Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.

Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.

Preconditioning with cortical spreading depression does not upregulate Cu/Zn-SOD or Mn-SOD in the cerebral cortex of rats.

Previous studies have demonstrated that preconditioning the brain with cortical spreading depression (CSD) induces tolerance to a subsequent episode of ischemia. In other models of preconditioning, induction of ischemic tolerance has been associated with increased expression of the antioxidant enzyme, superoxide dismutase (SOD). The objective of the present study was to determine whether CSD upregulates Cu/Zn-SOD or Mn-SOD. CSD was induced in one hemisphere by applying 2 M KCl to the frontal cortex in Wistar rats. After 2 or 24 h of recovery, Cu/Zn-SOD and Mn-SOD mRNA levels were determined in both hemispheres using Northern blot analysis. In separate rats, Cu/Zn-SOD and Mn-SOD protein levels were determined 24 and 72 h after CSD using Western blot analysis. In addition, total SOD, Cu/Zn-SOD and Mn-SOD enzymatic activities were measured 24 and 72 h after CSD using spectrophotometric and zymographic assays. At the times investigated, no significant differences in mRNA or protein levels for Cu/Zn-SOD or Mn-SOD were observed between the ipsilateral and contralateral cortex. Further, there were no significant differences in Cu/Zn-SOD or Mn-SOD enzymatic activities between the two hemispheres at 24 or 72 h after CSD. In addition, CSD did not alter the activities of Cu/Zn-SOD or Mn-SOD in either hemisphere, relative to those in unoperated animals. Taken together, these results fail to support the hypothesis that CSD-induced tolerance is mediated through the upregulation of Cu/Zn-SOD or Mn-SOD.

Dose-dependent induction of mRNAs encoding brain-derived neurotrophic factor and heat-shock protein-72 after cortical spreading depression in the rat.

Previous studies have demonstrated that cortical spreading depression (CSD) increases the expression of putative neuroprotective proteins. The objective of the present study was to elucidate the relationship between the number of episodes of CSD and steady-state levels of mRNAs encoding brain-derived neurotrophic factor (BDNF), heat-shock protein-72 (hsp72) and c-fos. Wistar rats were administered one, five, or twenty-five episodes of CSD evoked by application of 2 M KCl to the frontal cortex of one hemisphere. Animals were permitted to recover for 30 min, 2 h or 24 h prior to sacrifice. Total RNA was isolated from the parietal cortex of each hemisphere and analyzed using Northern blots. At 30 min recovery, levels of BDNF mRNA were not significantly elevated after 1 episode of CSD, but were increased 4-fold after five episodes of CSD and 11-fold after twenty-five episodes of CSD, relative to levels in the contralateral hemisphere. At 2 h recovery, BDNF mRNA levels increased 2-, 3- and 9-fold, respectively. At 24 h, BDNF mRNA had returned to control levels in all groups. Thus, CSD increased levels of BDNF mRNA in a dose-dependent fashion at the early recovery times. Hsp72 mRNA was below the level of detection after 1 and 5 episodes of CSD. However, after twenty-five episodes of CSD, hsp72 mRNA levels were increased in the ipsilateral hemisphere at 30 min and 2 h recovery. Unlike levels of BDNF and hsp72 mRNA, levels of c-fos mRNA were increased nearly to the same extent at 30 min and 2 h after one, five or twenty-five episodes of CSD before returning to control by 24 h recovery. These results demonstrate that CSD triggers a dose-dependent increase in the expression of genes encoding neuroprotective proteins, which may mediate tolerance to ischemia induced by CSD.

In vivo protein expression from mRNA delivered into adult rat brain.

The expression of proteins after local mRNA delivery has a great potential for analysis of protein function in vivo. To explore the feasibility of such a technique within the central nervous system (CNS), we delivered luciferase-encoding mRNA into the rat brain. The tissue distribution and stability of injected mRNA were analyzed using in situ detection and Northern hybridization, while luciferase expression was measured by enzymatic assay. Following intracerebral injection of lipofectin-complexed mRNA, expression of luciferase was detectable as early as 1 h, was maximal at 2-3 h, but was below the level of detection by 24 h. The extent of luciferase expression correlated with the amount of mRNA delivered. Luciferase expression was higher when lipofectin-complexed rather than naked mRNA was injected. In addition, the luciferase expression increased significantly by adding a 50 nt-long poly(A) tail to the 3'-end of the mRNA. Delivering mRNA to the cerebral cortex or hippocampus resulted in measurable luciferase activity at the injection sites but not in adjacent areas. Accordingly, the luciferase mRNA was also localized to the injection site, and the amount of intact transcript was significantly higher at 3 h compared to 24 h after injection. These results demonstrate that in vivo mRNA delivery is a feasible technique for immediate, transient overexpression of desired proteins in the CNS and, therefore, can serve as a model system to study the neurobiological effects of specific proteins.

HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response.

Dendritic cells (DC) are the major APCs involved in naive T cell activation making them prime targets of vaccine research. We observed that mRNA was efficiently transfected, resulting in superior translation in DC compared with other professional APCs. A single stimulation of T cells by HIV gag-encoded mRNA-transfected DC in vitro resulted in primary CD4(+) and CD8(+) T cell immune responses at frequencies of Ag-specific cells (5-12.5%) similar to primary immune responses observed in vivo in murine models. Additionally, mRNA transfection also delivered a maturation signal to DC. Our results demonstrated that mRNA-mediated delivery of encoded Ag to DC induced potent primary T cell responses in vitro. mRNA transfection of DC, which mediated efficient delivery of antigenic peptides to MHC class I and II molecules, as well as delivering a maturation signal to DC, has the potential to be a potent and effective anti-HIV T cell-activating vaccine.

Positive and negative elements mediate control of alternative splicing in the AMPD1 gene.

The second exon of the AMP deaminase (AMPD) 1 gene is alternatively spliced in response to stage-specific signals elaborated during myocyte differentiation. Since inheritance of the mutation in exon 2 of the AMPD1 gene has been recently shown to be associated with a better prognosis of congestive heart failure and the alternative splicing of exon 2 modulates the residual activity of AMPD1 in individuals with this mutant allele, the regulatory mechanism of alternative splicing in the AMPD1 gene is clinically intriguing. Retention or exclusion of exon 2 results from the interplay between negative and positive elements in the primary transcript. Exon 2 is intrinsically defective and difficult to recognize. Herein, we show that this property of exon 2 is the consequence of three defects; a suboptimal 3' splice acceptor site, a suboptimal 5' splice donor site and the small size of the exon. An improvement in any one of these defects relieves the masking of this exon. Further, this defective exon can only be identified in the presence of the adjacent downstream intron.

Overexpression of urokinase receptor in mammalian cells following administration of the in vitro transcribed encoding mRNA.

The ability to overexpress physiologically important proteins in cultured mammalian cells after delivering the encoding mRNAs could have important applications for analyzing their in vivo functions. To explore the potential of this approach, urokinase-type plasminogen activator receptor (uPAR), a membrane protein extensively modified post-translationally, was selected. The uPAR-encoding mRNAs, containing different 5' and 3' untranslated regions (UTR) were tested in cultured human osteosarcoma (HOS) cells following a cationic lipid-mediated delivery. The most effective structure was the capped and polyadenylated transcript containing Xenopus beta-globin 5' and 3' UTRs. Delivering this mRNA to HOS cells resulted in a significant increase of uPAR expression in 89% of the cells, measured by flow cytometry. Using a radioligand binding assay, the increase in functional uPAR levels was found to be up eight- to 11-fold between 8 and 48 h and up three-fold at 72 h after delivery. A similar increase in uPAR levels was achievable in a number of mammalian cell lines. Surprisingly, poly(A)-tailed mRNA leading to a uPAR production highest in magnitude and duration did not demonstrate increased intracellular stability compared with other tested mRNAs. Thus, the exceptional translational performance is not likely the result of an increased mRNA half-life. These results demonstrate that, after delivery of selected mRNAs into mammalian cells, immediate and significant overexpression of a post-translationally modified protein is achievable.

Effect of cortical spreading depression on the levels of mRNA coding for putative neuroprotective proteins in rat brain.

Previous studies have demonstrated that cortical spreading depression (CSD) induces neuronal tolerance to a subsequent episode of ischemia. The objective of the present investigation was to determine whether CSD alters levels of mRNA coding for putative neuroprotective proteins. Unilateral CSD was evoked in male Wistar rats by applying 2 mol/L KCl over the frontal cortex for 2 hours. After recovery for 0, 2, or 24 hours, levels of several mRNA coding for neuroprotective proteins were measured bilaterally in parietal cortex using Northern blot analysis. Levels of c-fos mRNA and brain-derived neurotrophic factor (BDNF) mRNA were markedly elevated at 0 and 2 hours, but not 24 hours after CSD. Tissue plasminogen activator (tPA) mRNA levels were also significantly increased at 0 and 2 hours, but not 24 hours after CSD. Levels of the 72-kDa heat-shock protein (hsp72) mRNA were not significantly increased by CSD, except for a small elevation (20%) at 2 hours recovery. Levels of the 73-kDa heat-shock cognate (hsc73) mRNA were slightly, but significantly, increased at 2 and 24 hours of recovery. Finally, levels of mRNA for protease nexin-1 and glutamine synthetase were not significantly altered by CSD at any time studied. The current results support the hypothesis that neuronal tolerance to ischemia after CSD may be mediated by increased expression of FOS, BDNF, or tPA, but not by increased expression of hsp72, hsc73, nexin-1, or glutamine synthetase.

Native atherosclerosis and vein graft arterialization: association with increased urokinase receptor expression in vitro and in vivo.

Interaction of proteases with cell surface receptors may modulate cell adhesion, migration, invasion, and matrix degradation. Since the plasminogen activator system has been hypothesized to play a role in intimal thickening after various types of vascular injury, we first studied the expression of urokinase receptor (u-PAR) protein and mRNA by smooth muscle cells (SMC) grown in explant cultures from normal and diseased vessels. Using equilibrium binding studies with radiolabeled 125I-labeled single chain urokinase-type plasminogen activator (scu-PA), we determined that SMC cultured from atherosclerotic arteries expressed a higher maximal number of binding sites/cell (3.6 +/- 0.4 x 10(5) sites/cell vs. 2.1 +/- 0.3 x 10(5), +/- SEM, p < 0.05) with a similar affinity (Kd = 1.5 +/- 0.1 vs. 1.2 +/- 0.2 nM, p = ns). However, SMC subcultured from diseased saphenous vein grafts expressed the highest levels of u-PAR compared to SMC from normal saphenous vein (4.8 +/- 0.6 x 10(5) sites/cell vs. 1.6 +/- 0.9 x 10(5), +/- SEM, p < 0.05). Using binding studies and Northern analysis, we demonstrated a dose and time dependent upregulation of u-PAR protein and mRNA expression respectively in human SMC in response to serum stimulation. Using a rabbit specific u-PAR cDNA probe, we demonstrated a similar upregulation of u-PAR mRNA both in rabbit aortic SMC in culture in response to serum stimulation and up to a 20 fold increase in u-PAR mRNA in rabbit jugular veins in response to implantation as arterial grafts in vivo. Finally, to confirm that u-PAR mRNA is upregulated in human vessels after injury, we performed immunohistochemistry and in situ hybridization studies on coronary arteries, normal saphenous veins and saphenous veins from 10 weeks to 13 years after implantation as grafts. u-PAR mRNA was found mainly in the periadventitial microcirculation in normal veins, but was found to be upregulated in the neointima and media of thickened veins in both macrophages and smooth muscle cells. SMC near the internal elastic laminae in diseased coronary arteries appeared to express increased u-PAR mRNA. These data suggest that this increased expression of u-PAR may contribute to early lesion development.

Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA.

Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n, n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS). Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50 microM), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30-80 mM phosphate, at pH 5.6-6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies.

The expression of urokinase-related genes in superficial and invasive transitional cell carcinoma.

Tumor invasion and metastasis is mediated in part by proteolytic enzymes including urokinase plasminogen activator (uPA). The object of this study was to quantitate the molecular expression of urokinase plasminogen activator-related components in human superficial and invasive transitional cell carcinoma tumors (TCC), as well as parental and invasive variant TCC cell lines. We examined 15 invasive and 14 superficial TCC tumors (from a total of 29 patients) and six bladder carcinoma cell lines for the steady state mRNA levels of uPA, the uPA receptor and uPA inhibitor-1 by quantitative RT-PCR normalized to the L7 ribosomal transcript (a housekeeping gene). Transcript levels were expressed in a ratio to the L7 housekeeping transcript. There was a three fold increase in uPA expression in invasive lesions compared to superficial tumors (p < 0.003). In addition, there was a concordant 2.7 fold increase in the uPA receptor transcript in invasive TCC (p < 0.008). However, there was no significant difference in the steady state levels of the PAI-1 mRNA between invasive and superficial tumors. Transcript levels for the urokinase-related genes were similar between normal mucosa and superficial tumors. Cultured cells (parental and invasive variants) were found to express higher levels of the three uPA-related genes overall. Invasive TCC cells selected by serial passage through a Boyden chamber demonstrated higher levels of uPA, uPA receptor and PAI-1 than parental cells (p < 0.05). These data from the human tumor specimens suggest that increased uPA and uPAr expression may be component of the invasive phenotype of TCC lesions.

Thermal preconditioning before rat arterial balloon injury: limitation of injury and sustained reduction of intimal thickening.

Heat shock proteins (HSPs) are a family of highly conserved proteins, essential to cell survival, that are induced during times of physiological stress. These proteins, when induced, can provide tolerance to subsequent injury. Several studies have documented that HSPs play an important role in the response of vascular cells to injury or stress. Whether the vasculature itself can be effectively preconditioned before arterial injury is unknown. Vascular HSP induction by whole-body hyperthermia (WBH) was evaluated with regard to its effects on the vascular response to balloon injury. WBH treatment of Sprague-Dawley rats (colonic temperatures of 41 to 42 degrees C for 15 minutes) resulted in maximal arterial HSP expression within 8 to 12 hours. Rats (male, 300 g, n=59) were randomly assigned to undergo either WBH or no treatment 8 hours before standard carotid balloon injury. At 14 (n=26) and 90 (n=21) days after balloon injury, histomorphometric analysis revealed a significant limitation of intimal accumulation in preconditioned arteries as compared to controls (intimal/medial area ratios+/-SEM: 14 days, 0.57+/-0.07 versus 0.86+/-0.08, P=0.01; 90 days, 0.78+/-0.12 versus 1.19+/-0.14, P<0.05). The medial cell proliferation index at 4 days (n=12) was significantly reduced in the treated group as well (3.6+/-0.9% versus 7.2+/-1.3%, P<0.05). Conversely, the mean total cell number in the media of heated arteries was higher (393+/-20 versus 328+/-17, P<0.05). Vascular preconditioning with brief WBH induces a heat shock response in the arterial wall that is associated with a significant and sustained reduction in intimal accumulation. This effect appears to be due in part to preservation of medial cell integrity and limitation of the proliferative response. These results suggest that thermal preconditioning of vascular tissue may be an effective strategy to improve long-term results after revascularization procedures.

Defensin modulates tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells.

Defensins are naturally occurring antimicrobial peptides that may participate in host defense against microorganisms. We previously reported that the amino acid sequence of leukocyte defensins resembles the lysine-binding site in the kringles of plasminogen and that defensin inhibits fibrinolysis mediated by tissue-type plasminogen activator (tPA) and plasminogen. In the present paper we analyze the mechanisms of this inhibition. Defensin binds specifically to cultured human umbilical vein endothelial cells (HUVEC) (half-maximal binding = 3 microM) as well as to fibrin. At saturating concentrations (5-10 microM), defensin stimulates the maximum binding of plasminogen to HUVEC and to fibrin approximately 10-fold. However, defensin inhibits plasminogen binding to both surfaces at concentrations >10 microM. Defensin also inhibits tPA and plasminogen-mediated fibrinolysis in a dose-dependent manner at all concentrations tested. Fibrinolysis is almost totally inhibited by 6 microM defensin, a concentration that stimulates the binding of plasminogen to fibrin. Discordance between the enhancement of plasminogen binding and its activation cannot be explained by an inhibitory effect of defensin on tPA binding nor by inhibition of plasmin activity, each of which occur only at higher concentrations. Rather, these results suggest that plasminogen bound to fibrin in the presence of defensin is less susceptible to activation by tPA.

Expression of very low density lipoprotein receptor in the vascular wall. Analysis of human tissues by in situ hybridization and immunohistochemistry.

The recently cloned very low density lipoprotein (VLDL) receptor binds triglyceride-rich, apolipoprotein-E-containing lipoproteins with high affinity. The observation that VLDL receptor mRNA is abundantly expressed in extracts of tissues such as skeletal muscle and heart, but not liver, has led to the hypothesis that this receptor may facilitate the peripheral uptake of triglyceride-rich lipoproteins. However, little information is available concerning the types of cells that express this receptor in vivo. As expression of the VLDL receptor in the vascular wall might have important implications for the uptake and transport of triglyceride-rich lipoproteins, and perhaps facilitate the development of atherosclerosis in hypertriglyceridemic individuals, we used in situ hybridization and immunohistochemistry to determine whether VLDL receptor mRNA and protein was expressed in human vascular tissue. We observed expression of the receptor by both endothelial and smooth muscle cells within normal arteries and veins, as well as within atherosclerotic plaques. In the latter, the VLDL receptor was also expressed by macrophage-derived foam cells. The widespread distribution of the VLDL receptor in vascular tissue suggests a potentially important role for this receptor in normal and pathophysiological vascular processes.

Sequence analysis of the 5.34-kb 5' flanking region of the human rhodopsin-encoding gene.

In order to define elements which may be involved in regulating human rhodopsin expression, we have isolated and sequenced a clone containing 5.34 kb of the 5'-upstream region of the human rhodopsin-encoding gene. The 5.34-kb human segment contains multiple potential transcription factor-binding sites and a subfamily of Alu repeats. The same subfamily of Alu repeats is found 5.8 kb upstream from the human red/green visual pigment-encoding gene.

Stimulatory effect of unsaturated fatty acids on the level of plasminogen activator inhibitor-1 mRNA in cultured human endothelial cells.

To determine whether unsaturated fatty acids induce changes in the mRNA level of plasminogen activator inhibitor type-1 (PAI-1), Northern analyses were performed on human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells that were treated with two common fatty acids. Supplementation of cultured HUVEC with docosahexanoic acid (DHA) or with dihomogamma linolenic acid (DGLA), resulted in a concentration dependent, specific increase of the PAI-1 transcript levels, which was detectable within 2 h. DHA and DGLA treatment of smooth muscle cells did not result in changes in the PAI-1 mRNA levels. Homology search of the upstream regulatory region of the PAI-1 gene sequences identified a consensus nucleotide sequence for a fatty acid-responsive element. Our results indicate that unsaturated fatty acids selectively increase PAI-1 mRNA levels in endothelial cells, the primary source of circulating PAI-1 in vivo.

Lipofectin-aided cell delivery of ribozyme targeted to human urokinase receptor mRNA.

A 37-mer hammerhead ribozyme has been designed to efficiently cleave the 1.4 kb mRNA of the urokinase plasminogen activator receptor (uPAR). Under in vitro conditions, the chemically synthesized ribozyme cleaved uPAR mRNA and inhibited its translation in a concentration-dependent fashion. The ribozymes were 5'-[35S]thiophosphorylated and used as a model to analyze conditions for RNA delivery in a cultured human osteosarcoma cell system. Ribozymes degraded immediately in cell-conditioned medium but ribozymes complexed with lipofectin were protected from RNases for up to 22 h. Lipofectin rapidly transported ribozyme into the cell, where it accumulated almost exclusively in the cytoplasm. Thus, lipofectin dramatically enhances stability and cytoplasmic delivery of ribozymes, potentially enabling targeting of mRNA in vivo.

RNase L and increased endoribonuclease activities in the mononuclear cells of patients with chronic myelogenous leukemia.

During investigations of the interferon-induced 2',5' oligoadenylate synthetase/RNase L system in malignancy, RNase L activity and an increased endoribonuclease activity were observed in peripheral blood mononuclear cell (PBMC) extracts from patients with chronic myelogenous leukemia. The cleavage of rRNA from intact ribosomes was used as the assay for both RNase L and the increased endoribonuclease activities. Novel rRNA cleavage products (NCP) were generated by extracts of Ficoll-purified mononuclear cells from chronic myelogenous leukemia (CML) patients and in the granulocytic fraction of both patients and healthy controls. Determination of the time course of rRNA degradation demonstrated that the novel cleavage products were rapidly derived from the further endoribonucleolytic degradation of the RNase L derived specific cleavage products. Prolonged incubation of mononuclear cell extracts from healthy controls also yielded the novel rRNA cleavage products. Comparisons of the kinetics of NCP production suggest that the novel endoribonuclease activity can be approximately 240-fold greater in PBMC extracts from CML patients than controls. Analysis of peripheral blood WBC count and differential indicated that the increased RNase activities were associated with the presence of immature granulocytic cells in the peripheral blood (p = 0.001, Fisher's exact test). However, these activities were also found in the mononuclear cells of a CML patient in lymphoid blast crisis. Since CML is a stem cell disease, the novel endoribonuclease activity may be indicative of active disease, rather than a marker for immature granulocytes. Thus, the RNase L and increased endoribonuclease activities may play a functional role in the biology of chronic myelogenous leukemia and may be important in the mechanism of action of interferon therapy in this disease.