Overexpression of HOXB4 confers a myelo-erythroid differentiation delay in vitro.
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Bone Marrow Cells↗
Biomedical subjects
R K Humphries
Publications and source records attributed to R K Humphries.
Transplantable cell lines generated with NUP98-Hox fusion genes undergo leukemic progression by Meis1 independent of its binding to DNA.
Hox genes have been identified in chromosomal translocations involving the nucleoporin gene NUP98. Though the resulting chimeric proteins directly participate in the development of leukemia, the long latency and monoclonal nature of the disease support the requirement for secondary mutation(s), such as those leading to overexpression of Meis1. Models to identify such events and to study leukemic progression are rare and labor intensive. Herein, we took advantage of the strong transforming potential of NUP98-HOXD13 or NUP98-HOXA10 to establish preleukemic myeloid lines from bone marrow cells that faithfully replicate the first step of Hox-induced leukemogenesis. These lines contain early granulomonocytic progenitors with extensive in vitro self-renewal capacity, short-term myeloid repopulating activity and low propensity for spontaneous leukemic conversion. We exploit such lines to show that Meis1 efficiently induces their leukemic progression and demonstrate a high frequency of preleukemic cells in the cultures. Furthermore, we document that the leukemogenic potential of Meis1 is independent of its direct binding to DNA and likely reflects its ability to increase the repopulating capacity of the preleukemic cells by increasing their self-renewal/proliferative capacity. The availability of lines with repopulating potential and capacity for leukemic conversion should open new avenues for understanding progression of Hox-mediated acute myeloid leukemia.
Acute Disease↗
NOD/SCID mice engineered to express human IL-3, GM-CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration.
Transplantation of immunodeficient mice with human hematopoietic cells has greatly facilitated studies of the earliest stages of human hematopoiesis. These include demonstration of the ability of injected 'human-specific' hematopoietic growth factors to enhance the production of human cells at multiple levels of differentiation. In contrast, the effects of continuous exposure to such molecules have not been well investigated. Here, we show that nonobese diabetic severe combined immunodeficiency mice genetically engineered to produce ng/ml serum levels of human interleukin-3 (IL-3), granulocyte/macrophage-stimulating factor (GM-CSF) and Steel factor (SF) display a complex phenotype when transplanted with primitive human bone marrow (BM) or fetal liver cells. This phenotype is characterized by an enhancement of terminal human myelopoiesis and a matched suppression of terminal human erythropoiesis, with a slight reduction in human B-lymphopoiesis in the BM of the engrafted mice. Human clonogenic progenitors are more prevalent in the blood of the transplanted growth factor-producing mice and this is accompanied by a very marked reduction of more primitive human cells in the BM. Our findings suggest that long-term exposure of primitive human hematopoietic cells to elevated levels of human IL-3, GM-CSF and SF in vivo may deleteriously affect the stem cell compartment, while expanding terminal myelopoiesis.
Animals↗
Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-microglobulin-deficient NOD/SCID mice and in NOD/SCID mice transgenic for human growth factors.
Primitive malignant progenitors defined as nonobese diabetic/severe combined immunodeficient (NOD/SCID) leukemia-initiating cells or NOD/SL-IC from patients with acute myeloid leukemia (AML) can be detected and quantitated in sublethally irradiated NOD/SCID mice. However, there is variability in the levels of bone marrow (BM) engraftment obtained after intravenous injection of cells from different AML samples. In the current study, AML cell engraftment in standard NOD/SCID mice was compared to that obtained with NOD/SCID mice transgenic for the human growth factor genes Steel factor (SF), interleukin-3 (IL-3) and granulocyte macrophage-colony-stimulating factor (GM-CSF) (N/S-S/GM/3) as well as beta 2 microglobulin-null NOD/SCID (N/S-beta 2m(-/-)) mice. Three of the eight AML samples that failed to engraft in standard NOD/SCID animals showed easily detectable and up to 70-fold increased in the number of leukemic cells in BM 8-12 weeks post-transplantation in each of the N/S-beta 2m(-/-) and N/S-S/GM/3 mouse strains. In two of the four AML samples studied at limiting dilution, the frequency of NOD/SL-IC detected was increased six- and seven-fold. Thus, in these novel mouse strains a broader spectrum of AML patient samples can be evaluated for their progenitor content and potentially studied for their response to innovative therapeutics in vivo.
Acute Disease↗
Correction of sickle cell disease in transgenic mouse models by gene therapy.
Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.
Anemia, Sickle Cell↗
Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10.
Recent studies show that several Hox transcription factors are important for regulation of proliferation and differentiation in hematopoiesis. Among these is H0XA10, which is selectively expressed at high levels in the most primitive subpopulation of human CD34(+) bone marrow cells. When overexpressed, H0XA10 increases the proliferation of early progenitor cells and can lead to the development of myeloid leukemia. To study the effects of H0XA10 on primitive hematopoietic progenitors in more detail, transgenic mice were generated with regulatable H0XA10 expression. The transgenic mouse model, referred to as tetO-HOXA10, contains the H0XA10 gene controlled by a tetracycline-responsive element and a minimal promoter. Thus, the expression of H0XA10 is inducible and reversible depending on the absence or presence of tetracycline or its analog, doxycycline. A retroviral vector containing the tetracycline transactivator gene (tTA) was used to induce expression of the H0XA10 gene in bone marrow cells from the transgenic mice. Reverse transcription-polymerase chain reaction analysis confirmed regulatable H0XA10 expression in several transgenic lines. H0XA10 induction led to the formation of hematopoietic colonies containing blastlike cells and megakaryocytes. Moreover, the induction of H0XA10 resulted in significant proliferative advantage of primitive hematopoietic progenitors (spleen colony-forming units [CFU-S(12)]), which was reversible on withdrawal of induction. Activation of H0XA10 expression in tet0-H0XA10 mice will therefore govern proliferation of primitive myeloid progenitors in a regulated fashion. This novel animal model can be used to identify the target genes of HOXA10 and better clarify the specific role of HOXA10 in normal and malignant hematopoiesis.
Animals↗
Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential.
In most human somatic cells telomeres progressively shorten with each cell division eventually leading to chromosomal instability and cell senescence. The loss of telomere repeats with cell divisions may also limit the replicative life span of antigen-specific T lymphocytes. Recent studies have shown that the replicative life span of various primary human cells can be prolonged by induced expression of the telomerase reverse transcriptase (hTERT) gene. To test whether introduction of hTERT can extend the life span of primary human T lymphocytes, naive CD8(+) T lymphocytes were transfected with retroviral vectors containing the hTERT gene. Transduced T-cell clones expressed high levels of telomerase and either maintained or elongated their telomere lengths upon culture for extended periods of time. Two of the transduced subclones retained a normal cloning efficiency for more than 170 population doublings (PDs). In contrast, T-cell clones transfected with control vectors exhibited progressive telomere length shortening and stopped proliferation at around 108 PDs. Telomerase-positive T clones had a normal 46,XY karyotype, maintained their cytotoxic properties, and showed very little staining for the apoptotic marker annexin-V. These results indicate that ectopic hTERT gene expression is capable of extending the replicative life span of primary human CD8(+) cytotoxic T lymphocytes. (Blood. 2001;98:597-603)
CD8-Positive T-Lymphocytes↗
Overexpression of HOXA10 perturbs human lymphomyelopoiesis in vitro and in vivo.
Several studies point to multiple members of the Hox transcription factor family as playing key roles in normal hematopoietic development, and they link the imbalanced expression of these transcription factors, in particular of the Abd-like A cluster HOX genes HOXA9 and HOXA10, to leukemogenesis. To test directly the hypothesis that HOXA10 is involved in human hematopoietic development, the gene was retrovirally overexpressed in human highly purified CD34(+)/GFP(+) hematopoietic progenitor cells derived from cord blood or fetal liver sources, and the impact of aberrant gene expression was analyzed on differentiation and proliferation in vitro and in vivo. HOXA10 misexpression profoundly impaired myeloid differentiation with a higher yield of blast cells in liquid culture and a greater than 100-fold increased generation of blast colonies after in vitro expansion or after replating of primary colonies first plated in methylcellulose directly after transduction (P < .01). Furthermore, aberrant HOXA10 expression almost completely blocked erythroid differentiation in methylcellulose (P < .02). HOXA10 deregulation also severely perturbed the differentiation of human progenitors in vivo, reducing B-cell development by 70% in repopulated NOD/SCID mice and enhancing myelopoiesis in the transduced compartment. The data provide evidence that the balanced expression of HOXA10 is pivotal for normal human hematopoietic development and that aberrant expression of the gene contributes to impaired differentiation and increased proliferation of human hematopoietic progenitor cells. These results also provide a framework to initiate more detailed analyses of HOX regulatory domains and HOX cofactors in the human system in vitro and in vivo.
Animals↗
HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation.
OBJECTIVE: Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, we have shown that overexpression of HOXB4 in mouse bone marrow can greatly enhance the level of hematopoietic stem cell (HSC) regeneration achieved at late times (> 4 months) posttransplantation. The objective of this study was to resolve if HOXB4 increases the rate and/or duration of HSC regeneration, and also to see if this enhancement was associated with impaired production of end cells or would lead to competitive reconstitution of all compartments. METHODS: Retroviral vectors were generated with the GFP reporter gene +/- HOXB4 to enable the isolation and direct tracking of transduced cells in culture or following transplantation. Stem cell recovery was measured by limit dilution assay for long-term competitive repopulating cells (CRU). RESULTS: HOXB4-overexpressing cells have enhanced growth in vitro, as demonstrated by their rapid dominance in mixed cultures and their shortened population doubling time. Furthermore, HOXB4-transduced cells have a marked competitive repopulating advantage in vivo in both primitive and mature compartments. CRU recovery in HOXB4 recipients was extremely rapid, reaching 25% of normal by 14 days posttransplant or some 80-fold greater than control transplant recipients, and attaining normal numbers by 12 weeks. Mice transplanted with even higher numbers of HOXB4-transduced CRU regenerated up to but not beyond the normal CRU levels. CONCLUSIONS: HOXB4 is a potent enhancer of primitive hematopoietic cell growth, likely by increasing self-renewal probability but without impairing homeostatic control of HSC population size or the rate of production and maintenance of mature end cells.
Animals↗
Regulation of SLAM-mediated signal transduction by SAP, the X-linked lymphoproliferative gene product.
Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative (XLP) disease. Although the exact role and mechanism of action of SAP are not known, it has the capacity to interact with the cytoplasmic region of SLAM and other related immune cell receptors. As SAP is composed almost exclusively of a Src homology 2 (SH2) domain, it has been proposed that it functions as a natural blocker of SH2 domain--mediated interactions. We report here that the SLAM receptor is capable of triggering a protein tyrosine phosphorylation signal in T cells via a mechanism that is strictly dependent on SAP expression. This signal involves the SH2 domain--containing inositol phosphatase (SHIP); the adaptor molecules Dok2, Dok1 and Shc; and Ras GTPase--activating protein RasGAP. SAP is essential for this pathway because it facilitates the selective recruitment and activation of the Src-related protein tyrosine kinase FynT. We also show that signaling via the SLAM-SAP pathway in an established T cell line can alter the profile of cytokine production during T cell activation. These findings identify a mechanism by which a putative adaptor molecule is required for receptor-mediated signaling events in the immune system. They also provide insights into the pathophysiology of a severe human lymphoproliferative disease.
Animals↗
Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin.
The low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have impeded studies to optimize the retroviral transduction of stem cells from adult sources. To address this problem, first a cytokine combination was defined that would both maximize the kinetics of adult BM CD34(+)CD38(-) cell mitogenesis and minimize the period of prestimulation required for the transduction of these cells by a MSCV-GFP/neo(r) virus in tissue culture dishes in the absence of fibronectin. Three days of stimulation with flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and sufficient to obtain 83% +/- 2% GFP(+) CD34(+)CD38(-) cells, 75% +/- 10% G418-resistant clonogenic progenitors, and 50% +/- 20% transduced long-term culture-initiating cells as recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this prestimulation protocol, repeated daily exposure to new virus (3x) did not alter the proportion of transduced cells over that obtained with a single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-beta(2)M(-/-)) mice were also efficiently transduced (10%-20% GFP(+) human lymphoid and myeloid cells present 6-8 weeks after transplant) using a 6-day prestimulation and infection protocol. A clinically useful efficiency of retrovirus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activation into cycle and concomitant increased expression of virus receptor transcripts before virus exposure.
ADP-ribosyl Cyclase↗
Functional cloning and characterization of a novel nonhomeodomain protein that inhibits the binding of PBX1-HOX complexes to DNA.
PBX1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during developmental and/or differentiation processes. A yeast two-hybrid screen of a fetal liver-hematopoietic cDNA library using PBX1a as bait led to the discovery of a novel non-homeodomain-containing protein that interacts with PBX1 as well as PBX2 and PBX3. RNA analysis revealed it to be expressed in CD34(+) hematopoietic cell populations enriched in primitive progenitors, as is PBX1; search of the expressed sequence tag data base indicated that it is also expressed in other early embryonic as well as adult tissues. The full-length cDNA encodes a 731-amino acid protein that has no significant homology to known proteins. This protein that we have termed hematopoietic PBX-interacting protein (HPIP) is mainly localized in the cytosol and in small amounts in the nucleus. The region of PBX that interacts with HPIP includes both the homeodomain and immediate N-terminal flanking sequences. Strikingly, electrophoretic mobility shift assays revealed that HPIP inhibits the ability of PBX-HOX heterodimers to bind to target sequences. Moreover, HPIP strongly inhibits the transactivation activity of E2A-PBX. Together these findings suggest that HPIP is a new regulator of PBX function.
Amino Acid Sequence↗
A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase.
Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk(-/-) mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85alpha form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85alpha haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C beta, Fyn, CD22, Galphaq, or Galpha11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.
Agammaglobulinaemia Tyrosine Kinase↗
Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice.
Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human beta-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked beta-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human beta-globin and the green fluorescent protein in 20-95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of beta-locus control region hypersensitive site 2 alone, human beta-globin mRNA expression levels ranged from 0.15% to 20% with human beta-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.
Animals↗
Differential regulation of B cell development, activation, and death by the src homology 2 domain-containing 5' inositol phosphatase (SHIP).
Although the Src homology 2 domain-containing 5' inositol phosphatase (SHIP) is a well-known mediator of inhibitory signals after B cell antigen receptor (BCR) coaggregation with the low affinity Fc receptor, it is not known whether SHIP functions to inhibit signals after stimulation through the BCR alone. Here, we show using gene-ablated mice that SHIP is a crucial regulator of BCR-mediated signaling, B cell activation, and B cell development. We demonstrate a critical role for SHIP in termination of phosphatidylinositol 3,4,5-triphosphate (PI[3,4,5]P(3)) signals that follow BCR aggregation. Consistent with enhanced PI(3,4,5)P(3) signaling, we find that splenic B cells from SHIP-deficient mice display enhanced sensitivity to BCR-mediated induction of the activation markers CD86 and CD69. We further demonstrate that SHIP regulates the rate of B cell development in the bone marrow and spleen, as B cell precursors from SHIP-deficient mice progress more rapidly through the immature and transitional developmental stages. Finally, we observe that SHIP-deficient B cells have increased resistance to BCR-mediated cell death. These results demonstrate a central role for SHIP in regulation of BCR signaling and B cell biology, from signal driven development in the bone marrow and spleen, to activation and death in the periphery.
Animals↗
A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice.
In this report, we demonstrate that the Src homology 2 domain-containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP(-/-) mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP(-/)- B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcgamma receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP(-/)- mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell-independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.
Animals↗
Huntingtin is required for normal hematopoiesis.
Huntington's disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in huntingtin, a widely expressed protein. The function of huntingtin is unknown although huntingtin plays a fundamental role in development since gene targeted HD (-) (/-)mouse embryos die shortly after gastrulation. Expression of huntingtin is detected in spleen and thymus but its role in hematopoiesis has not been examined. To determine the function of huntingtin and to provide insight into potential pathologic mechanisms in HD, we analyzed the role of huntingtin in hematopoietic development. Expression of huntingtin was analyzed in a variety of hematopoietic cell types, and in vitro hematopoiesis was assessed using an HD ( +/-)and several HD( -) (/-)embryonic stem (ES) cell lines. Although wild-type, HD ( +/-)and HD( -) (/-)ES cell lines formed primary embryoid bodies (EBs) with similar efficiency, the numbers of hematopoietic progenitors detected at various stages of the in vitro differentiation were reduced in HD ( +/-)and HD( -/-)() ()ES cell lines examined. Expression analyses of hematopoietic markers within the EBs revealed that primitive and definitive hematopoiesis occurs in the absence of huntingtin. However, further analysis using a suspension culture in the presence of hematopoietic cytokines demonstrated a highly significant gene dosage-dependent decrease in proliferation and/or survival of HD ( +/-)and HD( -) (/-)cells. Enrichment for the CD34(+)cells within the EB confirmed that the impairment is intrinsic to the hematopoietic cells. These obser- vations suggest that huntingtin expression is required for the generation and expansion of hematopoietic cells and provides an alternative system in which to assess the function of huntingtin.
Animals↗
High-efficiency retroviral transduction of mammalian cells on positively charged surfaces.
The efficiency of retroviruses as transducing agents has been appreciated for many years, particularly for hematopoietic cell targets for which alternative strategies applicable to adherent cells are not effective. Advances in vector design, pseudotyping, and infection conditions have eliminated the need to cocultivate the target cells with virus-producing cells. Nevertheless, improvements are still needed for many applications, including those with a therapeutic or clinical cell-tracking objective. In this study we show that more positively charged surfaces, including those designed for the culture of anchorage-dependent cells, allow measurable levels of adhesion by different pseudotypes of retroviruses, which can result in increased gene transfer efficiencies to a variety of target cells including normal primary human hematopoietic cells as well as human leukemic cell lines and rat and murine fibroblasts. In the experiments with primary human cells, equal aliquots of enriched CD34+ cord blood cells were first stimulated for 2 days with cytokines (Flt3 ligand, Steel factor, IL-3, IL-6, and G-CSF) and then exposed for 4 days to a green fluorescent protein (GFP)- and Neo(r)-encoding retrovirus produced in PG13 cells. Both the final yield (approximately 300% relative to initial numbers), and the proportion (approximately 60%) of transduced CD34+ cells, colony-forming cells, and long-term culture-initiating cells were the same for cells infected either in tissue culture dishes or in fibronectin-coated petri dishes. Similar proportions (approximately 10%) and absolute yields of GFP+ human cells were also found in multilineage engrafted NOD/SCID mice assessed 6 to 8 weeks after being transplanted with these two types of transduced, but unselected, cells. These findings suggest a new and simpler approach for achieving high gene transfer efficiencies to hematopoietic cells.
Animals↗