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B Lebleu

Publications and source records attributed to B Lebleu.

At least 73 records · Page 4Linked to original sources
PubMed1987

Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site.

Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression, but their efficient introduction within intact cells proved to be difficult to realize. As a step toward this goal, small (13- or 15-mer) synthetic oligodeoxyribonucleotides have been coupled at their 3' ends to epsilon-amino groups of lysine residues of poly(L-lysine) (Mr, 14,000). A 15-mer oligonucleotide-poly(L-lysine) conjugate complementary to the initiation region of vesicular stomatitis virus (VSV) N-protein mRNA specifically inhibits the synthesis of VSV proteins and exerts an antiviral activity against VSV when added in the cell culture medium at doses as low as 100 nM. Neither synthesis of cellular proteins nor multiplication of encephalomyocarditis virus was affected significantly by this oligonucleotide conjugate. The data suggest that oligonucleotide-poly(L-lysine) conjugates might become effective for studies on gene expression regulation and for antiviral chemotherapy.

Animals↗
PubMed1987

Requirement for endocytosis of poly(rI).poly(rC) to generate toxicity on interferon-treated LM cells.

Poly(rI).poly(rC) induces a lytic reaction in interferon-treated mouse LM cells. We have attempted to determine whether the intracellular penetration of poly(rI).poly(rC) is a prerequisite for cell lysis and to gain some insight into the pathway followed. We found that poly(rI).poly(rC) coupled to Sepharose beads was unable to generate lysis of interferon-treated cells whereas the cells underwent lysis after microinjection of poly(rI).poly(rC). Some inhibitors of endocytosis were found to inhibit the development of the lytic reaction. Lysosomotropic amines or a low temperature (19 degrees C) blocked endocytosis of poly(rI).poly(rC) but did not prevent its uptake. The internalization of poly(rI).poly(rC) was energy-dependent and was blocked when sodium azide and 2-deoxyglucose were added simultaneously. We conclude that poly(rI).poly(rC) is internalized and reaches an acidic compartment before triggering the lytic reaction in the cell.

Amines↗
PubMed1986

Transcriptional and post-transcriptional regulation of c-myc expression during the differentiation of murine erythroleukemia Friend cells.

c-myc RNA rapidly decreases to barely detectable levels in Friend erythroleukemia cells induced to differentiate upon the addition of dimethylsulfoxide. We show here that c-myc gene is down-regulated both at the transcriptional level presumably by a block in the elongation of primary transcripts and at the post-transcriptional level by an increase in the degradation of its mRNA.

Animals↗
PubMed1986

Activation of ribonuclease L by (2'-5')(A)4-poly(L-lysine) conjugates in intact cells.

Molecular hybrids were synthesized by coupling (2'-5')(A)n oligoadenylates or 2-5A, an intracellular mediator involved in antiviral activity of interferons (IFNs), with poly(L-lysine) used as a membrane carrier. (2'-5')(A)n in its free form was not taken up by cells, probably because of its ionic character. Conjugation with the polypeptide carrier overcame this problem and enabled its pharmacological properties to be developed. The alpha-glycol group of individual (2'-5')(A)n oligomers was oxidized by periodate oxidation and conjugated by an amino reductive reaction to poly(L-lysine), Mr 14 000, in a molar ratio of 5:1. These hybrid molecules left the biologically active 5' end moiety of the (2'-5')(A)n molecule unchanged, and in particular its triphosphate group, and stabilized the molecule by increasing its resistance to phosphodiesterase hydrolysis. A dose-dependent inhibition of virus growth was observed on concomitant incubation of (2'-5')(A)n-poly(L-lysine) conjugates with vesicular stomatitis virus infected L1210 cell cultures. This was a result of the activation of the (2'-5')(A)n-dependent endoribonuclease (RNase L) by intracellularly delivered (2'-5')(A)n as in some IFN-treated virus-infected cells. Indeed, (2'-5')(A)n-poly(L-lysine) conjugates bind RNase L effectively as can be seen from their ability to compete with authentic (2'-5')(A)n in a cell-free radiobinding assay. Moreover, (2'-5')(A)n-poly(L-lysine) conjugates promote transient inhibition of protein synthesis and a characteristic cleavage pattern of ribosomal RNAs in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1986

An antiviral state induced in HeLa cells by microinjected poly(rI).poly(rC).

The direct introduction with micropipettes of poly(rI).poly(rC) into the cytoplasm of several human cell lines inhibited the multiplication of vesicular stomatitis virus. This antiviral activity was at least partly due to interferon (IFN) production and secretion from the injected cells since it was species-specific, partly neutralized by IFN antibodies and was transmissible to non-adjacent cells. This suggests a mechanism of IFN induction involving the internalization of poly(rI).poly(rC) and its interaction with an intracellular target.

Animals↗
PubMed1985

Antiviral activity in L1210 cells of liposome-encapsulated (2'-5')oligo(adenylate) analogues.

Evidence is available for a role of a (2'-5')(A)n-activated endoribonuclease (RNase L) in the antiviral activity of interferon for several RNA viruses. (2'-5')(A)n and their analogues might thus provide an interesting alternative to exogenous interferons or their inducers in antiviral chemotherapy. In addition, the evaluation of the activity of (2'-5)(A)n as mediators of interferon's biological activities or as cell growth regulators requires biochemical studies using agonists or antagonists of the system. Non-disruptive techniques for the introduction of (2'-5')(A)n and their analogues into cell lines or tissues are required for these studies since these highly charged compounds are cell impermeable. (2'-5')(A)n oligomers and analogues of increased stability towards phosphodiesterases were derived by chemical modification of their 2' end and encapsulated in protein-A-bearing liposomes. The specific delivery of liposome contents into L1210 mouse leukemic cells was achieved with the help of monoclonal antibodies directed against the appropriate class I major histocompatibility complex-encoded proteins expressed by these cells. This intracellular delivery led to transient inhibition of protein synthesis and an antiviral activity, both compatible with activation of RNase L. This activity was enhanced for the analogues designed to resist degradation, with respect to the natural product.

Adenine Nucleotides↗
PubMed1985

Increased rate of degradation of c-myc mRNA in interferon-treated Daudi cells.

The recent observation made in our laboratory that cellular myc (c-myc) mRNA has a very short half-life in a variety of normal and transformed human cells emphasized the potential importance of post-transcriptional regulation of c-myc gene expression. Jonak and Knight [Jonak, G. J. & Knight, E., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, 1747-1750] have reported a selective reduction of c-myc mRNA accumulation in lymphoblastoid Daudi cells treated with human beta interferon. This provided a suitable situation in which to examine a possible action of negative modulators of c-myc expression at the level of mRNA stability. Our results confirm the observation by Jonak and Knight that c-myc mRNA level is depressed in cells treated with beta interferon and extend it to alpha 2 interferon. Furthermore, we now demonstrate that interferon has no effect on c-myc transcription rate in isolated nuclei but rather reduces the half-life of its mRNA. Conversely, we show that it increases the level of HLA-A2 mRNA by stimulating its transcription.

Gene Expression Regulation↗
PubMed1985

Subcellular distribution of 2',5'-oligoadenylate synthetase in differentiating Friend leukemia cells.

The occurrence of distinct (2'-5')(A)n-synthetase activities has recently been documented in cytoplasmic and nuclear extracts of several interferon (IFN)-treated cell lines. Since a role has been proposed for (2'-5')(A)n synthetase in the control of cell growth and differentiation, we examined the subcellular distribution of (2'-5')(A)n-synthetase activity both in IFN-treated undifferentiated Friend leukemia cells (FLCs) and during dimethyl-sulfoxide (DMSO)-induced erythroid differentiation of FLCs. Both the nuclear and cytoplasmic (2'-5')(A)n activities were modulated to the same extent by IFNs and DMSO. No evidence for a causal relationship between enzyme activation and FLC differentiation was found.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Increased stability and antiviral activity of 2'-O-phosphoglyceryl derivatives of (2'-5')oligo(adenylate).

Metabolically stable analogues of (2'-5')oligo(adenylate), (2'-5')(A)n, might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2'-O-phosphoglyceryl derivatives of (2'-5')(A)n oligomers, (2'-5')(A)n-PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell-free extracts. In line with its increased stability, (2'-5')(A)n-PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells. Since (2'-5')(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.

Adenine Nucleotides↗
PubMed1984

Role of interferon and 2',5'-oligoadenylate synthetase in erythroid differentiation of Friend leukemia cells. Studies with interferon-sensitive and -resistant variants.

It has been suggested that the interferon (IFN)-induced 2',5'-oligoadenylate (2-5A) synthetase, which polymerizes ATP into a series of 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, plays a general role in cell growth and terminal differentiation. For instance, an increase in 2-5A synthetase activity has been described during dimethyl sulfoxide (Me2SO)-induced erythroid differentiation of Friend leukemia cells (FLC). 2-5A synthetase has been measured in two Friend leukemia cell sublines by various techniques including a radioimmunoassay of its products which would detect 10(-16) mol of 2-5A cores. Although cells of both sublines fully differentiate (as measured by benzidine staining), only in one subline was there an increase in 2-5A synthetase activity upon treatment with Me2SO. Hexamethylenebisacetamide, another potent agent of differentiation in this system, did not increase 2-5A synthetase activity in either of these two sublines. An IFN-resistant FLC variant differentiated normally upon treatment with Me2SO or hexamethylenebisacetamide while it was noninducible for 2-5A synthetase activity by exogenous IFN or by the inducers themselves. A similar situation has been observed with regard to the level of phosphorylation of the IFN-induced Mr = 67,000 protein band. In addition, treatment of IFN-sensitive and resistant FLC sublines with mouse alpha beta IFN antiserum did not affect differentiation. Even though we have duplicated previous findings on the increase of 2-5A synthetase activity in Me2SO-induced FLC, the lack of any such increase with other inducers or other sublines indicates that there is no causal relationship between the enzyme activation and FLC differentiation.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Isolation and biological characterization of mouse LM fibroblast variants resistant to the cytotoxicity of polyriboinosinic polyribocytidylic acid after interferon treatment.

Mouse LM fibroblasts growing continuously in the absence of serum have an increased sensitivity to the cytotoxicity of poly(rI) X poly(rC) after interferon (IFN) exposure. This has allowed the isolation by an enrichment procedure of several independent and stable variant clones (IFN + I X C)R which are resistant to such a treatment. One of the resistant variants has been more extensively characterized as far as IFN action and IFN production are concerned. It behaves identically to the wild-type parent except for the spontaneous release of low amounts of IFN. The target of the mutation probably resides in a late step in the development of the cytotoxic response as revealed by microinjection techniques. The (IFN + I X C)R variants characterized here thus appear different from mutants in the IFN system isolated so far.

Animals↗
PubMed1983

Microinjected interferon does not promote an antiviral response in Hela cells.

Human fibroblast interferon (Hu IFN beta) was directly introduced with glass micropipets into the cytoplasm of Hela cells. Such an injection of more than 10(4) molecules per cell failed to induce any antiviral state when challenged with vesicular stomatitis virus (VSV). These findings are discussed in relation to the possible role of internalization in the mechanism of antiviral action of interferon.

HeLa Cells↗
PubMed1983

Isolation of Daudi cells with reduced sensitivity to interferon. II. On the mechanisms of resistance.

The mechanism of interferon resistance was studied in two clones of Daudi cells, DIF2 and DIF3, which exhibit respectively moderate and pronounced resistance to both the antiviral and antiproliferative actions of human interferons-alpha and -beta. Clones DIF2 and DIF3 were found to possess specific high affinity interferon receptors similar to those of parental Daudi cells. However, DIF2 cells, which have a tetraploid karyotype, had approximately twice as many interferon-binding sites as either DIF3 or parental Daudi cells. One of the first detectable changes in Daudi cells following interferon treatment is a rapid increase in the intracellular concentration of cyclic GMP. No increase in cyclic GMP was observed in DIF2 or DIF3 cells treated with interferon-alpha. However, neither DIF2 nor DIF3 cells respond to sodium azide, a nonphysiological inducer of cyclic GMP. Interferon treatment was found to induce the production of 2'-5'-oligo-isoadenylate synthetase in DIF2 and DIF3 cells in a manner similar to parental Daudi cells, indicating that these cells possess functional interferon receptors. The levels of 2'-5'-oligo-isoadenylate synthetase and 2'-5' A phosphodiesterase activity were similar in all three cell lines, suggesting that the interferon resistance of clones DIF2 and DIF3 was not due to a deficiency of pp(A2' p)nA.

Azides↗
PubMed1983

Responses to interferon of LM cells growing continuously in the absence of serum and exogenous lipids.

Mouse fibroblast LM cells growing continuously in the absence of serum and exogenous lipids are proposed as a model for studying interferon action. Several of its activities, namely the inhibition of virus and cell growth, and the increased cytotoxicity of poly(rI) X poly(rC), are shown to be potentiated in such conditions. Since cells cultured under these conditions are devoid of precursors of prostaglandin biosynthesis, the data suggest that none of the three effects of interferon depends on active prostaglandin synthesis.

Animals↗
PubMed1982

Effects of interferon on the expression of mouse mammary tumour virus in GR cells.

The effects of interferon treatment on the expression of murine mammary tumour virus (MuMTV) by a continuous mammary tumour cell line of GR mouse were investigated. Interferon markedly inhibited the excretion of MuMTV particles in the culture media as measured by hybridization of virus-specific RNA and virus-associated reverse transcriptase activity. There was no inhibition of virus RNA and protein synthesis in those conditions. The steady-state level of intracellular virus RNA and its rate of synthesis were not modified by interferon treatment. The intracellular levels of the major virus envelope and core proteins as measured by immunoprecipitation techniques remained unchanged. Interferon failed to inhibit the synthesis and the processing of the gp52 glycoprotein and p27 core protein precursors. However, the rate of maturation of the glycoprotein precursor was slowed down. Surface labelling of intact cells did not reveal accumulation of virus proteins on the cell membrane upon interferon treatment. The intracellular level of MuMTV-characteristic reverse transcriptase activity was reduced in interferon treated cells, although to a lower extent than extracellular virion-associated enzyme activity. MuMTV particles released from interferon-treated cells revealed no difference in their protein composition. These results are consistent with an inhibition by interferon of a late step in the replication of MuMTV.

Animals↗