme has been characterized as cyclizing endoribonuclease.

On the degradation of intracellular RNA by ribonucleases.

The kinetic and the specificity of two RNases purified from the insect. C. capitata have been studied. These two enzymes exhibit preference to degrade large polynucleotides. The alkaline enzyme is located in the soluble cellular fraction and the acid enzyme is also associated to microsomes and lysosomes. A hypothesis about the physiological role of these two insect enzymes in the degradation of the intracellular RNA is proposed.