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A R Thierry

Publications and source records attributed to A R Thierry.

18 recordsLinked to original sources
PubMed1998

Radiotoxicity of iodine-125-labeled oligodeoxyribonucleotides in mammalian cells.

UNLABELLED: We investigated the distribution, stability and radiotoxicity of 125I-oligodeoxyribonucleotides (125I-ODN) in human fibrosarcoma HT-1080 cells to study the radiotoxic effects of the Auger electron emitter 125I delivered to the cells by ODN. METHODS: We delivered 125I-ODN into the cells via complexing with a liposomal delivery system. To assess the intracellular distribution and stability of 125I-ODN delivered by the liposomal delivery system, we used autoradiography, fluorescent and confocal microscopy and electrophoresis. To study the radiotoxicity of the unbound 125I-ODN, we used a clonogenic assay. The radiotoxicity of 125I-ODN delivered by the liposomal delivery system was compared with that of freely diffusible 125I-antipyrine, membrane-excluded 125I-bovine serum albumin and DNA incorporated 125I-deoxyuridine (125I-UdR). RESULTS: Oligodeoxyribonucleotides accumulated in the cell nucleus within a few hours of incubation. On the basis of the number of decays at 37% survival, 125I-ODN are 2 times more radiotoxic than 125I-antipyrine, which is freely diffusible into cells, and 8 times more radiotoxic than 125I-bovine serum albumin, which remains outside cells. However, the radiotoxicity of unbound 125I-ODN is almost 3 orders of magnitude lower than that of DNA-incorporated 125I-UdR. The 125I-ODN are not significantly degraded by intracellular nucleases during the time of uptake incubation. CONCLUSION: The dramatic difference in radiotoxicity between 125I-ODN and 125I-UdR confirms that, despite the nuclear localization, 125I-ODN are not bound to or incorporated within the genomic DNA. Our data demonstrate that the radiotoxicity of Auger electron emitters is determined by the radiation dose delivered to nuclear DNA, not necessarily to the nucleus. Therefore, relatively high intracellular concentrations of unbound 125I-ODN can be achieved without causing significant cell death.

Animals↗
PubMed1997

Enhanced antisense inhibition of human immunodeficiency virus type 1 in cell cultures by DLS delivery system.

The relatively poor cell uptake of oligonucleotides and subsequent transport to the cytoplasm and nucleus is the main limitation in antisense therapeutics. The use of lipid-based carrier system is one of the most promising approaches to overcome these problems. In this study, we report the use of a new lipidic formulation to deliver a phosphorothioate oligonucleotide antisense directed against the regulatory gene rev of the HIV-1 genome and its application to the inhibition of HIV-1 in different cell culture models. Antiviral activity of either DLS-complexed or non-complexed oligonucleotides (ODNs) was compared in acutely and chronically infected cells. We have demonstrated that substantial antisense activity could be achieved at subnanomolar concentrations with DLS-complexed ODN in both acute and chronic infection systems. DLS-association highly improved inhibitory activity of the antisense ODN in acutely infected Molt-3 cells (100-fold) and primary cells (1000-fold) and in chronically infected H9 cells (1,500,000-fold). We have shown that anti-HIV activity of phosphorothioate ODNs can be strongly enhanced by using the DLS carrier system.

Anti-HIV Agents↗
PubMed1997

Characterization of liposome-mediated gene delivery: expression, stability and pharmacokinetics of plasmid DNA.

We have characterized a new synthetic gene delivery system, termed DLS, which may be suitable for systemic gene therapy. DLS constitutes a lipopolyamine and a neutral lipid and associated plasmid DNA in the formation of lamellar vesicles (DLS-DNA). The ratio of lipids and lipid to DNA as well as the method of preparation were optimized to yield a high in vitro transfection efficiency compared with that previously reported for cationic lipid systems. DLS-DNA showed a rapid cellular uptake and distribution in the cytoplasmic and nuclear (especially in the nucleoli) compartments as determined by laser-assisted confocal microscopy. There was little or no plasmid DNA degradation over a period of 20 min, relatively slow plasma clearance, and effective and rapid cellular uptake of DLS-DNA following intravenous administration in mice. Supercoiled plasmid DNA could be detected in blood cells up to 1 h after injection. Systemic administration of DLS-DNA yielded transgene expression in mouse tissues, such as in lung or liver. The ratio of DLS:DNA and the procedure used to form DLS-DNA affected both the level and cellular specificity of expression of a luciferase reporter gene showing that in vitro transfection efficiency of DLS-DNA formulations cannot be easily extrapolated to an in vivo setting. Optimization of the formulation of a DNA delivery system was critical to obtain a defined structure resulting in a preparation with high reproducibility and stability, greater homogeneity of particle size and high efficacy following systemic gene transfer. In addition, the DLS system may be formulated for specific target tissues and may have a wide range of applications for gene therapy.

Animals↗
PubMed1997

Delivery of oligoribonucleotides to human hepatoma cells using cationic lipid particles conjugated to ferric protoporphyrin IX (heme).

The receptor-ligand interaction between hepatocyte heme receptors and heme was evaluated as a basis for developing a targeted cationic lipid delivery reagent for nucleic acids. Heme (ferric protoporphyrin IX) was conjugated to the aminolipid dioleoyl phosphatidylethanolamine (DOPE) and used to form cationic lipid particles with dioleoyl trimethylammonium propane (DOTAP). These lipids particles (DDH) protect oligoribonucleotides from degradation in human serum and increase oligoribonucleotide uptake into 2.2.15 human hepatoma cells (to a level of 50-60 ng oligo/10(4) cells) when compared with the same lipid particles (DD) prepared identically without heme. The DDH heme level that was optimal for oligoribonucleotide delivery was also optimal for maximum expression of plasmid-encoded luciferase. The enhancing effect of heme was evident only at net particle negative charge. Fluorescence microscopy showed that DDH delivered oligoribonucleotides into both the 2.2.15 cell cytoplasm and nucleus. DDH may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in such liver diseases as viral hepatitis, hepatoma, and hypercholesterolemia.

Animals↗
PubMed1996

Expression of the human multidrug resistance and glucocerebrosidase cDNAs from adeno-associated vectors: efficient promoter activity of AAV sequences and in vivo delivery via liposomes.

Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5' end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analysis of low-molecular-weight DNA extracted from drug-resistant cells grown under continuous selection pressure indicated the persistence of nonintegrated AAV-MDR1 plasmids. Coordinate expression of Pgp and human glucocerebrosidase (hGC) was observed in drug-selected NIH-3T3 cells transfected with a bicistronic vector in which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis internal ribosome entry site sequence. Moreover, following a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved in all the organs we tested. Our results demonstrate that the efficiency of liposomes as vehicles for in vitro and in vivo gene delivery, the advantages of AAV-vectors, and the use of MDR1 as a selectable marker might be successfully combined in gene therapy protocols.

3T3 Cells↗
PubMed1996

In vitro and in vivo liposome-mediated gene transfer leads to human MDR1 expression in mouse bone marrow progenitor cells.

The ability to select bone marrow cells (BMC) expressing a selectable gene that confers resistance to anticancer drugs would be useful to protect bone marrow during chemotherapy. The human multidrug resistance (MDR1) gene encodes a 170-kD glycoprotein (P-gp), an ATP-dependent transmembrane efflux pump for many different cytotoxic drugs. In this work, we demonstrate efficient expression of the human MDR1 gene in mouse BMC after transfection with a liposomal delivery system (DLS-liposomes). The human MDR1 cDNA expression plasmid (pHaMDR1/A) was encapsulated in DLS-liposomes and delivered to mouse BMC using two approaches: (i) in vitro transfection of BMC followed by bone marrow transplantation and (ii) in vivo direct systemic gene transfer. After both the in vitro and the in vivo approaches, polymerase chain reaction (PCR) analysis confirmed that the human MDR1 gene was successfully transfected to bone marrow, spleen, and peripheral blood (PB) cells, with the human MDR1 gene detected in BMC for up to 30 days after bone marrow transplantation and 28 days after direct systemic administration. Efflux studies using rhodamine-123 demonstrated function of the MDR1 gene product in the in vitro-transfected BMC. Flow cytometry studies using the human MDR1-specific MRK16 monoclonal antibody confirmed the presence of P-gp in BMC after in vitro transfection, as well as in BMC from reconstituted or in vivo-transfected mice. Transgene expression in both lymphoid and myeloid subpopulations of BMC was demonstrated. Colony-forming units (CFU-Mix) were obtained after exposure of BMC to lethal doses of vincristine, demonstrating functional expression of the MDR1 gene in hematopoietic progenitor cells for up to 1 month.

3T3 Cells↗
PubMed1995

Systemic gene therapy: biodistribution and long-term expression of a transgene in mice.

We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA plasmids to effect long-term expression of a transgene in cells. A single i.v. injection of a plasmid DNA vector containing the luciferase gene as a marker was administered with the DLS liposomes in BALB/c mice. The luciferase gene and its product were found in all mouse tissues tested as determined by PCR analysis and immunohistochemistry. Luciferase activity was also detected in all tissues tested and was present in lung, liver, spleen, and heart up to 3 months postinjection. In contrast to the nonepisomal vectors tested (pRSV-luc and pCMVintlux), human papovavirus (BKV)-derived episomal vectors showed long-term transgene expression. We found that these episomal vectors replicated extrachromosomally in lung 2 weeks postinjection. Results indicated that transgene expression in specific tissues depended on the promoter element used, DNA/liposome formulation, dose of DNA per injection, and route of administration.

Animals↗
PubMed1995

Tumorigenesis and metastasis of neoplastic Kaposi's sarcoma cell line in immunodeficient mice blocked by a human pregnancy hormone.

Kaposi's sarcoma (KS) occurs more often in men than in women and HIV-1-associated KS has a high occurrence in homosexual men (over 30%). Most cultures of KS tumours yield cells with properties of hyperplastic (not malignant) endothelial cells under the control of several cytokines. The role of HIV-1 may be in promoting high levels of some cytokines and providing stimulation to angiogenesis by the HIV-1 Tat protein, which synergizes with basic fibroblast growth factor in promoting these effects. Here we describe an immortalized AIDS-KS cell line (KS Y-1) and show that these cells produce malignant metastatic tumours in nude mice and are killed in vitro and in vivo (apparently by apoptosis) by a pregnancy hormone, the beta-chain of human chorionic gonadotropin. Similarly, chorionic gonadotropin kills KS SLK, cells from another neoplastic cell line (established from a non-HIV-associated KS), as well as the hyperplastic KS cells from clinical specimens grown in short-term culture, but does not kill normal endothelial cells. These results provide evidence that KS can evolve into a malignancy and have implications for the hormonal treatment of this tumour.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Antisense oligodeoxynucleotide phosphorothioate complementary to Gag mRNA blocks replication of human immunodeficiency virus type 1 in human peripheral blood cells.

Gene-expression modulator 91 (GEM91) is a 25-nt antisense oligodeoxynucleotide phosphorothioate complementary to the Gag mRNA of human immunodeficiency virus type 1 (HIV-1). Cellular uptake and intracellular distribution of GEM91 within cells suggest that this oligomer is readily available for antisense activity. GEM91 inhibited HIV-1 replication in a dose-dependent and sequence-specific manner. In a comparative study, 2 microM GEM91 was as effective as 5 microM 3'-azido-3'-deoxythymidine in blocking virus replication during the 28-day treatment of an HIV-1-infected T-cell line. GEM91 also completely inhibited (> 99%) the growth of three different HIV-1 isolates in primary lymphocytes and prevented the cytopathic effect of the virus in primary CD4+ T cells. Similarly, treatment with GEM91 for 3 weeks of HIV-1/BaL-infected primary macrophages blocked virus replication. Based on GEM91 anti-HIV-activity, safety, and pharmacokinetic profile in animals, a clinical trial was started using this compound as an antisense oligonucleotide drug for the treatment of the acquired immunodeficiency syndrome.

Antigens, CD↗
PubMed1994

A new procedure for the preparation of liposomal doxorubicin: biological activity in multidrug-resistant tumor cells.

We describe a new procedure for the preparation of liposomal doxorubicin. Doxorubicin can be efficiently complexed to preformed or lyophilized cardiolipin-containing liposomes. Complex formation was performed by vigorous vortexing. As much as 96.8% of the initial drug quantity may be bound to those liposomes under optimal incubation conditions (4 h at 37 degrees C). The binding study showed the presence of two levels of specific binding (dissociation constants, 28 +/- 8 microM and 1.0 +/- 0.3 mM). The drug is firmly integrated in the liposome-membrane lipid bilayer rather than binding at the surface. Cytotoxicity studies using tumor cells revealed efficient drug delivery using liposome-complexed doxorubicin. This new liposomal doxorubicin preparation reverses multidrug resistance in MCF-7/ADR and CH LZ cells at levels equivalent to that obtained with a previously described liposome-encapsulated doxorubicin preparation, showing that the drug is integrated as well in the liposome carrier and is transported as well into cells. Increased concentration of liposomes at the subcytotoxic level in liposome-complexed doxorubicin enhances drug cytotoxicity in multidrug-resistant CH LZ cells as compared with liposome-encapsulated drug. This new preparation for liposomal doxorubicin may be carried out immediately prior to clinical administration, offering advantages in terms of cost and stability.

Breast Neoplasms↗
PubMed1993

Modulation of doxorubicin resistance in multidrug-resistant cells by liposomes.

In this study, we have confirmed the ability of liposome-encapsulated doxorubicin to modulate drug resistance, as previously observed in CH LZ cells (Thierry et al., Cancer Commun. 1, 311-316, 1989), in two human multidrug-resistant (MDR) cell lines, the breast cancer MCF-7/ADR cell line, and the ovarian carcinoma SKVLB cell line. This effect was specific to MDR cells, as liposomally encapsulated doxorubicin did not enhance cell sensitivity to the drug in the parental cell lines. Cytotoxicity assays demonstrated that empty liposomes in the presence of free doxorubicin (Dox) reversed resistance to the drug at a level that may be higher than that observed when liposome-encapsulated Dox is used. This effect seems to be due to the high affinity of Dox for cardiolipin, one of the liposome components, which leads to the association of the drug and the cardiolipin-containing liposomes in the culture medium before entry into the cells. Neither pretreatment of empty liposomes before drug treatment nor combined incubation of vincristine and empty liposomes alter MDR in CH LZ cells, suggesting that the drug must be encapsulated or complexed to the liposomes to overcome MDR. Because MDR in CH LZ cells does not seem to be related to GSH level, MDR modulation by liposome-encapsulated Dox apparently may not be effected by altering the GSH function. These results suggest that the enhancement of sensitivity of MDR cells using Dox encapsulated in liposomes or complexed with liposomes may be explained by an increase in cell drug incorporation and by an intracellular drug redistribution. Fluorescence confocal microscopy study indicated that Dox is transported and distributed mainly in intracytoplasmic vesicles in SKVLB and MCF-7/ADR cells, whereas in parental cells the drug is located mainly in the nucleus. In addition, presentation of Dox in liposomes modifies the drug distribution pattern in MDR cells by partially shifting the drug to nuclear compartments. Thus, liposome-associated Dox may bypass the vesicular drug transport in MDR cells, resulting in the enhancement of the drug biological activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Overcoming multidrug resistance in human tumor cells using free and liposomally encapsulated antisense oligodeoxynucleotides.

Antisense oligonucleotides offer a molecular targeting tool for overcoming cellular multidrug resistance. In order to improve the in vitro and the in vivo transport of oligodeoxynucleotides, we developed a new liposomal delivery system, using the minimal volume entrapment (MVE) technique. We have demonstrated that cellular uptake and intracellular release of oligodeoxynucleotides were facilitated by delivery in liposomes. 15 mers cap phosphorothioate oligodeoxynucleotides complementary to the 5' end of the coding region or to a loop-forming site in the mdr-1 mRNA were encapsulated in liposomes by the MVE method. P-glycoprotein synthesis and doxorubicin resistance were greatly reduced by exposure of the multidrug resistant SKVLB cells to 5 microM liposomally encapsulated oligonucleotide. A lower effect was observed when free oligodeoxynucleotides were used. Oligomers antisense to the loop-forming site appeared to be more effective and more specific in modulating multidrug resistance than oligomers with antisense sequence to the 5' end coding region.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity.

We have studied factors which may effect the intracellular availability of oligonucleotides to achieve antisense activity. 15-20 mer unmodified, phosphorothioate modified and liposomally encapsulated oligodeoxynucleotides have been tested in leukemia MOLT-3 cells. Phosphorothioate analogs penetrated and accumulated intact in cells in contrast to unmodified oligomers, which showed a high instability in cell culture medium. A slow decrease of intracellular concentration of undegraded phosphorothioate oligodeoxynucleotides was observed after cell treatment and could be predominantly explained by a significant efflux transport. Using laser-assisted confocal microscopy we have observed that fluorescein 5-end-labeled phosphorothioate derivatives predominantly distributed in intracytoplasmic endocytic vesicles following cell treatment. The end-capped version of phosphorothioate oligodeoxynucleotides exhibited greater cellular uptake than fully modified analogues while exhibiting similar biological stability. Liposome encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. Furthermore, the efflux rate of oligomer initially introduced within liposomes is 2-fold lower than that observed in cells which have been incubated with free oligonucleotides. Liposomal preparations of oligodeoxynucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. Furthermore, intracellular distribution studies demonstrate that intracellular transport of unmodified oligomers is effectively achieved using the liposomal carrier.

Base Sequence↗
PubMed1992

Effect of liposomes on P-glycoprotein function in multidrug resistant cells.

Presentation of doxorubicin in liposomes has shown to enhance the sensitivity of multidrug resistant CH LZ cells to the drug (Thierry et al. Cancer Commun. 1:311-316, 1989). We confirmed that liposomally encapsulated doxorubicin may partially overcome multidrug resistance in the human ovarian carcinoma SKVLB cell line and that this effect is, at least in part, due to an increase of cellular drug accumulation. When used at high concentration, empty liposomes appear to be specifically cytotoxic in the MDR SKVLB and CH LZ cells. As observed with certain multidrug resistance modulators, empty liposomes inhibited the specific [3H]-vincristine binding to P-glycoprotein-enriched membranes isolated from CH LZ cells (60% at 0.2 mg lipid/ml). Our data suggest that liposomes may alter the P-glycoprotein function by direct interaction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Modulation of multidrug resistance in Chinese hamster cells by liposome-encapsulated doxorubicin.

A Chinese hamster cell line (LZ), selected for multidrug resistance (MDR), exhibits a 3,000-fold resistance to doxorubicin, compared to parental V-79 cells. These drug resistant cells have amplified MDR genes, overexpress P-glycoprotein, and in the presence of doxorubicin show reduced intracellular drug accumulation. Using liposome-encapsulated doxorubicin (Rahman et al. Cancer Res. 45:796-803; 1985), we observed partial reversal of the resistance of LZ cells to this drug and a higher intracellular drug accumulation, compared to free drug. Parental V-79 cells, however, did not exhibit differences in survival or in drug accumulation when treated with encapsulated or free doxorubicin. Comparison of the effect of liposome-encapsulated doxorubicin with that of verapamil in reversing drug resistance showed that the liposomal preparation was as effective as verapamil used at its maximum clinically relevant concentration (1.5 microM). These results suggest that the use of liposomes as carriers of anticancer drugs may offer a strategy for overcoming MDR in tumor cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗