Pathways analysis of differential gene expression induced by engrafting doses of total body irradiation for allogeneic bone marrow transplantation in mice.

A major challenge in allogeneic bone marrow (BM) transplantation is overcoming engraftment resistance to avoid the clinical problem of graft rejection. Identifying gene pathways that regulate BM engraftment may reveal molecular targets for overcoming engraftment barriers. Previously, we developed a mouse model of BM transplantation that utilizes recipient conditioning with non-myeloablative total body irradiation (TBI). We defined TBI doses that lead to graft rejection, that conversely are permissive for engraftment, and mouse strain variation with regards to the permissive TBI dose. We now report gene expression analysis, using Agilent Mouse 8x60K microarrays, in spleens of mice conditioned with varied TBI doses for correlation to the expected engraftment phenotype. The spleens of mice given engrafting doses of TBI, compared with non-engrafting TBI doses, demonstrated substantially broader gene expression changes, significant at the multiple testing-corrected P <0.05 level and with fold change ≥2. Functional analysis revealed significant enrichment for a down-regulated canonical pathway involving B-cell development. Genes enriched in this pathway suggest that suppressing donor antigen processing and presentation may be pivotal effects conferred by TBI to enable engraftment. Regardless of TBI dose and recipient mouse strain, pervasive genomic changes related to inflammation was observed and reflected by significant enrichment for canonical pathways and association with upstream regulators. These gene expression changes suggest that macrophage and complement pathways may be targeted to overcome engraftment barriers. These exploratory results highlight gene pathways that may be important in mediating BM engraftment resistance.

Fine mapping of the Bmgr5 quantitative trait locus for allogeneic bone marrow engraftment in mice.

To identify novel mechanisms regulating allogeneic hematopoietic cell engraftment, we used forward genetics and previously described identification, in mice, of a bone marrow (BM) engraftment quantitative trait locus (QTL), termed Bmgr5. This QTL confers dominant and large allele effects for engraftment susceptibility. It was localized to chromosome 16 by quantitative genetic techniques in a segregating backcross bred from susceptible BALB.K and resistant B10.BR mice. We now report verification of the Bmgr5 QTL using reciprocal chromosome 16 consomic strains. The BM engraftment phenotype in these consomic mice shows that Bmgr5 susceptibility alleles are not only sufficient but also indispensable for conferring permissiveness for allogeneic BM engraftment. Using panels of congenic mice, we resolved the Bmgr5 QTL into two separate subloci, termed Bmgr5a (Chr16:14.6-15.8 Mb) and Bmgr5b (Chr16:15.8-17.6 Mb), each conferring permissiveness for the engraftment phenotype and both fine mapped to an interval amenable to positional cloning. Candidate Bmgr5 genes were then prioritized using whole exome DNA sequencing and microarray gene expression data. Further studies are warranted to elucidate the genetic interaction between the Bmgr5a and Bmgr5b QTL and identify causative genes and underlying gene variants. This may lead to new approaches for overcoming the problem of graft rejection in clinical hematopoietic cell transplantation.

The TspanC8 subgroup of tetraspanins interacts with A disintegrin and metalloprotease 10 (ADAM10) and regulates its maturation and cell surface expression.

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.

Tbl3 regulates cell cycle length during zebrafish development.

The regulation of cell cycle rate is essential for the correct timing of proliferation and differentiation during development. Changes to cell cycle rate can have profound effects on the size, shape and cell types of a developing organ. We previously identified a zebrafish mutant ceylon (cey) that has a severe reduction in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we find that the cey phenotype is due to absence of the gene transducin (beta)-like 3 (tbl3). The tbl3 homolog in yeast regulates the cell cycle by maintaining rRNA levels and preventing p53-induced cell death. Zebrafish tbl3 is maternally expressed, but later in development its expression is restricted to specific tissues. Tissues expressing tbl3 are severely reduced in cey mutants, including HSPCs, the retina, exocrine pancreas, intestine, and jaw cartilage. Specification of these tissues is normal, suggesting the reduced size is due to a reduced number of differentiated cells. Tbl3 MO injection into either wild-type or p53-/- mutant embryos phenocopies cey, indicating that loss of tbl3 causes specific defects in cey. Progression of both hematopoietic and retinal development is delayed beginning at 3 day post fertilization due to a slowing of the cell cycle. In contrast to yeast, reduction of Tbl3 causes a slowing of the cell cycle without a corresponding increase in p53 induced cell death. These data suggest that tbl3 plays a tissue-specific role regulating cell cycle rate during development.

Identification of a major susceptibility locus for lethal graft-versus-host disease in MHC-matched mice.

Graft-vs-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hemopoietic cell transplantation. From a genetic perspective, GVHD is a complex phenotypic trait. Although it is understood that susceptibility results from interacting polymorphisms of genes encoding histocompatibility Ags and immune regulatory molecules, a detailed and integrative understanding of the genetic background underlying GVHD remains lacking. To gain insight regarding these issues, we performed a forward genetic study. A MHC-matched mouse model was used in which irradiated recipient BALB.K and B10.BR mice demonstrate differential susceptibility to lethal GHVD when transplanted using AKR/J donors. Assessment of GVHD in (B10.BR x BALB.K)F(1) mice revealed that susceptibility is a dominant trait and conferred by deleterious alleles from the BALB.K strain. To identify the alleles responsible for GVHD susceptibility, a genome-scanning approach was taken using (B10.BR x BALB.K)F(1) x B10.BR backcross mice as recipients. A major susceptibility locus, termed the Gvh1 locus, was identified on chromosome 16 using linkage analysis (logarithm of the odds, 9.1). A second locus was found on chromosome 13, named Gvh2, which had additive but protective effects. Further identification of Gvh genes by positional cloning may yield new insight into genetic control mechanisms regulating GVHD and potentially reveal novel approaches for effective GVHD therapy.

A chromosome 16 quantitative trait locus regulates allogeneic bone marrow engraftment in nonmyeloablated mice.

Identifying genes that regulate bone marrow (BM) engraftment may reveal molecular targets for overcoming engraftment barriers. To achieve this aim, we applied a forward genetic approach in a mouse model of nonmyeloablative BM transplantation. We evaluated engraftment of allogeneic and syngeneic BM in BALB.K and B10.BR recipients. This allowed us to partition engraftment resistance into its intermediate phenotypes, which are firstly the immune-mediated resistance and secondly the nonimmune rejection of donor BM cells. We observed that BALB.K and B10.BR mice differed with regard to each of these resistance mechanisms, thereby providing evidence that both are under genetic control. We then generated a segregating backcross (n = 200) between the BALB.K and B10.BR strains to analyze for genetic linkage to the allogeneic BM engraftment phenotype using a 127-marker genome scan. This analysis identified a novel quantitative trait locus (QTL) on chromosome 16, termed Bmgr5 (logarithm of odds 6.4, at 11.1 cM). The QTL encodes susceptibility alleles, from the BALB.K strain, that are permissive for allogeneic BM engraftment. Further identification of Bmgr5 genes by positional cloning may reveal new and effective approaches for overcoming BM engraftment obstacles.

Stilbene derivatives that are colchicine-site microtubule inhibitors have antileukemic activity and minimal systemic toxicity.

Stilbenes are a group of natural compounds with many biological activities. Two highly potent stilbenes, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c) and cis-3,4',5-trimethoxy-3'-hydroxystilbene (stilbene 6c) induce G2/M cell-cycle arrest and leukemic cell death in nanomolarity range without affecting normal bone marrow progenitor cells. The mechanism of stilbenes is mediated by interfering with microtubule polymerization through the colchicine-binding site. Docking of the stilbenes into tubulin structure confirms that stilbenes fit into the colchicine-binding pocket. Animal studies show that stilbenes are well tolerated in mice and are capable of inducing more than 50% leukemic cell death by a single dose injection. A 5-day treatment with low-dose stilbenes suppresses tumor growth in mice with established tumor xenografts. No major organ damage was detected by histological section. Our results indicate that stilbene 5c is a microtubule-interfering agent and can be potentially useful in leukemic therapy.

Penumbra encodes a novel tetraspanin that is highly expressed in erythroid progenitors and promotes effective erythropoiesis.

In a search for new genes involved in the regulation of erythropoiesis, we identified murine Penumbra cDNA from a multipotent hematopoietic cell line based on its predominant expression in erythroblasts. Subsequently, we identified the human PENUMBRA from a bone marrow cDNA library. Penumbra is a new member of the tetraspanin superfamily of membrane proteins, many of which are thought to function as organizers of supramolecular signaling complexes. Human and murine Penumbras contain 283 amino acids and are 97% identical. The human PENUMBRA gene is mapped to chromosome 7q32, a hot spot for deletions in myelodysplastic syndromes and acute myelogenous leukemias. Penumbra is targeted to the cell surface and forms disulfide-bonded homodimers. To study the effects of Penumbra deletions, we created a knockout mouse model by gene targeting. Penumbra(-/-) mice develop massive splenomegaly, basophilic macrocytic red blood cells, and anemia as they age. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra(-/-) mouse. EMX exhibits ineffective erythropoiesis in the presence of erythropoietin, a defect that is reversed by reexpression of Penumbra. These findings indicate that Penumbra has a positive function in erythropoiesis and its deletion or mutation may result in anemia.

The hematopoietic transcription factor AML1 (RUNX1) is negatively regulated by the cell cycle protein cyclin D3.

The family of cyclin D proteins plays a crucial role in the early events of the mammalian cell cycle. Recent studies have revealed the involvement of AML1 transactivation activity in promoting cell cycle progression through the induction of cyclin D proteins. This information in combination with our previous observation that a region in AML1 between amino acids 213 and 289 is important for its function led us to investigate prospective proteins associating with this region. We identified cyclin D3 by a yeast two-hybrid screen and detected AML1 interaction with the cyclin D family by both in vitro pull-down and in vivo coimmunoprecipitation assays. Furthermore, we demonstrate that cyclin D3 negatively regulates the transactivation activity of AML1 in a dose-dependent manner by competing with CBFbeta for AML1 association, leading to a decreased binding affinity of AML1 for its target DNA sequence. AML1 and its fusion protein AML1-ETO have been shown to shorten and prolong the mammalian cell cycle, respectively. In addition, AML1 promotes myeloid cell differentiation. Thus, our observations suggest that the direct association of cyclin D3 with AML1 functions as a putative feedback mechanism to regulate cell cycle progression and differentiation.

The human Penumbra gene is mapped to a region on chromosome 7 frequently deleted in myeloid malignancies.

We previously cloned the murine Penumbra gene based on its differential expression in proerythroblasts/erythroblasts. Subsequently, we identified human Penumbra cDNA from a human bone marrow cDNA library and the human Penumbra gene from a BAC library. Penumbra is a new member of the tetraspanin protein family and exhibits growth-suppressive activity in vitro. In this study, we designed a human Penumbra probe contig and used fluorescent in situ hybridization (FISH) to analyze seven cases of myeloid malignancies with 7q deletions. Five patients with cytogenetic deletions involving 7q31.2 approximately q32 also showed deletions of Penumbra by FISH; these were not present in two patients with cytogenetic deletions not involving 7q31.2 approximately q32. Our findings provide the first FISH evidence supporting the mapping of human Penumbra to 7q31.2 approximately q32 and demonstrate the potential of the Penumbra probe in the detection of 7q31 approximately q32-related deletions in myeloid malignancies.

IL-3 induces inhibitor of DNA-binding protein-1 in hemopoietic progenitor cells and promotes myeloid cell development.

Hemopoiesis depends on the expression and regulation of transcription factors, which control the maturation of specific cell lineages. We found that the helix-loop-helix transcription factor inhibitor of DNA-binding protein 1 (Id1) is not expressed in hemopoietic stem cells (HSC), but is increased in more committed myeloid progenitors. Id1 levels decrease during neutrophil differentiation, but remain high in differentiated macrophages. Id1 is expressed at low levels or is absent in developing lymphoid or erythroid cells. Id1 expression can be induced by IL-3 in HSC during myeloid differentiation, but not by growth factors that promote erythroid and B cell development. HSC were transduced with retroviral vectors that express Id1 and were transplanted in vivo to evaluate their developmental potential. Overexpression of Id1 in HSC promotes myeloid but impairs B and erythroid cell development. Enforced expression of Id1 in committed myeloid progenitor cells inhibits granulocyte but not macrophage differentiation. Therefore, Id1 may be part of the mechanism regulating myeloid vs lymphoid/erythroid cell fates, and macrophage vs neutrophil maturation.

Jagged2 promotes the development of natural killer cells and the establishment of functional natural killer cell lines.

Emerging evidence indicates that Notch receptors and their ligands play important roles in the development of T cells and B cells. However, little is known about their possible roles in the development of other lymphoid cells. Here we demonstrate that Jagged2, a Notch ligand, stimulates the development of natural killer (NK) cells from Lin(-) Sca-1(+) c-kit(+) hematopoietic stem cells. Our culture system supports NK cell development for 2 to 3 months, often leading to the establishment of continuous NK cell lines. The prototype of such cell lines is designated as KIL. KIL depends on interleukin-7 for survival and proliferation and is NK1.1(+) CD3(-) TCRalphabeta(-) TCRdeltagamma(-) CD4(-) CD8(-) CD19(-) CD25(+) CD43(+) CD45(+) CD49b(-) CD51(+) CD94(+) NKG2D(+) Mac-1(-/low) B220(-) c-kit(+) perforin I(+) granzyme B(+) Notch-1(+), and cytotoxic. Like normal natural killer cells, the T-cell receptor-beta loci of KIL remain in the germ-line configuration. In response to interleukin-2, KIL proliferates extensively (increasing cell number by approximately 10(10)-fold) and terminally differentiates into adherent, hypergranular NK cells. Our findings indicate that Jagged2 stimulates the development of natural killer cells and the KIL cell line preserves most properties of the normal NK precursors. As such, KIL provides a valuable model system for NK cell research.

L-selectin defines a bone marrow analog to the thymic early T-lineage progenitor.

The recent description of an early T-lineage progenitor (ETP) population in adult mouse thymus implies the presence of a bone marrow predecessor that has not yet been identified. Here we describe a Lin(Neg) Sca-1(Pos) c-kit(Hi) Thy-1.1(Neg) L-selectin(Pos) adult mouse bone marrow population that resembles the thymic ETP in both antigen expression phenotype and posttransplantation lineage potential. These cells produce wavelike kinetics of thymic seeding and reconstitute the irradiated thymus with kinetics comparable to a thymocyte graft after intravenous transplantation. Transient B-lineage reconstitution is also observed, but little myeloid potential can be detected in transplant experiments. A second subset of progenitors is L-selectin(Neg) and is highly enriched for rapid and persistent T- and B-lineage potential, as well as some myeloid potential. L-selectin (CD62L) is therefore an effective marker for separating lymphoid progenitors from myeloid progenitors and hematopoietic stem cells in mouse bone marrow.

MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1.

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

OBJECTIVE: The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. METHODS: Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. RESULTS: Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. CONCLUSION: Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.