PubMed Health⌕ Search

Biomedical subjects

Robert G Hawley

Publications and source records attributed to Robert G Hawley.

At least 19 recordsLinked to original sources
PubMed2006

Stable gammaretroviral vector expression during embryonic stem cell-derived in vitro hematopoietic development.

Unlike conventional gammaretroviral vectors, the murine stem cell virus (MSCV) can efficiently express transgenes in undifferentiated embryonic stem cells (ESCs). However, a dramatic extinction of expression is observed when ESCs are subjected to in vitro hematopoietic differentiation. Here we report the construction of a self-inactivating vector from MSCV, MSinSB, which transmits an intron embedded within the internal transgene cassette to transduced cells. The internal transgene transcriptional unit in MSinSB comprises the composite cytomegalovirus immediate early enhancer-chicken beta-actin promoter and associated 5' splice site positioned upstream of the natural 3' splice site of the gammaretroviral envelope gene, and is configured such that the transgene translational initiation sequence is coincident with the envelope ATG. MSinSB could be produced at titers approaching 10(6) transducing units/ml and directed higher levels of transgene expression in ESCs than a splicing-optimized MSCV-derived vector, MSGV1. Moreover, when transduced ESCs were differentiated into hematopoietic cells in vitro, MSinSB remained transcriptionally active in greater than 90% of the cells, whereas MSGV1 expression was almost completely shut off. Persistent high-level expression of the MSinSB gammaretroviral vector was also demonstrated in murine bone marrow transplant recipients and following in vitro myelomonocytic differentiation of human CD34(+) cord blood stem/progenitor cells.

Animals↗
PubMed2006

Hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols, the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines.

Animals↗
PubMed2006

TLX1/HOX11-mediated disruption of primary thymocyte differentiation prior to the CD4+CD8+ double-positive stage.

The TLX1/HOX11 homeobox gene is frequently activated in T-cell acute lymphoblastic leukaemia (T-ALL) by the t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations or by as yet unknown transcriptional mechanisms in the absence of 10q24 cytogenetic abnormalities. Almost all TLX1(+) T-ALLs exhibit a CD4(+)CD8(+) double-positive (DP) phenotype. To investigate the role of TLX1 as an initiating oncogene in T-ALL pathogenesis, we assessed the consequences of retroviral vector-directed TLX1 expression during the differentiation of murine and human thymocytes in fetal thymic organ cultures. Interestingly, enforced expression of TLX1 disrupted the differentiation of murine fetal liver precursors and human cord blood CD34(+) stem/progenitor cells prior to the DP thymocyte stage. Although differentiation arrest was associated with an increased percentage of apoptotic thymocytes, it could only be partially bypassed by coexpression of transgenic BCL2. Mutation of the invariant asparagine residue at position 51 of the homeodomain - which is required for efficient DNA binding - released the block, consistent with the notion that TLX1 inhibits thymocyte differentiation and promotes T-cell oncogenesis by functioning as a transcription factor. The relevance of these findings is discussed in the context of activating NOTCH1 mutations and the other genetic lesions implicated in the multistep transformation process of TLX1(+) T-ALL.

Animals↗
PubMed2005

Correction of murine hemophilia A by hematopoietic stem cell gene therapy.

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally delivered FVIII transgene offers the possibility of permanently correcting the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based FVIII oncoretroviral vector into immunocompetent hemophilia A mice that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy), or a nonmyeloablative preparative regimen involving busulfan. Therapeutic levels of FVIII (42, 18, and 11% of normal, respectively) were detected in the plasma of the transplant recipients for the duration of the study (over 6 months). Moreover, subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII inhibitor antibody response in any of the experimental animals, in contrast to the vigorous neutralizing humoral reaction to FVIII that was stimulated in naive hemophilia A mice. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.

Animals↗
PubMed2005

Bypass of senescence, immortalization, and transformation of human hematopoietic progenitor cells.

We attempted to extend the lifespan of CD34+ stem/progenitor cells in human cord blood (CB) by transduction with lentiviral vectors carrying the human telomerase catalytic subunit (hTERT) and/or the human papillomavirus type 16 (HPV16) E6 and E7 oncogenes. We found that hTERT was incapable of prolonging the replicative capacity of CB cells maintained under serum-free conditions in the presence of stem cell factor, Flt3 ligand, thrombopoietin, and interleukin-3 beyond 4 months (n=3). However, transduced CB cells cultured in the same cytokine cocktail constitutively expressing HPV16 E6/E7 alone (n=2) or in concert with hTERT (n=9) continued to proliferate, giving rise to permanent (>2 years) cell lines with a CD45+ CD34- CD133+/- CD44+ CD235a+ CD71+ CD203+ CD33+ CD13+ myeloerythroid/mast cell progenitor phenotype. Notably, CB cell cultures expressing only HPV16 E6/E7 went through a crisis period, and the resulting oligoclonal cell lines were highly aneuploid. By comparison, the CB cell lines obtained by coexpression of HPV16 E6/E7 plus hTERT exhibited near-diploid karyotypes with minimal chromosomal aberrations, concomitant with stabilization of telomere length, yet were clonally derived. The immortalized E6/E7 plus hTERT-expressing CB cells were not tumorigenic when injected intravenously or subcutaneously into sublethally irradiated immunodeficient nonobese diabetic/severe combined immunodeficient mice but could be converted to a malignant state by ectopic expression of a v-H-ras or BCR-ABL oncogene. These findings provide new insights into the mechanisms governing the senescence checkpoint of primitive human hematopoietic precursors and establish a paradigm for studies of the multistep process of human leukemogenesis.

Antigens, CD34↗
PubMed2005

G1/S transcriptional networks modulated by the HOX11/TLX1 oncogene of T-cell acute lymphoblastic leukemia.

The HOX11/TLX1 homeobox gene is aberrantly expressed in a subset of T-cell acute lymphoblastic leukemia (T-ALL). Here, we employed oligonucleotide microarrays to compare the expression profiles of the K3P and Sil leukemic cell lines originating from patients with HOX11+ T-ALL to that of Jurkat cells, which originated from a distinct subtype of T-ALL (TAL1+). To distinguish potential HOX11 target genes from those characteristic of the stage of HOX11 leukemic arrest, we also performed gene expression analysis on Jurkat cells, genetically engineered to express exogenous HOX11. The resulting HOX11 gene expression signature, which was validated for representative signaling pathways by transient transfection of reporter constructs, was characterized by elevated expression of transcriptional programs involved in cell proliferation, including those regulated by E2F, c-Myc and cAMP response element-binding protein. We subsequently showed that ectopic HOX11 expression resulted in hyperphosphorylation of the retinoblastoma protein (Rb), which correlated with inhibition of the major Rb serine/threonine phosphatase PP1. HOX11 also inhibited PP2A serine/threonine phosphatase activity concomitant with stimulation of the AKT/PKB signaling cascade. These results suggest that transcriptional deregulation of G1/S growth-control genes, mediated in large part through blockade of PP1/PP2A phosphatase activity, plays an important role in HOX11 pathobiology.

Animals↗
PubMed2005

Specific pharmacological dimerization of KDR in lentivirally transduced human hematopoietic cells activates anti-apoptotic and proliferative mechanisms.

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context, little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional, biochemical, and microarray analyses. When deprived of natural ligands, GM-CSF, interleukin-3, or erythropoietin, AP20187 prevented apoptosis of transduced TF1 cells, induced dose-dependent proliferation, and supported long-term growth. In addition, AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably, mRNAs affiliated with heat stress, including Hsp70 and Hsp105, were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence, in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor, or KDR, signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes.

Apoptosis↗
PubMed2005

Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.

The genes for the alpha and beta chains of a highly reactive anti-MART-1 T-cell receptor were isolated from T-lymphocytes that mediated in vivo regression of tumor in a patient with metastatic melanoma. These genes were cloned and inserted into MSCV-based retroviral vectors. After transduction, greater than 50% gene transfer efficiency was demonstrated in primary T-lymphocytes stimulated by an anti-CD3 antibody. The specificity and biologic activity of TCR gene-transduced T-cells was determined by cytokine production after coculture of T-cells with stimulator cells pulsed with MART-1 peptide. The production of interferon-gamma and granulocyte macrophage-colony stimulating factor (GM-CSF) was comparable to highly active MART-1 specific peripheral blood lymphocytes (PBL) in the amount of cytokine produced and transduced cells recognized peptide pulsed cells at dilutions similar to cytotoxic T lymphocyte (CTL) clones. Human leukocyte antigen (HLA) class I restricted recognition was demonstrated by mobilization of degranulation marker CD107a, by cell lysis, by cytokine production, and by proliferation in the presence of HLA-A2-positive but not HLA-A2-negative melanoma cell lines. Similar data was obtained when tumor-infiltrating lymphocytes (TIL) were transduced with the TCR genes, converting previously nonreactive cells to tumor reactive cells. TCR-transduced T-cells are thus attractive candidates for evaluation in cell transfer therapies of patients with cancer.

Antigens, CD↗
PubMed2005

Retroviral transduction in fetal thymic organ culture.

T-cell development requires cytokines and intimate contact with stromal cells provided exclusively by the thymus. Consequently, an in vitro model of thymocyte differentiation, fetal thymic organ culture (FTOC), has been developed. FTOC recapitulates the normal development of T-cells derived from both mouse and human progenitor populations, providing a more rapid means to study T-cell development compared with alternative in vivo approaches. Furthermore, FTOC is easily amenable to genetic manipulation using retroviral gene transfer. In this chapter, we outline the basic FTOC technique and describe several applications, including retroviral transduction of mouse thymocyte subsets and human CD34+ stem/progenitor cells.

Animals↗
PubMed2004

Tissue inhibitor of matrix metalloproteinase-1 overexpression in M1 myeloblasts impairs IL-6-induced differentiation.

The balance between matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) is important for extracellular matrix interactions of hematopoietic cells. MMP-independent growth modulating activity for TIMP-1 on B lymphocytes and erythroid progenitors has also been described, but a role for TIMP-1 in myelomonocytic differentiation has not been previously reported. In this study, we demonstrate that TIMP-1 overexpression impairs differentiation of the myeloblastic M1 cell line following interleukin (IL)-6 stimulation. We generated retroviral vectors coexpressing human TIMP-1 and the green fluorescent protein (GFP) and stably transduced murine M1 myeloid cells. TIMP-1 expressing cells showed a large reduction in IL-6-induced macrophage differentiation in vitro that was reversible with a specific monoclonal antibody. The differentiation delay in M1/TIMP-1 cells was also specifically reversible by pharmacologic phosphatidylinositol-3 kinase (PI3-K) inhibition. Additionally, overexpression of a TIMP-1/GFP fusion protein also impaired M1 differentiation and this protein was localized to the cell surface, consistent with an autocrine receptor-mediated mechanism. Surprisingly, TIMP-1 transduced cells had a selective advantage for growth in IL-6, indicating that functional effects on growth and differentiation of M1 cells were primarily through an autocrine mechanism. Intrinsic TIMP-1 expression in myeloid leukemia cells might thus impact upon survival or differentiation.

Bone Marrow Cells↗
PubMed2004

Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells.

Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation. Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen. An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment. Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks). Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified. Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A.

Animals↗
PubMed2004

Hematopoietic cell fate and the initiation of leukemic properties in primitive primary human cells are influenced by Ras activity and farnesyltransferase inhibition.

The Ras pathway transduces divergent signals determining normal cell fate and is frequently activated in hematopoietic malignancies, but the manner in which activation contributes to human leukemia is poorly understood. We report that a high level of activated H-Ras signaling in transduced primary human hematopoietic progenitors reduced their proliferation and enhanced monocyte/macrophage differentiation. However, the exposure of these cells to a farnesyltransferase inhibitor and establishment of a moderate level of Ras activity showed increased proliferation, an elevated frequency of primitive blast-like cells, and progenitors with enhanced self-renewal capacity. These results suggest that the amplitude of Ras pathway signaling is a determinant of myeloid cell fate and that moderate Ras activation in primitive hematopoietic cells can be an early event in leukemogenesis.

Alkyl and Aryl Transferases↗
PubMed2004

SCID repopulating cells derived from unmobilised adult human peripheral blood.

Severe combined immunodeficient (SCID)-repopulating cells (termed SRC) with lymphohaematopoietic differentiation potential reside at an extremely low frequency in unmobilised adult human peripheral blood. Recently, an ex vivo method of increasing the relative numbers of at least four distinct human stem cell classes, that include CD34+ haematopoietic progenitor cells, in mononuclear cells (MNC) obtained from unmobilised adult human peripheral blood has been described. This process is triggered by a monoclonal antibody (mAb) against the human monomorphic region of the beta chain of HLA-DP, DQ and DR (clone CR3/43). Herein, we assess the ability of human male donor-derived MNC, following ex vivo culturing for 3 hr in haematopoietic-conducive conditions (HCC) (3-hr MNC/HCC), to form SRC in female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. All 3-hr MNC/HCC-recipient animals exhibited significant levels (> 0.5%) of human cell engraftment in the bone marrow, thymus and spleen when compared to animals receiving MNC cultured in the absence of CR3/43. Phenotypic characterisation of the bone marrow cell populations of engrafted mice demonstrated significant levels of human lymphohaematopoietic cell lineages, comprised of T lymphocytes, monocytes, erythrocytes and megakaryocytes, including platelets. In addition, significant levels of clonogenic human CD34+ cells were also detected by in vitro surrogate assay. The thymi of engrafted animals contained maturating human thymocytes, while the spleen consisted mainly of T lymphocytes. Fluorescence in situ hybridisation (FISH) further identified the presence of human male X and Y chromosomes at engrafted sites, whilst the human origin of the cells was confirmed by a specific PCR assay for the human Cart-1 gene. In conclusion, the conversion of MNC to SRC in response to treatment with CR3/43 for 3 hr could have far-reaching clinical implications especially where time and donor-histocompatibility are limiting factors.

Adult↗
PubMed2004

Detection and enrichment of hematopoietic stem cells by side population phenotype.

A flow cytometric procedure has recently been described to isolate hematopoietic stem cells from mouse bone marrow based on the efflux properties of the vital dye Hoechst 33342. The assay defines a subset of cells-termed the "side population" (SP)-by simultaneously measuring fluorescence of the dye at two wavelengths (~450 nm and >670 nm). In this chapter, SP protocols are provided to detect candidate hematopoietic stem cells in mouse bone marrow and human cord blood. In the standard method, SP profiles are readily observed on a stream-in-air cell sorter using 30 mW of 351-356 nm ultraviolet excitation from a krypton-ion laser. Alternatively, SP profiles can be resolved on an analytical flow cytometer with cuvette flow cell using 8 mW of 325-nm ultraviolet excitation from a helium-cadmium laser. The ability to perform the SP assay on an analytical instrument facilitates optimization of staining conditions to identify hematopoietic and other stem cells in a variety of tissues. It is also demonstrated that SP profiles of slightly lower resolution can be obtained on a stream-in-air cell sorter using 100 mW of 407-nm violet excitation from a krypton-ion laser, raising the possibility that with appropriate validation the SP assay could be performed on flow cytometers that are not equipped with ultraviolet lasers.

Animals↗
PubMed2004

Multiparameter flow cytometry of fluorescent protein reporters.

Reporters based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and GFP-like proteins from other marine organisms provide valuable tools to monitor gene transfer and expression noninvasively in living cells. Stable cell lines were generated from the Sp2/0-Ag14 hybridoma that express up to three spectral enhanced versions of GFP, the enhanced cyan fluorescent protein (ECFP), the enhanced green fluorescent protein (EGFP), and the enhanced yellow fluorescent protein (EYFP), and/or a variant of the Discosoma coral red fluorescent protein (DsRed). The panel of lines was used to demonstrate a flow cytometric procedure for simultaneous analysis of all four fluorescent proteins that utilizes dual-laser excitation at 488 nm and 407 nm. Additional schemes for simultaneous detection of two, three or four of these fluorescent proteins are also presented.

Animals↗
PubMed2003

Catalytic-dependent and -independent roles of SHP-2 tyrosine phosphatase in interleukin-3 signaling.

SHP-2 tyrosine phosphatase is highly expressed in hematopoietic cells, however, the function of SHP-2 in hematopoietic cell signaling is not well understood. Here we focus on the role of SHP-2 phosphatase in the signal transduction of interleukin (IL)-3, a cytokine involved in hematopoietic cell survival, proliferation, and differentiation. We established immortalized SHP-2(-/-) hematopoietic cell pools and showed that IL-3-induced proliferative response was diminished in SHP-2(-/-) cells. Moreover, inhibition of the catalytic activity of SHP-2 in wild-type (WT) bone marrow hematopoietic progenitor cells and Ba/F3 cells by overexpression of catalytically inactive SHP-2 mutant suppressed their differentiative and proliferative responses to IL-3, demonstrating an important positive role for SHP-2 in IL-3 signal transduction. Further biochemical analyses revealed that IL-3-induced Jak/Stat, Erk, and PI3 kinase pathways in SHP-2(-/-) cells were impaired and reintroduction of WT SHP-2 into mutant cells partially restored IL-3 signaling. Interestingly, in catalytically inactive SHP-2-overexpressing Ba/F3 cells, although IL-3-induced activation of Jak2 and Erk kinases was reduced and shortened, PI3 kinase activation remained unaltered. Taken together, these results suggest that SHP-2 tyrosine phosphatase plays multiple roles in IL-3 signal transduction, functioning in both catalytic-dependent and -independent manners in the Jak/Stat, Erk, and PI3 kinase pathways.

Adaptor Proteins, Signal Transducing↗