Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery.

Rapidly generated high-titer Semliki Forest virus (SFV) vectors can infect numerous mammalian cell lines and primary cell cultures, and result in high levels of transgene expression. SFV-based expression of transmembrane receptors has been characterized by specific ligand-binding activity and functional responses. Adaptation of the SFV technology for mammalian suspension cultures has allowed the production of hundreds of milligrams of recombinant receptor for purification and structural studies. The same SFV stock solutions used for the infection of mammalian cells in culture have also been successfully applied for efficient transgene expression in organotypic hippocampal slices, as well as in vivo in rodent brain.

Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest Virus, adenovirus, adeno-associated virus, lentivirus, and measles virus.

Viral vectors are useful for transferring genes into neurons. Here, we characterized recombinant Semliki Forest virus (SFV), adenovirus type 5 (Ad5), adeno-associated virus type 2 (AAV), lentivirus, and measles virus (MV) by their expression of green fluorescent protein (GFP) in rat hippocampal slice cultures. SFV infected more neurons (>90% of all GFP-positive cells) than AAV, lentivirus, and MV (71, 69, and 62%, respectively), whereas no infected neurons were identified with Ad5. AAV-mediated GFP expression was neuron-specific when the platelet-derived growth factor beta-chain promoter rather than cytomegalovirus promoter was used. Transgene expression occurred rapidly but transiently for SFV, increased slowly but remained stable with AAV and lentivirus, and was fast with MV. Resting membrane potential and conductance, action potentials, firing accommodation, and H-current appeared normal in infected CA1 pyramidal cells. Thus, SFV is useful for short-term and AAV and lentivirus for long-term transduction of hippocampal slices, while MV constitutes a novel vector.

Modulation of early growth response (EGR) transcription factor-dependent gene expression by using recombinant adenovirus.

Early growth response (EGR) transcription factors link initial cytoplasmic events to long-term alterations of cellular gene expression and are induced by various stimuli. To test their roles in cell physiology, we constructed adenoviral recombinants encoding NGFI-A binding protein 2 (NAB2, a repressor of EGR1, EGR2, and EGR3), EGR1, NAB-insensitive EGR1(I293F) (EGR1*), EGR2, and the NAB-binding, repressive domain 1 (R1) of EGR1. These viruses regulated EGR-dependent expression of GFP and luciferase reporter genes in heterologous expression assays. Infection of a myoblast cell line with EGR1 and EGR1* adenovirus induced the endogenous gene for platelet-derived growth factor A chain (PDGF-A). In addition, in neuroblastoma cells, the two novel EGR1 target genes EGR3 and NAB2 were identified by using adenoviral transfer of EGR1 and EGR1*. Our results demonstrate that recombinant adenovirus is useful to regulate heterologous and endogenous EGR target gene expression, and suggest that EGR transcription factors can autoregulate themselves.

Adenoviral gene transfer to the injured spinal cord of the adult rat.

We have investigated gene transfer to the injured adult rat spinal cord by the use of a recombinant adenovirus. 105 or 5 x 106 plaque-forming units (pfu) of a replication-defective adenoviral vector carrying the green fluorescent protein (GFP) reporter gene were injected into a dorsal hemisection lesion at spinal level T8. Gene expression and inflammatory responses were studied 4, 8 and 21 days after surgery. Numerous cells within 3 mm on each side of the lesion were found to express high levels of GFP at 4 days after infection as shown by GFP fluorescence and immunohistochemistry. At 8 days, expression was still strong although weaker than at 4 days. After 21 days, transgene expression had almost ceased. Expression was neither higher nor more prolonged in animals that had received the higher vector dose. Delayed injection 1 week after spinal injury also did not increase transgene expression. Infected cell types were identified immunohistochemically. The most prominent transduced cells were spinal motoneurons. Additionally, we could identify other neurons, astrocytes, oligodendrocytes and peripheral cells infiltrating the lesion site. The glial and inflammatory reaction at and around the lesion was studied by cresyl violet histology, alpha-GFAP, OX42 and alpha-CD-8 immunohistochemistry. No significant differences from controls were found in the low virus group; in the high virus group a strong invasion of CD-8-positive lymphocytes was found. Open-field locomotion analysis showed virus-infected animals performing as well as control animals. Adenoviral gene transfer may be an efficient way to introduce factors to the injured spinal cord in paradigms of research or therapy.

EGR1 target genes in prostate carcinoma cells identified by microarray analysis.

The EGR1 transactivator is overexpressed in prostate cancer, and its expression pattern suggests that EGR1 could potentially regulate a number of steps involved in initiation and progression of prostate cancer, such as mitogenesis, invasiveness, angiogenesis, and metastasis. To identify potential EGR1 target genes in an unbiased manner, we have utilized adenovirus-mediated expression of EGR1 in a prostate cancer cell line to identify specific genes that are induced by EGR1. Using oligonucleotide arrays, a number of EGR1-regulated genes were identified and their regulation was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. One of the largest gene classes identified in this screen includes several neuroendocrine-associated genes (neuron-specific enolase, neurogranin), suggesting that EGR1 overexpression may contribute to the neuroendocrine differentiation that often accompanies prostate cancer progression. This screen also identified several growth factors such as insulin-like growth factor-II, platelet-derived growth factor-A, and transforming growth factor-beta1, which have previously been implicated in enhancing tumor progression. The insulin-like growth factor-II gene lies within the 11p15.5 chromosomal locus, which contains a number of other imprinted genes, and EGR1 expression was found to induce at least two other genes in this locus (IPL, p57(KIP2)). Based on our results, coupling adenoviral overexpression with microarray and quantitative reverse transcription-polymerase chain reaction analyses could be a versatile strategy for identifying target genes of transactivators.

The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells.

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21(WAF1), a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

Activation of heteromeric G protein-gated inward rectifier K+ channels overexpressed by adenovirus gene transfer inhibits the excitability of hippocampal neurons.

G protein-gated inward rectifier K+ channel subunits 1-4 (GIRK1-4) have been cloned from neuronal and atrial tissue and function as heterotetramers. To examine the inhibition of neuronal excitation by GIRKs, we overexpressed GIRKs in cultured hippocampal neurons from 18 day rat embryos, which normally lack or show low amounts of GIRK protein and currents. Adenoviral recombinants containing the cDNAs for GIRK1, GIRK2, GIRK4, and the serotonin 1A receptor were constructed. Typical GIRK currents could be activated by endogenous GABAB, serotonin 5-HT1A, and adenosine A1 receptors in neurons coinfected with GIRK1+2 or GIRK1+4. Under current clamp, GIRK activation increased the cell membrane conductance by 1- to 2-fold, hyperpolarized the cell by 11-14 mV, and inhibited action potential firing by increasing the threshold current for firing by 2- to 3-fold. These effects were not found in non- and mock-infected neurons, and were similar to the effects of muscarinic stimulation of native GIRK currents in atrial myocytes. Two inhibitory effects of GIRK activation, hyperpolarization and diminution of depolarizing pulses, were simulated from the experimental data. These inhibitory effects are physiologically important in the voltage range between the resting membrane potential and the potential where voltage-gated Na+ and K+ currents are activated; that is where GIRK currents are outward.

ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils.

Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP-ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N-formyl-Met-Leu-Phe. Regulation of these oscillations may be important for the control of neutrophil motility.

Binding to heparan sulfate or heparin enhances neutrophil responses to interleukin 8.

The interaction of interleukin 8 (IL-8) with heparin was studied by using synthetic IL-8 analogs with C- and N-terminal truncations. Elimination of the N-terminal region preceding the first cysteine, which constitutes the IL-8 receptor binding site, did not affect the affinity to heparin-Sepharose. Affinity, however, decreased with progressive truncation at the C terminus, and no binding was observed when the C-terminal alpha-helix was eliminated. The effect of heparin and other glycosaminoglycans on IL-8 activity was also tested. When IL-8 was applied together with heparan sulfate, neutrophil chemotaxis in vitro was enhanced up to 4-fold, and the stimulus-dependent increase in cytosolic free Ca2+ increased markedly in both rate and peak value. Heparin had a similar effect on the Ca2+ response but did not enhance chemotaxis. The glycosaminoglycans by themselves did not elicit neutrophil responses. Their enhancing effect was restricted to stimulation with IL-8 and was not observed when the unrelated chemoattractant fMet-Ile-Phe-Leu was used as the stimulus. Elastase released from stimulated neutrophils was inhibited by heparin, heparan sulfate, and, to a lesser extent, chondroitin sulfate B, confirming previous observations. Taken together, these results suggest that heparan sulfate, which is present on the endothelial cell surface and in the basement membrane, may have a dual function in diapedesis, promotion of IL-8-dependent transmigration of neutrophils, and protection of the tissue microenvironment from damage by lytic enzymes released from the migrating cells.

The interleukin-8-related chemotactic cytokines GRO alpha, GRO beta, and GRO gamma activate human neutrophil and basophil leukocytes.

GRO alpha, a protein structurally related to interleukin-8 (IL-8) and originally described as a melanoma growth stimulatory factor, possesses potent neutrophil-stimulating activity. Recently, two closely related genes, gro beta and gro gamma, were identified. In the present work, the three GRO proteins were chemically synthesized, and their biological activities on human neutrophils and other leukocytes were compared. GRO alpha, GRO beta, and GRO gamma, like IL-8, induced chemotaxis, shape change, a rise in intracellular free calcium levels, exocytosis, and the respiratory burst in neutrophils. The GRO proteins were also active toward basophils as shown by chemotaxis and intracellular calcium concentration changes. The order of potency in neutrophils and basophils was IL-8 > GRO alpha > or = GRO gamma > GRO beta. Of the two IL-8 receptors expressed on human neutrophils, one binds GRO alpha with high and the other with low affinity. Competition binding experiments using radiolabeled IL-8 and GRO alpha revealed the same characteristics for GRO beta and GRO gamma. Similarly, cross-desensitization, as assessed by the stimulus-dependent changes in intracellular calcium concentration, indicated that all three GRO proteins interact with common receptors. From these results, it can be concluded that GRO alpha, GRO beta, and GRO gamma have the same pattern of activity toward human granulocytes and that the differences in amino acid sequence among these proteins have only minor effects on biological activity.

Arachidonic acid and other unsaturated fatty acids alter membrane potential in PC12 and bovine adrenal chromaffin cells.

The action of arachidonic acid and other fatty acids on membrane potential in PC12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1-40 microM) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K(+)-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC12 and bovine chromaffin cells by modulating K+ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca2+ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca2+ influx and secretion. We propose a model where internally generated fatty acids act as a feedback to desensitize the stimulated cell via inhibition of receptor-dependent Ca2+ influx and induction of membrane hyperpolarization.