Polyriboinosinic Acid-polyribocytidylic Acid Research Articles

Liposomes-Encapsulating Double-Stranded Nucleic Acid (Poly I:C) for Head and Neck Cancer Treatment.

Polyriboinosinic acid-polyribocytidylic acid (Poly I:C) serves as a synthetic mimic of viral double-stranded dsRNA, capable of inducing apoptosis in numerous cancer cells. Despite its potential, therapeutic benefits, the application of Poly I:C has been hindered by concerns regarding toxicity, stability, enzymatic degradation, and undue immune stimulation, leading to autoimmune disorders. To address these challenges, encapsulation of antitumor drugs within delivery systems such as cationic liposomes is often employed to enhance their efficacy while minimizing dosages. In this study, we investigated the potential of cationic liposomes to deliver Poly I:C into the Head and Neck 12 (HN12) cell line to induce apoptosis in the carcinoma cells and tumor model. Cationic liposomes made by the hydrodynamic focusing method surpass traditional methods by offering a continuous flow-based approach for encapsulating genes, which is ideal for efficient tumor delivery. DOTAP liposomes efficiently bind Poly I:C, confirmed by transmission electron microscopy images displaying their spherical morphology. Liposomes are easily endocytosed in HN12 cells, suggesting their potential for therapeutic gene and drug delivery in head and neck squamous carcinoma cells. Activation of apoptotic pathways involving MDA5, RIG-I, and TLR3 is evidenced by upregulated caspase-3, caspase-8, and IRF3 genes upon endocytosis of Poly(I:C)-encapsulated liposomes. Therapeutic evaluations revealed significant inhibition of tumor growth with Poly I:C liposomes, indicating the possibility of MDA5, RIG-I, and TLR3-induced apoptosis pathways via Poly I:C liposomes in HN12 xenografts in J:NU mouse models. Comparative histological analysis underscores enhanced cell death with Poly I:C liposomes, warranting further investigation into the precise mechanisms of apoptosis and inflammatory cytokine response in murine models for future research.

Surface-assembled poly(I:C) on PEGylated PLGA microspheres as vaccine adjuvant: APC activation and bystander cell stimulation

Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres are potential vehicles to deliver antigens for vaccination. Because they lack the full capacity to activate professional antigen presenting cells (APCs), combination with an immunostimulatory adjuvant may be considered. A candidate is the synthetic TLR3 ligand polyriboinosinic acid-polyribocytidylic acid, poly(I:C), which drives cell-mediated immunity. However, poly(I:C) has also been linked to the pathogenesis of autoimmunity, as affected by widespread stimulation of non-hematopoietic bystander cells. To address this aspect, we propose to minimize the poly(I:C) dose as well as to control the stimulation of non-immune bystander cells by poly(I:C). To facilitate the maturation of APCs with minimal poly(I:C) doses, we surface-assembled poly(I:C) onto PLGA microspheres. The microspheres’ surface was further modified by poly(ethylene glycol) (PEG) coronas with varying PEG-densities. PLGA microspheres loaded with tetanus toxoid (tt) as model antigen were manufactured by microextrusion-based solvent extraction. The negatively charged PLGA(tt) microspheres were coated with polycationic poly(l-lysine) (PLL) polymers, either PLL itself or PEG-grafted PLL (PLL-g-PEG) with varying grafting ratios (g=2.2 and g=10.1). Stable surface assembly of poly(I:C) was achieved by subsequent incubation of polymer-coated PLGA microspheres with aqueous poly(I:C) solutions. We evaluated the immunostimulatory potential of such PLGA(tt) microsphere formulations on monocyte-derived dendritic cells (MoDCs) as well as human foreskin fibroblasts (HFFs) as model for non-hematopoietic bystander cells. Formulations with surface-assembled poly(I:C) readily activated MoDCs with respect to the expression of maturation-related surface markers, proinflammatory cytokine secretion and directed migration. When surface-assembled, poly(I:C) enhanced its immunostimulatory activity by more than one order of magnitude as compared to free poly(I:C). On fibroblasts, surface-assembled poly(I:C) upregulated class I MHC but not class II MHC. Phagocytosis of PLGA(tt) microsphere formulations by MoDCs and HFFs remained mostly unaffected by PEG-grafted PLL coatings. In contrast, high concentrations of free poly(I:C) led to a marked drop of microsphere phagocytosis by HFFs. Overall, surface assembly on PEGylated PLGA microspheres holds promise to improve both efficacy and safety of poly(I:C) as vaccine adjuvant.

Surface assembly of poly(I:C) on PEGylated microspheres to shield from adverse interactions with fibroblasts

By expressing an array of pattern recognition receptors (PRRs), fibroblasts play an important role in stimulating and modulating the response of the innate immune system. The TLR3 ligand polyriboinosinic acid-polyribocytidylic acid, poly(I:C), a mimic of viral dsRNA, is a vaccine adjuvant candidate to activate professional antigen presenting cells (APCs). However, owing to its ligation with extracellular TLR3 on fibroblasts, subcutaneously administered poly(I:C) bears danger towards autoimmunity. It is thus in the interest of its clinical safety to deliver poly(I:C) in such a way that its activation of professional APCs is as efficacious as possible, whereas its interference with non-immune cells such as fibroblasts is controlled or even avoided. Complementary to our previous work with monocyte-derived dendritic cells (MoDCs), here we sought to control the delivery of poly(I:C) surface-assembled on microspheres to human foreskin fibroblasts (HFFs). Negatively charged polystyrene (PS) microspheres were equipped with a poly(ethylene glycol) (PEG) corona through electrostatically driven coatings with a series of polycationic poly(L-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG, of varying grafting ratios g from 2.2 up to 22.7. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres with aqueous poly(I:C) solutions. Notably, recognition of both surface-assembled and free poly(I:C) by extracellular TLR3 on HFFs halted their phagocytic activity. Ligation of surface-assembled poly(I:C) with extracellular TLR3 on HFFs could be controlled by tuning the grafting ratio g and thus the chain density of the PEG corona. When assembled on PLL-5.7-PEG-coated microspheres, poly(I:C) was blocked from triggering class I MHC molecule expression on HFFs. Secretion of interleukin (IL)-6 by HFFs after exposure to surface-assembled poly(I:C) was distinctly lower as compared to free poly(I:C). Overall, surface assembly of poly(I:C) may have potential to contribute to the clinical safety of this vaccine adjuvant candidate.

The Deficiency of PGI 2 Signaling Induces STAT6-independent Allergic Airway Inflammation

S A T U R D A Y 209 Peripheral Blood Natural Killer Cells from Asthmatic Subjects Respond Differently to Stimulation with Poly(I:C) and Diesel Exhaust Particles C. V. E. Chehrazi, K. M. Horvath, I. Jaspers; University of North Carolina, Chapel Hill, NC. RATIONALE: It has been suggested that NK cells in asthmatic and healthy subjects differ, but it is unknown whether these differences alter NK responses to virus and contribute to increased asthmatic susceptibility to viral infections. Data from our lab suggest that diesel exhaust exposure enhances virally-induced exacerbation of allergic inflammation, yet the role of NK cells in these responses is unknown; therefore, we examined the response of asthmatic peripheral NK cells to polyriboinosinic acid-polyribocytidylic acid [poly(I:C)a mimetic of virus-derived dsRNA] and diesel exhaust particles (DEP). METHODS: NK cells were isolated from the blood of asthmatic subjects and subsequently incubated with vehicle, poly(I:C), DEP, or poly (I:C)+DEP. Flow cytometry and cytokine analysis were performed to evaluate changes in cell surface markers and mediator profiles, respectively. RESULTS: As compared to vehicle control, poly(I:C) and DEP stimulation significantly augmented surface expression of CD16, which mediates antibody-dependent cell-mediated cytotoxicity. In contrast, only DEP stimulation significantly enhanced surface expression of CXCR3, the receptor for CXCL10, which is expressed by virally-infected cells and promotes NK chemotaxis. Analysis of cytokines and other cell surface receptors will be performed. CONCLUSIONS: Stimulation with poly(I:C) and DEP alters the expression of cell surface markers involved in NK activity. Whereas NK cytotoxicity appears enhanced by stimulation with dsRNA, NK chemotactic potential appears enhanced by DEP, suggesting that the NK response of asthmatics may be altered by air pollution. These novel findings offer insight into potential mechanisms by which pollutant exposure amplifies asthmatic morbidity due to viral infection.

Tuning the immune response of dendritic cells to surface-assembled poly(I:C) on microspheres through synergistic interactions between phagocytic and TLR3 signaling

The artificial dsRNA polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a potent adjuvant candidate for vaccination, as it strongly drives cell-mediated immunity. However, because of its effects on non-immune bystander cells, poly(I:C) administration may bear danger for the development of autoimmune diseases. Thus poly(I:C) should be applied in the lowest dose possible. We investigated microspheres carrying surface-assembled poly(I:C) as a two-in-one adjuvant formulation to stimulate maturation of monocyte-derived dendritic cells (MoDCs). Negatively charged polystyrene microspheres were equipped with a poly(ethylene glycol) corona through electrostatically driven surface assembly of a library of polycationic poly( l-lysine)- graft-poly(ethylene glycol) copolymers, PLL- g-PEG. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres in an aqueous poly(I:C) solution. Surface-assembled poly(I:C) exhibited a strongly enhanced efficacy to stimulate maturation of MoDCs by up to two orders of magnitude, as compared to free poly(I:C). Multiple phagocytosis events were the key factor to enhance the efficacy. The cytokine secretion pattern of MoDCs after exposure to surface-assembled poly(I:C) differed from that of free poly(I:C), while their ability to stimulate T cell proliferation was similar. Overall, phagocytic signaling plays an important role in defining the resulting immune response to such two-in-one adjuvant formulations.

Diesel exhaust enhances virus- and poly(I:C)-induced Toll-like receptor 3 expression and signaling in respiratory epithelial cells

Prior exposure of respiratory epithelial cells to an aqueous-trapped solution of diesel exhaust (DE(as)) enhances the susceptibility to influenza infections. Here, we examined the effect of DE(as) on the Toll-like receptor 3 (TLR3) pathway, which is responsible for the recognition of and response to viruses and double-stranded RNA. Flow cytometric and confocal microscopy analyses showed that TLR3 is predominantly expressed in the cytoplasm of respiratory epithelial cells. To examine the effect of DE on TLR3 expression and function, differentiated human bronchial or nasal epithelial cells as well as A549 cells were exposed to DE(as) and then infected with influenza A or treated with polyriboinosinic acid-polyribocytidylic acid [poly(I:C)], a synthetic form of double-stranded RNA. Exposure to DE(as) before infection with influenza or stimulation with poly(I:C) significantly upregulated the expression of TLR3. Additionally, preexposure to DE(as) significantly increased the poly(I:C)-induced expression of IL-6. Overexpression of a dominant-negative mutant form of TNF receptor-associated factor 6 reversed the effects of DE(as) on poly(I:C)-induced IL-6 expression, suggesting that the response was TLR3 dependent. Similarly, preexposure to DE(as) significantly increased nuclear levels of interferon regulatory factor 3 and the expression of IFN-beta in response to poly(I:C). Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, was able to abate the effect of DE(as) on poly(I:C)-induced IFN-beta expression. Together, these results indicate that exposure of respiratory epithelial cells to DE(as) could potentially alter the response to viral infections by increasing the expression and function of TLR3.

In vitro translational activity of messenger-RNA isolated from mice treated with the interferon inducer, polyriboinosinic acid·polyribocytidylic acid

Treatment of mice with interferon and interferon inducers causes down regulation of a number of hepatic proteins. In a previous publication it was demonstrated that these treatments depress hepatic protein synthesis and increase protein degradation, particularly of the endoplasmic reticulum Gooderham NJ and Mannering GJ, Arch Biochem Biophys 250: 418–425, 1986. In the present study the effects of polyriboinosinic acid-polyribocytidylic acid (poly IC) treatment on mouse hepatic RNA levels and the translation of this RNA in a cell-free system were examined. Poly IC treatment of mice increased hepatic poly (A +)RNA levels. The translation of isolated poly(A +)RNA was evaluated at various intervals after the administration of poly IC. Translation was marginally increased at 3–6 hr after treatment and depressed after 12–18 hr. Antibodies were employed to examine the effects of poly IC treatment on specific polypeptides in order to evaluate the in vitro translation of mRNAs for tyrosine aminotransferase and albumin; translation of these proteins was biphasic with pronounced depression. These studies indicate that in vivo interferon may regulate gene expression by altering levels of hepatic proteins via increased transcription and decreased translation.

Effect of interferon inducers on carbon tetrachloride toxicity in congenic strains of mice

Conference Article| August 01 1989 Effect of interferon inducers on carbon tetrachloride toxicity in congenic strains of mice MAHTA JAHANSHAHI; MAHTA JAHANSHAHI 1Department of Clinical Pharmacology, Royal Postgraduate Medical School, Du Cane Road, London W12 0NN, U.K. Search for other works by this author on: This Site PubMed Google Scholar NIGEL J. GOODERHAM NIGEL J. GOODERHAM * *To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1989) 17 (4): 733–734. https://doi.org/10.1042/bst0170733 Article history Received: November 28 1988 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation MAHTA JAHANSHAHI, NIGEL J. GOODERHAM; Effect of interferon inducers on carbon tetrachloride toxicity in congenic strains of mice. Biochem Soc Trans 1 August 1989; 17 (4): 733–734. doi: https://doi.org/10.1042/bst0170733 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: poly(I), poly(C), polyriboinosinic acid-polyribocytidylic acid, LPS, lipopolysaccharide This content is only available as a PDF. © 1989 Biochemical Society1989 Article PDF first page preview Close Modal You do not currently have access to this content.

The Kinetics of Interferon Induction by Poly ICLC in Humans

A preliminary trial of the interferon (IFN) inducer polyriboinosinic acid-polyribocytidylic acid in poly-L-lysine and carboxymethylcellulose (poly ICLC) was conducted in patients with chronic progressive multiple sclerosis (MS). Because IFN induction in men is greater than in women, the kinetics of IFN induction were studied. As no significant differences were found in the serum half-life of IFN, sex-linked differences in IFN clearance or degradation are not likely to be responsible for the differences noted. Because fever was the major limiting side effect in MS patient, the relationship of fever to IFN levels was assessed. Changes in temperature were not related to changes in IFN levels, suggesting that fever might be blocked without reducing IFN levels.

Pyrogenicity of interferon and its inducer in rabbits

The effects of intracerebral administration of interferon (IFN) or its inducer polyriboinosinic acid-polyribocytidylic acid (poly I:C) on thermoregulatory responses were assessed in conscious rabbits. Administration of IFN (10(2)-10(6) IU) or poly I:C (0.012-12 micrograms) into the preoptic anterior hypothalamus or the third cerebral ventricle caused a dose-dependent fever in rabbits at three ambient temperatures (Ta) tested. In the cold (Ta = 8 degrees C), the fever was due to increased metabolism, whereas in the heat (Ta = 32 degrees C) the fever was due to a reduction in respiratory evaporative heat loss and ear skin blood flow. At the moderate environmental temperature (Ta = 22 degrees C), the fever was due to increased metabolism and cutaneous vasoconstriction. Compared with the febrile responses induced by cerebroventricular route injection of IFN or poly I:C, the hypothalamic route of injection required a much lower dose of IFN or poly I:C to produce a similar fever. Furthermore, the fever induced by intrahypothalamic injection of IFN or poly I:C was reduced by pretreatment of animals with a systemic dose of indomethacin (an inhibitor of all prostaglandins formation) or cycloheximide (an inhibitor of protein synthesis). The data indicate that IFN or its inducer may act through the endogenous release of a prostaglandin or a protein factor of an unknown chemical nature in the preoptic anterior hypothalamic region to induce fever in rabbits. The fever induced by IFN or its inducer is brought about by a decrease in heat loss and/or an increase in heat production in rabbits.

Effect of co-administration of interferon inducer, polyriboinosinic acid-polyribocytidylic acid, with SKF 525-A on hepatic drug metabolizing enzymes of rats.

The protective effect of SKF 525-A on the suppression of cytochrome P-450 content and monooxygenase activities by treatment with CoCl2 and polyriboinosinic acid-polyribocytidylic acid [poly(I.C] was compared as a part of studies of suppression of drug metabolizing enzymes by interferon inducers. Induction of heme oxygenase activity by CoCl2 and poly (I.C) was not altered by simultaneous treatment with SKF 525-A. Depression of cytochrome P-450 content and benzphetamine N-demethylase activity by treatment with CoCl2 was prevented by co-treatment with SKF 525-A. This effect was explained by the prevention of release of heme from cytochrome P-450 by forming metabolic intermediate complexes with metabolites of SKF 525-A. On the other hand, poly(I.C) significantly suppressed P-450 content and benzphetamine N-demethylase and benzo [a] pyrene hydroxylase activities, even under simultaneous treatment with SKF 525-A. This inhibition by poly (I.C) was accompanied by weak staining of proteins corresponding to cytochrome P-450 in SDS gel electrophoresis. In addition, the activity of non-heme enzyme, 4-hydroxybiphenyl glucuronyltransferase, was suppressed by treatment with poly (I.C) but not by CoCl2-treatment. These findings strongly suggested that, unlike CoCl2, poly (I.C) suppressed cytochrome P-450 content and monooxygenase activities due to decreased synthesis or increased degradation of the apoprotein of cytochrome P-450 with slight contribution of the induced heme oxygenase.

Suppressive effect of interferon inducer, polyriboinosinic acid-polyribocytidylic acid on induction of uridine diphosphate-glucuronyltransferases and monooxygenases in liver microsomes of rats.

The effect of polyriboinosinic acid-polyribocytidylic acid [poly(I).poly(C)] on glucuronyltransferase activities toward 4-nitrophenol and 4-hydroxybiphenyl in liver microsomes of Wistar rats was examined by its single or co-administration with 3-methylcholanthrene and phenobarbital. The increased 4-nitrophenol glucuronyltransferase activity by treatment with 3-methylcholanthrene was significantly suppressed following the co-administration with poly(I).poly(C), although the activity was not affected by the treatment with poly(I).poly(C) alone. In addition, 4-hydroxybiphenyl glucuronyltransferase activity decreased or tended to decrease by the treatment with poly(I).poly(C) alone, and the activity induced by phenobarbital was strikingly decreased following the co-administration with poly(I).poly(C). This result suggested that poly(I).poly(C) comprehensively decrease the induction of glucuronyltransferases regardless of their multiple forms. Furthermore, contents of cytochromes P-450 and b5 were also decreased by the treatment with poly(I).poly(C) alone or the co-administration with the inducers. Concomitantly, arylhydrocarbon hydroxylase and benzphetamine N-demethylase activities were significantly decreased by the treatment alone or the co-administration with the inducers. These findings supported a view that the suppressive effect of poly(I).poly(C) may be derived from the prevention of de novo synthesis of the apoprotein of the enzymes and/or the increased degradation.

Treatment with poly I.C. enhances lipid peroxidation and the activity of xanthine oxidase, and decreases hepatic P-450 content and activities in mice and rats

Treatment of mice and rats with polyriboinosinic acid-polyribocytidylic acid (poly I.C., 5 mg/kg i.p.), a potent interferon inducer, decreased hepatic cytochrome P-450 system content and activities without influencing P-450-independent xenobiotic metabolizing enzymes. Treatment with poly I.C. decreased the content of P-450 by 28% in mice (P less than 0.05) and 30% in rats (P less than 0.05) but did not alter the activity of cytochrome c reductase. With treatment of poly I.C., the activity of XO increased 87% in mice (P less than 0.01) and 30% in rats (P less than 0.01). Lipid peroxidation was enhanced by 82% in mice (P less than 0.01) and 95% in rats (P less than 0.05). These results raise the possibility that a part of the depression of P-450 system content and activities by poly I.C. might be caused by enhanced lipid peroxidation associated with increased activity of XO.

Intensification of galactosamine hepatotoxicity by polyriboinosinic acid-polyribocytidylic acid

The effects of polyriboinosinic acid-polyribocytidylic acid (poly IC) on galactosamine-induced liver cell injury in rats were studied. Treatment of rats with D-galactosamine-HCl (400 mg/kg) increased their serum glutamate-oxaloacetate transaminase (E.C.2.6.1.1, GOT) activities, indicating that hepatocyte injury was induced by galactosamine. Rapid and intense elevations of serum GOT activities were observed when galactosamine (200 mg/kg) was administered simultaneously with poly IC (10 mg/kg) but not with poly I or poly C. Acute increase in serum GOT activities caused by the simultaneous administration of poly IC and galactosamine was prevented by the simultaneous administration of uridine (1 g/kg), which is known to inhibit the early biochemical alterations caused by the amino sugar in the hepatocyte. These findings suggest that poly IC intensifies the hepatotoxic effect of galactosamine in rats. This poly IC-induced sensitization was inhibited by pretreatment with poly IC (10 mg/kg) itself, when injected 24 hr before the administration of the hepatotoxin together with poly IC.

Inducible expression of amplified human beta interferon genes in CHO cells.

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 10(10) U/liter medium in culture supernatants. Constitutive production of IFN-beta rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-beta had acquired resistance to cytotoxic antiviral effects of IFN-beta. Two forms of human IFN-beta were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-beta production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-beta production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-beta-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-beta.

Influence of one virus infection on a second concurrent primary in vivo antiviral cytotoxic T-cell response.

The influence of one virus on the in vivo cytotoxic T-cell response to a different concurrent viral infection was analyzed. Lymphocytic choriomeningitis and Newcastle disease viruses, known to induce high interferon titers, and the synthetic interferon inducer polyriboinosinic acid-polyribocytidylic acid inhibited the cytotoxic T-cell response against the second virus. In contrast, vaccinia and vesicular stomatitis viruses failed to induce inhibition. Inhibition directly correlated with the interferon titers; similarly, the interferon titers directly correlated with macrophage and natural killer cell activation. The involvement in vivo of interferon in macrophage and natural killer cell activation and the possible mechanisms of inhibition of the cytotoxic responses are shown by the inhibition of the effect by antibodies against interferon.

Cycloheximide induces expression of the human interferon beta 1 gene in mouse cells transformed by bovine papillomavirus-interferon beta 1 recombinants

Mouse cells transformed by a bovine papillomavirus recombinant vector containing the human interferon (IFN) beta 1 (IFN-beta 1) gene could be induced to produce human as well as mouse IFNs. The optimal conditions for induction of human IFN and of its mRNA in these transformants resembled those needed for mouse IFN: high concentrations of DEAE-dextran and low concentrations of polyriboinosinic acid-polyribocytidylic acid. Superinduction by inhibitors of protein synthesis which strongly stimulate IFN-beta 1 induction in human cells had only a small effect on human IFN induction in bovine papillomavirus IFN-beta 1-transformed mouse cells. In contrast, cycloheximide without double-stranded RNA could induce significant levels of human IFN in the bovine papillomavirus IFN-beta 1 mouse transformants. After cycloheximide treatment, these cells contained IFN-beta 1 mRNA whose 5' ends originated in the authentic start site of the human IFN-beta 1 gene, as shown by S1 nuclease mapping. The transferred human gene, propagated extrachromosomally in the mouse cells, was, therefore, inducible under conditions different from those in human cells. The results also confirmed that the inhibitor of protein synthesis, cycloheximide, can induce expression of a human IFN gene.

Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells.

A 1.6-kilobase DNA segment of the genomic human interferon beta 1 (IF-beta 1) gene was inserted into each of two possible orientations at the single HindIII site of a recombinant plasmid pBPV69T, consisting of the 69% transforming region of the bovine papilloma virus type 1 (BPV-1) and a modified SalI-SalI fragment of plasmid pBR322. After cleavage of the pBR322 sequences from this recombinant, BPV69T-IF-beta 1 hybrid DNAs were transfected onto C127 mouse cells by the standard calcium precipitation technique. Mouse cells transformed by this hybrid DNA produced low levels of human IF-beta 1 constitutively and responded to induction with either inactivated Newcastle disease virus or polyriboinosinic acid-polyribocytidylic acid. The BPV69T-IF-beta 1 hybrid DNA was nonintegrated in the transformed mouse cells but had acquired DNA sequences as a result of the transfection. Accurate transcripts of the IF-beta 1 mRNA were detected in cells only after induction. When the IF-beta 1 gene was oriented in the plasmid in the same direction of transcription as the BPV-1 genome, transcription was promoted from within the BPV-1 sequences. These results indicate that the regulatory sequences responsible for the inducible expression of the human IF-beta 1 gene are present in the 1.6-kilobase genomic segment and that these sequences can function in a free extrachromosomal state linked to BPV-1 sequences.

Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells.

A 1.6-kilobase DNA segment of the genomic human interferon beta 1 (IF-beta 1) gene was inserted into each of two possible orientations at the single HindIII site of a recombinant plasmid pBPV69T, consisting of the 69% transforming region of the bovine papilloma virus type 1 (BPV-1) and a modified SalI-SalI fragment of plasmid pBR322. After cleavage of the pBR322 sequences from this recombinant, BPV69T-IF-beta 1 hybrid DNAs were transfected onto C127 mouse cells by the standard calcium precipitation technique. Mouse cells transformed by this hybrid DNA produced low levels of human IF-beta 1 constitutively and responded to induction with either inactivated Newcastle disease virus or polyriboinosinic acid-polyribocytidylic acid. The BPV69T-IF-beta 1 hybrid DNA was nonintegrated in the transformed mouse cells but had acquired DNA sequences as a result of the transfection. Accurate transcripts of the IF-beta 1 mRNA were detected in cells only after induction. When the IF-beta 1 gene was oriented in the plasmid in the same direction of transcription as the BPV-1 genome, transcription was promoted from within the BPV-1 sequences. These results indicate that the regulatory sequences responsible for the inducible expression of the human IF-beta 1 gene are present in the 1.6-kilobase genomic segment and that these sequences can function in a free extrachromosomal state linked to BPV-1 sequences.

The protective effect of polyriboinosinic acid-polyribocytidylic acid against the occurrence of galactosamine-induced liver cell injury in rat.

A marked increase of serum transaminase activities, histological changes of livers similar to those seen in viral hepatitis in man, and inhibition of hepatic protein synthesis were observed in rats following a single injection of D-galactosamine-HCl. These galactosamine-induced phenomena were prevented by the pretreatment of polyriboinosinic acid-polyribocytidylic acid 24 h before the galactosamine administration.