Recombinant HoxB4 fusion proteins enhance hematopoietic differentiation of human embryonic stem cells.

Advanced Cell Technology, Worcester, MA 01605, USA.

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.

PMID: 17784829 [PubMed - indexed for MEDLINE]

Effects of HOXB4 overexpression on ex vivo expansion and immortalization of hematopoietic cells from different species.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.

Overexpression of the human HOXB4 has been shown to induce the expansion and self-renewal of murine hematopoietic stem cells. In preparation for clinical studies, we wished to investigate the effects of HOXB4 on cells from other species, in particular preclinical large animals such as dogs and nonhuman primates. Thus, we transduced CD34(+) cells from nonhuman primates, dogs, and humans with a HOXB4-expressing gammaretroviral vector and a yellow fluorescent protein-expressing control vector. Compared with the control vector, HOXB4 overexpression resulted in a much larger increase in colony-forming cells in dog cells (28-fold) compared with human peripheral blood, human cord blood, and baboon cells (two-, four-, and fivefold, respectively). Furthermore, we found that HOXB4 overexpression resulted in immortalization with sustained growth (>12 months) of primitive hematopoietic cells from mice and dogs but not from monkeys and humans. This difference correlated with increased levels of retrovirally overexpressed HOXB4 in dog and mouse cells compared with human and nonhuman primate cells. The immortalized cells did not show any evidence of insertional mutagenesis or chromosomal abnormalities. Competitive congenic transplantation experiments showed that HOXB4-expanded mouse cells engrafted well after 1 or 3 months of expansion, and no leukemia was observed in mice. Our findings suggest that the growth promoting effects of HOXB4 are critically dependent on HOXB4 expression levels and that this can result in important species-specific differences in potency. Disclosure of potential conflicts of interest is found at the end of this article.

PMID: 17510218 [PubMed - indexed for MEDLINE]

Immunocytochemical detection of the homeobox B3, B4, and C6 gene products within the human thymic cellular microenvironment.

Department of Pathology, University of Southern California, Los Angeles, USA. Bodey18@aol.com

The homeobox (HOX) was originally described as a conserved DNA motif of about 180 base pairs. The protein domain encoded by the homeobox, the homeodomain, is thus about 60 amino acids long. The homeodomain is a DNA-binding domain, and many homeobox genes have now been shown to bind to DNA and regulate the transcription of other genes. Thus homeodomain proteins are basically transcription factors, most of which play a role in development. The homeobox genes seem to represent another class of oncofetal antigens involved in both normal development and carcinogenesis, as well as tumor progression. The expression pattern of three homeobox gene products (HOX-B3, HOX-B4, and HOX-C6) was examined immunocytochemically in human thymuses of different ages and developmental stages (prenatal: 16 weeks and postnatal: 3 years, 5 years, and 21 years) employing an indirect alkaline phosphatase conjugated antigen detection technique on formalin-fixed, paraffin-embedded tissue sections. The immunoreactivity was located in the thymic RE cellular network (cortical and medullar), showing different intensity (+3 to +4 or 50% to 90% and over 90% in the prenatal thymic tissue and +2 to +3 or 50% to 90% during the three different postnatal stages). Intense expression was identified in the thymic medulla, including very strong immunoreactivity in the immigrating, committed hematopoietic stem cells (HSCs) present within the interlobular connective tissue (ICT). Strong presence of the HOX-B3 and HOX-B4 proteins was detected in the thymic Hassall's bodies (HBs), suggesting an intensive functional activity of the RE cells present within these unique formations within the thymic medulla. The precise role of these and other HOX gene products in the various steps of intrathymic T lymphopoieis should be elucidated through further basic molecular biological research.

PMID: 10904875 [PubMed - indexed for MEDLINE]

HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.

Department of Dermatology, VA Medical Center and University of California-San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA.

The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha-2-integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA-positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human epidermis and in hyperproliferation conditions, including cancers, in adult epidermis. Published 2002 Wiley-Liss, Inc.

PMID: 11984874 [PubMed - indexed for MEDLINE]

Hematopoietic expression of HOXB4 is regulated in normal and leukemic stem cells through transcriptional activation of the HOXB4 promoter by upstream stimulating factor (USF)-1 and USF-2.

Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34(+) cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.

PMID: 11085749 [PubMed - indexed for MEDLINE]

PMCID: PMC2193192

Antisense knockout of HOXB4 blocks 1,25-dihydroxyvitamin D3 inhibition of c-myc expression.

Department of Pharmacology, University of Michigan, School of Medicine, 1301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0632, USA.

The expression of c-myc is decreased by 1,25-(OH)2D(3) during HL-60 cell differentiation. Concomitantly, 1,25-(OH)2D(3) increases the expression and DNA binding activity of HOXB4, a homeobox gene. HOXB4 binds to the c-my c gene at sites involved in blocking c-myc transcription elongation. In this study, a phosphorothioate antisense oligonucleotide targeted against HOXB4 was examined for its effect on 1,25-(OH)2D(3) inhibition of c-myc expression. Alone, 1,25-(OH)2D(3) (20 nM) increased HOXB4 levels by 103+/-7% (mean+/-s.e., n=3) and decreased c-myc levels by 89+/-5% (mean+/-s.e.m., n=3) at 48 h of treatment. HOXB4 antisense treatment completely blocked the induction of HOXB4 by 1,25-(OH)2D(3). In addition, HOXB4 antisense partially blocked 1,25-(OH)2D(3)-mediated decrease in c-myc levels (46+/-6% inhibition) and promotion of HL-60 cell differentiation (20+/-2% and 25+/-3% inhibition as assessed by nitroblue tetrazolium and non-specific esterase assays respectively). The data further establish that HOXB4 levels are regulated by 1,25-(OH)2D(3) and reveal that HOXB4 participates in the down-regulation of c-myc expression.

PMID: 11250656 [PubMed - indexed for MEDLINE]

The HOXB4 homeoprotein differentially promotes ex vivo expansion of early human lymphoid progenitors.

Institut Cochin, Département d'Hématologie, Paris, France.

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34(+) hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.

PMID: 17962697 [PubMed - indexed for MEDLINE]

HOXB13 homeodomain protein is cytoplasmic throughout fetal skin development.

Department of Dermatology, VA Medical Center and University of California, San Francisco, California, USA.

Substantial evidence suggests that HOX homeobox genes regulate aspects of body development, including hair formation. We initially isolated the HOXB13 gene from human fetal skin in experiments designed to identify candidate genes that regulate scarless fetal wound healing. Although the HOX homeodomain proteins have been proposed to function as transcription factors, we have demonstrated previously that substantial fractions of the HOXB6 and HOXB4 proteins are localized to the cytoplasm throughout epidermal development. The purpose of the current study was to identify HOXB13 protein expression patterns in developing skin to elucidate potential mechanisms by which this protein might regulate aspects of tissue development and healing. HOXB13 protein expression was detected throughout the developing epidermis, with weaker signal observed in the early developing dermis. Epidermal HOXB13 signal was detected over the entire body surface, but surprisingly, essentially all of the signal was cytoplasmic in developing skin. Low-level HOXB13 protein expression was detected in adult skin and within the telogen hair follicle, and a portion of the residual signal in adult epidermis was nuclear. Expression in hyperproliferative skin conditions remained cytoplasmic with the exception of epidermis associated with Kaposi's sarcoma, which showed strong HOXB13 expression that was partially localized to the nucleus. Copyright 2003 Wiley-Liss, Inc.

PMID: 12761847 [PubMed - indexed for MEDLINE]

Differential and common leukemogenic potentials of multiple NUP98-Hox fusion proteins alone or with Meis1.

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver V5Z 1L3, Canada.

NUP98-Hox fusion genes are newly identified oncogenes isolated in myeloid leukemias. Intriguingly, only Abd-B Hox genes have been reported as fusion partners, indicating that they may have unique overlapping leukemogenic properties. To address this hypothesis, we engineered novel NUP98 fusions with Hox genes not previously identified as fusion partners: the Abd-B-like gene HOXA10 and two Antennepedia-like genes, HOXB3 and HOXB4. Notably, NUP98-HOXA10 and NUP98-HOXB3 but not NUP98-HOXB4 induced leukemia in a murine transplant model, which is consistent with the reported leukemogenic potential ability of HOXA10 and HOXB3 but not HOXB4. Thus, the ability of Hox genes to induce leukemia as NUP98 fusion partners, although apparently redundant for Abd-B-like activity, is not restricted to this group, but rather is determined by the intrinsic leukemogenic potential of the Hox partner. We also show that the potent leukemogenic activity of Abd-B-like Hox genes is correlated with their strong ability to block hematopoietic differentiation. Conversely, coexpression of the Hox cofactor Meis1 alleviated the requirement of a strong intrinsic Hox-transforming potential to induce leukemia. Our results support a model in which many if not all Hox genes can be leukemogenic and point to striking functional overlap not previously appreciated, presumably reflecting common regulated pathways.

PMID: 14966272 [PubMed - indexed for MEDLINE]

PMCID: PMC350554

Expression of homebox-containing genes in human preimplantation development and in embryos with chromosomal aneuploidies.

Reproductive Genetics Institute, Chicago, Illinois, USA.

PURPOSE: The purpose of the study was to investigate homeobox gene expression in human oocytes and preembryos and in postimplantation embryos with impaired embryonic development determined by chromosomal abnormalities. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets for Homeobox gene sequences was used. RESULTS: The homeobox genes HoxA4, HoxA7, HoxB4, and HoxB5 were present in human oocytes and cleaving normal and triploid embryos. The expression pattern was different between chromosomally abnormal and normal first-trimester embryos. Of four homeobox transcripts (HoxA7, HoxB4+ ++, HoxB5, and HoxC6) that are expressed in diploid embryos, only HoxA7, HoxB4 and HoxC6 were present in a trisomy 7 embryo, and only HoxB4 and HoxB 5 in triploid embryos and an embryo with trisomy 9. Cloning experiments revealed differences in the number of homeobox clones obtained from trisomy 7 and control embryos. CONCLUSIONS: The transcripts of homeobox genes, HoxA4, HoxA7, HoxB4, and HoxB5, were present in oocytes and cleaving embryos. The pattern of expression of homeobox genes in cultured fibroblasts derived from spontaneously aborted embryos with aneuploidies was different from that in control diploid cells.

PMID: 8688592 [PubMed - indexed for MEDLINE]

The HOX homeodomain proteins block CBP histone acetyltransferase activity.

Department of Medicine, VA Medical Center and University of California, San Francisco, California 94121, USA. wfshen@itsa.ucsf.edu

Despite the identification of PBC proteins as cofactors that provide DNA affinity and binding specificity for the HOX homeodomain proteins, HOX proteins do not demonstrate robust activity in transient-transcription assays and few authentic downstream targets have been identified for these putative transcription factors. During a search for additional cofactors, we established that each of the 14 HOX proteins tested, from 11 separate paralog groups, binds to CBP or p300. All six isolated homeodomain fragments tested bind to CBP, suggesting that the homeodomain is a common site of interaction. Surprisingly, CBP-p300 does not form DNA binding complexes with the HOX proteins but instead prevents their binding to DNA. The HOX proteins are not substrates for CBP histone acetyltransferase (HAT) but instead inhibit the activity of CBP in both in vitro and in vivo systems. These mutually inhibitory interactions are reflected by the inability of CBP to potentiate the low levels of gene activation induced by HOX proteins in a range of reporter assays. We propose two models for HOX protein function: (i) HOX proteins may function without CBP HAT to regulate transcription as cooperative DNA binding molecules with PBX, MEIS, or other cofactors, and (ii) the HOX proteins may inhibit CBP HAT activity and thus function as repressors of gene transcription.

PMID: 11585930 [PubMed - indexed for MEDLINE]

PMCID: PMC99922

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation.

Heinrich-Pette-Institute, Department of Cell and Virus Genetics, Hamburg, Germany.

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P <.03) and in vivo (P =.01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P <.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

PMID: 12406897 [PubMed - indexed for MEDLINE]

Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells.

Terry Fox Laboratory, British Columbia Cancer Agency, and the Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

Identification of the molecular mechanisms that can promote human hematopoietic stem cell amplification is a major goal in experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. One important class of early developmental genes determining hematopoietic development are homeobox transcription factors. Here, we report that retrovirally mediated expression of the homeobox gene HOXB4 rapidly triggers an increase in the number of human hematopoietic cord blood cells with stem cell and progenitor cell properties detected both by in vitro and in vivo assays. This growth enhancement extended across primitive myeloid-erythroid and B-lymphoid progenitors but did not lead to alterations in the balance of lymphomyeloid reconstitution in vivo, suggesting that HOXB4 does not affect control of end-cell output. These findings reveal HOXB4 as a novel, positive regulator of the primitive growth activity of human hematopoietic progenitor cells and underline the relevance of early developmental factors for stem cell fate decisions.

PMID: 12130496 [PubMed - indexed for MEDLINE]

NF-Y cooperates with USF1/2 to induce the hematopoietic expression of HOXB4.

Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

The transcription factor homeobox B4 (HOXB4) is preferentially expressed in immature hematopoietic cells and implicated in the transition from primitive hematopoiesis to definitive hematopoiesis as well as in immature hematopoietic cell proliferation and differentiation. We previously identified Hox response element 1 (HxRE-1) and HxRE-2/E-box as 2 critical DNA-binding sites of the HOXB4 promoter active in hematopoietic cells and demonstrated that upstream stimulating factor 1 and 2 (USF1/2) activate HOXB4 transcription through their binding to the E-box site. Here we report that the trimeric regulatory complex nuclear factor Y (NF-Y) is the factor that recognizes HxRE-1 and activates the HOXB4 promoter in hematopoietic cells. We further show that NF-Y interacts biochemically with USF1/2 on the HOXB4 promoter, and that the formation of this NF-Y/USF1/2 complex is required for the full activity of the HOXB4 promoter. Most important, NF-Ya subunit protein levels are found to be lower in c-Kit-Gr-1+ granulocytic bone marrow (BM) cells than in c-Kit+ immature BM cells, in parallel with a reduction of NF-Y occupancy on the HOXB4 promoter as shown by chromatin immunoprecipitation (ChIP) assay. These results suggest that NF-Y is a developmentally regulated inducer of the HOXB4 gene in hematopoietic cells.

PMID: 12791656 [PubMed - indexed for MEDLINE]

Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein.

Institut Cochin, Département d'Hématologie, INSERM U567, CNRS UMR 8104, Université Paris 5, Maternité Port-Royal, 123, Bd de Port-Royal, 75014 Paris, France.

Expansion of human hematopoietic stem cells (HSCs) is a major challenge in cellular therapy, and currently relies on the use of recombinant cytokines or on gene transfer of transcription factors. Of these, the HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. To eliminate any deleterious effects that might be associated with stable HOXB4 gene transfer into human cells, we took advantage of the ability of HOX proteins to passively translocate through cell membranes. Here we show that when cultured on stromal cells genetically engineered to secrete HOXB4, human long-term culture-initiating cells (LTC-ICs) and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse repopulating cells (SRCs) were expanded by more than 20- and 2.5-fold, respectively, over their input numbers. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. This method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

PMID: 14578882 [PubMed - indexed for MEDLINE]

TALE class homeodomain gene Irx5 is an immediate downstream target for Hoxb4 transcriptional regulation.

Department of Anatomy and Developmental Biology, St. George's Hospital Medical School, London, United Kingdom.

The Hox genes are a family of homeodomain-containing transcription factors that determine anteroposterior identity early on in development. Although much is now known about their regulation and function, very little is known of their effector (downstream target) genes. Here, we show that the TALE class homeodomain transcription factor Irx5 is a direct, positively regulated target of Hoxb4. Copyright 2003 Wiley-Liss, Inc.

PMID: 12701098 [PubMed - indexed for MEDLINE]