Biomedical subjects

R K Humphries

Publications and source records attributed to R K Humphries.

At least 55 records · Page 3Linked to original sources

PubMed1997

Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis.

Several homeobox genes of the HOXA and HOXB clusters are expressed in primitive blood cells, suggesting a role for HOX genes in normal hematopoiesis. The HOXA9 gene is expressed in CD34+ marrow cells and in developing lymphocytes. We examined blood-forming organs of mice homozygous for an interrupted HOXA9 allele to determine if loss of HOX gene function is deleterious to hematopoiesis. HOXA9-/- mice have approximately 30% to 40% reductions in total leukocytes and lymphocytes (P < .001) and a blunted granulocytic response to granulocyte colony-stimulating factor (G-CSF). Homozygous mice have significantly smaller spleens and thymuses. Myeloid/erythroid and pre-B progenitors in the marrow are significantly reduced, but no significant decreases are noted in mixed colonies, day 12 colony-forming units-spleen (CFU-S), or long-term culture-initiating cells (LTC-IC), suggesting little or no perturbation in earlier progenitors. Heterozygous animals display no hematopoietic defects. The abnormalities in leukocyte production are transplantable, indicating that the defect resides in the hematopoietic cells. These studies demonstrate a physiologic role for a HOX gene in blood cell differentiation, with the greatest apparent influence of HOXA9 at the level of the committed progenitor.

Animals↗

PubMed1997

Hox homeobox genes as regulators of normal and leukemic hematopoiesis.

Hox genes, first recognized for their role in embryonic development, may also play lineage-specific functions in a variety of somatic tissues including the hematopoietic system. Expression of these transcription factors has been demonstrated both in normal and leukemic human and hematopoietic cells, suggesting functional roles in hematopoietic cell growth and differentiation. Several recent studies have shown that Hox proteins are involved in controlling proliferation of primitive bone marrow cells and also in altering differentiation of myeloid as well as lymphoid progenitors, alterations that also can contribute to leukemic transformation. Hox genes, together with their upstream regulators and downstream target genes, may play key roles in fundamental processes controlling hematopoietic stem cell properties.

Animals↗

PubMed1997

Overexpression of HOXB3 in hematopoietic cells causes defective lymphoid development and progressive myeloproliferation.

HOXB3 mRNA levels are high in the earliest CD34+ lineage- bone marrow cells and low to undetectable in later CD34+/CD34- cells. To gain some insight into the role this gene may play in hematopoiesis, HOXB3 was overexpressed in murine bone marrow cells using retroviral gene transfer. Thymi of HOXB3 marrow recipients were reduced in size compared with control transplant recipients, with a 24-fold decrease in the absolute number of CD4+ CD8+ cells and a 3-fold increase in the number of CD4- CD8- thymocytes that contained a high proportion of gammadelta TCR+ cells. B cell differentiation was also perturbed in these mice, as indicated by the virtual absence of transduced IL-7-responsive pre-B clonogenic progenitors. Recipients of HOXB3-transduced cells also had elevated numbers of mature granulocyte macrophage colony-forming cells in their bone marrow and spleen. Together these results suggest roles for HOXB3 in proliferation and differentiation processes of both early myeloid and lymphoid developmental pathways.

Animals↗

PubMed1997

Isolation and analysis of different subpopulations of normal human breast epithelial cells after their infection with a retroviral vector encoding a cell surface marker.

The use of gene transfer procedures has greatly facilitated the investigation of cell lineage relationships and other developmental processes in a variety of primary tissues. In this report we described the infection and selection of primary human breast epithelial cells using retroviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing the complete 228 bp coding sequence of a murine cell surface marker (Heat Stable Antigen, HSA) as well as the neomycin resistance (neo(r)) gene. Expression of the transduced HSA gene was detectable using either flow cytometry or immunohistochemistry after staining cells with an anti-murine HSA-specific antibody (M1/69). Expression of the transduced neo(r) gene conferred resistance to G418. In initial experiments with the MCF-7 breast cancer cell line, continued expression of both markers was demonstrated in a high proportion of cells for at least 4 weeks after their infection by positive M1/69 staining of cells that had been selected by prior incubation in G418. Evidence of gene transfer to early stage (< 9 days old) primary cultures of normal human breast epithelial cells (15 experiments with cells from 12 normal individuals) was also obtained using an infection protocol in which these calls were exposed to helper-free viral supernatants (2 incubations, 4 to 6 hr each) after being subcultured for 12 to 18 hr to increase their rate of proliferation. The presence of 5-50% (mean = 26%) HSA+ cells was demonstrated in these experiments within 5 days after their infection and the HSA+ populations included both myoepithelial and luminal phenotypes. The transduced (HSA+) cells within both of these subpopulations could also be separately isolated by FACS and subcultured. These results should provide an important starting point for future studies of genetically modified or marked primary human breast epithelial cell populations.

Animals↗

PubMed1997

Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia.

Multiple members of the A, B, and C clusters of Hox genes are expressed in hematopoietic cells. Several of these Hox genes have been found to display distinctive expression patterns, with genes located at the 3' side of the clusters being expressed at their highest levels in the most primitive subpopulation of human CD34+ bone marrow cells and genes located at the 5' end having a broader range of expression, with downregulation at later stages of hematopoietic differentiation. To explore if these patterns reflect different functional activities, we have retrovirally engineered the overexpression of a 5'-located gene, HOXA10, in murine bone marrow cells and demonstrate effects strikingly different from those induced by overexpression of a 3'-located gene, HOXB4. In contrast to HOXB4, which causes selective expansion of primitive hematopoietic cells without altering their differentiation, overexpression of HOXA10 profoundly perturbed myeloid and B-lymphoid differentiation. The bone marrow of mice reconstituted with HOXA10-transduced bone marrow cells contained in high frequency a unique progenitor cell with megakaryocytic colony-forming ability and was virtually devoid of unilineage macrophage and pre-B-lymphoid progenitor cells derived from the transduced cells. Moreover, and again in contrast to HOXB4, a significant proportion of HOXA10 mice developed a transplantable acute myeloid leukemia with a latency of 19 to 50 weeks. These results thus add to recognition of Hox genes as important regulators of hematopoiesis and provide important new evidence of Hox gene-specific functions that may correlate with their normal expression pattern.

Acute Disease↗

PubMed1997

The role of erythropoietin receptor tyrosine phosphorylation in erythropoietin-induced proliferation.

Although studies with truncated erythropoietin receptors (EpoRs) have suggested the tyrosine phosphorylation (Yphos) of the EpoR may not play a significant role in Epo-induced proliferation, we found, using a full length EpoR mutant designed Null, in which all 8 of the intracellular tyrosines (Ys) were substituted with phenylalanines (Fs), that Null cells required 5-10 fold more Epo than wild type (WT) EpoR containing cells in order to proliferate as well. Moreover, a comparison of Epo-induced proliferation with Epo-induced Yphos patterns, using DA-3 cells expressing WT, Null and various Y to F EpoR point mutants revealed that Stat5 Yphos and activation correlated directly with proliferation and was mediated primarily throuhg the most membrane proximal Y, i.e., Y343, although other tyrosines (most likely Y401 and Y431) within the EpoR could activate Stat5 in its absence. We also found that EpoR Yphos was essential for the Yphos of Shc and for the Yphos and association of a 145 kDa protein with Shc. We purified and cloned this Shc-associated 145 kDa protein and found that it was a unique SH2 containing inositol polyphosphate-5-phosphatase. This novel enzyme, which we have called SHIP for SH2-containing inositol-phosphatase, may modulate both Ras and inositol signaling pathways.

Amino Acid Sequence↗

PubMed1996

Interleukin-3 (IL-3) inhibits erythropoietin-induced differentiation in Ba/F3 cells via the IL-3 receptor alpha subunit.

Introduction of erythropoietin receptors (EpoRs) into the interleukin-3 (IL-3)-dependent murine hemopoietic cell line, Ba/F3, enables these cells to not only proliferate, after an initial lag in G1, but also to increase beta-globin mRNA levels in response to erythropoietin (Epo). With IL-3 and Epo costimulation, IL-3-induced signaling appears to be dominant since no increase in beta-globin mRNA occurs. Differentiation and proliferation signals may be uncoupled since EpoRs lacking all eight intracellular tyrosines were compromised in proliferative signaling but retained erythroid differentiation ability. Intriguingly, a chimeric receptor of the extracellular domain of the EpoR and the transmembrane and intracellular domains of IL-3RbetaIL-3 chain (EpoR/IL-3RbetaIL-3) was capable of Epo-induced proliferative and differentiating signaling, suggesting either the existence of a second EpoR subunit responsible for differentiation or that the alpha subunit of the IL-3 receptor (IL-3R) prevents it. Arguing against the former, a truncated EpoR lacking an intracellular domain was incapable of promoting proliferation or differentiation. An EpoR/IL-3Ralpha chimera, in contrast, was capable of transmitting a weak Epo-induced proliferative signal but failed to stimulate accumulation of beta-globin mRNA. Most significantly, coexpression of the EpoR/IL-3Ralpha chimera with either EpoR/IL-3Rbeta or wild-type EpoRs suppressed Epo-induced beta-globin mRNA accumulation. Taken together, these results suggest an active role for the IL-3Ralpha subunit in inhibiting EpoR-specific differentiating signals.

Animals↗

PubMed1996

Cross-linking the murine heat-stable antigen induces apoptosis in B cell precursors and suppresses the anti-CD40-induced proliferation of mature resting B lymphocytes.

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes, the co-stimulation of CD4+ T cells, and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells, but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together, these data implicate HSA as a potent negative regulator of B cell development and activation.

Animals↗

PubMed1996

Cloning and characterization of human SHIP, the 145-kD inositol 5-phosphatase that associates with SHC after cytokine stimulation.

We recently cloned and sequenced a cDNA encoding a 145-kD protein from the murine hematopoietic cell line B6SUtA, that becomes tyrosine phosphorylated and associated with Shc after cytokine stimulation. Based on its domains and enzymatic activity, we named this protein SHIP for SH2-containing inositol phosphatase (Damen et al, Proc Natl Acad Sci USA 93:1689, 1996). We describe here the cloning of the human homologue of murine SHIP (mSHIP) from a human megakaryocytic cell line (MO7e) lambda gt11 cDNA library using two nonoverlapping mSHIP cDNA fragments as probes. Northern blot analysis suggests that human SHIP (hSHIP) is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of this cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mSHIP. Contained within the defined open reading frame is an N-terminal, group l src homology 2 (SH2) domain; three NXXY motifs that, if phosphorylated, could be bound by phosphotyrosine binding (PTB) domains; a C-terminal proline-rich region; and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization, using the full-length hSHIP cDNA as a probe, mapped hSHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo.

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.

Animals↗

PubMed1996

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro.

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

Base Sequence↗

PubMed1996

Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.

Eight of the nine homeobox genes of the Hoxb locus encode proteins which contain a conserved hexapeptide motif upstream from the homeodomain. All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but different flanking regions, form complexes of similar stability. These data suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.

Amino Acid Sequence↗

PubMed1996

A bicistronic therapeutic retroviral vector enables sorting of transduced CD34+ cells and corrects the enzyme deficiency in cells from Gaucher patients.

Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral-producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models.

3T3 Cells↗

PubMed1996

The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase.

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Reduction of early B lymphocyte precursors in transgenic mice overexpressing the murine heat-stable antigen.

To study the role of the murine heat-stable Ag (HSA) in lymphocyte maturation, we generated transgenic mice in which the HSA cDNA was under the transcriptional control of the TCR V beta promoter and Ig mu enhancer. The HSA transgene was expressed during all stages of B lymphocyte maturation. Expression was first detected in the earliest lymphoid-committed progenitors, which normally do not express HSA, and subsequently reached the highest levels in pro- and pre-B cells. In bone marrow, the number of IL-7-responsive clonogenic progenitors was < 4% of normal, whereas the frequency of earlier B lymphocyte-restricted precursors, detectable as Whitlock-Witte culture-initiating cells, was normal. Pro- and pre-B cells detected by flow cytometry were reduced by approximately 50% relative to controls. Mature splenic B cells were also reduced but to a lesser extent than in marrow, and their response to LPS stimulation was impaired. Reconstitution of SCID and BALB/c-nu/nu mice with HSA transgenic marrow indicated that the perturbations in B lymphopoiesis were not caused by a defective marrow microenvironment or by abnormal T cells. Our previous studies showed elevated HSA expression throughout thymocyte development, which resulted in a profound depletion of CD4+CD8+ double-positive and single-positive thymocytes. Together, these results indicate that HSA levels can determine the capacity of early T and B lymphoid progenitors to proliferate and survive. Therefore, HSA could serve as an important regulator during the early stages of B and T lymphopoiesis.

Animals↗

PubMed1996

Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer.

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL-3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony-forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony-forming cells in the unsorted population was < or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.

Animals↗

PubMed1996

The role of HOX homeobox genes in normal and leukemic hematopoiesis.

A sizable amount of new data points to a role for the HOX family of homeobox genes in hematopoiesis. Recent studies have demonstrated that HOXA and HOXB genes are expressed in human CD34+ cells, and are downregulated as cells leave the CD34+ compartment. In addition, expression of certain genes, including HOXB3 and HOXB4, is largely restricted to the long-term culture-initiating cell enriched pool, containing the putative stem cell population. Studies have also shown that HOX genes appear to be important for normal T lymphocyte and activated natural killer cell function. Overexpression of Hox-b4 in transplanted murine marrow cell results in a dramatic expansion of stem cells, while maintaining normal peripheral blood counts. In contrast, overexpression of Hox-a10 resulted in expansion of progenitor pools, accompanied by unique changes in the differentiation patterns of committed progenitors. Overexpression of Hox-a10 or Hox-b8 led to the development of myeloid leukemias, while animals transfected with marrow cells overexpressing Hox-b4 do not appear to develop malignancies. Blockade of HOX gene function using antisense oligonucleotides has revealed that several HOX genes appear to influence either myeloid or erythroid colony formation. Mice homozygous for a targeted disruption of the HOX-a9 gene show reduced numbers of granulocytes and lymphocytes, smaller spleens and thymuses, and reduced numbers of committed progenitors. These studies demonstrate that HOX homeobox genes play a role in both the early stem cell function as well as in later stages of hematopoietic differentiation, and that perturbations of HOX gene expression can be leukemogenic.

Animals↗

PubMed1995

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

3T3 Cells↗