Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R₉F₂ was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R₉ conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R₉F₂ and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.     

Mitogen-activated protein kinase phosphatase 1 activity is necessary for oxidized phospholipids to induce monocyte chemotactic activity in human aortic endothelial cells

Entrapment and oxidation of low density lipoproteins (LDL) in the sub-endothelial space is a key process in the initiation of atherosclerotic lesion development. Functional changes induced by oxidized lipids in endothelial cells are early events in the pathogenesis of atherosclerosis. Oxidized-l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC), a major component of minimally modified/oxidized-LDL (MM-LDL) mimics the biological activities assigned to MM-LDL both in vitro in a co-culture model as well as in vivo in mice. We hypothesized that ox-PAPC initiates gene expression changes in endothelial cells that result in enhanced endothelial/monocyte interactions. To analyze the gene expression changes that oxidized lipids induce in endothelial cells, we used a suppression subtractive hybridization procedure to compare mRNA from PAPC-treated human aortic endothelial cells (HAEC) with that of ox-PAPC-treated cells. We report here the identification of a gene, mitogen-activated protein kinase phosphatase 1 (MKP-1), that is rapidly and transiently induced in ox-PAPC-treated HAEC. Inhibition of MKP-1 using either the phosphatase inhibitor sodium orthovanadate or antisense oligonucleotides prevents the accumulation of monocyte chemotactic activity in ox-PAPC-treated HAEC supernatants. Furthermore, we show that decreased monocyte chemotactic activity in HAEC treated with sodium orthovanadate or MKP-1 antisense oligonucleotides is due to decreased MCP-1 protein. Our results implicate a direct role for MKP-1 in ox-PAPC-induced signaling pathways that result in the production of MCP-1 protein by ox-PAPC-treated HAEC.     

An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer.

Direct RNA-PCR analyses of T-cell lymphomas that developed in rhesus macaques during a gene transfer experiment revealed the presence of several different recombinant murine leukemia viruses (MuLV). Most prominent was the expected MuLV recombinant, designated MoLTRAmphoenv in which the amphotropic env of the helper packaging virus was joined to the long terminal repeat (LTR) of the Moloney MuLV-derived vector. This retrovirus does not exist in nature. An additional copy of the core enhancer acquired from the vector LTR may have augmented the replicative properties of MoLTRAmphoenv MuLV in several different rhesus cell types compared with the prototype amphotropic MuLV4070A. Unexpectedly, at least two types of mink cell focus-forming MuLV elements, arising from endogenous retroviral sequences expressed in the murine packaging cell line, were also transmitted and highly expressed in one of the macaques. Furthermore, murine virus-like VL-30 sequences were detected in the rhesus lymphomas, but these were not transcribed into RNA. The unanticipated presence of an array of MuLV-related structures in a primate gene transfer recipient demands ever-vigilant scrutiny for the existence of transmissible retroviral elements and replication-competent viruses possessing altered tropic or growth properties in packaging cells producing retroviral vectors.     

Expression of theJE/MCP-1 gene suppresses metastatic potential in murine colon carcinoma cells

The purpose of this study was to determine whether the expression of theJE/MCP-1 gene encoding for the monocyte chemottractant protein, MCP-1 (also known as monocyte chemotactic and activating factor MCAF, TDCF, and SMC-CF) can influence the metastatic properties of tumor cells. The highly metastatic murine colon carcinoma CT-26 cells, syngeneic to BALB/c mice that do not produce endogenous JE/MCP-1 protein, were transfected with a BCMGS-Neo expression vector (control) or a vector containing full-lengthJE cDNA. CT-26 parental cells, CT-26 Neo, and CT-26 JE/MCP-1-positive cells were injected into syngeneic or nude mice. The CT-26 JE/MCP-1-positive cells produced significantly fewer lung metastases. The decrease in incidence of metastasis was not due to the inability of the transfected cells to arrest in the lung vasculature or to differences in cell cycle time. CT-26 cells producing JE/MCP-1 were highly susceptible to lysis by syngeneic macrophages treated with subthreshold concentrations of lipopolysaccharide. In addition, culture supernatants of JE/MCP-1-expressing cells plus lipopolysaccharide synergistically activated tumoricidal properties in syngeneic macrophages. This activity was blocked by anti-JE/MCP-1 antibodies, indicating the involvement of the JE/MCP-1 molecule in this process. Moreover, purified JE/MCP-1 added to lipopolysaccharide-containing medium resulted in significant activation of macrophages against parental CT-26 cells. These data suggest that, in addition to its chemotactic properties, JE/MCP-1 can synergize with bacterial endotoxins to activate macrophages to become tumoricidal and, hence, could suppress metastasis.     

Ecotropic and mink cell focus-forming murine leukemia viruses integrate in mouse T, B, and non-T/non-B cell lymphoma DNA.

Structures of somatically acquired murine leukemia virus (MuLV) genomes present in the DNA of a large panel of MuLV-induced C57BL and BALB/c B and non-T/non-B cell lymphomas were compared with those present in MuLV-induced T-cell lymphomas induced in the same low-"spontaneous"-lymphoma-incidence mice. Analyses were performed with probes specific for the gp70, p15E, and U3-long terminal repeat (LTR) regions of ecotropic AKV MuLV and a mink cell focus-forming virus (MCF)-LTR probe annealing with U3-LTR sequences of a unique endogenous xenotropic MuLV, which also hybridizes with U3-LTR sequences of a substantial portion of somatically acquired MCF genomes in spontaneous AKR thymomas. The DNAs of both T- and B-cell tumors induced by neonatal inoculation with the highly oncogenic C57BL-derived MCF 1233 virus predominantly contain integrated MCF proviruses. In contrast, the DNAs of more slowly developing B and non-T/non-B cell lymphomas induced by poorly oncogenic ecotropic or MCF C57BL MuLV isolates mostly contain somatically acquired ecotropic MuLV genomes. Approximately 50% of the spontaneous C57BL lymphoma DNAs contain somatically acquired MuLV genomes. None of the integrated MuLV proviruses annealed with the MCF-LTR probe, which indicates a clear difference in LTR structure with a substantial portion of the somatically acquired MuLV genomes present in the DNA of spontaneous AKR thymomas. This study stresses a dominant role of MuLV with ecotropic gp70 and LTR sequences in the development of slowly arising MuLV-induced B and non-T/non-B cell lymphomas.     

Thrombin impairs the angiogenic activity of extravillous trophoblast cells via monocyte chemotactic protein-1 (MCP-1): A possible link with preeclampsia

Cytokines’ secretion from the decidua and trophoblast cells has been known to regulate trophoblast cell functions, such as Extravillous trophoblasts (EVTs) cell migration and invasion and remodeling of spiral arteries. Defective angiogenesis and spiral arteries transformation are mainly caused by proinflammatory cytokines and excessive thrombin generation during preeclampsia. Monocyte chemotactic protein-1 (MCP-1), a crucial cytokine, has a role in maintaining normal pregnancy. In this study, we explored whether thrombin regulates the secretion of MCP-1 in HTR-8/SVneo cells; if yes, what is its function? We used HTR-8/SVneo cells, developed from ?rst trimester villous explants of early pregnancy, as the model of EVTs. MCP-1 gene silencing was performed using gene-specific siRNA. qPCR and ELISA were performed to estimate the expression and secretion of MCP-1. Here, we found that thrombin enhanced the secretion of MCP-1 in HTR-8/SVneo cells. Proteinase-activated receptor-1 (PAR-1) was found as the primary receptor, regulating MCP-1 secretion in these cells. Furthermore, MCP-1 secretion is modulated via protein kinase C (PKC) α, β, and Rho/Rho-kinase-dependent pathways. Thrombin negatively regulates HTR-8/SVneo cells’ ability to mimic tube formation in an MCP-1 dependent manner. In conclusion, we propose that thrombin-controlled MCP-1 secretion may play an essential role in normal placental development and successful pregnancy maintenance. Improper thrombin production and MCP-1 secretion during pregnancy might cause inadequate vascular formation and transformation of spiral arteries, which may contribute to pregnancy disorders, such as preeclampsia.     

Evaluation of monocyte chemotactic responsiveness in uraemic patients undergoing haemodialysis with different dialytic membranes

Recent data show that monocyte chemotactic peptide-1 (MCP-1), a chemotactic factor specific for monocytes, may play a central role in regulating the activation of these cells. For this reason, the production of MCP-1 in peripheral blood mononuclear cell (PBMC) cultures of eight healthy subjects, six chronic uraemic subjects under conservative treatment and six chronic uraemic patients undergoing haemodialysis (HD), was assessed. In the latter group of individuals, complement-activating membranes such as cuprophan (CU) were used for 1 month followed by biocompatible non-complement-activating membranes, like polymethylmetacrylate (PMMA) for the next 30 days. The chemotactic index (CI) elicited by PBMC supernatants from patients undergoing dialysis was found to be significantly higher than that obtained by supernatants recovered from normal subjects or uraemic patients on conservative therapy. Furthermore, the CI from PBMC supernatants having had contact with CU membranes was higher than that obtained from PBMC activated by PMMA. Finally, the increased chemotactic ability in the supernatants was closely correlated with the augmented MCP-1 gene expression and production, as assessed by in vitro hybridization studies.     

A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells.

The expression of Moloney murine leukemia virus (Mo-MuLV) and Mo-MuLV-derived vectors is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have previously described the isolation of retroviral mutants with host range properties expanded to embryonal cell lines. One of these mutants, the murine embryonic stem cell virus (MESV), is expressed in ES cell lines. Expression of MESV in these cells relies on DNA sequence motifs within the enhancer region of the viral long terminal repeat (LTR). Here we show that replacement of the Mo-MuLV enhancer region by sequences derived from the MESV LTR results in the activation of the Mo-MuLV LTR in ES cells. The enhancer regions of MESV and Mo-MuLV differ by seven point mutations. Of these, a single point mutation at position -166 is sufficient to activate the Mo-MuLV LTR and to confer enhancer-dependent expression to Mo-MuLV-derived retroviral vectors in ES cells. This point mutation creates a recognition site for a sequence-specific DNA-binding factor present in nuclear extracts of ES cells. This factor was found by functional assays to be the murine equivalent to human Sp1.     

Functional differences between monocyte chemotactic protein-1 receptor A and monocyte chemotactic protein-1 receptor B expressed in a Jurkat T cell

The monocyte chemotactic protein-1 (MCP-1) receptor (MCP-1R) is expressed on monocytes, a subpopulation of memory T lymphocytes, and basophils. Two alternatively spliced forms of MCP-1R, CCR2A and CCR2B, exist and differ only in their carboxyl-terminal tails. To determine whether CCR2A and CCR2B receptors function similarly, Jurkat T cells were stably transfected with plasmids encoding the human CCR2A or CCR2B gene. Nanomolar concentrations of MCP-1 induced chemotaxis in the CCR2B transfectants that express high, intermediate, and low levels of MCP-1R. Peak chemotactic activity was shifted to the right as receptor number decreased. Five-fold more MCP-1 was required to initiate chemotaxis of the CCR2A low transfectant, but the peak of chemotaxis was similar for the CCR2A and CCR2B transfectants expressing similar numbers of receptors. MCP-1-induced chemotaxis was sensitive to pertussis toxin, implying that both CCR2A and CCR2B are G(i)alpha protein coupled. MCP-1 induced a transient Ca(2+) flux in the CCR2B transfectant that was partially sensitive to pertussis toxin. In contrast, MCP-1 did not induce Ca(2+) flux in the CCR2A transfectant. Since MCP-1 can stimulate chemotaxis of the CCR2A transfectant without inducing Ca(2+) mobilization, Ca(2+) flux may not be required for MCP-1-induced chemotaxis in the Jurkat transfectants. These results indicate that functional differences exist between the CCR2A and CCR2B transfectants that can be attributed solely to differences in the carboxyl-terminal tail.     

Efficient expression of pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells using Moloney retroviral promoter

We have compared the level of expression of several enhancer/promoters in human lymphoblastoid Namalwa KJM-1 cells when fused to a common reporter gene. A cassette containing the pro-urokinase (pro-UK) coding sequence followed by the rabbit -globin and simian virus 40 (SV40) 3 nontranslated region was used for evaluation of the enhancer activity. Cells containing Moloney murine leukemia virus (Mo-MuLV) promoter had an average of 10–20 fold higher expression levels of pro-UK than those containing other promoters, such as SV40 early gene promoter, human cytomegalovirus (hCMV) major immediate-early gene promoter, Rous sarcoma virus (RSV) promoter and chicken -actin gene promoter. The expression level of pro-UK under the control of Mo-MuLV promoter was 2–3 g/10⁶ cells/day and was constant for more than 6 months. Furthermore, the production of a high producer clone, obtained by using dhfr gene coamplification, reached 30–40 g/10⁶ cells/day. Thus, Mo-MuLV promoter showed the desired characteristics for efficient expression of foreign genes in Namalwa KJM-1 cells.Abbreviations dhfr dihydrofolate reductase - G-CSF granulocyte colony-stimulating factor - hCMV human cytomegalovirus - LTR long terminal repeat - Mo-MuLV Moloney murine leukemia virus - MTX methotrexate - pro-UK pro-urokinase - RSV Rous sarcoma virus - SV40 simian virus 40 - T3 triiodo-thyronine - TRE thyroid-hormone responsive element     

Functional role of the cis-acting elements in human monocyte chemotactic protein-1 gene in the regulation of its expression by phorbol ester in human glioblastoma cells

The monocyte chemotactic protein-1 (MCP-1) is a 76 amino acid protein that specifically attracts monocytes. The expression of MCP-1 gene can be induced by lipopolysaccharides (LPS), phorbol esters (TPA) and several cytokines. However, how they regulate MCP-1 gene expression is not known. We tested whether the two putative TPA-responsive elements (TREs) and one B enhancer-like region found in the MCP-1 promoter region, are involved in this regulation of MCP-1 gene expression. The 5 untranslated region of MCP-1 gene was linked to chloramphenicol acetyl transferase (CAT) reporter gene and transfected into human glioblastoma cells in which endogenous MCP gene expression was found to be stimulated by TPA and tumor necrosis factor- (TNF-). The 128 bp 5-flanking region containing one TRE was adequate for basal promoter activity but the presence of both TREs in the MCP-1 promoter region were needed to give TPA responsive enhancement (2.5 fold) of expression of the marker gene. Mutations in either of the TRE's could abolish the TPA induction of CAT expression. Replacement of the B enhancer-like element with a TRE-like sequence caused a 10-fold enhancement of CAT expression by TPA treatment. Random mutation of B enhancer-like element did not affect CAT expression or its TPA induction. None of the MCP promoter constructs showed significant increase in CAT expression by treatment with tumor necrosis factor- (TNF-). This result suggested that the TNF regulation of MCP-1 gene involves other parts of the gene besides the proximal 5 flanking region. (Mol Cell Biochem141: 121–128, 1994)     

A newly identified chemotactic sexual pheromone from Closterium ehrenbergii

 In sexual reproduction of Closterium ehrenbergii, pairing with the sexual partner cells is the first process observed. A cell migration-inducing activity, specific for mating-type plus (mt⁺; NIES-228) cells, was detected in the culture medium of mating-type minus (mt^–; NIES-229) cells. Light was necessary for production of the active substance by mt^– cells and for migration of mt⁺ cells. The active substance was heat-labile and had an apparent molecular mass of 20 kDa, as determined by gel filtration. A protein of 20 kDa was detected in the active fraction of gel filtration after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on these results, it is proposed that a chemotactic sexual pheromone involved in the formation of sexual pairs of cells is secreted by mt^– cells of C. ehrenbergii and is proteinaceous, like other sexual pheromones secreted by Closterium species. Received: 31 July 1997 / Revision accepted: 25 November 1997     

Differential requirements for activation of integrated and transiently transfected human T-cell leukemia virus type 1 long terminal repeat

Adult T-cell leukemia (ATL) cells contain integrated human T-cell leukemia virus type 1 (HTLV-1) proviruses. Although the exact sequence of events leading to the development of ATL remains incompletely resolved, expression of the integrated HTLV-1 long terminal repeat (LTR) is likely required at some point during the process of T-cell transformation. While much has been learned about the regulated expression of transiently transfected LTR reporter plasmids, an analysis of factors required for expression of chromosomally integrated HTLV-1 LTR has not been done. Here, we have constructed CHOK1 and HeLa cells that contain an integrated HTLV-1 LTR-luciferase gene. Using these cells, we have compared the requirements for activation of transiently transfected versus stably integrated HTLV-1 LTR. We observed different requirements for CREB, p300, and P/CAF in the expression of transiently transfected versus stably integrated HTLV-1 LTR. Furthermore, with dominant-negative mutants of CREB, p300, and P/CAF, we found that activation of integrated HTLV-1 LTR by an ambient stress signal, UV-C, proceeds through a path mechanistically distinct from that used by viral oncoprotein, Tax. Our findings point to additional complexities in the regulated expression of HTLV-1 proviruses compared with those hitherto revealed through transfection studies.     

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.