Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer

BACKGROUND: The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo. METHODS: A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis. RESULTS: The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells. CONCLUSIONS: This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.     

Induction of cyclooxygenase (COX)-2 but not COX-1 gene expression in apoptotic cell death

In this study we assessed the regulation of cyclooxygenase (COX)-2 in models of apoptotic cell death in vivo and in vitro. By 6 h after hippocampal colchicine injection in rat, COX-2 (but not COX-1) mRNA expression was elevated. The induction of COX-2 mRNA expression preceded temporally and overlapped anatomically the cellular morphological features of apoptosis in the granule cell layer of the dentate gyrus. Similarly, in an established in vitro model of apoptosis in P19 cells, COX-2 induction preceded apoptosis in response to serum deprivation by 12 h. These studies suggest that COX-2 may be involved in the early mechanisms leading to apoptosis.     

Antisense oligonucleotides targeting protein kinase C-alpha, -beta I, or -delta but not -eta inhibit lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages: involvement of a nuclear factor kappa B-dependent mechanism

The signaling pathway for protein kinase C (PKC) activation and the role of PKC isoforms in LPS-induced nitric oxide (NO) release were studied in RAW 264.7 macrophages. The tyrosine kinase inhibitor genestein attenuated LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression, as did the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 and the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609. LPS stimulated phosphatidylinositol (PI) hydrolysis and PKC activity in RAW cells; both were inhibited by genestein. The PKC inhibitors (staurosporine, calphostin C, Ro 31-8220, or Go 6976) or long-term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment also resulted in inhibition of LPS-induced NO release and iNOS expression. Western blot analysis showed expression of PKC-alpha, -betaI, -delta, -eta, and -zeta in RAW cells; down-regulation of PKC-alpha, -betaI, and -delta, but not -eta, was seen after long-term TPA treatment, indicating the possible involvement of one or all of PKC-alpha, -betaI, and -delta, but not -eta, in LPS-mediated effects. Treatment with antisense oligonucleotides for these isoforms further demonstrated the involvement of PKC-alpha, -betaI, and delta, but not -eta, in LPS responses. Stimulation of cells with LPS for 1 h caused activation of NF-kappaB in the nuclei by detection of NF-kappaB-specific DNA-protein binding; this was inhibited by genestein, U73122, D609, calphostin C, or antisense oligonucleotides for PKC-alpha, -betaI, and -delta, but not -eta. These data suggest that LPS activates PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, resulting in the stimulation of NF-kappaB DNA-protein binding, then initiated the expression of iNOS and NO release. PKC isoforms alpha, betaI, and delta were shown to be involved in the regulation of these LPS-induced events.     

Polyprotein processing in African swine fever virus: a novel gene expression strategy for a DNA virus

This report shows that African swine fever virus (ASFV)--a large DNA-containing virus--synthesizes a polyprotein to produce several of its structural proteins. By immunoprecipitation analysis, we have found that ASFV polyprotein is a 220 kDa myristoylated polypeptide (pp220) which, after proteolytic processing, gives rise to four major structural proteins: p150, p37, p34 and p14. Processing of the ASFV polyprotein takes place at the consensus sequence Gly-Gly-X and occurs through an ordered cascade of proteolytic cleavages. So far, polyprotein processing as a mechanism of gene expression had been found only in positive-strand RNA viruses and retroviruses. According to the results presented here, ASFV is the first example of a DNA virus that synthesizes a polyprotein as a strategy of gene expression.     

Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation

Maternal smoking during pregnancy causes reduction of fetal breathing movements, an effect attributed to nicotine in fetal blood. Nicotine is metabolized to cotinine which has a long plasma half-life and exhibits slow clearance across membrane barriers. It is also known to activate placental phospholipase-A2-like enzymes, resulting in formation of prostaglandins. Therefore, we studied transport of nicotine in isolated perfused cotyledon of normal human term placenta. The placental cotyledon was perfused with aerated (21% O2, 5% CO2) Krebs-Ringer bicarbonate buffer (pH 7.4, 37 degrees C) containing 2% albumin on both maternal (230 ml, 15 ml/min, 35 mm Hg) and fetal (93 ml, 1.75 ml/min, 70 mm Hg) sides in a closed recirculating system. Nicotine (2 mg) was added to the maternal perfusate; perfusate samples (1 ml) were collected from both sides at regular intervals and analyzed for nicotine and cotinine by high-pressure liquid chromatography. This study gave the following results: (1) In about 60-80 min, 18.6% of the nicotine added to the maternal perfusate was transferred to the fetal perfusate, and the maternal/fetal concentration ratio reached 1.0. These results show rapid placental transfer of nicotine, consistent with its high lipid solubility. (2) Less than 1% is metabolized to cotinine in placenta. The ratio of cotinine concentrations in maternal and fetal perfusates reached 1.0 in about 40 min. These studies were also verified using 14C-nicotine. (3) Maximal reduction in fetal breathing movements occurs at about 30 min, and recovery occurs at 90 min after tobacco smoking by the mother. These observations agree with the rate of placental transfer of nicotine. (4) When nicotine was added on the fetal side, part of it was metabolized to cotinine. However, the maximal concentration of cotinine was twice higher on fetal than on maternal side. These observations suggest that accumulation of cotinine on fetal side may activate prostaglandin formation and trigger spontaneous abortions in pregnant smokers.     

Rats alternate on a dry-land but not swimming-pool (Morris task) place task: Implications for spatial processing.

Groups of rats were rewarded with food for traveling from a start point to 2 different locations while their alternations in choice between those locations on 2 daily trials were recorded. In one experimental condition, the rats swam and received food once they climbed upon a platform that was hidden just below the surface of the water at the food location. In the other condition, the rats walked to reach the food. It was found that the rats did not alternate their choices between target locations when swimming but that they did alternate target choices when walking. Even experience in alternating when walking did not produce reliable alternation when swimming. It is proposed that rats treat escape (swimming) and search (walking) tasks in different ways, and this difference is discussed with respect to the possibility that different central processes may be used in the task solutions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The gamma interferon gene knockout mouse: a highly sensitive model for evaluation of therapeutic agents against Cryptosporidium parvum

Cryptosporidiosis is a serious disease in malnourished children and in people with malignancies or AIDS. Current rodent models for evaluating drug therapy against cryptosporidiosis have many limitations, including the need for a high inoculum, the absence of symptoms resembling those seen in humans, and the need to maintain exogenous immunosuppression. We have developed a gamma interferon knockout (GKO) mouse model with which to evaluate therapies against C. parvum and have used paromomycin for evaluation of this model. The GKO model offers considerable improvements over other systems, since it requires no additional immunosuppression and adult mice can be infected with as few as 10 oocysts (compared with 10(7) for SCID mice). Infected mice develop profound gastrointestinal dysfunction due to extensive infection and severe mucosal damage involving the entire small intestine. Clinical symptoms, which include depression, anorexia, weight loss, and wasting, result in death within 2 to 4 weeks. The time of death depends on the oocyst challenge dose. Paromomycin modulated parasitological and clinical parameters in highly predictable and significant ways, including prevention of death. In addition, examination of the extensively infected gut provided an important insight into the dynamics between a specific drug treatment, its impact on the extent and the site of parasite distribution, and clinical outcome. These uniform symptoms of weight loss, wasting, and death are powerful new parameters which bring this model closer to the actual disease seen in humans and other susceptible mammalian species.     

Cell division gene ftsQ is required for efficient sporulation but not growth and viability in Streptomyces coelicolor A3(2)

We show that the cell division gene ftsQ of Streptomyces coelicolor A3(2) is dispensable for growth and viability but is needed during development for the efficient conversion of aerial filaments into spores. Combined with our previous demonstration that ftsZ of S. coelicolor is not needed for viability, these findings suggest that cell division has been largely co-opted for development in this filamentous bacterium. This makes S. coelicolor an advantageous system for the study of cell division genes.     

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2) d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.     

(R)-methanandamide, but not anadamide, substitutes for Δ–9-THC in a drug-discrimination procedure.

Fourteen male rats were trained to discriminate between injections of 2 mg/kg delta-9 tetrahydrocannabinol (Δ–9-THC) and vehicle in a 2-lever operant drug-discrimination paradigm. Following training, substitution tests using a cumulative dosing procedure revealed that anandamide (0.5-16 mg/kg ip), the putative endogenous cannabinoid receptor ligand, failed to generalize to the discriminative stimulus properties of the training dose of Δ–9-THC. However, dose-dependent generalization to the Δ–9-THC cue was observed following administration of both CP-55,940 (0.05-0.8 mg/kg ip), a synthetic cannabinoid, and (R)-methanandamide (0.5-8 mg/kg ip), a metabolically stable analog of anandamide. Collectively, these results demonstrate a cannabinoid-specific in vivo effect of an anandamide compound and suggest that the naturally occurring form of anandamide may be metabolized too rapidly to produce a cannabimimetic interoceptive state when administered peripherally. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The flavin-containing monooxygenase 2 gene (FMO2) of humans, but not of other primates, encodes a truncated, nonfunctional protein

Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoenzymes that catalyze the oxidation of heteroatom centers in numerous drugs and xenobiotics. FMO2, or "pulmonary" FMO, one of five forms of the enzyme identified in mammals, is expressed predominantly in lung and differs from other FMOs in that it can catalyze the N-oxidation of certain primary alkylamines. We describe here the isolation and characterization of cDNAs for human FMO2. Analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the major FMO2 allele of humans encodes a polypeptide that, compared with the orthologous protein of other mammals, lacks 64 amino acid residues from its C terminus. Heterologous expression of the cDNA revealed that the truncated polypeptide was catalytically inactive. The nonsense mutation that gave rise to the truncated polypeptide, a C --> T transition in codon 472, is not present in the FMO2 gene of closely related primates, including gorilla and chimpanzee, and must therefore have arisen in the human lineage after the divergence of the Homo and Pan clades. Possible mechanisms for the fixation of the mutation in the human population and the potential significance of the loss of functional FMO2 in humans are discussed.     

Recombinant factor VIIa (eptacog alfa): a new therapeutic option for patients with severe bleeding disorder due to inhibitory antibodies against a coagulation factor

In 2 patients with severe haemorrhage (a 63-year-old man with haemophilia A (the factor VIII level was 29%) and a 44-year-old woman), of an inhibitory antibody against factor VIII was diagnosed. The development of recombinant factor VIIa (eptacog alpha) has made available a new therapeutic option for patients with an inhibitory antibody against a coagulation factor. Both patients were treated successfully with the new factor after other forms of treatment had failed. The new concept of the coagulation cascade on which the treatment with eptacog alpha is based assumes that the lack of an amplifying loop in the coagulation which takes place via factor IX (in combination with factor VIII) can be compensated by extra stimulation of the principal route (tissue factor-factor VIIa --> factor X) by pharmacological amounts of factor VIIa.     

Recombinant gene expression and 1H NMR characteristics of the kringle (2 + 3) supermodule: spectroscopic/functional individuality of plasminogen kringle domains

The plasminogen kringle 2 (K2HPg) and kringle 3 (K3HPg) modules occur in tandem within the polypeptide segment that affords the heavy chain of plasmin. The K2HPg and K3HPg are unique among the plasminogen kringle domains in that they also are linked to each other via the Cys169-Cys297 (Cys4 of K2HPg to Cys43 of K3HPg, kringle numbering convention) disulfide bridge, thus generating a K2HPg-K3HPg "supermodule". The kringle (2 + 3) sequence of human plasminogen (r-EE[K2HPgK3HPg]DS) was expressed in Escherichia coli, using an expression vector containing the phage T5 promoter/operator N250PSN250P29 and the codons for an N-terminal hexahistidine tag to ensure the isolation of the recombinant protein by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose under denaturing and reducing conditions. Kringle (2 + 3) was refolded in the presence of glutathione redox buffer. By taking advantage of the lysine affinity of kringle 2, the protein was purified by affinity chromatography on lysine-Bio-Gel. Recombinant kringle (2 + 3) was identified by amino acid composition, N-terminal sequence and mass determination. The 1H NMR spectrum shows that the intact r-K2HPgK3HPg is properly folded. By reference to spectra of the individual kringles, r-K2HPg and r-K3HPg, resonances of the K2HPg and K3HPg components in the spectrum of the intact r-K2HPgK3HPg can be readily distinguished. The strictly conserved Leu46 residue (kringle residue number convention) yields delta-methyl signals that are characteristic for K2HPg and K3HPg, exhibiting chemical shifts of -0.87 and -0.94 ppm, respectively, which are distinct from those of K1HPg, K4HPg, and K5HPg, (-1.04 to -1.05 ppm). Thus, the high-field Leu46 signals from K2HPg and K3HPg are well resolved from those of other kringles and can be identified unambiguously in spectra of the K1HPgK2HPgK3HPg elastolytic fragment of plasminogen as well as in spectra of Glu-plasminogen. Overall, r-K2HPgK3HPg exhibits broader resonance line widths than does the K1HPg component, consistent with a lesser mobility of the K2HPgK3HPg segment within the K1HPgK2HPgK3HPg fragment, a reflection of the extra structural constraint imposed by the disulfide bridge linking K2HPg to K3HPg. The ligand 6-aminohexanoic acid (6-AHA), which is known to interact with r-K2HPg but not with r-K3HPg, selectively perturbs K2 aromatic signals in the intact r-K2HPgK3HPg spectrum while leaving K3 resonances largely unaffected. Association constant (K(a)) values for 6-AHA determined from 1H NMR ligand titration experiments yield K(a) approximately 2.2 +/- 0.3 mM(-1) for the intact r-K2HPgK3HPg, comparable to K(a) approximately 2.3 +/- 0.2 mM(-1) determined for the isolated r-K2HPg, which demonstrates that the interactions of 6-AHA with the K2HPg ligand-binding site are not significantly affected by the neighboring K3HPg domain within the intact r-K2HPgK3HPg supermodule.     

Transcriptional activation of NF-kappa B activity by Epstein-Barr virus (EBV) LMP1 as a selective therapeutic strategy for EBV-associated diseases

Epstein-Barr virus (EBV) has been known to be associated with many malignant tumors, including nasopharyngeal carcinoma (NPC). Previous studies have indicated that an EBV-encoded oncoprotein, latent membrane protein 1 (LMP1), is expressed in many NPC tissues. LMP1 has been shown to stimulate HIV LTR through the two NF-kappa B binding sites within this promoter. In this study, we examined the feasibility of using this property of LMP1 as a therapeutic strategy for the treatment of NPC. This therapy consists of the preferential killing of the LMP1-expressing cells by gene transfer using the NF-kappa B-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system. The 800-bp HIV-LTR, which contains two NF-kappa B binding sites, was used to drive the HSVtk gene. Stable C33A cell clones expressing the LMP1 and the HSVtk genes were subjected to the GCV sensitivity test. Results showed that cells expressing both the LMP1 and the HSVtk genes were highly sensitive to GCV treatment. These cells were introduced into nude mice subcutaneously and tumors became palpable within 2 weeks. GCV was then introduced intraperitoneally to these mice and the sizes of the tumors were measured daily. Results showed that the tumors regressed in the group of mice carrying cells that stably expressed both the LMP1 and the HSVtk genes, but not in mice carrying cells containing LMP1 or HSVtk alone. Our data indicate that the HSVtk gene expressed from a NF-kappa B-binding motif-containing promoter that is regulated by LMP1 may be used as an in vivo gene therapy strategy of EBV LMP1-expressing cancers such as NPC.     

High level expression of the multidrug resistance (MDR1) gene in the normal bladder urothelium: a potential involvement in protection against carcinogens?

It has been suggested that expression of the P-glycoprotein transmembrane efflux pump (PGP), encoded by the multi-drug resistance (MDR1) gene, may play a role in the protection of epithelial tissues from a variety of local and systemic toxins. We report that in approximately 50% (6/11) of the population, MDR1 messenger RNA levels in the normal urinary epithelium are comparable to those found in the highest expressing tissues in the body, and suggest a role for PGP in the normal bladder urothelium. MDR1 mRNA levels in the normal urothelium do, however, vary over a 60-fold range between individuals, and furthermore are uniformly significantly lower (about 6-fold, P相似文献   

Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue- and urokinase-type plasminogen activators, is considered a critical regulator of the fibrinolytic system. We previously reported a child with abnormal bleeding and complete PAI-1 deficiency caused by a frame-shift mutation in exon 4 of the PAI-1 gene. The purpose of this study was to provide genetic and clinical data on the extended pedigree of the original proband to better define the phenotype associated with PAI-1 deficiency. Allele-specific oligonucleotide hybridization was used to genotype individuals, and serum PAI-1 antigen was measured by enzyme-linked immunosorbent assay. By this approach we have identified 19 individuals who are heterozygous for the PAI-1 null allele and 7 homozygous individuals with complete PAI-1 deficiency. Clinical manifestations of PAI-1 deficiency were restricted to abnormal bleeding, which was observed only after trauma or surgery in homozygous affected individuals. A spectrum of bleeding patterns was observed, including intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe menstrual bleeding, and frequent bruising. Fibrinolysis inhibitors, including epsilon-aminocaproic acid and tranexamic acid, were effective in treating and preventing bleeding episodes. Other than abnormal bleeding, no significant developmental or other abnormalities were observed in homozygous PAI-1-deficient individuals. Heterozygous PAI-1 deficiency was not associated with abnormal bleeding, even after trauma or surgery. These observations define the clinical spectrum of PAI-1 deficiency and provide additional evidence to support the hypothesis that the primary function of plasminogen activator inhibitor-1 in vivo is to regulate vascular fibrinolysis.     

Continuous update with random encoding (CURE): a new strategy for dynamic imaging

Although dynamic imaging is presently used for various applications, it is still limited by the temporal resolution. In this paper, we present a new technique that uses a random phase-encoding strategy to facilitate faster and smoother update of images and to improve the temporal resolution in dynamic studies. The technique was implemented on a conventional clinical scanner and demonstrated with various in vivo studies. Technical details, simulations, and experimental results are described. Images from experimental studies indicate that the new technique is robust in generating dynamic images and can be potentially utilized for clinical applications.     

Pigeons (Columba livia) associate time intervals with symbols in a touch screen task: Evidence for ordinality but not summation.

The present experiments examined whether pigeons can sum symbols that are associated with various temporal consequences in a touch screen apparatus. Pigeons were trained to discriminate between two visual symbols that were associated with 0, 1, 2, 3, 4, or 5 s either of delay to 4 s of hopper access (delay group) or duration of hopper access (reward group). In Experiment 1, the pigeons in both groups learned to select the symbol associated with the more favorable outcome, and they successfully transferred this discrimination to novel symbol pairs. However, when tested with 2 pairs of symbols associated with different summed durations, they responded on the basis of a simple response rule rather than the sum of the symbol pair. In Experiment 2, the reward group was presented with four symbols at once and was allowed to successively choose one symbol at a time. All pigeons chose the symbols in order from largest to smallest. This indicates that pigeons formed an ordered representation of symbols associated with different time intervals, even though they did not sum the symbols. (PsycINFO Database Record (c) 2010 APA, all rights reserved)