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G Symonds

Publications and source records attributed to G Symonds.

At least 19 recordsLinked to original sources
PubMed2000

Induced p21WAF1 expression acts to reverse myc myelomonocytic cell transformation.

Two murine myelomonocytic cells lines were used to examine p21WAF1 expression in myc-induced cell transformation. tEMmyc4 and FDLV are two v-myc-transformed immortalised myeloid cell lines exhibiting different transformed phenotypes. FDLV cells were derived from the transduction of v-myc into FDC-P1 cells and retain growth factor (IL-3) dependence, whereas tEMmyc4 cells were derived from the transduction of embryonal monocytes with v-myc and are growth factor-independent, constitutively express endogenous CSF-1, and are highly tumorigenic in syngeneic mice. Both cell lines were found to exhibit low p21WAF1 expression. When examined in tEMmyc4 cells, neither the p53-dependent pathway (mitomycin C or exogenous p53) nor p53-independent pathway (TPA or growth factor, CSF-1, stimulation) acted to increase p21WAF1 levels. Growth factor (IL-3) withdrawal, shown to reduce p21WAF1 levels in parental FDC-P1 cells, failed to do this in FDLV cells. The dependence of p21WAF1 expression on v-myc was further demonstrated by showing that a v-myc-targeted ribozyme, which acts to decrease v-myc RNA, increased p21WAF1 levels in tEMmyc4 cells. Enforced expression of exogenous p21WAF1 in tEMmyc4 cells with dysfunctional growth cycle (including growth arrest and increased susceptibility to apoptosis) was examined. p21WAF1 partially restored cell cycle regulation and apoptosis as well as inhibited the delayed cell cycle progression and apoptosis induced by mitomycin C or serum withdrawal. These results show p21WAF1 expression to be affected by v-myc and a restoration of p21WAF1 expression to partially reverse myc-mediated transformation.

Animals↗
PubMed2000

The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model.

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.

Animals↗
PubMed1999

Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice.

Mutations that activate the N-ras oncogene are among the most frequently detected genetic alterations in human acute myeloid leukemias (AMLs), Philadelphia chromosome-negative myeloproliferative disorders (MPDs), and myelodysplastic syndromes (MDSs). However, because N-ras has not been shown to induce these disorders in an in vivo model, the role of N-ras in the evolution of myeloid leukemia is unclear. To investigate the potential of N-ras to induce myeloid leukemia, lethally irradiated mice were reconstituted with bone marrow (BM) cells infected with a retroviral vector carrying activated N-ras. Approximately 60% of these mice developed hematopoietic disorders, including severe MPDs resembling human chronic myelogenous leukemia (CML) or AML with differentiation (French-American-British [FAB] classification M2). Other reconstituted mice succumbed to hematopoietic defects that were pathologically similar to human MDSs. The latter disorders appeared to be due to a myeloid impairment that was demonstrated by enumeration of day-12 colony-forming units-spleen (CFU-S) and by in vitro colony assays. A high level of apoptosis associated with thymic atrophy and peripheral blood (PB) lymphopenia was also evident in N-ras reconstituted mice. Our results are consistent with a model in which antiproliferative effects are a primary consequence of N-ras mutations and secondary transforming events are necessary for the development of myeloid leukemia. This is the first report of an in vivo model for N-ras induced MPD and leukemia.

Animals↗
PubMed1999

Artificial capillary culture: expansion and retroviral transduction of CD4+ T-lymphocytes for clinical application.

An artificial capillary culture/transduction technique has been developed for application in a phase I gene therapy clinical trial for HIV. The trial protocol involves isolation of CD4+ T-lymphocytes from a genetically matched HIV negative twin, retroviral transduction of equal numbers of cells with the ribozyme therapeutic and control genes, and expansion in Cellmax artificial capillary modules. Preclinical studies showed transduction efficiencies in the range of 3-30%, with preferential expansion of CD4+ lymphocytes over a culture period of 10-14 days. Over this time period, an average yield of 1.7 x 10(9) lymphocytes was readily attainable from 5 x 10(7) CD8-depleted lymphocytes. In addition, a sensitive and reliable quantitative competitive PCR method was developed to assess the levels of transduction before infusion into the recipient. The transduction data suggest that the efficiency of retroviral transduction was affected by the presence of inhibitory factors present in the virus preparations or generated as a result of the transduction process itself. It is hypothesised that the method of transduction could significantly affect the extent of this inhibition, and thus impact on clinical efficacy of retrovirus mediated gene therapy.

CD4-Positive T-Lymphocytes↗
PubMed1998

Preclinical characterization of an anti-tat ribozyme for therapeutic application.

A hammerhead ribozyme retroviral construct, denoted RRz2, targeting the coding region of the human immunodeficiency virus type 1 (HIV-1) tat gene, has shown itself to be effective in a range of test systems. Inhibition of the replication of HIV-1 IIIB and primary drug-resistant strains in pooled transduced CEMT4 cells was consistently found to be more than 80% compared with the control-vector transduced cells, whereas a mutant RRz2 gave approximately 45% inhibition. A multiple HIV-1 passage assay showed the absence of emergence of mutations within the specific viral RNA ribozyme target sequences. This lack of generation of ribozyme "escape mutants" occurred despite the almost complete disappearance of a HIV-1 quasi-species in the testing virus. When RRz2 was tested in peripheral blood lymphocytes (PBLs) from HIV-1-infected patients, paired analysis showed that cell viability in the ribozyme-transduced HIV-1-infected PBLs was significantly higher than that in the vector-transduced cells. This difference in viability (vector versus RRz2) was not observed in PBLs from non-HIV-1-infected donors. Taken together, these results indicate that the transfer of an anti-HIV-1 ribozyme gene into human T lymphocytes could have major impact on viral replication and T cell viability in the HIV-1-infected individual.

Base Sequence↗
PubMed1998

Effects of megakaryocyte growth and development factor on survival and retroviral transduction of T lymphoid progenitor cells.

Murine retroviral vectors have the potential to mediate stable gene transfer into hematopoietic progenitor cells. A known drawback to the use of these vectors is that transduction can only take place in cells actively progressing through the cell cycle. Thrombopoietin, the c-mpl ligand, is known to support division of hematopoietic precursors of primitive origin. Polyethylene glycol (PEG)-conjugated recombinant human megakaryocyte growth and development factor (MGDF) is a polypeptide related to thrombopoietin that stimulates megakaryocyte production. To investigate whether MGDF would also induce stem cell division and support retroviral transduction of CD34+ cells, we compared the effects of MGDF, stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, alone or in combination, using amphotropic and vesicular stomatitis virus (VSV-G) pseudotyped murine retroviral vectors. Similar transduction efficiency was observed when CD34+ cells were transduced in the presence of SCF and MGDF as compared to SCF, IL-3, and IL-6. Using the SCID-hu mouse model of thymopoiesis, we investigated whether CD34+ cells transduced in the presence of these cytokines could reconstitute irradiated thymic implants, and whether vector sequences were present in mature thymocytes. At early timepoints, no significant differences were observed on engraftment of donor progenitors incubated with each cytokine combination. However, a significant difference in the percentage of donor derived CD4+/CD8+ immature thymocytes was observed 9 weeks after implantation of CD34+ cells exposed to the combination of SCF and MGDF as compared to SCF, IL-3, and IL-6 (p = 0.04), indicating that MGDF/SCF better supported the survival of thymocyte precursor cells. Approximately 4% of thymocytes in both cytokine groups harbored vector sequences. These studies provide evidence that MGDF and SCF in combination can mediate transduction of hematopoietic progenitors capable of contributing to long-term thymopoiesis. These results may have important applications for the implementation of gene therapy strategies in disorders affecting the T lymphoid system.

Animals↗
PubMed1998

V-myc in a simple, single gene retroviral vector causes rapid induction of leukemia and concomitant apoptosis following bone marrow transplantation.

We have previously developed an in vivo model of leukemogenesis utilizing mice reconstituted with genetically modified bone marrow cells. Based on those studies, a new single gene retroviral vector has been engineered which efficiently transfers v-myc into immature murine bone marrow cells. All reconstituted mice developed leukemia with a short latency period (5-11 weeks). In addition to hyperproliferation associated with elevated levels of PCNA, extensive apoptosis was also observed in all leukemic animals with p53 accumulating in the apoptotic cells. Whereas bax encoded protein, an effector of p53 apoptotic activity was detected in apoptotic cells, p21Waf1 protein, a potential mediator of p53 growth suppression was not detected in these cells suggesting that v-myc-induced apoptosis was independent of the ability of p53 to induce p21Waf1. These results indicate that apoptosis, a part of the cellular response to v-myc expression, does not prevent leukemia development and that hyperproliferation rather than abrogation of oncogene-induced apoptosis appears to be a critical event in v-myc-induced leukemia.

Animals↗
PubMed1997

Anti-HIV ribozymes.

HIV is an RNA virus that replicates intracellularly through various RNA intermediates. Several of these can be targeted by ribozymes (catalytic RNA molecules), and a number of investigators, including this group, have demonstrated the ability of ribozymes to suppress HIV replication in this way. It is argued that this gene therapy approach may be viewed as an adjunct to chemotherapeutic drugs, which may allow not just viral suppression, but also immune restoration. This can only finally be tested in clinical trials, and several are planned. The basic ribozyme unit, the potential of which was described less than 10 years ago, is about to be tested in an amunable disease state.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Myeloproliferative disease and myelodysplastic syndrome induced by transplantation of bone marrow cells expressing mutant p53.

p53 mutations are the most common genetic alterations observed in human cancers including lymphomas and leukemias. We have previously shown that transduction of normal murine hematopoietic cells with mutant p53 alone in vitro results in an enhanced proliferative capacity and modified differentiation potential of transduced cells. In order to investigate further the role of mutant p53 in hematopoietic cell transformation, mutant p53-transduced bone marrow cells were used to reconstitute the hematopoietic system of lethally irradiated mice. The results show that overexpression of mutant p53 can initiate the transformation of immature murine hematopoietic cells in vivo and induce two types of hematopoietic disorders, myeloproliferative disease and myelodysplastic syndrome.

Animals↗
PubMed1997

Inhibition of Moloney murine leukaemia virus by a retroviral vector, LNL6, carrying ribozymes targeted to the 5' non-coding sequence.

The packaging region psi is the RNA sequence which directs the inclusion of genomic viral RNA into virions. As such it represents an apparently accessible site for ribozyme action. Ribozymes directed against the psi site of Moloney murine leukaemia virus (MoMLV) were delivered to target cells using a related retroviral vector, LNL6. LNL6 is a vector predominantly derived from MoMLV and, like all retroviruses, requires the psi region to be packaged. By exploiting the heterogeneity between nucleotide sequences of the MoMLV target and LNL6 vector in the psi packaging region, two ribozymes were designed and shown to selectively cleave the target but not the vector sequence. Clonally derived cell lines expressing ribozymes showed inhibition of virus replication. These results show the utility of catalytic RNA as specific antiviral agents.

3T3 Cells↗
PubMed1996

Apoptosis in v-myc transformation of myelomonocytic cells and its modulation by CSF-1.

c-myc is a proto-oncogene essential for cell growth. When activated, its expression can lead to uncontrolled cell proliferation, transformation and tumorigenesis. The cell line tEMmyc4 is a murine myelomonocytic cell line that was established following transformation by v-myc. It has a high level of v-myc expression and constitutively expresses endogenous CSF-1, the monocytic growth and viability factor. Under growth restricting conditions (high cell density serum deprivation, heat shock, or dexamethasone addition) cells of this line were found to undergo cell death through apoptosis. The induction of apoptosis by dexamethasone was associated with a decrease in constitutive CSF-1 expression without significant change in v-myc expression. Exogenous CSF-1 rescued these cells from dexamethasone induced-apoptosis. In vivo studies showed that tEMmyc4 cells were tumorigenic in syngeneic animals despite exhibiting some spontaneous apoptosis within the tumour mass. Co-administration of dexamethasone with the tumour cells significantly inhibited tumor development and the administration of dexamethasone to mice with established tumors resulted in tumor regression in all mice. This regression was associated with a high level of apoptosis and necrosis in the tumors. This study shows a correlation between the in vitro and in vivo induction of apoptosis and indicates that cancer cells bearing activated oncogenes may be more sensitive to apoptosis induction by chemotherapeutic agents.

Animals↗
PubMed1996

Retroviral transduction of hematopoietic progenitor cells with mutant p53 promotes survival and proliferation, modifies differentiation potential and inhibits apoptosis.

Mutations in the p53 tumor suppressor gene have been shown to be associated with many human tumors and various leukemias and lymphomas. To examine whether constitutive overexpression of mutant p53 can effect transformation of normal hematopoietic cells, a mutant p53 gene was introduced into normal murine bone marrow hematopoietic cells by retroviral gene transfer. Compared to vector alone-infected cells, hematopoietic progenitor cells transduced with mutant p53 showed increased proliferative potential, enhanced cloning efficiencies and a modified differentiation pattern in vitro. In addition, mutant p53-transduced hematopoietic cells were more resistant to loss of viability and/or induction of apoptosis when cultured in a low concentration of serum or in the absence of both growth factors and serum. These effects occurred rapidly with no apparent contributory secondary events. No permanent cell lines or growth factor-independent cell strains were obtained. The results indicate that introduction of mutant p53 into normal hematopoietic cells in vitro contributes to transformation, including enhanced proliferative potential, modified differentiation and the suppression of apoptosis in these cells.

Animals↗
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↗