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Biomedical subjects

R G Hawley

Publications and source records attributed to R G Hawley.

At least 73 records · Page 4Linked to original sources
PubMed1996

Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells.

Two mutants of the green fluorescent protein (GFP), RSGFP4 and GFPS65T, have been recently created which differ from the wildtype GFP of A. victoria in their excitation maxima. Here we show that human fibroblasts transfected with either of the two mutant GFP genes emit a green fluorescence that is 18-fold brighter than the cells transfected with the wildtype GFP gene. Retroviral vectors expressing the improved GFP gene were also constructed to determine their suitability for stable gene transduction into mammalian cells. The inclusion of the RSGFP4 gene in a retroviral vector did not reduce the viral titer and resulted in a fluorescent signal in viable transduced cells detectable by both fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Therefore, the improved mutant GFP provides a vital marker for monitoring gene transfer and expression in mammalian cells.

Animals↗
PubMed1995

Adhesion molecules involved in the binding of murine myeloma cells to bone marrow stromal elements.

In previous work, we reported the development of the B9/BMI syngeneic murine bone marrow metastasis model. Interleukin (IL)-6-dependent, IL-I-producing B9/BMI cells, which preferentially home to and colonise the vertebral and femoral marrow after i.v. injection, exhibit striking similarity in cell surface phenotype to human myeloma cells, especially the expression of 3 adhesion molecules, CD44, VLA-4 and ICAM-I. Because the haematopoietic microenvironment consists of different cell types, such as endothelial cells, fibroblasts, adipocytes and macrophages, we investigated the functional significance of these adhesion molecules in heterotypic binding assays between B9/BMI cells and a newly established bone marrow-derived endothelial cell line (BMEC), a fibroblastoid pre-adipocyte cell line (BMS2.2) and primary bone marrow-derived macrophages. B9/BMI cells adhered well to all stromal elements: a combination of monoclonal antibodies (MAbs) against CD44 and VLA-4 significantly inhibited the adherence of B9/BMI cells to BMEC and BMS2.2 cells, whereas binding of B9/BMI cells to macrophages was partially blocked with an anti-ICAM-I MAb. Our results implicate multiple recognition mechanisms, including those involving CD44, VLA-4 and ICAM-I, in the retention of B9/BMI cells in the bone marrow.

Animals↗
PubMed1995

Chronic exposure to retroviral vector encoded MGDF (mpl-ligand) induces lineage-specific growth and differentiation of megakaryocytes in mice.

Megakaryocyte growth and development factor (MGDF) has recently been identified as a ligand for the c-mpl receptor. Using retroviral-mediated gene transfer, MGDF has been overexpressed in mice to evaluate the systematic effects due to chronic exposure to this growth factor. MGDF overexpressing mice had more rapid platelet recovery than control mice after transplantation. Following this recovery, the platelet levels continued increasing to fourfold to eightfold above normal baseline levels and remained elevated (five-fold above control mice) in these animals, which are alive and well at more than 4 months posttransplantation. Increased megakaryocyte numbers were detected in a number of organs in these mice including bone marrow, spleen, liver, and lymph nodes. Prolonged overexpression of MGDF led to decreased marrow hematopoiesis, especially erythropoiesis, with a shift to extramedullary hematopoiesis in the spleen and liver. All the MGDF overexpressing mice analyzed to date developed myelofibrosis and osteosclerosis, possibly induced by megakaryocyte and platelet produced cytokines. No significant effect on other hematopoietic lineages was seen in the MGDF overexpressing mice, showing that the stimulatory effect of MGDF in vivo is restricted to the megakaryocyte lineage.

Animals↗
PubMed1995

Prevention of autoimmune disease by retroviral-mediated gene therapy.

T lymphocytes have been implicated in a variety of autoimmune diseases, and therefore one potential therapeutic approach would be to tolerize the pathogenic self-reactive T cells. In this study, we examined whether retroviral gene therapy could be used to induce tolerance and prevent autoimmunity using a transgenic mouse model for experimentally induced diabetes. In this model, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) is expressed on the beta-islet cells of the pancreas under the control of the rat insulin promoter (RIP). Previous work showed that the T cells specific for the gp remain unaware of the transgenic gp Ag expressed by the iselt cells, and infection with LCMV leads to immune-mediated diabetes. To tolerize the gp-specific pathogenic T cells, a retroviral vector (RV) expressing the LCMV gp was constructed, RV-gp. Replication-defective recombinant retroviruses were used to transduce bone marrow cells, which were subsequently infused into host RIP-gp transgenic animals. Unlike control animals, RV-gp chimeric animals did not possess T cells specific for the gp Ag as measured by proliferation and cytotoxic function, and further analysis suggested that tolerance of the gp-specific self-reactive T cells occurred by clonal deletion. Further experiments demonstrated that chimeric RIP-gp transgenic animals generated using bone marrow transduced with RV-gp did not develop experimentally induced diabetes. Our animal model demonstrates that retroviral gene therapy may cure immune-mediated diabetes by providing long lasting Ag-specific tolerance.

Animals↗
PubMed1995

Hematopoietic transforming potential of activated ras in chimeric mice.

Although activating mutations in ras genes are the most common genetic abnormality in human hematologic malignancies, the role of ras mutations as an initiating event in leukemogenesis remains unclear. To assess the consequences of ectopic expression of an activated ras gene in normal hematopoietic cells in vivo, lethally irradiated mice were reconstituted with bone marrow cells infected with a mutant ras-containing retrovirus [murine stem cell virus (MSCV)-v-H-ras] based on the MSCV retroviral vector which efficiently transduces functional genes into hematopoietic stem/progenitor cells. Despite a marked myeloid leukocytosis detectable in the peripheral blood within 4 weeks of engraftment, none of 22 primary or secondary transplant recipients studied for longer periods of time presented with myeloid neoplasms. Instead, 18 of the MSCV-v-H-ras mice developed pre-T-cell thymic lymphomas and/or pre-B-cell lymphoblastic leukemia/lymphomas between 7 and 12 weeks post-transplantation. The pre-B and pre-T lymphoid tumors that arose in one animal were shown to harbor a common MSCV-v-H-ras provirus, indicating that the target cell for transformation was a bipotential lymphoid precursor. To more precisely examine the effects of activated ras expression on the behavior of hematopoietic progenitors, infected bone marrow cells were assayed in methylcellulose cultures under conditions favorable for growth of multilineage myeloid colonies or were passaged as bulk suspension cultures in the presence of various hematopoietic growth factors, including interleukin (IL)-3, IL-4, IL-6 and IL-7. MSCV-directed expression of v-H-ras selectively promoted the formation of large dense colonies comprised of monocyte-macrophages in methylcellulose cultures. When transferred to liquid cultures, the vast majority of the cells underwent terminal macrophage differentiation. By comparison, tumorigenic B-lymphoid and mixed lymphoid/myeloid cell lines were routinely established from the bulk suspension cultures, with cell lines of predominantly myeloid phenotype emerging only in IL-6-supplemented cultures. These results, considered together with previous findings, suggest that activating ras mutations could be an initiating genetic alteration in human acute lymphoblastic leukemia but are more likely to be a post-initiation change in human acute myeloid leukemia.

Animals↗
PubMed1995

Prevention of myeloma cell apoptosis by ectopic bcl-2 expression or interleukin 6-mediated up-regulation of bcl-xL.

Upon cytokine withdrawal, interleukin (IL) 6-dependent murine plasmacytoma/hybridoma (myeloma) cells die in a way characteristic of apoptosis. Although gene transfer-mediated elevation in Bcl-2 protein levels has been demonstrated to repress a number of apoptotic death programs, it has been reported that ectopic bcl-2 expression is unable to prolong the survival of IL-6-deprived myeloma cells. In view of the recent identification of Bax as a protein that antagonizes the anti-apoptotic function of Bcl-2, we sought to determine whether the inability of transfected bcl-2 to protect against myeloma cell apoptosis might simply be due to insufficient levels of Bcl-2 protein produced to counteract this inhibitor. We show here that high-level expression of an exogenous bcl-2 gene, introduced into IL-6-dependent B9 myeloma cells via retroviral or bovine papilloma virus-based vectors, is indeed able to suppress apoptotic death following cytokine deprivation, with the extent of protection provided correlating with the amount of Bcl-2 protein synthesized in relation to the amount of endogenous Bax protein present in the cells. Of note, however, we found that IL-6-mediated suppression of B9 apoptosis does not involve induction of endogenous bcl-2 expression but is associated instead with the upregulation of cellular bcl-x mRNA and Bcl-xL protein. These results thus extend the apoptotic death mechanisms that are inhibitable by both bcl-2 and bcl-xL to include that operative in IL-6-dependent cells and suggest that apoptosis in other cell types using the gp130 subunit of the IL-6 receptor might also be bcl-2 regulable or bcl-xL dependent.

Animals↗
PubMed1995

SBH-1, a novel Reed-Sternberg-like cell line capable of inducing tumors in SCID mice: immunophenotypic, cytogenetic, and cytokine expression profiles.

A new cell line, SBH-1, with the morphologic, immunophenotypic, and karyotypic features consistent with those of Reed-Sternberg (RS) and Hodgkin (H) cells, has been established from the pleural effusion of a patient. The cytologic appearance of SBH-1 cells is characteristic of multinucleate RS and mononuclear H cells, all containing inclusion-like nucleoli. The SBH-1 cells express CD30, CD15, CD25, CD71, CD45, CD20, CD22, and bcl-2 protein and are negative for epithelial membrane antigen. Cytogenetic analysis showed multiple clonal abnormalities with breakpoints at 14q32, 6q21, and 11q23. The Ig heavy chain genes and both Ig light chain genes were rearranged in SBH-1 cells, whereas the bcl-2 gene was in germline configuration. Messages for the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha, and transforming growth factor-beta and the cytokine receptors IL-2R, IL-4R, IL-6R, and IL-7R were detected by reverse transcription-polymerase chain reaction analysis. Xenotransplantation of SBH-1 cells into severe combined immunodeficient (SCID) mice led to local and disseminated tumor growth. The cytologic, histologic, and immunohistochemical features of SBH-1 cells in SCID mouse tumors were typical of RS and H cells. The SBH-1 cell line will be useful in the study of RS and H cell biology, inasmuch as it represents a cell line obtained from a previously untreated patient.

Aged↗
PubMed1995

Retroviral insertional mutagenesis as a strategy for the identification of genes associated with cis-diamminedichloroplatinum(II) resistance.

Expression of resistance to cis-diamminedichloroplatinum(II) (CDDP), one of the most effective chemotherapeutic drugs used to treat a variety of malignancies, remains a serious obstacle for improving cancer treatment. To study possible genetic mechanisms underlying the development of CDDP resistance, we have adopted the approach of retroviral insertional mutagenesis. An early-stage CDDP-sensitive human melanoma cell line, WM35, was infected with a defective amphotropic murine retrovirus (murine stem cell virus), and the pooled cells were subsequently selected for CDDP-resistant variants. Nine CDDP-resistant clones independently derived from murine stem cell virus-infected WM35 cells were analyzed and it was found that five of these clones acquired an identical retroviral integration site, designated as CDDP resistance locus 1 (CRL-1), as revealed by isolation of retroviral flanking sequences. Furthermore, using the flanking sequence as probe, we have detected a 3.5-4.0-kilobase message, the expression of which is strongly increased in clones carrying a rearranged CRL-1 locus. These results strongly suggest that overexpression of CRL-1 confers resistance to CDDP in these clones. In addition, the present study indicates that retroviral insertional mutagenesis represents a potential strategy to identify genes responsible for CDDP resistance and possibly other chemotherapeutic drugs as well.

Cisplatin↗
PubMed1994

Tyrosine phosphorylation of JAK-TYK kinases in malignant plasma cell lines growth-stimulated by interleukins 6 and 11.

The pleiotropic cytokine interleukin (IL-6) is a major growth factor for murine plasmacytomas/hybridomas and human myeloma cells. Here we report that IL-6 stimulated different patterns of tyrosine phosphorylation of JAK-TYK kinases in IL-6-responsive murine (B9E and T10D) and human (ANBL-6 and OCI-My4) plasma cell tumor lines. Interestingly, the Stat91 transcription factor essential for interferon signaling mediated by JAK-TYK kinases was significantly tyrosine phosphorylated in response to IL-6 in ANBL-6 cells but not in the other cell lines. We further show that IL-11, a cytokine that signals via the gp130 subunit of the IL-6 receptor, induced similar profiles of JAK-TYK tyrosine phosphorylation as IL-6 in B9E and T10D cells. These results suggest that functionally redundant JAK-TYK kinase cascades triggered through gp130 are involved in the growth regulation of plasma cell neoplasms.

Animals↗
PubMed1994

Inhibition of nuclear hormone receptor activity by calreticulin.

We have shown that a polypeptide of M(r) 60,000 (60K) that shares N-terminal homology with a calcium-binding protein, calreticulin, can bind to an amino-acid sequence motif, KXGFFKR, found in the cytoplasmic domains of all integrin alpha-subunits. The homologous amino-acid sequence, KXFFKR (where X is either G, A or V), is also present in the DNA-binding domain of all known members of the steroid hormone receptor family; amino acids in this sequence make direct contact with nucleotides in their DNA-responsive elements and are crucial for DNA binding. Here we show that both the 60K protein (p60), purified on a KLGFFKR-Sepharose affinity matrix, and recombinant calreticulin can inhibit the binding of androgen receptor to its hormone-responsive DNA element in a KXFFKR-sequence-specific manner. Calreticulin can also inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation. Our results indicate that calreticulin can act as an important modulator of the regulation of gene transcription by nuclear hormone receptors.

Amino Acid Sequence↗
PubMed1994

Versatile retroviral vectors for potential use in gene therapy.

A set of retroviral vectors is described whose capacity for high efficiency transduction of functional genes into undifferentiated murine embryonic and haematopoietic cells makes them ideally suited for preclinical studies with murine models. Multiple unique cloning sites permit insertion of genes into the vectors such that no selectable marker exists or either the neomycin phosphotransferase (neo) gene, the hygromycin B phosphotransferase (hph) gene or the puromycin N-acetyl transferase (pac) gene is included as a dominantly acting selectable marker. Because the sequences in the viral gag region shown to improve the encapsidation of viral RNA have been modified to prevent viral protein synthesis and all env sequences have been removed to eliminate helper virus production by homologous recombination with packaging DNA, these vectors might prove useful in human gene therapy protocols.

Acetyltransferases↗
PubMed1994

Hematopathology of interleukin 6-type cytokines.

Interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) constitute a subfamily of cytokines on the basis of similar predicted structures and functional redundancy in their actions on a wide spectrum of cell types. Among the diverse targets of these pleiotropic factors are adipocytes, embryonic stem cells, hepatocytes, and cells belonging to the hematopoietic, immune and nervous systems. An explanation for the shared biologic activities of IL-6 subfamily members is provided by the finding that these cytokines trigger overlapping signal transduction pathways in responsive cells through a common receptor subunit, termed gp130. With the exception of CNTF, which appears to function primarily within the nervous system, IL-6-type cytokines differ from classical hormones in that they are synthesized by many tissues, frequently in association with disease states or in response to infection or injury. This article discusses the hematologic effects of dysregulated expression of IL-6 and related cytokines, highlighting results obtained in our laboratory with murine models of cytokine excess created by retroviral gene delivery to the hematopoietic system.

Amino Acid Sequence↗
PubMed1994

The HOX11 homeobox-containing gene of human leukemia immortalizes murine hematopoietic precursors.

The t(10;14) chromosomal translocation of T-cell acute lymphoblastic leukemia joins the T-cell receptor delta gene to a region upstream of a diverged homeobox-containing gene called HOX11. To better understand the pathogenetic role of HOX11 in leukemogenesis, post 5-fluorouracil-treated murine bone marrow cells were infected with a replication-defective retrovirus bearing the gene. Constitutive expression of HOX11 in hematopoietic precursors yielded cell lines at high frequency consisting of immature cells belonging to the myeloid lineage. HOX11-transformed cell lines displayed a strict dependence on IL-3 for their survival and proliferation in culture and were not leukemogenic. The results support the hypothesis that the transforming capacity of HOX11 derives from its ability to alter the expression of genes regulating hematopoietic differentiation and that secondary mutations promoting cell survival or stimulating proliferation are required for progression to malignancy. The findings further suggest that the oncogenic activity of HOX11 might not be restricted to T-cell leukemias in humans.

Animals↗
PubMed1993

Progenitor cell hyperplasia with rare development of myeloid leukemia in interleukin 11 bone marrow chimeras.

Post 5-fluorouracil-treated murine bone marrow cells infected with a recombinant retrovirus (murine stem cell virus-interleukin 11 [MSCV-IL-11]) bearing a human IL-11 gene were transplanted into lethally irradiated syngeneic mice. Analysis of proviral integration sites in DNA prepared from hematopoietic tissues and purified cell populations of long-term reconstituted primary and secondary recipients demonstrated polyclonal engraftment by multipotential stem cells. High levels (100-1,500 U/ml) of IL-11 were detected in the plasma of the MSCV-IL-11 mice. Systemic effects of chronic IL-11 exposure included loss of body fat, thymus atrophy, some alterations in plasma protein levels, frequent inflammation of the eyelids, and often a hyperactive state. A sustained rise in peripheral platelet levels (approximately 1.5-fold) was seen throughout the observation period (4-17 wk). No changes were observed in the total number of circulating leukocytes in the majority of the transplanted animals (including 10 primary and 18 secondary recipients) despite a > 20-fold elevation in myeloid progenitor cell content in the spleen. The exceptions were members of one transplant pedigree which presented with myeloid leukemia during the secondary transplant phase. A clonal origin of the disease was determined, with significant expansion of the MSCV-IL-11-marked clone having occurred in the spleen of the primary host. Culturing of leukemic spleen cells from a quaternary recipient led to the establishment of a permanent cell line (denoted PGMD1). IL-11-producing PGMD1 myeloid leukemic cells are dependent on IL-3 for continuous growth in vitro and they differentiate into granulocytes and macrophages in response to granulocyte/macrophage colony-stimulating factor. The inability of autogenously produced IL-11 to support autonomous growth of PGMD1 cells argues against a mechanism of transformation involving a classical autocrine loop.

Animals↗