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1.
Leukemia ; 31(11): 2515-2522, 2017 11.
Article in English | MEDLINE | ID: mdl-28360415

ABSTRACT

With combined antiretroviral therapy (cART), the risk for HIV-infected individuals to develop a non-Hodgkin lymphoma is diminished. However, the incidence of Burkitt lymphoma (BL) remains strikingly elevated. Most BL present a t(8;14) chromosomal translocation which must take place at a time of spatial proximity between the translocation partners. The two partner genes, MYC and IGH, were found colocalized only very rarely in the nuclei of normal peripheral blood B-cells examined using 3D-FISH while circulating B-cells from HIV-infected individuals whose exhibited consistently elevated levels of MYC-IGH colocalization. In vitro, incubating normal B-cells from healthy donors with a transcriptionally active form of the HIV-encoded Tat protein rapidly activated transcription of the nuclease-encoding RAG1 gene. This created DNA damage, including in the MYC gene locus which then moved towards the center of the nucleus where it sustainably colocalized with IGH up to 10-fold more frequently than in controls. In vivo, this could be sufficient to account for the elevated risk of BL-specific chromosomal translocations which would occur following DNA double strand breaks triggered by AID in secondary lymph nodes at the final stage of immunoglobulin gene maturation. New therapeutic attitudes can be envisioned to prevent BL in this high risk group.


Subject(s)
B-Lymphocytes/metabolism , Burkitt Lymphoma/genetics , Gene Products, tat/physiology , Genes, myc , Immunoglobulin Heavy Chains/genetics , Aged , Female , Humans , Male , Middle Aged
2.
J Membr Biol ; 249(5): 593-600, 2016 10.
Article in English | MEDLINE | ID: mdl-26920545

ABSTRACT

This paper proposes to monitor the spheroid's permeabilization within a dedicated microfluidic device using electrorotation analyses. The combination of two electric solicitations, the negative dielectrophoresis force (nDEP) for the spheroid trapping and the electrorotation torque for its dielectric characterization, is used. An estimation of the spheroid dielectric parameters is obtained through the analysis of the rotational velocity curve versus the electric field frequency before and after the PEF application. An observation set-up includes a fast camera that allows time controlled image sequence acquisition. Frames are then digitalized and from the analysis of the rotational velocity of the spheroid, its complex permittivity is determined. Different models, involving the variation of the dielectric properties of the concentric shells that constitute the spheroid, as well as the heterogeneity of cells within each shell, are proposed and used to determine its dielectric properties.


Subject(s)
Electrophoresis, Microchip , Electroporation , Spheroids, Cellular , Electrodes , Humans , Micro-Electrical-Mechanical Systems , Tumor Cells, Cultured
3.
Chem Commun (Camb) ; 51(80): 14881-4, 2015 Oct 14.
Article in English | MEDLINE | ID: mdl-26303028

ABSTRACT

Triphenylamines are on/off fluorescent DNA minor groove binders, allowing nuclear staining of fixed cells. By contrast, they accumulate in the cytoplasm of living cells and efficiently trigger cell apoptosis upon prolonged visible light irradiation. This process occurs concomitantly with their subcellular re-localization to the nucleus, enabling fluorescence imaging of apoptosis.


Subject(s)
Aniline Compounds/pharmacology , Cell Death , Cations , Cell Line, Tumor , Humans
4.
Cell Death Differ ; 17(5): 811-20, 2010 May.
Article in English | MEDLINE | ID: mdl-19876065

ABSTRACT

p53 binding protein-1 (53BP1) participates in checkpoint signaling during the DNA damage response (DDR) and during mitosis. In this study we report that 53BP1 aggregates in nuclear foci within syncytia elicited by the human immunodeficiency virus (HIV)-1 envelope. 53BP1 aggregation occurs as a consequence of nuclear fusion (karyogamy (KG)). It colocalizes partially with the promyelomonocytic leukemia protein (PML), and the ataxia telangiectasia mutated kinase (ATM), the two components of the DDR that mediate apoptosis induced by the HIV-1 envelope. ATM-dependent phosphorylation of 53BP1 on serines 25 and 1778 (53BP1S25P and 53BP1S1778P) occurs at these DNA damage foci. 53BP1S25P was also detected in syncytia present in the lymph nodes or frontal brain sections from HIV-1-infected carriers, as well as in peripheral blood mononucleated cells from HIV-1-infected individuals, correlating with viral load. Knockdown of 53BP1 caused HIV-1 envelope-induced syncytia to enter abnormal mitoses, leading to their selective destruction through mitochondrion-dependent and caspase-dependent pathways. In conclusion, depletion of 53BP1 triggers the demise of HIV-1-elicited syncytia through mitotic catastrophe.


Subject(s)
HIV-1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Adult , Apoptosis/genetics , Apoptosis/physiology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Damage/genetics , DNA Damage/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Giant Cells/metabolism , HeLa Cells , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/genetics , Male , Mitosis/genetics , Mitosis/physiology , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1 , env Gene Products, Human Immunodeficiency Virus/metabolism , env Gene Products, Human Immunodeficiency Virus/physiology
5.
Br J Cancer ; 101(1): 7-11, 2009 Jul 07.
Article in English | MEDLINE | ID: mdl-19536093

ABSTRACT

BACKGROUND: KIT exon 11 mutations are observed in 60% of gastrointestinal stromal tumours (GIST). Exon 11 codes for residues Tyr568 and Tyr570, which play a major role in signal transduction and degradation of KIT. Our aim was to compare the outcome of patients with deletion of both Tyr568-570 (delTyr) and the most frequent deletion delWK557-558 (delWK). METHODS: Pathology and clinical characteristics of 68 patients with delTyr (n=26) or delWK (n=42) were reviewed and compared. RESULTS: GISTs with delTyr were more frequently extragastric than those with delWK (69 vs 26%, P<0.0005). After curative surgery, median relapse-free survival were 10.8 and 11.1 months for patients with delTyr (n=14) and delWK (n=29), respectively (P=0.92). All patients treated with imatinib for a non-resectable or metastatic GIST had an objective response (n=15) or a stable disease (n=21) as best response, regardless of mutation. Median progression-free survival with imatinib were 21.9 and 18.9 months for patients with GIST with delTyr (n=14) and delWK (n=22), respectively (P=0.43). CONCLUSION: In this large retrospective series, the type of KIT exon 11 mutation was correlated with the origin of GIST, but not with prognosis or response to imatinib.


Subject(s)
Exons , Gastrointestinal Stromal Tumors/genetics , Gene Deletion , Proto-Oncogene Proteins c-kit/genetics , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/therapeutic use , Benzamides , Disease-Free Survival , Female , Gastrointestinal Stromal Tumors/drug therapy , Gastrointestinal Stromal Tumors/pathology , Gastrointestinal Stromal Tumors/surgery , Humans , Imatinib Mesylate , Male , Middle Aged , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Retrospective Studies , Survival Rate , Young Adult
6.
Cell Death Differ ; 16(2): 298-311, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19023333

ABSTRACT

Promyelomonocytic leukemia (PML) is a prominent oncosuppressor whose inactivation is involved in the pathogenesis of hematological and epithelial cancers. Here, we report that PML aggregated in nuclear bodies in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. PML aggregation occurred after the fusion of nuclei (karyogamy) within syncytia but before the apoptotic program was activated. The aggregation of PML was detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Using a range of specific inhibitors of PML (the oncogenic PML/RARalpha fusion product or specific small interfering RNAs), we demonstrated that, in Env-elicited syncytia, PML was required for activating phosphorylation of ataxia telangiectasia mutated (ATM), which colocalized with PML in nuclear bodies, in a molecular complex that also involved topoisomerase IIbeta-binding protein 1. PML knockdown thus inhibited the ATM-dependent DNA damage response that culminates in the activation of p53, p53-dependent transcription of pro-apoptotic genes and cell death. Infection of CD4-expressing cells with HIV-1 also induced syncytial apoptosis, which could be suppressed by inhibiting PML. Altogether, these data indicate that PML activation is a critical early event that participates in the apoptotic demise of HIV-1-elicited syncytia.


Subject(s)
Apoptosis , HIV-1 , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , env Gene Products, Human Immunodeficiency Virus/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Giant Cells/virology , HeLa Cells , Humans , Promyelocytic Leukemia Protein , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering , Signal Transduction , Tumor Suppressor Protein p53/metabolism
7.
Biochem Biophys Res Commun ; 279(2): 401-6, 2000 Dec 20.
Article in English | MEDLINE | ID: mdl-11118299

ABSTRACT

EWS Fli-1, a fusion gene resulting from a t(11;22) translocation is found in 90% of both Ewing's sarcoma and primitive neuroectodermal tumor (PNET). In the present study, we show that recently developed polyisobutylcyanoacrylate nanocapsules with an aqueous core were able to encapsulate efficiently high amounts of phosphorothioate oligonucleotides (ODN) directed against EWS Fli-1 chimeric RNA. Release of these ODN in serum medium was shown to be biphasic which was explained by the presence of two types of nanocapsules able to release ODN with different kinetics. In addition, nanocapsules were found to provide protection of these oligonucleotides from the degradation in serum. These ODN nanocapsules permitted to obtain inhibition of Ewing sarcoma-related tumor in mice after intratumoral injection of a cumulative dose as low as 14.4 nanomoles. This new type of non viral vector shows great potential for in vivo administration of oligonucleotides.


Subject(s)
DNA-Binding Proteins/genetics , Oligodeoxyribonucleotides, Antisense/therapeutic use , Proto-Oncogene Proteins , Sarcoma, Ewing/genetics , Sarcoma, Ewing/therapy , Trans-Activators/genetics , 3T3 Cells , Animals , Capsules , Drug Carriers , Humans , Mice , Mice, Nude , Neuroectodermal Tumors, Primitive/genetics , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligodeoxyribonucleotides, Antisense/blood , Proto-Oncogene Protein c-fli-1 , Thionucleotides , Transfection
8.
J Med Chem ; 43(8): 1533-40, 2000 Apr 20.
Article in English | MEDLINE | ID: mdl-10780910

ABSTRACT

Our prior studies showed that polyhydroxylated styrylquinolines are potent HIV-1 integrase (IN) inhibitors that block the replication of HIV-1 in cell culture at nontoxic concentrations. To explore the mechanism of action of these inhibitors, various novel styrylquinoline derivatives were synthesized and tested against HIV-1 IN and in cell-based assays. Regarding the in vitro experiments, the structural requirements for biological activity are a carboxyl group at C-7, a hydroxyl group at C-8 in the quinoline subunit, and an ancillary phenyl ring. However the in vitro inhibitory profile tolerates deep alterations of this ring, e.g. by the introduction of various substituents or its replacement by heteroatomic nuclei. Regarding the ex vivo assays, the structural requirements for activity are more stringent than for in vitro inhibition. Thus, in addition to an o-hydroxy acid group in the quinoline, the presence of one ortho pair of substituents at C-3' and C-4', particularly two hydroxyl groups, in the ancillary phenyl ring is imperatively required for inhibitory potency. Starting from literature data and the SARs developed in this work, a putative binding mode of styrylquinoline inhibitors to HIV-1 IN was derived.


Subject(s)
Anti-HIV Agents/chemical synthesis , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase/metabolism , HIV-1/drug effects , Quinolines/chemical synthesis , Styrenes/chemical synthesis , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Catalytic Domain , Cell Line , HIV Integrase/chemistry , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , Humans , Protein Binding , Quinolines/chemistry , Quinolines/pharmacology , Structure-Activity Relationship , Styrenes/chemistry , Styrenes/pharmacology , Virus Replication
9.
Nucleosides Nucleotides ; 18(6-7): 1721-2, 1999.
Article in English | MEDLINE | ID: mdl-10474256

ABSTRACT

We studied the uptake and intracellular distribution of an FITC labelled phosphodiester oligodeoxynucleotide (ODN) vectorized by a dendrimeric structure in cell culture.


Subject(s)
Oligonucleotides/metabolism , Polyamines/chemistry , 3T3 Cells , Animals , Fluorescein-5-isothiocyanate , HeLa Cells , Humans , Mice , Microscopy, Confocal
10.
Biochem Pharmacol ; 58(1): 95-107, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10403523

ABSTRACT

Factors limiting the use of antisense phosphodiester oligodeoxynucleotides (ODNs) as therapeutic agents are inefficient cellular uptake and intracellular transport to RNA target. To overcome these obstacles, ODN carriers have been developed, but the intracellular fate of ODNs is controversial and strongly depends on the means of vectorization. Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electrostatically. These complexes have been demonstrated to protect phosphodiester ODNs from nuclease degradation and also to increase their cellular uptake and pharmacological effectiveness. We studied the intracellular distribution of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer vector and found that intracellular ODN distribution was dependent on the phase of the cell cycle, with a nuclear localization predominantly in the G2/M phase. In addition, in order to evaluate the relevance of ODN vectors in enhancing the inhibition of the targeted genes' expression, we developed a rapid screening system which measures the transient expression of two reporter genes, one used as target, the other as control and vice versa. This system was validated through investigating the effect of the dendrimer vector on ODN biological activity. Antisense sequence-specific inhibition of more than 70% of one reporter gene was obtained with a chimeric ODN containing four phosphorothioate groups, two at each end.


Subject(s)
Cell Cycle/physiology , Oligonucleotides, Antisense/metabolism , Oligonucleotides, Antisense/pharmacology , 3T3 Cells , Animals , Cell Culture Techniques , Flow Cytometry/methods , Gene Expression/drug effects , Genes, Reporter , Genetic Vectors/metabolism , Genetic Vectors/pharmacology , Green Fluorescent Proteins , HeLa Cells , Humans , Kinetics , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice , Microscopy, Confocal , Oligonucleotides, Antisense/genetics , RNA, Messenger/biosynthesis
11.
Blood ; 93(5): 1511-23, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10029579

ABSTRACT

The identification of stromal cell-derived factor (SDF)-1alpha as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1alpha during megakaryocytopoiesis. CD34(+) cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41(+) cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34(+) cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41(+) cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1alpha acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1alpha is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1alpha induced the migration of colony forming unit-megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1alpha as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1-related thrombocytopenia.


Subject(s)
Cell Lineage , Hematopoiesis , Megakaryocytes/metabolism , Receptors, CXCR4/biosynthesis , Cell Differentiation , Fetal Blood , Flow Cytometry , Humans , Infant, Newborn , Megakaryocytes/cytology , Platelet Glycoprotein GPIIb-IIIa Complex/biosynthesis , Platelet Glycoprotein GPIb-IX Complex/biosynthesis , Receptors, Virus/biosynthesis
12.
J Mol Biol ; 285(3): 945-54, 1999 Jan 22.
Article in English | MEDLINE | ID: mdl-9887261

ABSTRACT

Previously, we have observed a strong restriction of the Moloney murine leukemia virus (MoMLV) replicative cycle in a cell line displaying resistance to topoisomerase II (topo II)-interactive drugs. Resistance towards these antitumoral inhibitors was associated with decreased expression and activity of topo II, suggesting that such a decrease may be responsible for MoMLV restriction. To more specifically assess the role of topo II during the retroviral cycle, we have used the antisense strategy to obtain a selective decrease of cellular topo II expression. The RNA antisense was isolated from a retroviral library expressing random fragments of human topo II (alpha form). This system allowed us to investigate the HIV-1 replicative cycle in two related human CEM cell lines expressing different levels of topo II. Expression of the enzyme is decreased four- to sixfold following formation of a sense-antisense RNA hybrid. Repression of the topo II enzyme results in an impairment of the HIV-1 replicative cycle. Using the polymerase chain reaction, we showed that the number of integration events was decreased in cells repressing the enzyme, although viral DNA synthesis and circularization were equivalent to those in the parent cells.


Subject(s)
DNA Topoisomerases, Type II/genetics , Gene Expression Regulation, Enzymologic/genetics , HIV-1/genetics , RNA, Antisense/genetics , Virus Replication/genetics , Cell Line , Cell Survival/genetics , DNA, Viral/genetics , Enzyme Inhibitors/pharmacology , Etoposide/pharmacology , Humans , Polymerase Chain Reaction , RNA, Messenger/genetics , Topoisomerase II Inhibitors
13.
J Med Chem ; 41(15): 2846-57, 1998 Jul 16.
Article in English | MEDLINE | ID: mdl-9667973

ABSTRACT

On the basis of the fact that several polynucleotidyl transferases, related to HIV integrase, contain in their active site two divalent metal cations, separated by ca. 4 A, new potential HIV integrase inhibitors were designed, in which a quinoline substructure is linked to an aryl nucleus possessing various hydroxy substitution patterns, by means of an ethylenic spacer. Although the most active compounds contain the catechol structure, this group is not essential for the activity, since compound 21 that lacks such a moiety is a potent drug, implicating the presence of a different pharmacophore. The most promising styrylquinolines thus synthesized inhibit HIV-1 integrase in vitro at micromolar or submicromolar concentrations and block HIV replication in CEM cells, with no significant cellular toxicity in a 5-day period assay. These inhibitors are active against integrase core domain-mediated disintegration, suggesting that fragment 50-212 is their actual target. These new styrylquinolines may provide lead compounds for the development of novel antiretroviral agents for AIDS therapeutics, based upon inhibition of HIV integrase. They might also be used in the elucidation of the mechanism of inhibition of this enzyme; e.g., they could serve as candidates for cocrystallization studies with HIV integrase.


Subject(s)
Anti-HIV Agents , HIV Integrase Inhibitors , HIV-1/drug effects , Quinolines , Styrenes , Virus Replication/drug effects , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Anti-HIV Agents/toxicity , Cell Line, Transformed , HIV Integrase/genetics , HIV Integrase/metabolism , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacology , HIV Integrase Inhibitors/toxicity , HIV-1/enzymology , HIV-1/physiology , Humans , Mutation , Quinolines/chemical synthesis , Quinolines/pharmacology , Quinolines/toxicity , Sequence Deletion , Styrenes/chemical synthesis , Styrenes/pharmacology , Styrenes/toxicity , Tumor Cells, Cultured
14.
C R Seances Soc Biol Fil ; 191(3): 473-85, 1997.
Article in French | MEDLINE | ID: mdl-9295969

ABSTRACT

We have used a biological test on the microplates of cellular cultures in order to investigate the toxicity and the antiviral properties against different viruses: defective Moloney Murine Leukemia virus (MoMLV) derived from the SVX shuttle and expressing resistance to the G418 antimitotic, and Human Immunodeficiency Virus (HIV) of a hydroalcoholic extract from Haemanthus albiflos (Amaryllidacae). The toxicity was assessed through coloric test evaluation of fixed cells stained with crystal violet. In a population of NIH 3T3 cells (Fibroblasts mouse), the toxicity found with 2, 7, 14 and 28 microliters/ml of lyophilisat extract corresponding at: 0.23, 0.81, 1.62 and 3.24 mg of plant dry, was 32, 50, 63 and 70% respectively. With regards to the antiviral properties, the plant extracts showed an inhibition of 88% on the formation of G418 resistant 3T3 clones. The assay on HIV infected lymphotic cells (P4) showed an IC50 of 4 microliters/ml for this extract plant. Therefore, the toxic effect was similar to the antiviral response.


Subject(s)
Antiviral Agents/pharmacology , HIV/drug effects , Moloney murine leukemia virus/drug effects , Plant Extracts/pharmacology , 3T3 Cells/drug effects , 3T3 Cells/virology , Animals , Anti-HIV Agents/pharmacology , Anti-HIV Agents/toxicity , Antiviral Agents/toxicity , HeLa Cells/virology , Humans , In Vitro Techniques , Mice , Plant Extracts/toxicity
15.
Cancer Res ; 55(14): 3211-7, 1995 Jul 15.
Article in English | MEDLINE | ID: mdl-7606743

ABSTRACT

The possible intervention of nuclear proteins as cofactors of integrase-catalyzed integration of retroviral DNA into the host cell genome is not fully understood. Among various nuclear proteins, DNA topoisomerase II appears to be a plausible candidate. This hypothesis is supported by a series of evidence, including the fact that integration is markedly affected by the topology of the target DNA and mainly occurs in transcribed regions in which topoisomerase II is preferentially located. In an attempt to confirm the validity of this hypothesis, we have comparatively investigated the early stages of a recombinant Moloney murine leukemia virus (psi neo) in two related Chinese hamster cell lines (DC3F and R/DC3F) expressing different levels of both isoforms of topoisomerase II. R/DC3F is derived from the parental cell line DC3F and displays a resistant phenotype towards the usual anticancer topoisomerase II inhibitors (actinomycin D, doxorubicin, and taxol). Results show that the early stages of the retroviral cycle are markedly impaired in cells underexpressing topoisomerase II (R/DC3F). This alteration mimics Fv-1 restriction and is characterized by about a 6-fold decrease in viral DNA synthesis and total inhibition of viral genome integration. The specific impairment of integration in R/DC3F cells compared to DC3F cells is assessed by the absence of G418-resistant colonies upon viral infection and a lack of the viral genome in cellular nuclear DNA as detected by the PCR procedure. These features are observed in relevant infecting conditions leading, in both cell lines, to the same amount of linear viral DNA and to the occurrence of two long terminal repeats containing circular DNA in the nuclear fractions.


Subject(s)
DNA Topoisomerases, Type II/metabolism , Moloney murine leukemia virus/genetics , Virus Integration , 3T3 Cells/physiology , 3T3 Cells/virology , Animals , Base Sequence , Cell Line , Cricetinae , Cricetulus , DNA, Viral/genetics , Lung/enzymology , Lung/virology , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Transcription, Genetic , Tumor Cells, Cultured
16.
J Biol Chem ; 269(34): 21635-8, 1994 Aug 26.
Article in English | MEDLINE | ID: mdl-8063805

ABSTRACT

Integration of human immunodeficiency virus (HIV) DNA into the genome of host cells is an obligatory step in the replicative cycle of the virus. The overall process is carried out in vitro by a single viral protein, the integrase, which binds to short sequences located at the ends of viral DNA long terminal repeats (LTRs). These end sequences are highly conserved in all HIV genomes and are therefore attractive targets for selective DNA binding compounds. The integrase-binding site located in U3 LTR contains a purine motif, 5'-GGAAGGG-3' which can be selectively targeted by oligonucleotide-intercalator conjugates. Under neutral pH and physiological temperature, these conjugates readily form a stable complex with the viral DNA which involves a short DNA triplex. Triple-helix formation prevents the catalytic functions of the integrase in vitro which results in a sequence-specific inhibition of the U3 integration process.


Subject(s)
Carbazoles/pharmacology , HIV-1/drug effects , Intercalating Agents/pharmacology , Oligonucleotides/pharmacology , Virus Integration/drug effects , Base Sequence , Cell-Free System , DNA/metabolism , DNA Nucleotidyltransferases/metabolism , DNA, Viral/metabolism , HIV Long Terminal Repeat/drug effects , HIV-1/genetics , HIV-1/growth & development , Integrases , Molecular Sequence Data
17.
C R Acad Sci III ; 317(5): 430-6, 1994 May.
Article in English | MEDLINE | ID: mdl-7994621

ABSTRACT

Combinations of ddC with either the ribonucleotide reductase inhibitor hydroxyurea (HU) or with the natural nucleoside thymidine have been investigated on the cycle of a defective (psi neo) Moloney Leukemia Virus (MoMLV) using 3T3 fibroblasts as host cells. In this experimental model, ddC displayed very poor antiviral action which was obvious given an IC50 value close to 100 microM, i.e. an efficiency about thirty thousand fold lower than that of AZT. Both HU and thymidine alone resulted in significant inhibition of MoMLV replication with IC50 values of 40 microM and 100 microM respectively. The combination of ddC with 50 microM HU lowered the IC50 of ddC by a factor of 10. A similar but more pronounced effect was obtained by combining ddC and 100 microM thymidine, which decreases the IC50 value of ddC by a factor of 50. Combining 40 microM ddC and 100 microM thymidine resulted in the quite complete inhibition of viral replication. These results show that in cell types with strongly restricted ddC action, combination treatment with compounds known to ultimately decrease dCTP biosynthesis leads to the restoration of efficient antiviral activity.


Subject(s)
Hydroxyurea/pharmacology , Moloney murine leukemia virus/physiology , Thymidine/pharmacology , Virus Replication/drug effects , Zalcitabine/pharmacology , 3T3 Cells/virology , Animals , Drug Synergism , Mice , Transcription, Genetic/drug effects , Zidovudine/pharmacology
18.
Biochem Pharmacol ; 47(10): 1821-6, 1994 May 18.
Article in English | MEDLINE | ID: mdl-8204098

ABSTRACT

In search of potential inhibitors of integration of retroviral DNA into host cells genome, we have investigated the effect of the external DNA binder netropsin on the in vitro insertion of long terminal repeat (LTR) ends of Moloney murine leukemia virus (M.MuLV) as catalysed by integrase purified from baculovirus strain expression vector. In agreement with the preferential binding of netropsin to A+T rich sequences, footprinting experiments have shown that this drug selectively binds to the 5'-TTTCAT LTR end sequence which is included in the DNA binding site of integrase. This feature results in the potent inhibition of both reactions involved in the insertion process, namely, nucleolytic cleavage and strand transfer. The relation between netropsin binding to A+T rich region of M.MuLV LTR end and inhibition of insertion is strongly suggested from the inability of the drug to inhibit the insertion of HIV U3 LTR end which displays a G+C rich sequence. Selective inhibition of integration of viral DNA appears to be feasible using drugs recognizing LTR end sequences.


Subject(s)
DNA, Viral/metabolism , Moloney murine leukemia virus/drug effects , Netropsin/pharmacology , Repetitive Sequences, Nucleic Acid , Amino Acid Sequence , Base Sequence , DNA Nucleotidyltransferases/antagonists & inhibitors , DNA Nucleotidyltransferases/isolation & purification , In Vitro Techniques , Integrases , Molecular Sequence Data , Moloney murine leukemia virus/enzymology , Moloney murine leukemia virus/genetics , Netropsin/chemistry
19.
Virology ; 200(1): 87-97, 1994 Apr.
Article in English | MEDLINE | ID: mdl-7510441

ABSTRACT

We studied Moloney murine leukemia virus replication in newly infected Balb c/3T3 cells brought to the G0 phase by serum depletion. Using the polymerase chain reaction method, we showed that Moloney murine leukemia virus can be efficiently internalized in nonproliferating fibroblasts, although reverse transcription of the viral RNA in these cells remains incomplete. It seems likely that a lower availability of deoxyribonucleotides in G0-arrested cells is responsible for this premature termination of the reverse transcription step. Accordingly, the addition of high concentrations of nucleosides to the culture medium of nondividing cells simultaneously with infection enables them to complete the reverse transcription process, without re-initiating the cell cycle. Inhibition of reverse transcription by hydroxyurea confirms the dependence of this retroviral step on the intracellular nucleotide pool rather than on the precise arrest point of the host cell cycle. Furthermore, the pyrimidine nucleotide pool, and more particularly the cytidine pool, appears to play a central regulatory role in this step.


Subject(s)
DNA, Viral/biosynthesis , Moloney murine leukemia virus/growth & development , Nucleosides/pharmacology , RNA-Directed DNA Polymerase/metabolism , Resting Phase, Cell Cycle , Transcription, Genetic/drug effects , Animals , Base Sequence , Cytidine/metabolism , Fibroblasts , Hydroxyurea/pharmacology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Pyrimidine Nucleotides/metabolism , RNA-Directed DNA Polymerase/drug effects , Virus Replication
20.
Biochemistry ; 33(14): 4187-96, 1994 Apr 12.
Article in English | MEDLINE | ID: mdl-8155634

ABSTRACT

In an attempt to target short purine sequences in view of pharmacological application, we have synthesized three new TFO (triple-helix-forming oligonucleotide) conjugates in which an intercalating oxazolopyridocarbazole (OPC) chromophore is linked by a pentamethylene linker to a 7-mer oligonucleotide matching the polypurine/polypyrimidine sequence located in the HIV-1 U3 LTR end region. The TFO moiety of conjugates are 5'CCTTCCC, 5'GGGAAGG, and 5'GGGTTGG. Their ability to bind to double-stranded DNA targets was examined. This binding is demonstrated by a footprinting technique using DNase I as a cleaving agent. The complex involved intermolecular pyr-pur*pyr or pur-pur*pyr triple helix. Pyrimidine TFO-OPC binds in a pH-dependent manner, whereas the others do not. The formation of the complex has been investigated at neutral pH and increasing temperature. We observed that the protection due to the purine and mixed TFO-OPC was pH independent and remained identical up to 40 degrees C. To determine the position of the OPC chromophore, molecular modeling was undertaken on the purine-conjugate/target complex. It has been suggested that the complex involved the intercalation of the OPC at the triplex-duplex junction with a small unwinding at the next excluded site.


Subject(s)
DNA, Viral/chemistry , HIV Long Terminal Repeat , HIV-1/genetics , Intercalating Agents/chemistry , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Base Sequence , Carbazoles/chemistry , DNA, Viral/drug effects , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Data , Temperature
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