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Biomedical subjects

R G Hawley

Publications and source records attributed to R G Hawley.

At least 19 recordsLinked to original sources
PubMed2007

TLX1/HOX11-induced hematopoietic differentiation blockade.

Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.

Acetylation↗
PubMed2003

Improved therapeutic outcome following combination immunogene vaccination therapy in murine myeloma.

Increasing evidence suggests a role for immunologic vaccination and therapy in the management of minimal residual myeloma. We have previously demonstrated a synergistic effect of combining the Th1 stimulating cytokine IL-12 with the co-stimulatory molecule CD80 in murine myeloma vaccination therapy. We reasoned that the efficacy of such treatment might be further improved by incorporating additional gene products which enhance the function of antigen presenting cells. Studies were therefore conducted with murine myeloma BM1 cells expressing Flt3L (membrane bound or soluble forms) or GM-CSF and the IL-12 x CD80 combination. Single agent and combined therapeutic approaches were explored. All gene-modified BM1 cells, except BM1/IL-12 x CD80, developed tumors when subcutaneously injected into BALB/c mice. As prophylactic tumor vaccines, the combined use of gene-modified BM1/sFlt3L+GM-CSF+IL-12 x CD80 was most effective, providing 100% protection against subsequent parental BM1 tumor challenge. By comparison, only partial protection was observed with any single gene-engineered tumor vaccine. Notably, IL-12 x CD80 coexpressing BM1 cell vaccines were the most effective therapeutic vaccine in a minimal disease model. Such protective vaccination was achieved by stimulation of lymphocyte proliferation and enhancement of cytotoxic lymphocyte activity.

Adjuvants, Immunologic↗
PubMed2001

Rhesus monkey placental transgene expression after lentiviral gene transfer into preimplantation embryos.

Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal-fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture of in vitro fertilized ova were transduced with a self-inactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription-PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term = 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.

Animals↗
PubMed2001

The myeloma-associated oncogene fibroblast growth factor receptor 3 is transforming in hematopoietic cells.

Translocations involving fibroblast growth factor receptor 3 (fgfr3) have been identified in about 25% of patients with myeloma. To directly examine the oncogenic potential of fgfr3, murine bone marrow (BM) cells were transduced with retroviral vectors containing either wild-type fgfr3 or an activated mutant form of the receptor, fgfr3-TD. Mice transplanted with FGFR3-TD-expressing BM developed a marked leukocytosis and lethal hematopoietic cell infiltration of multiple tissues within 6 weeks of transplantation. Secondary and tertiary recipients of spleen or BM from primary fgfr3-TD mice also developed tumors within 6 to 8 weeks. Analysis of the circulating tumor cells revealed a pre-B-cell phenotype in most mice, although immature T-lymphoid or mature myeloid populations also predominated in some animals. Enhanced lymphoid but not myeloid colony formation was observed in the early posttransplantation period and only interleukin 7 and FGF-responsive pre-B-cell lines could be established from tumors. Cell expansions in primary recipients appeared polyclonal, whereas tumors in later passages exhibited either clonal B- or T-cell receptor gene rearrangements. Mice transplanted with wild-type FGFR3-expressing BM developed delayed pro-B-cell lymphoma/leukemias approximately 1 year after transplantation. These studies confirm that FGFR3 is transforming and can produce lymphoid malignancies in mice.

Animals↗
PubMed2001

Allelic exclusion and differentiation by protein kinase C-mediated signals in immature thymocytes.

Pre-T cell receptor (preTCR)-derived signals mediate the transition of thymocytes from the CD4(-) CD8(-) double-negative (DN) to CD4(+) CD8(+) double-positive stage of T lymphocyte development. This progression, termed beta-selection, is limited to thymocytes that have generated a functional TCR-beta chain able to associate with pTalpha to form the preTCR complex. Formation of the preTCR complex not only induces differentiation, survival, and proliferation of DN thymocytes; it also inhibits further TCR-beta gene rearrangement through an ill-defined process known as allelic exclusion. The signaling pathways controlling this critical developmental checkpoint have not been characterized. Here we demonstrate that formation of the preTCR complex leads to the activation of protein kinase C (PKC), and that activation of PKC is necessary for the differentiation and expansion of DN thymocytes. Importantly, we also show that allelic exclusion at the TCR-beta gene loci is enforced by PKC-mediated signals. These results define PKC as a central mediator of both differentiation and allelic exclusion during thymocyte development.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Fibronectin fragment CH-296 inhibits apoptosis and enhances ex vivo gene transfer by murine retrovirus and human lentivirus vectors independent of viral tropism in nonhuman primate CD34+ cells.

The fibronectin fragment CH-296 improved gene transfer to cytokine-mobilized nonhuman primate CD34+ cells irrespective of tropism to the MoMLV, GaLV, and VSV-G envelope proteins using murine stem cell virus (MSCV) and human immunodeficiency virus-1 (HIV-1)-based retrovirus vectors. For the HIV-1 lentivirus vector, CH-296 enhanced gene transfer in the absence of added hematopoietic growth factors necessary for ex vivo stem cell expansion. In the presence of CH-296, apoptosis of CD34+ cells was inhibited, and in mobilized peripheral blood CD34+ cells, cell division was stimulated as measured by cell history/tracking experiments.

Animals↗
PubMed2001

Identification and characterization of Magmas, a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction.

OBJECTIVE: The aim of this study was to identify granulocyte-macrophage colony-stimulating factor (GM-CSF) responsive genes. MATERIALS AND METHODS: Potential GM-CSF responsive genes were identified by comparing the mRNA expression pattern of the murine myeloid cell line PGMD1 grown in either interleukin-3 (IL-3) or GM-CSF by differential display. Human and murine cDNA clones of one of the bands having increased expression in GM-CSF were isolated. mRNA expression of the gene was examined by Northern blot. Immunohistochemistry and studies with a green fluorescent fusion protein were used to determine its intracellular location. Growth factor-stimulated proliferation of PGMD1 cells transfected with constitutively expressed sense and anti-sense cDNA constructs of the gene was measured by 3H-thymidine incorporation. RESULTS: A gene, named Magmas (mitochondria-associated granulocyte macrophage CSF signaling molecule), was shown to be rapidly induced when cells were switched from IL-3 to GM-CSF. Analysis of the amino acid sequence of Magmas showed it contained a mitochondrial signal peptide, but not any other known functional domains. The human and murine clones encode nearly identical 13-kDa proteins that localized to the mitochondria. Magmas mRNA expression was observed in all tissues examined. PGMD1 cells that overexpressed Magmas proliferated similarly to untransfected cells when cultured in IL-3 or GM-CSF. In contrast, cells with reduced protein levels grew normally in IL-3, but had impaired proliferation in GM-CSF. CONCLUSION: Magmas is a mitochondrial protein involved in GM-CSF signal transduction.

Amino Acid Sequence↗
PubMed2001

Tetracycline-regulatable adenovirus vectors: pharmacologic properties and clinical potential.

Stringent control of gene expression in human gene therapy strategies is important for both therapeutic and safety reasons. Replication-defective vectors derived from adenoviruses have been shown to be capable of highly efficient in vivo gene delivery to a wide variety of dividing and nondividing human cells. Here, we review the progress in the development of regulatable adenovirus vectors that allow gene expression to be tightly controlled by low concentrations of tetracyclines. As an example of the potential clinical utility of this technology, we highlight our results obtained in an immunotherapy model for prostate cancer with a tetracycline-regulatable adenovirus vector expressing the cytokine interleukin-12. Recombinant adenovirus vectors with tetracycline-regulatable gene expression provide new opportunities and improved safety for gene therapy applications in humans.

Adenoviridae↗
PubMed2001

Tricistronic viral vectors co-expressing interleukin-12 (1L-12) and CD80 (B7-1) for the immunotherapy of cancer: preclinical studies in myeloma.

Synergy between interleukin-12 (IL-12) and B7-1 (CD80) for cancer immunotherapy has previously been demonstrated in animal models of breast cancer, lymphoma, and multiple myeloma. With a view to human clinical application, tricistronic retroviral and adenovirus vectors co-expressing IL-12 (IL-12p40 plus IL-12p35) and CD80 were constructed by utilizing two internal ribosome entry site (IRES) sequences to link the three cDNAs. A murine stem cell virus (MSCV)-based retroviral vector (MSCV-hIL12.B7) utilized distinct IRES sequences from the encephalomyocarditis virus (EMCV) and the foot-and-mouth disease virus (FMCV), whereas Ad5-based adenovirus vectors contained transcriptional units with two EMCV IRES sequences under the control of murine (AdMh12.B7) or human (AdHh12.B7) cytomegalovirus promoters. AdMh12.B7 was found to consistently direct higher levels of IL-12 and CD80 expression than AdHh12.B7 following infection of a number of human tumor cell lines. In preclinical studies, the human myeloma cell line U266 was infected with MSCV-hIL12.B7 and a resulting clonal cell line, U/MSCV-h12.B7, was generated with stable expression of CD80 and secreting IL-12 at 1 ng/24 h/10(6) cells. By comparison, following AdMh12.B7 infection, 81% of infected U266 cells (U/AdMh12.B7) expressed CD80 and secreted IL-12 at 25-50 ng/24 h/10(6) cells. Both engineered myeloma cell lines stimulated enhanced allogeneic mixed lymphocyte proliferation and provoked increases in cytotoxic T-lymphocyte responses and gamma-interferon release from normal donor lymphocytes exposed to parental U266 cells. These results suggest potential clinical utility of AdMh12.B7 in immunotherapy strategies for the treatment of multiple myeloma and other cancers.

Adenoviridae↗
PubMed2001

"Rainbow" reporters for multispectral marking and lineage analysis of hematopoietic stem cells.

Hematologic diseases potentially benefiting from gene-based therapies involving hematopoietic stem cells (HSCs) include hereditary hemoglobinopathies, immunodeficiency syndromes, and congenital bleeding disorders such as hemophilia A, as well as acquired diseases like AIDS. Successful treatment of these blood diseases with gene-modified HSCs requires high efficiency gene delivery to the target cell population and persistence of transgene expression following differentiation. We review flow cytometric procedures that permit simultaneous, noninvasive measurements of transgene expression and phenotypic discrimination of hematopoietic cell subsets. Central to this approach has been the recent development of a spectrum of blue, cyan, and yellowish-green fluorescent reporters based on the jellyfish Aequorea victoria green fluorescent protein and the discovery of a red fluorescent protein in DISCOSOMA: coral. This methodology should facilitate the optimization of oncoretroviral and lentiviral vectorology and HSC transduction protocols for the ultimate purpose of HSC-directed gene therapy.

Animals↗
PubMed2001

High levels of transgene expression following transduction of long-term NOD/SCID-repopulating human cells with a modified lentiviral vector.

Both oncoretroviral and lentiviral vectors have been shown to transduce CD34(+) human hematopoietic stem cells (HSC) capable of establishing human hematopoiesis in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice that support partially human hematopoiesis. We and others have reported that murine stem cell virus (MSCV)-based oncoretroviral vectors efficiently transduced HSC that had been cultured ex vivo for 4-7 days with cytokines, resulting in transgene expression in lymphoid and myeloid progenies of SCID-engrafting cells 4-8 weeks post-transplantation. Although lentiviral vectors have been demonstrated to transduce HSC under minimal ex vivo culture conditions, concerns exist regarding the level of transgene expression mediated by these vectors. We therefore evaluated a novel hybrid lentiviral vector (GIN-MU3), in which the U3 region of the HIV-1 long terminal repeat was replaced by the MSCV U3 region (MU3). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G envelope protein-pseudotyped lentiviruses during a 48-hour culture period. After a total of 4 days in culture, transduced cells were transplanted into NOD/SCID mice to examine gene transfer and expression in engrafting human cells. Fifteen weeks post-transplantation, 37% +/- 12% of engrafted human cells expressed the green fluorescence protein (GFP) gene introduced by the lentiviral vector. High levels of GFP expression were observed in lymphoid, myeloid and erythroid progenies, and in engrafted human cells that retained the CD34(+) phenotype 15 weeks post-transplantation. This study provides evidence that lentiviral vectors transduced both short-term and long-term engrafting human cells, and mediated persistent transgene expression at high levels in multiple lineages of hematopoietic cells.

Animals↗
PubMed2001

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins.

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.

3T3 Cells↗
PubMed2001

Progress toward vector design for hematopoietic stem cell gene therapy.

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy because of their capacity for self renewal and the wide systemic distribution of their progeny. Sustained expression of transgenes at clinically relevant levels in the progeny of HSCs would provide novel and potentially curative treatments for a wide range of inherited and acquired blood diseases. Recent improvements in retroviral transduction protocols have resulted in the first successful amelioration of a human hematologic disease--a form of severe combined immunodeficiency--by HSC gene transfer. However, continued advances in gene transfer technology are necessary if the inherent promise of HSC gene therapy is to be fully realized. Ongoing efforts are focused on modifying oncoretroviral vector designs and pseudotyping with alternative envelope proteins. In addition, because of their ability to transduce non-divided cells, safety-modified human immunodeficiency virus-1-based lentiviral vectors have emerged as promising tools for gene modification of HSCs, which reside primarily in the G0/G1 phase of the cell cycle. Irrespective of these advances, accumulated data indicate that stably integrated transgenes are frequently subject to position-effect variegation and extinction of expression. Therefore, the extent to which genetic control elements such as chromatin domain insulators and scaffold/matrix attachment regions in conjunction with posttranscriptional regulatory elements will result in enhanced probability and level of transgene expression is under active investigation. Collectively, these developments increase the likelihood that HSC gene transfer will ultimately become an effective therapeutic strategy.

Binding Sites↗
PubMed2000

The NFAT-related protein NFATL1 (TonEBP/NFAT5) is induced upon T cell activation in a calcineurin-dependent manner.

NFAT DNA binding complexes regulate programs of cellular activation and differentiation by translating receptor-dependent signaling events into specific transcriptional responses. NFAT proteins, originally defined as calcium/calcineurin-dependent regulators of cytokine gene transcription in T lymphocytes, are expressed in many different cell types and represent critical signaling intermediates that mediate an increasingly wide spectrum of biologic responses. Recent studies have identified a novel protein containing a region of similarity to the NFAT DNA binding domain. Here we demonstrate that this protein, designated NFATL1 (also known as tonicity enhancer binding protein and NFAT5) is expressed at high levels in the thymus but is undetectable in mature lymphocytes. However, NFATL1 can be induced in both primary quiescent T lymphocytes and differentiated Th1 and Th2 cell populations upon mitogen- or Ag receptor-dependent activation. The induction of NFATL1 protein, as well as NFATL1-dependent transcription, is inhibited by cyclosporin A and FK506, and expression of constitutively active calcineurin induces NFATL1-dependent transcription. Overexpression of NFATc1 and inhibition of NFATc activity through the use of a dominant negative NFATc1 protein have no affect on NFATL1-dependent transcription, indicating that NFATc proteins do not play a role in the calcineurin-dependent induction of NFATL1. Interestingly, induction of NFATL1 by a hyperosmotic stimulus is not blocked by the inhibition of calcineurin. Moreover, osmotic stress response genes such as aldose reductase are not induced upon T cell activation. Thus inducible expression of NFATL1 represents a mechanism by which receptor-dependent signals as well as osmotic stress signals are translated into transcriptional responses that regulate cell function.

Aldehyde Reductase↗
PubMed2000

Ectopic expression of fibroblast growth factor receptor 3 promotes myeloma cell proliferation and prevents apoptosis.

The t(4;14) translocation occurs in 25% of multiple myeloma (MM) and results in both the ectopic expression of fibroblast growth factor receptor 3 (FGFR3) from der4 and immunoglobulin heavy chain-MMSET hybrid messenger RNA transcripts from der14. The subsequent selection of activating mutations of the translocated FGFR3 by MM cells indicates an important role for this signaling pathway in tumor development and progression. To investigate the mechanism by which FGFR3 overexpression promotes MM development, interleukin-6 (IL-6)-dependent murine B9 cells were transduced with retroviruses expressing functional wild-type or constitutively activated mutant FGFR3. Overexpression of mutant FGFR3 resulted in IL-6 independence, decreased apoptosis, and an enhanced proliferative response to IL-6. In the presence of ligand, wild-type FGFR3-expressing cells also exhibited enhanced proliferation and survival in comparison to controls. B9 clones expressing either wild-type FGFR3 at high levels or mutant FGFR3 displayed increased phosphorylation of STAT3 and higher levels of bcl-x(L) expression than did parental B9 cells after cytokine withdrawal. The mechanism of the enhanced cell responsiveness to IL-6 is unknown at this time, but does not appear to be mediated by the mitogen-activated protein kinases SAPK, p38, or ERK. These findings provide a rational explanation for the mechanism by which FGFR3 contributes to both the viability and propagation of the myeloma clone and provide a basis for the development of therapies targeting this pathway.

Apoptosis↗
PubMed2000

Expansion of human cord blood CD34(+)CD38(-) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function.

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin(-) CD34(+)CD38(-) cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin(-)CD34(+) CD38(-) cells and SRC, CD34(+)-enriched lineage-depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34(+)CD38(-) cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP(+) human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34(+)CD38(-) phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34(+)CD38(-) phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34(+)CD38(+) cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

ADP-ribosyl Cyclase↗
PubMed2000

Lentiviral vectors for enhanced gene expression in human hematopoietic cells.

Accumulated data indicate that current generation lentiviral vectors, which generally utilize an internal human cytomegalovirus (CMV) immediate early region enhancer-promoter to transcribe the gene of interest, are not yet optimized for efficient expression in human hematopoietic stem/progenitor cells (HSPCs). As a first step toward this goal, we constructed self-inactivating derivatives of the HIV-1-based transfer vector pHR' containing the enhanced green fluorescent protein (GFP) gene as reporter and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). GFP expression was driven by a variety of strong viral and cellular promoters, including the murine stem cell virus (MSCV) long terminal repeat (LTR), a Gibbon ape leukemia virus (GALV) LTR, the human elongation factor 1alpha (EF1alpha) promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), and the human phosphoglycerate kinase 1 (PGK) promoter. In contrast to results obtained in human embryonic kidney 293T cells and fibrosarcoma HT1080 cells, in which the CMV promoter expressed GFP at the highest levels, significantly higher levels of GFP expression (3- to 5-fold) were achieved with the MSCV LTR, the EF1alpha promoter, and the CAG promoter in the human HSPC line KG1a. Removal of the WPRE indicated that it stimulated GFP expression from all of the vectors in KG1a cells (up to 3-fold), although it only marginally improved the performance of the intron-containing EF1alpha and CAG promoters (<1.5-fold stimulation). The vectors using the MSCV LTR, the GALV LTR, and the PGK promoter were the most efficient at transducing primary human CD34+ cord blood progenitors under the conditions employed. High-level GFP expression in the NOD/SCID xenograft model was demonstrated with the pHR' derivative bearing the MSCV LTR. These new lentiviral vector backbones provide a basis for the rational design of improved delivery vehicles for human HSPC gene transfer applications.

Antigens, CD34↗