Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides.

A 21-mer oligodeoxynucleotide complementary to the polyadenylation signal for human hepatitis B virus (HBV) was complexed to a soluble DNA-carrier system that is targetable to hepatocytes via asialoglycoprotein receptors present on those cells. A cell line, HepG2 (2.2.15) that possesses asialoglycoprotein receptors and is permanently transfected with hepatitis B virus (ayw subtype) was exposed to complexed antisense DNA or controls. In the presence of complexed antisense DNA, the concentration of hepatitis B surface antigen in medium was 80% lower than controls after 24 h. Furthermore, during the next 6 days, there was no significant increase in surface antigen concentration in the presence of complexed antisense DNA. The inhibition could be effectively blocked by competition with an excess of free asialoglycoprotein. Total protein synthesis remained unchanged by exposure to complexed antisense sequences under identical conditions. In addition, HBV DNA in the medium and cell layers after 24-h exposure to complexed antisense sequences was 80% lower than in controls. The data indicate that antisense oligonucleotides complexed by a soluble DNA-carrier system can be targeted to cells via asialoglycoprotein receptors resulting in specific inhibition of hepatitis B viral gene expression and replication.     

Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis.

A phosphorothioate-oligonucleotide-based antisense strategy for depleting MAP kinase was developed. The 17mer antisense probe, EAS 1, caused a potent and concentration-dependent decrease in the steady state expression of p42 and p44 MAP kinase in 3T3 L1 fibroblasts and adipocytes with submicromolar concentrations effective. Antisense EAS 1 elicited a dose-dependent inhibition of insulin- and serum-stimulated DNA synthesis. Elimination of p42 MAP kinase by > 95% and p44 MAP kinase to levels undetected blocked the ability of serum in 3T3 L1 fibroblasts and insulin in 3T3 L1 adipocytes to stimulate DNA synthesis by 87-95%. The differentiation of 3T3 L1 fibroblasts into adipocytes was prevented by 1 microM antisense EAS 1. The corresponding sense, scrambled or sense plus antisense EAS 1 phosphorothioate oligonucleotides did not deplete the p42 or p44 MAP kinase from either cell type, did not inhibit stimulation of DNA synthesis and did not interfere with differentiation. Two kinases on different MAP kinase activation pathways were not depleted by antisense EAS 1 whereas the ability of insulin to activate p90 S6 kinase was > 90% eliminated in 3T3 L1 adipocytes by 4.5 microM antisense EAS 1. In conclusion these results show that MAP kinase is required for insulin and serum stimulation of DNA synthesis, for insulin stimulation of p90 S6 kinase activity and for differentiation of 3T3 L1 cells. Moreover, the development of the antisense probe EAS 1 against a target sequence of p42 MAP kinase that is conserved in p44 MAP kinase and across a range of species provides a molecular tool of general applicability for further dissecting the precise targets and roles of MAP kinase.     

Changes in DNA and RNA synthesis and associated enzyme activities after the stimulation of serum-depleted BHK21-C13 cells by the addition of serum

BHK21/C13 cells placed in medium containing low (1%) serum ceased DNA synthesis within 4 days. DNA synthesis recommenced 10 h after the readdition of serum (to 10%) to cells incubated for 6 days in serum-depleted medium. Two peaks of thymidine incorporation were observed at 12–13 h and 15–17 h, followed by a single peak of dividing cells at 25 h. The two peaks of incorporation represent variation in the extent of DNA replication during a single synchronous S phase.Uridine, deoxyadenosine and deoxyguanosine kinase activities did not decline in serum-depleted cells and, after the addition of serum, their activities showed cyclical variation about a mean involving two-fold changes in enzyme specific activity. All other enzyme activities examined were markedly decreased in resting cells.Ornithine decarboxylase activity increased 15-fold within 5 h of serum addition, but had returned to the resting level by 8 h. There was no apparent correlation between this alteration of enzyme activity and the rate of RNA synthesis.DNA polymerase, thymidine kinase and deoxycytidine kinase activities all decreased further within 4 h of the addition of serum, followed by several-fold increases in activity. The peak of DNA polymerase activity corresponded to, and encompassed, both peaks of DNA synthesis. However, thymidine and deoxycytidine kinase activities, although exhibiting two activity maxima corresponding to the peaks of DNA synthesis, were at their highest levels in G2.     

Expression of human papilloma virus E7 protein causes apoptosis and inhibits DNA synthesis in primary hepatocytes via increased expression of p21(Cip-1/WAF1/MDA6)

The impact of human papilloma virus (HPV16) E7 proteins and retinoblastoma (RB) antisense oligonucleotides upon mitogen-activated protein kinase (MAPK)-mediated inhibition of DNA synthesis via p21(Cip-1/WAF1/MDA6) (p21) was determined in primary hepatocytes. Prolonged activation of the MAPK pathway in p21(+/+) or p21(-/-) hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Either transfection with RB antisense oligonucleotides, expression of wild type E7, or RB binding mutant E7 (C24S) proteins increased p21 levels and reduced DNA synthesis in p21(+/+) hepatocytes. RB antisense oligonucleotides and E7 proteins increased apoptosis in p21(+/+), but not p21(-/-), hepatocytes. Expression of wild type E7 increased DNA synthesis above control levels in p21(-/-) cells, which was additive with prolonged MAPK activation. In contrast, expression of mutant E7 did not alter DNA synthesis above control levels in p21(-/-) cells and was supra-additive with prolonged MAPK activation. Antisense ablation of RB in p21(-/-) hepatocytes had a weak stimulatory effect upon DNA synthesis itself but enhanced the capacity of mutant E7 protein to stimulate DNA synthesis to the same level observed using wild type E7. The ability of prolonged MAPK activation to stimulate DNA synthesis in the presence of mutant E7 and antisense RB was additive. Collectively, the present data demonstrate that loss of RB function together with loss of p21 function plays an important role in the E7- and MAPK-dependent modulation of apoptosis and DNA synthesis in primary hepatocytes.     

Herpes Simplex Virus Type 2 Functions Expressed During Stimulation of Human Cell DNA Synthesis

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.     

Consequences of herpes simplex virus type 2 and human cell interaction at supraoptimal temperatures.

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.     

Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on g protein-mediated activation of mitogen-activated protein kinases

Hypoxia has been shown to act as a proliferative stimulus for adventitial fibroblasts of the pulmonary artery. The signaling pathways involved in this growth response, however, remain unclear. We tested the hypothesis that hypoxia-induced proliferation of fibroblasts would be dependent on distinct (compared with serum) activation and utilization patterns of mitogen-activated protein (MAP) kinases initiated by Galpha(i/o) proteins. We found that hypoxia stimulated increases in DNA synthesis and growth of quiescent fibroblasts in the absence of exogenous mitogens and also markedly augmented serum-stimulated growth responses. Hypoxia caused a transient activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the time course and pattern of which was somewhat similar to that induced by serum but which was of lesser magnitude. On the other hand, hypoxia-induced activation of p38 MAP kinase was biphasic, whereas serum-stimulated activation of p38 MAP kinase was transient, and the magnitude of activation was greater for hypoxia compared with that of serum stimulation. ERK1/2, JNK1, and p38 MAP kinase but not JNK2 were necessary for hypoxia-induced proliferation because PD98059, SB202190, and JNK1 antisense oligonucleotides nearly ablated the growth response. JNK2 appeared to act as a negative modulator of hypoxia-induced growth because JNK2 antisense oligonucleotides led to an increase in DNA synthesis. In serum-stimulated cells, antisense JNK1 oligonucleotides and PD98059 had inhibitory effects on proliferation, whereas SB202190 led to an increase in DNA synthesis. Pertussis toxin, which blocks Galpha(i/o)-mediated signaling, markedly attenuated hypoxia-induced DNA synthesis and activation of ERK and JNK but not p38 MAP kinase. We conclude that hypoxia itself can act as a growth promoting stimulus for subsets of bovine neonatal adventitial fibroblasts largely through Galpha(i/o)-mediated activation of a complex network of MAP kinases whose specific contributions to hypoxia-induced proliferation differ from traditional serum-induced growth signals.     

Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation.     

Antiproliferative action of protein kinase C in cultured rabbit aortic smooth muscle cells

In rabbit aortic smooth muscle cells (SMC), protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the whole blood serum (WBS)-induced DNA synthesis. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate (PDBu), but not by 4 alpha-phorbol-12,13- didecanoate known to be inactive for this enzyme. Prolonged treatment of the cells with PDBu caused the down-regulation of protein kinase C. In these cells, WBS still induced DNA synthesis but the inhibitory action of TPA was abolished. DNA synthesis started at 18 h and reached a maximal level 24 h after the addition of WBS. TPA inhibited the WBS-induced DNA synthesis even when added 12 h after the addition of WBS. These results suggest that protein kinase C has an antiproliferative action in rabbit aortic SMC and that this action is attributed to the inhibition of the progression from the late G1 into S phase of the cell cycle. TPA also inhibited the phospholipase C-mediated hydrolysis of phosphoinositides which was induced by WBS within several minutes, but the relevance of this effect on the antiproliferative action of TPA is uncertain.     

Inhibition of Thymidine Kinase Activity and Deoxyribonucleic Acid Synthesis in L Cells Infected with the Meningopneumonitis Agent

The activities of enzymes related to deoxyribonucleic acid (DNA) synthesis were studied in uninfected L cells and in L cells infected with Chlamydia psittaci (strain meningopneumonitis). The meningopneumonitis agent multiplied normally but failed to induce the synthesis of thymidine kinase in LM (TK(-)) cells which contain no thymidine kinase in the uninfected state. It was concluded that this microorganism has no thymidine kinase of its own and that it does not depend on the functioning of the host enzyme for synthesizing its DNA. Exposure of clone 5b L cells to the meningopneumonitis agent was followed by a decline in their thymidine kinase activity to nearly zero levels, whereas the levels of uridine kinase and thymidylate synthetase remained unchanged. Inhibition of thymidine kinase activity in L cells occurred soon after infection and required new protein synthesis by the meningopneumonitis agent. This inhibition occurred before inhibition of host DNA synthesis, but it was not an essential prelude to the latter inhibition. On the basis of this and previous investigations and in light of present knowledge of the mammalian cell cycle, it was postulated that the meningopneumonitis agent inhibits macromolecular synthesis in L cells by preventing the initiation of a new cell cycle.     

Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus

In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus.     

DNA polymerase, thymidine kinase and DNA synthesis in erythropoietic mouse spleen cells separated on bovine serum albumin gradients.

A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-alpha. The activity of low molecular weight DNA polymerase-beta does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-alpha showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-alpha activity is the young normblast.     

c-Jun N-terminal kinase is essential for growth of human T98G glioblastoma cells

The c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway is activated by numerous cellular stresses. Although it has been implicated in mediating apoptosis and growth factor signaling, its role in regulating cell growth is not yet clear. Here, the influence of JNK on basal (unstimulated) growth of human tumor glioblastoma T98G cells was investigated using highly specific JNK antisense oligonucleotides to inhibit JNK expression. Transient depletion of either JNK1 or JNK2 suppressed cell growth associated with an inhibition of DNA synthesis and cell cycle arrest in S phase. The growth-inhibitory potency of JNK2 antisense ((JNK)2 IC(50) = 0.14 micrometer) was greater than that of JNK1 antisense ((JNK)1 IC(50) = 0.37 micrometer), suggesting that JNK2 plays a dominant role in regulating growth of T98G cells. Indeed, JNK2 antisense-treated populations exhibited greater inhibition of DNA synthesis and accumulation of S-phase cells than did the JNK1 antisense-treated cultures, with a significant proportion of these cells detaching from the tissue culture plate. JNK2 (but not JNK1) antisense-treated cultures exhibited marked elevation in the expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1) accompanied by inhibition of Cdk2/Cdc2 kinase activities. Taken together, these results indicate that JNK is required for growth of T98G cells in nonstress conditions and that p21(cip1/waf1) may contribute to the sustained growth arrest of JNK2-depleted T98G cultures.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Control of expression of the vaccinia virus thymidine kinase gene.

mRNA extracted from vaccinia virus-infected cells early after infection directs cell-free synthesis of enzymatically active viral thymidine kinase (Hruby and Ball, Virology, in press). We used this assay for a specific vaccinia virus mRNA to study the induction and repression of the viral thymidine kinase gene during infection of thymidine kinase-deficient L-cells. As observed previously by other workers, the synthesis of thymidine kinase occurred immediately after infection but was switched off after 4 h later. We observed similar kinetics of accumulation and shutoff under conditions where viral DNA synthesis and late gene expression were inhibited. Cell-free translation of mRNA from infected cells showed that the concentration of functional message for viral thymidine kinase reached a peak 3 to 4 h after infection and then decreased with a half-life of about 1 h. These kinetics indicated that significant levels of thymidine kinase mRNA persisted in cells which had stopped synthesizing the enzyme. Under conditions where late gene expression was inhibited, high concentrations of functional mRNA could be isolated from cells at late times after infection. On the basis of these results, we conclude that the repression of thymidine kinase expression is mediated at the translational level by one or more early or delayed early viral genes. Repression is accompanied by, but does not depend on, the inactivation or degradation of thymidine kinase mRNA, which is a late gene function.     

Anti-sense transforming growth factor alpha oligonucleotides inhibit autocrine stimulated proliferation of a colon carcinoma cell line.

Many carcinoma cells secrete transforming growth factor alpha (TGF alpha). A 23 base anti-sense oligonucleotide that recognizes the TGF alpha mRNA inhibits both DNA synthesis and the proliferation of the colon carcinoma cell line LIM 1215. The effects of the anti-sense TGF alpha oligonucleotide are reversed by epidermal growth factor (EGF) at 20 ng/ml. When the LIM 1215 cells are grown under serum free conditions, the anti-sense TGF alpha oligonucleotides have their greatest effects at high cell density (2 x 10(5) cells/cm2), indicating that the secreted TGF alpha is acting as an exogenous growth stimulus. In addition, at higher cell densities, the kinase activity of the EGF receptor is activated and the receptor is down-modulated. The cell density dependent activation of the EGF receptor is inhibited by the application of the antisense TGF alpha oligonucleotides.     

Cell Cycle-Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)