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G Symonds

Publications and source records attributed to G Symonds.

At least 37 records · Page 2Linked to original sources
PubMed1995

Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA.

The structural motif formed between a hammerhead ribozyme and its substrate consists of three RNA double helices in which the sequence 5' to the XUY is termed helix I and the sequence 3' to the XUY helix III. Two hammerhead ribozymes targeted to the tat gene of HIV-1SF2 were designed to study target specificity and the potential effect of helix I mismatch on ribozyme efficacy both in vitro and in vivo. The first ribozyme (Rz1) targeted to the 5' splicing region of the tat gene was designed to cleave GUC*A. In HIV-1IIIB the A is changed to a G. The second ribozyme (Rz2) was targeted to the translational initiation region of the tat gene which is highly conserved among a variety of HIV-1 isolates, including both HIV-1SF2 and HIV-1IIIB. In vitro cleavage studies demonstrated that Rz1 efficiency cleaved HIV-1SF2 substrate RNA, but not HIV-1IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1SF2 or HIV-1IIIB substrate with equal efficiency. Both ribozymes were cloned into the 3' untranslated region of the neomycin gene (neo) within the pSV2neo vector and transfected into the SupT1 human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1SF2 or HIV-1IIIB virus. While Rz1-expressing cells were significantly protected from HIV-1SF2 infection, they exhibited no protection when infected with HIV-1IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1SF2 and HIV-1IIIB in SupT1 cells. Expression of both ribozymes in these cells was demonstrated by Northern analysis. RT-PCR sequencing analysis confirmed the respective HIV-1 target sequence integrity. These data demonstrate the importance of the first base pair distal to the XUY within helix I of the hammerhead structure for both in vitro and in vivo ribozyme activities and imply that the effectiveness of the anti-HIV-1 ribozymes against appropriate target sequences is due to their catalytic activities rather than any antisense effect.

Base Sequence↗
PubMed1995

Resistance to human immunodeficiency virus type 1 infection conferred by transduction of human peripheral blood lymphocytes with ribozyme, antisense, or polymeric trans-activation response element constructs.

Human peripheral blood lymphocytes (PBLs) were transduced with a number of recombinant retroviruses including RRz2, an LNL6-based virus with a ribozyme targeted to the human immunodeficiency virus (HIV) tat gene transcript inserted within the 3' region of the neomycin-resistance gene; RASH5, and LNHL-based virus containing an antisense sequence to the 5' leader region of HIV-1 downstream of the human cytomegalovirus promoter; and R20TAR, an LXSN-based virus with 20 tandem copies of the HIV-1 trans-activation response element sequence driven by the Moloney murine leukemia virus long terminal repeat. After G418 selection, transduced PBLs were challenged with the HIV-1 laboratory strain IIIB and a primary clinical isolate of HIV-1, 82H. Results showed that PBLs from different donors could be transduced and that this conferred resistance to HIV-1 infection. For each of the constructs, a reduction of approximately 70% in p24 antigen level relative to the corresponding control-vector-transduced PBLs was observed. Molecular analyses showed constitutive expression of all the transduced genes from the retroviral long terminal repeat, but no detectable transcript was seen from the internal human cytomegalovirus transcript was seen from the internal human cytomegalovirus promoter for the antisense construct. Transduction of, and consequent transgene expression in, PBLs did not impact on the surface expression of either CD4+/CD8+ (measured by flow cytometry) or on cell doubling time (examined by [3H]thymidine uptake). These results indicate the potential utility of these anti-HIV-1 gene therapeutic agents and show the preclinical value of this PBL assay system.

Animals↗
PubMed1995

Isolation of a novel macrophage-specific gene by differential cDNA analysis.

To analyze myelomonocytic differentiation we have used the approach of differential cDNA analysis to isolate novel genes that are preferentially expressed in mature macrophages. Differential screening of a macrophage cDNA library led to the identification of a novel cDNA that showed macrophage lineage- and differentiation stage-specific expression. Transcripts from the gene, which we have termed Mpg-1, are found at a high level in mature human and murine macrophages and at a moderate level in certain myelomonocytic cell lines. The expression of Mpg-1 was found to increase when murine fetal liver hematopoietic progenitor cells were induced to differentiate into macrophages. An Mpg-1-specific transcript was not detected in a wide variety of other tissues and cell lines. The DNA sequence of Mpg-1 (4,214 bp) was obtained from a series of overlapping cDNA, 3' rapid amplification of cDNA ends (RACE), and genomic clones. Primer extension analysis predicted the existence of multiple transcription start sites, ranging from 26 to 117 bp upstream of the 5' proximal ATG of the open reading frame. The predicted 669-amino acid, Mpg-1-encoded protein has potential glycosylation and phosphorylation sites in addition to a signal sequence. The core protein is predicted to have a molecular weight of 71 to 74 kD. Computer-assisted local similarity searches indicate that Mpg-1 is a novel gene that may share a distant ancestry to perforin, a lytic protein found in cytotoxic T lymphocytes and natural killer cells.

Amino Acid Sequence↗
PubMed1995

Transformation of FDC-P1 cells to IL-3 independence by a recombinant murine retrovirus containing v-erb-B.

The oncogene v-erb-B has been shown to transform pre-B lymphocytes and early erythroid precursor cells and has been implicated in leukemogenesis. We have examined the effect of this oncogene on the growth of a murine myeloid interleukin-3 (IL-3)-dependent cell line, FDC-P1, FDC-P1 cells were infected with a recombinant murine retrovirus containing v-erb-B. As a result, clonal IL-3-independent cell lines (FI-v-erb-B) were generated with a high level of v-erb-B expression and an altered morphology compared to parental FDC-PI cells. The F1-v-erb-B cells were tumorigenic and did not express or secrete IL-3, suggesting the acquisition of IL-3 independence by a non-autocrine mechanism. In addition to the formation of myeloid colonies, FI-v-erb-B cells, when grown in semi-solid medium with exogenous IL-3, erythropoietin (Epo), or IL-3 plus Epo, could also form erythroid and mixed-erythroid colonies. By contrast, parental FDC-P1 cells formed only myeloid colonies under the same conditions. Our results indicate that v-erb-B abrogates growth factor dependence in these cells and may cause lineage modulation by acting to allow the induction of erythroid differentiation in FI-v-erb-B cells.

Animals↗
PubMed1994

Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.

Several hammerhead ribozymes targeted to different sites within the retroviral packaging (psi) sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV psi packaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV psi site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro cleavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 psi packaging site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a significant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the psi packaging site with hammerhead ribozymes.

3T3 Cells↗
PubMed1994

An internal deletion enhances the transcriptional activity of a recombinant retrovirus in hematopoietic cells in vivo.

Lv-myc is a recombinant retrovirus that spontaneously arose during experiments designed to express the provirus LNAv-myc in the hematopoietic system of bone marrow-reconstituted mice (L. Bonham, K. MacKenzie, S. Wood, P. B. Rowe, and G. Symonds, Oncogene 7:2219-2229, 1992). The recombinant provirus is of interest because it is able to promote long terminal repeat-initiated transcription in hematopoietic cells in vivo, whereas the parental provirus, LNAv-myc, is transcriptionally repressed in the same cells. Here we report that Lv-myc was generated by precise deletion of the neomycin resistance gene (neo) and the human gamma-actin promoter from LNAv-myc. In comparison with LNAv-myc, no sequence alterations in the viral regulatory regions of Lv-myc were detected. Thus, it appears that neo and/or the gamma-actin promoter exerted a cis-acting repressor effect on the long terminal repeat of LNAv-myc in vivo. The origin of Lv-myc was also investigated, and it was shown that Lv-myc was harbored as a productive provirus in a G418-resistant subpopulation of the LNAv-myc producer cell line, psi 2AV. It appears that Lv-myc arose during propagation of the psi 2AV cell line. Repeated sequence detected at the sites of the deletion suggest that Lv-myc was generated by a template misalignment during reverse transcription of LNAv-myc.

Actins↗
PubMed1994

In vitro transformation of Li-Fraumeni syndrome fibroblasts by SV40 large T antigen mutants.

Transfection of SV40 early region DNA into normal human diploid fibroblasts (NHDFs) increases their proliferative potential to a limited extent. We have investigated the roles of the SV40 large T antigen (LTAg) regions responsible for binding to the protein products of the retinoblastoma (Rb) and p53 genes in this temporary escape from senescence. Patients encoding LTAg mutants were transfected into NHDFs and into Li-Fraumeni syndrome (LFS) fibroblasts which are heterozygous wild-type (wt)/null-mutant for p53. A LTAg mutated in the p53-binding region (T402DE) had greatly reduced efficiency of focus formation, and a p110Rb-binding mutant was unable to induce any foci. T402DE-induced NHDF foci senesced at the same time as untransfected cells, but the equivalent LFS foci all had increased proliferative potentials, with the greatest increase being seen in clones that lost the wt p53 allele. One LFS clone expressed the T402DE mutant during focus formation, but later lost both the T402DE DNA and the wt p53 allele. We conclude that SV40-induced focus formation in NHDFs requires the LTAg p110Rb-binding region, and is enhanced by loss of normal p53 function. In contrast, increased proliferative potential is primarily due to loss of p53 function.

Antigens, Polyomavirus Transforming↗
PubMed1993

Immortalization and characterization of human cell lines with mast cell and monocytic properties.

We have previously derived a cell strain which had both mast cell and monocytic properties from the bone marrow of a child with diffuse cutaneous mastocytosis. This cell strain, termed HBM-M, consisted of two cell populations both of which possessed certain ultrastructural, cytochemical and surface phenotypic features of degranulated mast cells. The cells also displayed cytochemical and surface phenotypic features of monocytes. These cells may represent a common bone marrow derived mast cell/monocyte precursor. Studies of human mast cells have been hindered by the fact that it is difficult to establish such cells in long-term culture. Thus, we sought to immortalize HBM-M cells by introducing Simian virus 40 large T-antigen. Following transfection by the strontium phosphate technique, transformed cells were selected, expanded and passaged until the cells entered a non-proliferative phase termed crisis. Certain clones passed through crisis 3 months later and by this means two immortal cell lines, HBM-MI-1 and HBM-MI-2, were obtained. The criterion for immortality was growth for greater than 100 population doublings. The immortal cell lines retained some, but not all, of the mast cell and monocytic properties of the original HBM-M cell strain. The immortalization of the cell strain HBM-M provides an opportunity to investigate the mast cell and monocytic properties of these cells, and the apparent relationship between mast cells and monocytes.

Animals↗
PubMed1993

Induction of pre-B lymphoid leukemia following reconstitution of lethally irradiated mice with v-erb-B virus-infected bone marrow progenitor cells.

We have previously shown that v-erb-B contained within a recombinant murine retroviral vector is capable of transforming pre-B lymphocytes (M. Miller, A. K. Kennewell, and G. Symonds, Leukemia, 6: 18-28, 1992) and early erythroid precursor cells [blast-forming units (erythroid) (M. Miller, A. Kennewell, Y. Takayama, A. Bruskin, J. M. Bishop, G. Johnson, and G. Symonds, Oncogene, 5: 1125-1131, 1990)] in vitro. To determine the sites and nature of v-erb-B-induced transformation in vivo, the hematopoietic systems of lethally irradiated mice were repopulated with v-erb-B-infected bone marrow. All mice became moribund within 4-12 weeks of reconstitution, with a median onset of disease at 6 weeks. Histopathological and flow cytometric evaluation of tissues from diseased mice, as well as morphological and phenotypic analysis (cytochemical as well as molecular) of the cell lines established from the mice, revealed that all but one of the mice examined at postmortem had developed a pre-B lymphoid leukemia or lymphoma. Abnormally high levels of mast cells in the spleen and bone marrow of the remaining mouse indicated a mast cell disease. The development of pre-B lymphoid malignancy in the majority of the reconstituted mice indicates a marked predisposition of v-erb-B to transform cells of the pre-B lymphoid lineage. The reconstitution of lethally irradiated mice with v-erb-B virus-infected bone marrow provides a model system for the analysis of events involved in the initiation and maintenance of acute lymphoid leukemia.

Animals↗
PubMed1992

An in vitro fibroblast model system to study myc-driven tumour progression.

We have utilized fibroblast cell lines to investigate myc-mediated cell transformation and tumour progression. Deregulated myc alleles were introduced into the rat fibroblast cell line, Rat-1, and a partially-transformed derivative of this cell line termed Rat-1 (PT). A human c-myc gene coupled to the Moloney murine leukemia virus long terminal repeat was introduced into both cell lines by transfection. The avian MC29 v-myc gene was introduced into the Rat-1 cell line by retroviral infection using a Moloney murine leukemia recombinant retrovirus. For both cell lines, the introduction of exogenous myc genes resulted in an increased degree of transformation. For the non-tumorigenic Rat-1 cell line, this also resulted in the acquisition of tumorigenicity, while for the Rat-1 (PT) cell line the degree of tumorigenicity was increased. Various clones were isolated and, for both human c-myc and avian v-myc, the level of myc expression correlated with the degree of transformation and the tumorigenic potential of the cell lines. In addition, both these parameters could be increased by passaging through syngeneic recipients. Our data show that tumour progression may be driven by the deregulated expression of myc genes; that transformation and tumorigenicity correlate with the level of exogenous myc expression; that additional events involving both in vitro and in vivo selection are involved in this process; and that myc expression may increase the cells' metastatic capacity.

Animals↗
PubMed1992

Both myeloproliferative disease and leukemia are induced by transplantation of bone marrow cells expressing v-myc.

An in vivo system has been established to investigate v-myc-induced hematopoietic neoplasia in mice. A Moloney murine leukemia virus (Mo-MLV)-derived recombinant retrovirus containing v-myc was used to infect immature bone marrow cells, and these cells were then transplanted into lethally irradiated recipients. All provirus-positive reconstituted mice were found to develop hematopoietic proliferative disorders and, in certain cases, overt leukemia--myeloblastic, myelomonocytic and T lymphocytic. In all cases expression of v-myc was high and the disease type did not correlate with the level of expression. We have isolated immortalized monocytes, myeloid progenitors and T lymphocytes from several of these mice and shown tumorigenicity in secondary syngeneic recipients. This system provides a model for investigating the progression from a pre-leukemic disease to malignancy. In addition, we describe a recombinant v-myc-containing retrovirus that directs high-level v-myc expression from the Mo-MLV promoter in all the hematopoietic cell types examined.

Animals↗
PubMed1992

Germ-line splicing mutation of the p53 gene in a cancer-prone family.

Li-Fraumeni syndrome is a rare autosomal dominant susceptibility to a variety of cancers including carcinomas of the breast and the adrenal cortex, tumors of brain and muscle tissue, and leukemias. Affected individuals develop cancer at a young age and often at multiple primary sites. A study has been conducted into the genetic basis of cancer in a particular Li-Fraumeni syndrome family. Examination of p53 as a candidate susceptibility gene revealed that, in two affected individuals, there was an aberrant larger transcript of 3.6 kilobases present in both tumor and constitutional material in addition to the normal-sized 2.8-kilobase transcript. The additional transcript was not found in three unaffected family members. S1 nuclease mapping localized the insertion toward the 5' end of the p53 transcript near exons 4 and 5, and sequencing revealed a point mutation in the splice donor site of intron 4 in the germ-line of the two affected individuals, which accounted for the presence of the larger transcript. The same splicing mutation was also detected in two obligate carriers and was not found in two unaffected individuals. As no mutations were detected in exons 5-8 in either tumor examined, the second p53 allele was most likely lost during tumorigenesis in both tumors. The demonstration of a germ-line splicing mutation in affected individuals from a Li-Fraumeni syndrome family provides for a novel mechanism of p53 inactivation not seen previously in other affected families, in whom the mutations have all been missense.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Neoplasms↗
PubMed1992

Constitutive expression of exogenous myc in myelomonocytic cells: acquisition of a more transformed phenotype and inhibition of differentiation induction.

The effects of deregulated expression of the human c-myc and MC29 v-myc oncogenes have been examined in a murine myelomonocytic cell line J774 (c-myc) and in a variety of myelomonocytic cell lines of different degrees of maturity generated from primary hematopoietic tissue (v-myc). Introduction of a Moloney murine leukemia virus long terminal repeat (LTR) c-myc construct into J774 cells resulted in constitutive expression of the exogenous myc gene and a concomitant increase in the degree of transformation and tumorigenicity of the cells. In addition, constitutive expression of exogenous myc inhibited induced differentiation of these cells by a variety of treatments including addition to the medium of lipopolysaccharide (LPS) or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) as well as complete withdrawal of serum from the medium. The degree of increased transformation, tumorigenicity and inhibition of terminal differentiation was dependent upon the level of exogenous myc expression. For the v-myc-generated myelomonocytic cell lines, introduction of v-myc resulted in a high degree of transformation and, irrespective of the differentiation status of the cells, a block of induced differentiation. These results indicate that the level of constitutive myc expression can affect the transformed phenotype, tumorigenicity and differentiation inducibility of myelomonocytic cells.

Animals↗
PubMed1992

Transformation of murine pre-B-lymphocytes by v-erb-B: progression to growth factor independence and tumorigenicity.

v-erb-B is the principal transforming gene of avian erythroblastosis virus, a replication defective retrovirus that transforms erythroid and fibroblast cells in vitro and causes erythroleukemia and fibrosarcoma in vivo. We have used a recombinant murine retrovirus, based on the truncated genome of Moloney murine leukemia virus and containing a chimeric gag-v-erb-B gene, to determine the murine hematopoietic cells transformed by v-erb-B. Infection of day 16.5 murine embryonal cells in liquid culture with this virus resulted in the outgrowth of foci of loosely to non-adherent hematopoietic cells which grew in close association with an adherent monolayer. After several weeks in culture a clonal population of transformed pre-B-lymphocytes emerged from this transformation initiated, though still growth factor dependent, population. These transformed cells were growth factor independent, and were tumorigenic in syngeneic mice. The results indicate that although v-erb-B can initiate transformation of murine hematopoietic cells, additional events are required for establishment of the fully transformed growth factor independent, tumorigenic phenotype. This v-erb-B induced progression from growth factor dependence to independence provides an in vitro model system for analysing events involved in the initiation and maintenance of leukemia.

Animals↗