Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.     

Ubiquitination of protein kinase C-alpha and degradation by the proteasome

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.     

Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.     

Regulation of serotonin2A receptor expression by an antisense oligodeoxynucleotide

The regulation of 5-HT2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A1A1 variant cells, which express the 5-HT2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5-HT2A receptor sites as measured by the binding of 2-[125I]-iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5-HT2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT2A receptor density and an increase in headshake behavior induced by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT2A receptor expression.     

Drug delivery system (DDS) for the use of antisense oligodeoxynucleotide phosphorothioate in controlling gene expression]

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.     

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.     

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.     

Differential membrane-binding and activation mechanisms of protein kinase C-alpha and -epsilon

To elucidate the mechanisms of membrane binding and activation of conventional and novel protein kinase C (PKC), we measured the interactions of rat PKC-alpha and -epsilon with phospholipid monolayers and vesicles of various compositions. Besides the established difference in calcium requirement, the two isoforms showed major differences in their membrane-binding and activation mechanisms. For PKC-alpha, diacylglycerol (DG) specifically enhanced the binding of PKC-alpha to phosphatidylserine (PS)-containing vesicles by 2 orders of magnitude, allowing PKC-alpha high specificity for PS. Also, PKC-alpha could penetrate into the phospholipid monolayer with a packing density comparable to that of the cell membrane only in the presence of Ca2+ and PS. When compared to PKC-alpha, PKC-epsilon had lower binding affinity for PS-containing vesicles both in the presence and in the absence of DG. As a result, PKC-epsilon did not show pronounced specificity for PS. Also, PKC-epsilon showed reduced penetration into PS-containing monolayers, which was comparable to the Ca2+-independent penetration of PKC-alpha into the same monolayers. Taken together, these results suggest the following: (1) The role of Ca2+ in the membrane binding of PKC-alpha is to expose a specific PS-binding site. (2) Once bound to membrane surfaces, PS specifically induces the partial membrane penetration of PKC-alpha that allows its optimal interactions with DG, hence the enhanced membrane binding and activation. (3) PKC-epsilon, due to the lack of Ca2+ binding, cannot specifically interact with PS and DG, which implies the presence of other physiological activator(s) for this isoform.     

Evidence for posttranscriptional regulation of transgenic protein kinase C-alpha in T cells

This study investigates the activities and interactions of elderly patients in an acute medical geriatric unit and a psychiatric unit. The Clifton Assessment Procedures for the Elderly were used to measure cognitive and behavioural functioning, and the 24 subjects studied were divided into three groups: lucid, confused, and demented. Information about subjects' activities and interactions with nursing staff was gained from time sampling by non-participant direct observation. The results reveal very low levels of staff-patient interaction outside of expected routines of patient care. At no time during the observation periods did staff engage patients in social activities or prolonged informal conversations. Yet 306 nurses, of various grades, completed a questionnaire in which they ranked talking to patients as 'enjoyable', 'important', 'rewarding', and an 'objective' for themselves and the unit.     

Feedback regulation of extracellular ATP-stimulated phosphoinositide hydrolysis by protein kinase C-alpha in bovine glomerular endothelial cells

In glomerular endothelial cells, extracellular ATP stimulates a phospholipase C with subsequent hydrolysis of polyphosphoinositides and an increase in cytosolic free Ca2+ concentration ([Ca2+]i). Short-term (30 min) pretreatment of endothelial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C (PKC), decreases the ATP-stimulated phosphoinositide degradation and Ca2+ mobilization. However, this inhibition was lost after incubating the cells for four hours with TPA. Longer-term pretreatment (10 to 48 hr) even potentiated ATP-induced phosphoinositide breakdown and Ca2+ mobilization. In addition, pretreating the cells for 30 minutes with the specific PKC inhibitor Ro 31-8220 dose-dependently increased ATP-stimulated phosphoinositide hydrolysis, thus clearly indicating a regulatory role for PKC in the inositol lipid signaling pathway in glomerular endothelial cells. By using specific antibodies recognizing the different PKC isoenzymes, it is observed that glomerular endothelial cells express five isoenzymes: PKC-alpha, -delta, -epsilon, -zeta and -theta. No PKC-beta, -gamma, -eta and -mu isoenzymes were detected. On exposure to TPA, a complete depletion of PKC-alpha is observed within four hours. In contrast, PKC-epsilon was more resistant to phorbol ester, and even after 48 hours of TPA treatment, only 60% of PKC-epsilon was down-regulated. PKC-theta decreased very slowly from the cytosol (47% left after 24 hr of phorbol ester treatment) and translocated to the Triton X100-insoluble fraction. Moreover, PKC-delta and PKC-zeta were not significantly affected by 48 hours of phorbol ester incubation. Thus, only PKC-alpha is depleted with a kinetic that corresponds to the loss of feedback inhibition of ATP-stimulated phosphoinositide turnover. In the next step, [Ca2+]i changes were measured in single cells loaded with Fura-2 after microinjection of neutralizing PKC isoenzyme-specific antibodies. Injection of antibodies specific for PKC-alpha potently increased Ca2+ mobilization in response to ATP stimulation when compared to cells injected with buffer only or antibodies specific for PKC-epsilon. These results provide evidence that PKC-alpha mediates feedback inhibition of ATP-stimulated phosphoinositide hydrolysis in glomerular endothelial cells.     

Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C-alpha

It is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-alpha to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-alpha translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1 to S-phase transition.     

Pharmacokinetics and metabolism in mice of a phosphorothioate oligonucleotide antisense inhibitor of C-raf-1 kinase expression

The plasma and tissue disposition of CGP 69846A (ISIS 5132) was characterized in male CD-1 mice following iv bolus injections administered every other day for 28 days (total of 15 doses). The doses ranged from 0.8 mg/kg to 100 mg/kg. Urinary excretion of oligonucleotide was also monitored over a 24-hr period following single dose administration over the same dose range. Pharmacokinetic plasma profiles were determined following single dose administration (dose 1) and after multiple doses (dose 15) at doses of 4 and 20 mg/kg. Concentrations in kidney, liver, spleen, heart, lung, and lymph nodes were characterized following doses 1, 8, and 15 for all doses. Capillary gel electrophoresis was used to quantitate intact (full-length) oligonucleotide and its metabolites (down to N - 11 base deletions) in both plasma and tissue at all time points. The plasma and tissue disposition of CGP 69846A was characterized by a rapid distribution into all tissues analyzed. Rapid plasma clearance of the parent oligonucleotide (9.3-14.3 ml/min/kg) was predominantly the result of distribution to tissue and, to a lesser extent, metabolism. Appearance and pattern of chain-shortened metabolites seen in plasma and tissue were consistent with predominantly exonuclease-mediated base deletion. No measurable accumulation of oligonucleotide was observed in plasma following multiple-dose administration, but both the liver and the kidney exhibited 2-3-fold accumulations. In general, the tissues exhibited half-lives for the elimination of parent oligonucleotide of 16-60 hr compared with plasma half-lives of 30-45 min. After repeated administrations, significant decreases in plasma clearance and volume of distribution at steady state (Vss) were observed following dose 15 at the dose of 20 mg/kg but not at the dose of 4 mg/kg. Changes in tissue accumulation and evidence for saturation of tissue distribution at the high doses may explain the plasma disposition changes observed in the absence of alteration of metabolism or plasma accumulation. Urinary excretion was a minor pathway for elimination of oligonucleotide over the 24-hr period immediately following iv administration. However, the amount of oligonucleotide excreted in the urine increased as a function of dose from less than 1% to approximately 13% of the administered dose over a dose range of 0.8 mg/kg to 100 mg/kg.     

Selective inhibition of [D-Ala2, Glu4]deltorphin antinociception by supraspinal, but not spinal, administration of an antisense oligodeoxynucleotide to an opioid delta receptor

Evidence in vivo has suggested the existence of subtypes of the delta opioid receptor (DOR), which have been termed delta 1 and delta 2. These proposed DOR subtypes are thought to be activated by [D-Pen2, D-Pen5]enkephalin (DPDPE, delta 1) and [D-Ala2, Glu4]deltorphin (delta 2). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin, but not of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO, mu agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonists selective for delta, mu and kappa receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin but not of i.th. DAMGO or U69,593 (kappa agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, selectively inhibited the antinociceptive response to i.c.v. [D-Ala2, Glu4]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as delta 2 and further, suggest that this delta receptor subtype may play a major role in eliciting spinal delta-mediated antinociception.     

Alzheimer's-specific effects of soluble beta-amyloid on protein kinase C-alpha and -gamma degradation in human fibroblasts

Alzheimer's disease (AD) is a multifactorial disease in which beta-amyloid peptide (betaAP) plays a critical role. We report here that the soluble fraction 1-40 of betaAP differentially degrades protein kinase C-alpha and -gamma (PKCalpha and PKCgamma) isoenzymes in normal (age-matched controls, AC) and AD fibroblasts most likely through proteolytic cascades. Treatment with nanomolar concentrations of betaAP(1-40) induced a 75% decrease in PKCalpha, but not PKCgamma, immunoreactivity in AC fibroblasts. In the AD fibroblasts, a 70% reduction of the PKCgamma, but not PKCalpha, immunoreactivity was observed after betaAP treatment. Preincubation of AC or AD fibroblasts with 50 microM lactacystine, a selective proteasome inhibitor, prevented beta-AP(1-40)-mediated degradation of PKCalpha in the AC cells, and PKCgamma in the AD fibroblasts. The effects of betaAP(1-40) on PKCalpha in AC fibroblasts were prevented by inhibition of protein synthesis and reversed by PKC activation. A 3-hr treatment with 100 nM phorbol 12-myristate 13-acetate restored the PKCalpha signal in treated AC cells but it did not reverse the effects of betaAP(1-40) on PKCgamma in the AD fibroblasts. Pretreatment with the protein synthesis inhibitor, cycloheximide (CHX, 100 microM), inhibited the effects of betaAP(1-40) on PKCalpha and blocked the rescue effect of phorbol 12-myristate 13-acetate in AC fibroblasts but did not modify PKCgamma immunoreactivity in AD cells. These results suggest that betaAP(1-40) differentially affects PKC regulation in AC and AD cells via proteolytic degradation and that PKC activation exerts a protective role via de novo protein synthesis in normal but not AD cells.     

Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence

BACKGROUND: The emergence of resistance to chemotherapy remains a major problem in the treatment of patients with small-cell lung cancer. Elevated expression of Bcl-2, a protein that inhibits programmed cell death or apoptosis, has been associated with radiation and drug resistance and has been observed in the majority of small-cell lung cancer specimens and cell lines. PURPOSE: To test the hypothesis that Bcl-2 expression levels are critical for inhibiting apoptosis in small-cell lung cancer cells, we used an antisense strategy to reduce Bcl-2 expression in these cells in an attempt to restore the natural occurrence of apoptosis. METHODS: Thirteen antisense oligodeoxynucleotides (ODNs) targeting various regions of the bcl-2 messenger RNA and a control scrambled-sequence ODN were tested to identify the most effective sequence(s) for reducing Bcl-2 protein levels. Northern and western blot analyses were used to examine basal bcl-2 messenger RNA and protein levels, respectively, in four human small-cell lung cancer cell lines (SW2, NCI-H69, NCI-H82, and NCI-N417). SW2 cells were treated with the antisense ODNs in the presence of cationic lipids (to facilitate uptake), and cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis of DNA fragmentation and cell morphology was also performed. The cytotoxic effect of the most potent antisense ODN was also tested on the three other cell lines. RESULTS: The viability of SW2 cells was effectively reduced by ODNs that targeted the translation initiation and termination sites of the bcl-2 messenger RNA, but ODN 2009 that targeted the coding region was the most cytotoxic. Treatment of SW2 cells with 0.15 microM ODN 2009 for 96 hours reduced their viability by 91% (95% confidence interval [CI] = 88%-94%) and caused a dose-dependent reduction in Bcl-2 levels that became detectable 24 hours after treatment and persisted up to 96 hours; analysis of cellular morphology demonstrated that viability was reduced through apoptosis. Moreover, ODN 2009 at 0.15 microM was cytotoxic to NCI-H69, NCI-H82, and NCI-N417 cells, resulting in decreases in cell viability of 82% (95% CI = 78%-86%), 100%, and 100%, respectively, after 96 hours of treatment. The cytotoxic effects were inversely correlated with the basal Bcl-2 levels in the cell lines (r = -9964). A control scrambled-sequence oligodeoxynucleotide had no statistically significant effect on the cell lines (P values ranging from .38 to .89). CONCLUSION: We have identified a novel antisense ODN sequence (ODN 2009) that effectively reduces the viability of small-cell lung cancer cells by reducing Bcl-2 levels and facilitating apoptosis.     

Suppression of postischemic hippocampal nerve growth factor expression by a c-fos antisense oligodeoxynucleotide

We examined the uptake and distribution of an antisense phosphorothioated oligodeoxynucleotide (s-ODN) to c-fos, rncfosr115, infused into the left cerebral ventricle of male Long-Evans rats and the effect of this s-ODN on subsequent Fos, NGF, neurotrophin-3 (NT-3), and actin expression. To establish the uptake and turnover of s-ODN in the brain, we studied the copurification of the immunoreactivity of biotin with biotinylated s-ODN that was recovered from different regions of the brain. A time-dependent diffusion and the localization of s-ODN were further demonstrated by labeling the 3'-OH terminus of s-ODN in situ with digoxigenin-dUTP using terminal transferase and detection using anti-digoxigenin IgG-FITC. Cellular uptake of the s-ODN was evident in both the hippocampal and cortical regions, consistent with a gradient originating at the ventricular surface. Degradation of the s-ODN was observed beginning 48 hr after delivery. The effectiveness of c-fos antisense s-ODN was demonstrated by its suppression of postischemic Fos expression, which was accompanied by an inhibition of ischemia-induced NGF mRNA expression in the dentate gyrus. Infusion of saline, the sense s-ODN, or a mismatch antisense s-ODN did not suppress Fos expression. That this effect of c-fos antisense s-ODN was specific to NGF was demonstrated by its lack of effect on the postischemic expression of the NT-3 and beta-actin genes. Our results demonstrate that c-fos antisense s-ODN blocks selected downstream events and support the contention that postischemic Fos regulates the subsequent expression of the NGF gene and that Fos expression may have a functional component in neuroregeneration after focal cerebral ischemia-reperfusion.     

Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-alpha

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp-like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.     

Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC8) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC8 or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-espilon to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C2-Cer also inhibited the activation of phospholipase D activity induced by either diC8 (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.