Inhibition of spinal cytosolic phospholipase A(2) expression by an antisense oligonucleotide attenuates tissue injury-induced hyperalgesia.

Activation of the spinal phospholipase A(2) (PLA(2)) -cyclooxygenase (COX) -prostaglandin signaling pathway is widely implicated in nociceptive processing. Although the role of spinal COX isoforms in pain signal transmission has been extensively characterized, our knowledge of PLA(2) enzymes in this cascade is limited. Among all PLA(2) groups, cytosolic calcium-dependent PLA(2) group IVA (cPLA(2)IVA) appears to be the predominant PLA(2) enzyme in the spinal cord. In the present study we sought to (i) characterize anatomical and cellular distribution and localization of cPLA(2)IVA in dorsal horn of rat spinal cord, (ii) verify efficacy and selectivity of intrathecal (IT) delivery of an antisense oligonucleotide (AS) targeting rat cPLA(2)IVA mRNA on spinal expression of this enzyme, and (iii) examine the effect of down-regulation of spinal cPLA(2)IVA on peripheral tissue injury-induced pain behavior. Here we demonstrate that cPLA(2)IVA is constitutively expressed in rat spinal cord, predominantly in dorsal horn neurons and oligodendrocytes but not in astrocytes or microglia. Intrathecal injection of AS significantly down-regulated both protein and gene expression of cPLA(2)IVA in rat spinal cord, while control missense oligonucleotide (MS) had no effect. Immunocytochemistry confirmed that the reduction occurred in neurons and oligodendrocytes. cPLA(2)IVA AS did not alter expression of several other PLA(2) isoforms, such as secretory PLA(2) (groups IIA and V) and calcium-independent PLA(2) (group VI), indicating that the AS was specific for cPLA(2)IVA. This selective knockdown of spinal cPLA(2)IVA did not change acute nociception (i.e. paw withdrawal thresholds to acute thermal stimuli and intradermal formalin-induced first phase flinching), however, it significantly attenuated formalin-induced hyperalgesia (i.e. second phase flinching behavior), which reflects spinal sensitization. Thus the present findings suggest that cPLA(2)IVA may specifically participate in spinal nociceptive processing.

Inhibition of telomerase by 2'-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells.

2'-O-(2-methoxyethyl) (2'-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2'-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC(50) values of 5-10 nM at 37 degrees C. IC(50) values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.

Inhibition of C-raf expression by antisense oligonucleotides extends heart allograft survival in rats.

BACKGROUND: C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival. METHODS: Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2'-methoxyethyl (ME) substitutions at the 2'-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides. RESULTS: Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos. CONCLUSIONS: C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity.

Oncogenic homeodomain transcription factor E2A-Pbx1 activates a novel WNT gene in pre-B acute lymphoblastoid leukemia.

A large fraction of pediatric pre-B acute lymphoblastoid leukemias (ALL) consistently contain a t(1;19) chromosomal translocation. The t(1;19) translocation results in the production of a chimeric transcription factor containing the N-terminal transactivation domain of E2A fused to the C-terminal DNA-binding homeodomain of Pbx1. Here, we show that the E2A-Pbx1 fusion protein activates the expression of a novel WNT gene, WNT-16. WNT-16 normally is expressed in peripheral lymphoid organs such as spleen, appendix, and lymph nodes, but not in bone marrow. In contrast, high levels of WNT-16 transcripts are present in bone marrow and cell lines derived from pre-B ALL patients carrying the E2A-Pbx1 hybrid gene. Inhibition of E2A-Pbx1 expression leads to a significant decrease in WNT-16 mRNA levels, suggesting that WNT-16 is a downstream target of E2A-Pbx1. Three putative WNT receptors, FZ-2, FZ-3, and FZ-5, are expressed in cells of the B lineage, including pre-B ALL cells aberrantly expressing WNT-16. We propose that a WNT-16-mediated autocrine growth mechanism contributes to the development of t(1;19) pre-B ALL.

2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

Inhibition of endotoxin-mediated activation of endothelial cells by a perfluorocarbon emulsion.

Endothelial cell (EC) activation plays a key role in the inflammatory response by promoting the margination of leukocytes in inflamed loci. Augmented leukocyte margination to activated EC is mediated by the increased display of leukocyte adhesion molecules on EC surface membranes. The biocompatibility of synthetic oxygen-transport fluids is intimately linked to EC function, since one of the first tissues encountered by such fluids is the vascular endothelium. We investigated the effect of one such agent, a phospholipid-based perfluorocarbon emulsion containing 90% w/v perfluorooctyl bromide (perflubron, PFOB) on EC activation. Human umbilical vein EC (HUVEC) were activated by 5 U/ml interleukin-1 (IL-1), 20 U/ml tumor necrosis factor (TNF), or 50 ng/ml E coli endotoxin (LPS) in the presence or absence of up to 20%, w/v perflubron. HUVEC activation was monitored by the extent of up-regulation of expression of intercellular adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM). Exposure of HUVEC to perflubron did not alter the upregulation of ICAM or ELAM in response to IL-1 or TNF (n = 20). However, at 10% perflubron ICAM upregulation in response to LPS was inhibited by 95 +/- 6% (n = 9; p < .05). ELAM expression was similarly affected. The concentration of perflubron required to diminish LPS-induced up-regulation by 50% was 6.0 +/- 0.6% (n = 3). The inhibitory effect of 10% perflubron was overcome by > 1 microgram/ml LPS (n = 3) and the inhibitory effect was attenuated by adding perflubron to the cultures after LPS. In agreement with the above, additional experiments showed that incubation of LPS with perflubron prevented LPS-induced stimulation of TNF synthesis by a murine macrophage (RAW) cell line (n = 3). We conclude: 1) perflubron neither activates HUVEC nor interferes with HUVEC activation by IL-1 or TNF, 2) perflubron prevented HUVEC activation by LPS in a dose and time-dependent manner, 3) perflubron prevented LPS-induced activation of more than one cell line. Taken together, the data suggest that perflubron may bind and sequester limited concentrations of LPS. Whether this property of perflubron also occurs upon in vivo infusion and whether it might be clinically useful in preventing some of the adverse effects of endotoxemia are unknown.

Inhibition of endotoxin-mediated activation of endothelial cells by a perfluorocarbon emulsion.

Endothelial cell (EC) activation plays a key role in the inflammatory response by promoting the margination of leukocytes in inflamed loci. Augmented leukocyte margination to activated EC is mediated by the increased display of leukocyte adhesion molecules on EC surface membranes. The biocompatibility of synthetic oxygen-transport fluids is intimately linked to EC function, since one of the first tissues encountered by such fluids is the vascular endothelium. We investigated the effect of one such agent, a phospholipid-based perfluorocarbon emulsion containing 90% w/v perflurooctyl bromide (perflubron, PFOB) on EC activation. Human umbilical vein EC (HUVEC) were activated by 5 U/ml interleukin-1 (IL-1), 20 U/ml tumor necrosis factor (TNF), or 50 ng/ml E coli endotoxin (LPS) in the presence or absence of up to 20%, w/v perflubron. HUVEC activation was monitored by the extent of up-regulation of expression of intercellular adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM). Exposure of HUVEC to perflubron did not alter the up-regulation of ICAM or ELAM in response to IL-1 or TNF (n = 20). However, at 10% perflubron ICAM up-regulation in response to LPS was inhibited by 95 +/- 6% (n = 9; p less than .05). ELAM expression was similarly affected. The concentration of perflubron required to diminish LPS-induced up-regulation by 50% was 6.0 +/- 0.6% (n = 3). The inhibitory effect of 10% perflubron was overcome by greater than 1 ug/ml LPS (n = 3) and the inhibitory effect was attenuated by adding perflubron to the cultures after LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C inhibitors block the enhanced expression of intercellular adhesion molecule-1 on endothelial cells activated by interleukin-1, lipopolysaccharide and tumor necrosis factor.

Interleukin 1 (IL-1), bacterial lipopolysaccharide (LPS) and tumor necrosis factor (TNF alpha) enhance the adherence properties of endothelial cells (EC) for neutrophils (PMN). This is mediated in part by the up-regulation of Intercellular Adhesion Molecule 1 (ICAM-1) on EC. Phorbol esters, which activate protein kinase c (PKC) and enhance the adherence properties of EC for PMN also up-regulate the ICAM-1 expression on EC. We investigated the effect of PKC inhibitors on ICAM-1 expression of human umbilical vein EC (HUVEC). Staurosporine (STS) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) prevented inflammatory mediator-dependent stimulation of both ICAM-1 expression and PMN adherence by HUVEC (ID50 for STS = 2.7-2.9 microM; for H-7 = 7.6-8.8 microM). Inhibition was dose and time-dependent and was not due to HUVEC toxicity. The STS analog K252a and the H-7 analog W-7 were less potent inhibitors of ICAM-1 up-regulation and adherence promotion. Prolonged exposure of HUVEC to phorbol myristate acetate down-regulated PKC activity and inhibited subsequent ICAM-1 up-regulation by this agent and by IL-1. We conclude that inflammatory mediator induced stimulation of HUVEC expression of ICAM-1 and promotion of adherence properties are mediated in part by activation of PKC.

Modulation of endothelial cell expression of intercellular adhesion molecule 1 by protein kinase C activation.

Inflammatory stimuli enhance the adherence properties of human umbilical vein endothelial cells (HUVEC) for neutrophils (PMN). This is mediated in part by the up-regulation on HUVEC of Intercellular Adhesion Molecule 1 (ICAM 1). Phorbol esters, which activate protein kinase c (PKC), have also been reported to enhance the adherence properties of HUVEC for PMN. We investigated the effect of agents which activate PKC on the expression of ICAM 1 by HUVEC. Both phorbol myristate acetate (PMA) and Mezerein, a non-phorbol which also stimulates PKC, enhanced both the expression of ICAM 1 on HUVEC and the adherence of HUVEC for PMN. The PKC inhibitors staurosporine and H-7 prevented both PMA and Mezerein-induced stimulation of HUVEC expression of ICAM 1 and adherence for PMN. We conclude that activation of PKC in HUVEC is associated with increased expression of ICAM 1 on HUVEC. PKC-mediated up-regulation of ICAM 1 may be responsible, in part, for the promotion of endothelial cell adherence properties toward PMN.

Neutral triglyceride lipase in macrophages.

High levels of neutral triglyceride lipase activity have been demonstrated in several types of macrophages (J774 cells, human monocyte/macrophages, rabbit alveolar macrophages, and resident mouse peritoneal macrophages). The pH optima ranged from 6.5 to 7.4 depending upon the buffer and the conditions of incubation. The addition of bovine serum albumin stimulated activity at low concentrations, as expected for a fatty acid-releasing reaction, but strongly inhibited at higher concentrations; maximal activity was observed in the presence of 0.625 mg/ml of bovine serum albumin. The enzyme was remarkably thermostable, showing no apparent loss of activity at 50 degrees C for as long as 6 hours. The lipase was inhibited 80% by 0.1 M NaCl. Assayed under optimal conditions, the specific activity of the neutral triglyceride lipase from J774 cells was more than 100-fold greater than the activity of lipoprotein lipase or neutral cholesterol esterase from those cells; this activity was 10-fold greater than the levels of hormone-sensitive lipase from 3T3-L1 adipocytes. This neutral triglyceride lipase may play an important role in the degradation and mobilization of cytosolic triglyceride in macrophage-derived foam cells.

Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase.

Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation.