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1.
J Clin Invest ; 124(8): 3551-65, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24960165

ABSTRACT

Mutations within the gene encoding the DNA helicase RECQL4 underlie the autosomal recessive cancer-predisposition disorder Rothmund-Thomson syndrome, though it is unclear how these mutations lead to disease. Here, we demonstrated that somatic deletion of Recql4 causes a rapid bone marrow failure in mice that involves cells from across the myeloid, lymphoid, and, most profoundly, erythroid lineages. Apoptosis was markedly elevated in multipotent progenitors lacking RECQL4 compared with WT cells. While the stem cell compartment was relatively spared in RECQL4-deficent mice, HSCs from these animals were not transplantable and even selected against. The requirement for RECQL4 was intrinsic in hematopoietic cells, and loss of RECQL4 in these cells was associated with increased replicative DNA damage and failed cell-cycle progression. Concurrent deletion of p53, which rescues loss of function in animals lacking the related helicase BLM, did not rescue BM phenotypes in RECQL4-deficient animals. In contrast, hematopoietic defects in cells from Recql4Δ/Δ mice were fully rescued by a RECQL4 variant without RecQ helicase activity, demonstrating that RECQL4 maintains hematopoiesis independently of helicase activity. Together, our data indicate that RECQL4 participates in DNA replication rather than genome stability and identify RECQL4 as a regulator of hematopoiesis with a nonredundant role compared with other RecQ helicases.


Subject(s)
Hematopoiesis/physiology , RecQ Helicases/genetics , RecQ Helicases/metabolism , Rothmund-Thomson Syndrome/enzymology , Rothmund-Thomson Syndrome/genetics , Animals , Apoptosis , Bone Marrow Transplantation , DNA Damage , DNA Replication , Disease Models, Animal , Genomic Instability , Hematopoiesis/genetics , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/pathology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Multipotent Stem Cells/enzymology , Multipotent Stem Cells/pathology , Mutation , Phenotype , RecQ Helicases/deficiency
2.
Eur J Immunol ; 44(9): 2617-24, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24935715

ABSTRACT

Friend leukemia integration 1 (Fli-1) is a member of the Ets transcription factor family and is expressed during T-cell development; however, the role Fli-1 plays in early T-cell differentiation has not been elucidated. In this report, we demonstrate that in mouse, Fli-1 overexpression retards the CD4(-) CD8(-) double-negative (DN) to CD4(+) CD8(+) double-positive (DP) transition by deregulating normal DN thymocyte development. Specifically, Fli-1 expression moderates the DN2 and DN3 developmental transitions. We further show that Fli-1 overexpression partially mimics strong TCR signals in developing DN thymocytes and thereby enhances γδ T-cell development. Conversely, Fli-1 knockdown by small hairpin RNA reverses the lineage bias from γδ T cells and directs DN cells to the αß lineage by attenuating TCR signaling. Therefore, Fli-1 plays a critical role in both the DN2 to DN3 transition and αß/γδ lineage commitment.


Subject(s)
Proto-Oncogene Protein c-fli-1/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Thymocytes/immunology , Animals , Cells, Cultured , Mice , Proto-Oncogene Protein c-fli-1/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Antigen, T-Cell, gamma-delta/genetics , Signal Transduction/genetics , T-Lymphocytes/cytology , Thymocytes/cytology
3.
Int Immunol ; 25(10): 589-99, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23988615

ABSTRACT

The majority of T-cell development occurs in the thymus. Thymic epithelial cells are specialized cells that express NOTCH ligands and secrete specific cytokines required for normal T-cell lymphopoiesis. It has been demonstrated that OP9 cells derived from macrophage colony-stimulating factor (M-CSF)-deficient mice can support T-cell development when transduced with a NOTCH ligand, Delta-like 1 (Dll1). In this report, we have tested CSF-deficient mouse fibroblasts transduced with Dll1 for their ability to support T-cell differentiation. The data provided here demonstrate that CSF-deficient fibroblasts expressing DLL1 can support T-cell development. Indeed, co-cultures with these fibroblasts produced more T-cell progenitors compared with OP9-DL1 cultures. Addition of myeloid cytokines to OP9-DL1 co-cultures significantly inhibited T-cell development while CSF-deficient DLL1(+) fibroblasts retained partial T-cell differentiation. Taken together, these data imply that their lack of myeloid cytokines allows DLL1(+) fibroblasts to more efficiently generate T-cells. Development of this fibroblast system suggests that there is potential for generating human T-cell precursors via co-culture with human fibroblasts expressing DLL1 or DLL4. These T-cell precursors could be used for treating immunodeficient patients.


Subject(s)
Epithelial Cells/metabolism , Fibroblasts/immunology , Immunologic Deficiency Syndromes/therapy , T-Lymphocytes/immunology , Thymus Gland/immunology , Animals , Calcium-Binding Proteins , Cell Differentiation , Cell Line , Cellular Microenvironment , Coculture Techniques , Cytokines/metabolism , Epithelial Cells/immunology , Hematopoietic Stem Cell Transplantation , Humans , Immunologic Deficiency Syndromes/immunology , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Myeloid Cells/metabolism , Receptors, Notch/genetics , Receptors, Notch/metabolism
4.
PLoS One ; 8(5): e62346, 2013.
Article in English | MEDLINE | ID: mdl-23667468

ABSTRACT

The Ets transcription factor Fli-1 is preferentially expressed in hematopoietic tissues and cells, including immature T cells, but the role of Fli-1 in T cell development has not been closely examined. To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development. We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo. Surprisingly, Fli-1 overexpression in vivo eventuated in development of pre-T cell lymphoblastic leukaemia/lymphoma (pre-T LBL). Known Fli-1 target genes such as the pro-survival Bcl-2 family members were not found to be upregulated. In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours. These data show a novel function for Fli-1 in T cell development and leukaemogenesis and provide a new mouse model of pre-T LBL to identify treatment options that target the Fli-1 and Notch1 signalling pathways.


Subject(s)
Carcinogenesis/immunology , Hematopoietic Stem Cells/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Protein c-fli-1/genetics , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Animals , Gene Expression , Humans , Intracellular Space/genetics , Mice , Mice, Inbred C57BL , Organ Specificity , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , Receptor, Notch1/genetics , Up-Regulation/immunology
5.
J Exp Med ; 209(9): 1629-39, 2012 Aug 27.
Article in English | MEDLINE | ID: mdl-22891272

ABSTRACT

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn(2+)-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , B-Lymphocytes/physiology , Carrier Proteins/metabolism , Dyneins/metabolism , Membrane Proteins/metabolism , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , B-Lymphocytes/pathology , Bcl-2-Like Protein 11 , Carrier Proteins/genetics , Cytoplasmic Dyneins , DNA Damage , Dyneins/genetics , Gene Expression Regulation , Lymphopenia/genetics , Lymphopenia/pathology , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Transcription Factors , V(D)J Recombination
6.
Genes Dev ; 26(2): 120-5, 2012 Jan 15.
Article in English | MEDLINE | ID: mdl-22279045

ABSTRACT

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.


Subject(s)
Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival , Gene Deletion , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred C57BL , Myeloid Cell Leukemia Sequence 1 Protein , Tamoxifen/pharmacology , Tumor Cells, Cultured
7.
Cell Stem Cell ; 8(6): 602-3, 2011 Jun 03.
Article in English | MEDLINE | ID: mdl-21624800

ABSTRACT

The Notch signaling pathway is activated in the majority of T cell acute lymphoblastic leukemias (T-ALL). Adding to the complexity of Notch signaling in hematopoiesis, recently in Nature, Klinakis et al. (2011) demonstrate a tumor-suppressor function for the Notch pathway in myeloid malignancy.

8.
Blood ; 117(21): 5631-42, 2011 May 26.
Article in English | MEDLINE | ID: mdl-21421837

ABSTRACT

Erythropoietin (Epo) has been used in the treatment of anemia resulting from numerous etiologies, including renal disease and cancer. However, its effects are controversial and the expression pattern of the Epo receptor (Epo-R) is debated. Using in vivo lineage tracing, we document that within the hematopoietic and mesenchymal lineage, expression of Epo-R is essentially restricted to erythroid lineage cells. As expected, adult mice treated with a clinically relevant dose of Epo had expanded erythropoiesis because of amplification of committed erythroid precursors. Surprisingly, we also found that Epo induced a rapid 26% loss of the trabecular bone volume and impaired B-lymphopoiesis within the bone marrow microenvironment. Despite the loss of trabecular bone, hematopoietic stem cell populations were unaffected. Inhibition of the osteoclast activity with bisphosphonate therapy blocked the Epo-induced bone loss. Intriguingly, bisphosphonate treatment also reduced the magnitude of the erythroid response to Epo. These data demonstrate a previously unrecognized in vivo regulatory network coordinating erythropoiesis, B-lymphopoiesis, and skeletal homeostasis. Importantly, these findings may be relevant to the clinical application of Epo.


Subject(s)
B-Lymphocytes/metabolism , Bone Marrow/drug effects , Bone and Bones/metabolism , Erythropoiesis/physiology , Erythropoietin/pharmacology , Homeostasis , Lymphopoiesis/physiology , Animals , Bone Marrow/metabolism , Bone Remodeling/physiology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Erythroblasts/metabolism , Flow Cytometry , Gene Expression , Humans , Male , Mesoderm/cytology , Mesoderm/metabolism , Mice , Mice, Inbred C57BL , Receptors, Erythropoietin/metabolism , Recombinant Proteins , Spleen/cytology , Spleen/metabolism
9.
J Immunol ; 185(1): 367-75, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20530266

ABSTRACT

Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8(+) T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.


Subject(s)
Antigen Presentation/immunology , Cell Division/immunology , Conserved Sequence/immunology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , Animals , Antigen-Presenting Cells/cytology , Antigen-Presenting Cells/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Adhesion/immunology , Cell Polarity/immunology , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Transgenic , T-Lymphocyte Subsets/metabolism
11.
Mol Immunol ; 45(10): 2888-96, 2008 May.
Article in English | MEDLINE | ID: mdl-18321577

ABSTRACT

Cytokine signals are central to the differentiation of thymocytes and their stepwise progression through defined developmental stages. The intensity and duration of cytokine signals are regulated by the suppressor of cytokine signalling (SOCS) proteins. A clear role for SOCS1 during the later stages of thymopoiesis has been established, but little is known about its role during early thymopoiesis, nor the function of its closest relative, SOCS3. Here, we find that both SOCS1 and SOCS3 are expressed during early thymopoiesis, with expression coincident during the double negative (DN)2 and DN3 stages. We examined thymocyte differentiation in vitro by co-culture of SOCS-deficient bone marrow cells with OP9 cells expressing the Notch ligand Delta-like1 (OP9-DL1). Cells lacking SOCS1 were retarded at the DN3:DN4 transition and appeared unable to differentiate into double positive (DP) thymocytes. Cells lacking both SOCS1 and SOCS3 were more severely affected, and displayed an earlier block in T cell differentiation at DN2, the stage at which expression of SOCS1 and SOCS3 coincides. This indicates that, in addition to their specific roles, SOCS1 and SOCS3 share overlapping roles during thymopoiesis. This is the first demonstration of functional redundancy within the SOCS family, and has uncovered a vital role for SOCS1 and SOCS3 during two important checkpoints in early T cell development.


Subject(s)
Cell Differentiation , Suppressor of Cytokine Signaling Proteins/deficiency , Thymus Gland/cytology , Animals , Cell Line , Coculture Techniques , Flow Cytometry , Lymphoid Tissue/cytology , Mice , Stem Cells/cytology , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling 3 Protein
12.
Leuk Res ; 32(6): 873-83, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18082256

ABSTRACT

TLX1/HOX11 is an oncogenic transcription factor in human T-cell leukemia, however, the molecular basis for its transforming activity has remained elusive. The ALDH1A1 gene, whose product participates in retinoic acid synthesis, was previously identified as a TLX1-responsive gene. Here, we confirm regulation of ALDH1A1 transcription by TLX1 and show that ALDH1A1 can profoundly perturb murine hematopoiesis by promoting myeloid differentiation at the expense of lymphopoiesis. Together, these data demonstrate that ALDH1A1 plays a key role in normal hematopoiesis, and confirm ALDH1A1 as a TLX1 transcriptional target that may contribute to the ability of this homeoprotein to alter cell fate and induce tumor growth.


Subject(s)
Aldehyde Dehydrogenase/genetics , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/physiology , Leukemia, Erythroblastic, Acute/pathology , Leukemia-Lymphoma, Adult T-Cell/pathology , Lymphopoiesis/physiology , Myelopoiesis/physiology , Proto-Oncogene Proteins/physiology , Aldehyde Dehydrogenase/antagonists & inhibitors , Aldehyde Dehydrogenase/metabolism , Aldehyde Dehydrogenase 1 Family , Animals , Blotting, Northern , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Differentiation , Cell Proliferation , Cells, Cultured , DNA Primers , Female , Flow Cytometry , Gene Expression Regulation , Hematopoietic Stem Cells/pathology , Humans , Leukemia, Erythroblastic, Acute/metabolism , Leukemia-Lymphoma, Adult T-Cell/metabolism , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retinal Dehydrogenase , Reverse Transcriptase Polymerase Chain Reaction , Spleen/cytology , Spleen/metabolism
13.
Mol Immunol ; 45(3): 682-9, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17826832

ABSTRACT

Functional genetic screens on mutant backgrounds have been successfully used in lower organisms to investigate biological processes. However, few identical screens have been performed in mice. Recombinase activating gene-1 deficient (Rag1-/-) mice have a severe T-cell developmental block owing to lack of rearrangement of their T-cell receptor (TCR) genes. Using a retroviral cDNA library derived from wild-type embryonic thymocytes we performed a suppressor screen in Rag1-/- hematopoietic cells and recovered TCRbeta. This is the first demonstration that targeted genetic screens are feasible using transduced primary cells in vivo. Consequently, this technique can be used to interrogate multiple blood lineages using diverse hematopoietic mouse mutants.


Subject(s)
Cell Differentiation/genetics , Genes, T-Cell Receptor beta/genetics , Homeodomain Proteins/genetics , T-Lymphocytes , Animals , Cell Differentiation/immunology , Cloning, Molecular , Genes, T-Cell Receptor beta/immunology , Hematopoietic Stem Cells/immunology , Homeodomain Proteins/immunology , Mice , Retroviridae , T-Lymphocytes/immunology , Transduction, Genetic
14.
Int Immunol ; 19(12): 1421-30, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17981791

ABSTRACT

Notch1 signalling is essential for the commitment of multipotent lymphocyte precursors towards the alphabeta T-cell lineage and plays an important role in regulating beta-selection in CD4(-)CD8(-) double-negative (DN) thymocytes. However, the role played by Notch in promoting the development of CD4(+)CD8(+) double-positive (DP) thymocytes is poorly characterized. Here, we demonstrate that the introduction of a constitutively active Notch1 (ICN1) construct into RAG(-/-) lymphocyte precursors resulted in the generation of DP thymocytes in in vitro T-cell culture systems. Notably, developmental rescue was dependent not only on the presence of an intact Notch1 RAM domain but also on Delta-like signals, as ICN1-induced DP development in RAG(-/-) thymocytes occurred within an intact thymus or in OP9-DL1 co-cultures, but not in OP9-control co-cultures. Interestingly, ICN1 expression in SLP-76(-/-) precursors resulted in only a minimal developmental rescue to the immature CD8(+) single-positive stage, suggesting that Notch is utilizing the same signalling pathway as the pre-TCR complex. In support of this, ICN1 introduction resulted in the activation of the ERK-MAPK-signalling cascade in RAG(-/-) thymocytes. Taken together, these studies demonstrate that constitutive Notch signalling can bypass beta-selection during early T-cell development by inducing pre-TCR-like signals within a T-cell-promoting environment.


Subject(s)
Lymphoid Progenitor Cells/cytology , Lymphoid Progenitor Cells/metabolism , Receptor, Notch1/metabolism , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Thymus Gland/immunology , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation , Lymphocyte Activation , Lymphoid Progenitor Cells/immunology , Mice , Mice, Mutant Strains , Signal Transduction , T-Lymphocytes/cytology , Thymus Gland/cytology , Thymus Gland/metabolism
15.
Blood ; 110(10): 3753-62, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17698635

ABSTRACT

The acquired activation of stem cell leukemia (SCL) during T lymphopoiesis is a common event in T-cell acute lymphoblastic leukemia (T-ALL). Here, we generated tamoxifen (TAM)-inducible transgenic mice (lck-ER(T2)-SCL) to study the consequences of acquired SCL activation during T-cell development. Aberrant activation of SCL in thymocytes resulted in the accumulation of immature CD4(+)CD8(+) (double-positive, DP) cells by preventing normal surface expression of the T-cell receptor alphabeta (TCRalphabeta) complex. SCL-induced immature DP cells were further characterized by up-regulated NOTCH1 and generated noncycling polyclonal CD8(+)TCRbeta(low) cells. The prevalence of these cells was SCL dependent because TAM withdrawal resulted in their disappearance. Furthermore, we observed that SCL activation led to a dramatic up-regulation of NOTCH1 target genes (Hes-1, Deltex1, and CD25) in thymocytes. Strikingly, NOTCH1 target gene up-regulation was already observed after short-term SCL induction, implying that enhanced NOTCH signaling is mediated by SCL and is not dependent on secondary genetic events. These data represent the basis for a novel pathway of SCL-induced leukemogenesis and provide a functional link between SCL and NOTCH1 during this process.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Transformation, Neoplastic/metabolism , Leukemia, T-Cell/genetics , Proto-Oncogene Proteins/genetics , Receptor, Notch1/metabolism , Animals , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Cell Survival , Cell Transformation, Neoplastic/genetics , Genes, T-Cell Receptor beta , Mice , Mice, Transgenic , Models, Biological , Organ Culture Techniques , Signal Transduction/physiology , T-Cell Acute Lymphocytic Leukemia Protein 1 , Thymus Gland/cytology , Thymus Gland/embryology
16.
Br J Haematol ; 138(1): 54-67, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17555447

ABSTRACT

The TLX/HOX11 subfamily of divergent homeobox genes are involved in various aspects of embryogenesis and, in the case of TLX1/HOX11 and TLX3/HOX11L2, feature prominently as oncogenes in human T-cell acute lymphoblastic leukaemia. TLX1 possesses immortalising activity in a wide variety of blood cell lineages, however, the effect of this oncogene on haemopoietic cell differentiation has not been fully investigated. We therefore constitutively expressed TLX1 in murine bone marrow or fetal liver cells using retroviral transfer followed by transplantation and/or in vitro culture. TLX1 was found to dramatically alter haemopoiesis, promoting the emergence of a non-haemopoietic CD45(-) CD31(+) cell population while markedly inhibiting erythroid and granulocytic cell differentiation. To identify genetic programs perturbed by TLX1, a comparison of transcript profiles from J2E erythroid cells with and without enforced TLX1 expression was undertaken. This revealed a pattern of gene expression indicative of enhanced proliferation coupled to differentiation arrest. Of the genes identified, two, KIT and VEGFC, were found to be potential TLX1 targets based on transcriptional assays. These results demonstrate that TLX1 can act broadly to impair haemopoiesis and divert differentiation to an alternative fate. This may account for its ability to promote the pre-leukaemic state via perturbation of specific gene expression programs.


Subject(s)
Bone Marrow Cells/pathology , Gene Expression Regulation, Leukemic , Homeodomain Proteins/genetics , 3T3 Cells , Animals , Cell Differentiation/genetics , Female , Flow Cytometry , Gene Expression , Gene Expression Profiling , Hematopoiesis/genetics , Liver/embryology , Luciferases/genetics , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-kit/genetics , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/pathology , Transduction, Genetic , Vascular Endothelial Growth Factor C/genetics , beta-Galactosidase/genetics
17.
Proc Natl Acad Sci U S A ; 103(44): 16460-5, 2006 Oct 31.
Article in English | MEDLINE | ID: mdl-17060613

ABSTRACT

Mixl1, the sole murine homologue of the Xenopus Mix/Bix family of homeobox transcription factors, is essential for the patterning of axial mesendodermal structures during early embryogenesis. Gene targeting and overexpression studies have implicated Mixl1 as a regulator of hematopoiesis arising in differentiating embryonic stem cells. To assess the role of Mixl1 in the regulation of adult hematopoiesis, we overexpressed Mixl1 in murine bone marrow using a retroviral transduction/transplantation model. Enforced expression of Mixl1 profoundly perturbed hematopoietic lineage commitment and differentiation, giving rise to abnormal myeloid progenitors and impairing erythroid and lymphoid differentiation. Moreover, all mice reconstituted with Mixl1-transduced bone marrow developed fatal, transplantable acute myeloid leukemia with a mean latency period of 200 days. These observations establish a link between enforced Mixl1 expression and leukemogenesis in the mouse.


Subject(s)
Cell Differentiation , Hematopoiesis , Homeodomain Proteins/metabolism , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Animals , Bone Marrow/metabolism , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Gene Expression Regulation , Homeodomain Proteins/genetics , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Transgenic , Myeloid Cells/cytology , Myeloid Cells/metabolism , Phenotype , Survival Rate
18.
Blood ; 105(7): 2724-32, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15598809

ABSTRACT

Evidence for the lineage relationship between embryonic and adult hematopoietic stem cells (HSCs) in the mouse is primarily indirect. In order to study this relationship in a direct manner, we expressed the tamoxifen-inducible Cre-ER(T) recombinase under the control of the stem cell leukemia (Scl) stem-cell enhancer in transgenic mice (HSC-SCL-Cre-ER(T)). To determine functionality, HSC-SCL-Cre-ER(T) transgenics were bred with Cre reporter mice. Flow cytometric and transplantation studies revealed tamoxifen-dependent recombination occurring in more than 90% of adult long-term HSCs, whereas the targeted proportion within mature progenitor populations was significantly lower. Moreover, the transgene was able to irreversibly tag embryonic HSCs on days 10 and 11 of gestation. These cells contributed to bone marrow hematopoiesis 5 months later. In order to investigate whether the de novo HSC generation is completed during embryogenesis, HSC-SCL-Cre-ER(T)-marked fetal liver cells were transplanted into adult recipients. Strikingly, the proportion of marked cells within the transplanted and the in vivo-remaining HSC compartment was not different, implying that no further HSC generation occurred during late fetal and neonatal stages of development. These data demonstrate for the first time the direct lineage relationship between midgestation embryonic and adult HSCs in the mouse. Additionally, the HSC-SCL-Cre-ER(T) mice will provide a valuable tool to achieve temporally controlled genetic manipulation of HSCs.


Subject(s)
Enhancer Elements, Genetic , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Integrases/genetics , Age Factors , Animals , Antineoplastic Agents, Hormonal/pharmacology , Biomarkers , Cell Lineage , Gene Expression/drug effects , Hematopoietic Stem Cell Transplantation , Lac Operon , Mice , Mice, Inbred C57BL , Mice, Transgenic , Recombination, Genetic , Tamoxifen/pharmacology , Transgenes/physiology
19.
Blood ; 104(6): 1769-77, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15187022

ABSTRACT

Tumor growth is dependent in part on "neoangiogenesis." Functional involvement of bone marrow (BM)-derived cells in this process has been demonstrated. However, it remains controversial as to whether tumor endothelium itself is BM derived. Here we sought to address this issue with an endothelial-specific, inducible transgenic model. We generated Cretransgenic mice (endothelial-SCL-Cre-ER(T)) using the tamoxifen-inducible Cre-ER(T) recombinase driven by the 5' endothelial enhancer of the stem cell leukemia (SCL) locus. These mice were intercrossed with Cre reporter strains in which beta-galactosidase (LacZ) or enhanced yellow fluorescent protein (EYFP) are expressed upon Cre-mediated recombination. After tamoxifen administration, endothelial LacZ staining was observed in embryonic and adult tissues. Cre-mediated recombination was also observed in newly generated tumor endothelium. In adult BM cells we could only detect trace amounts of recombination by flow cytometry. Subsequently, BM from endothelial-SCL-Cre-ER(T);R26R mice was transplanted into irradiated recipients. When tumors were grown in recipient mice, which received tamoxifen, no tumor LacZ staining was detected. However, when tumors were grown in endothelial-SCL-Cre-ER(T);R26R mice 3 weeks after the cessation of tamoxifen treatment, there was widespread endothelial LacZ staining present. Thus, this genetic model strongly suggests that BM cells do not contribute to tumor endothelium and demonstrates the lineage relation between pre-existing endothelium and newly generated tumor endothelial cells.


Subject(s)
Bone Marrow Cells/cytology , Cell Lineage , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium/metabolism , Endothelium/pathology , Neoplasms/genetics , Neoplasms/pathology , Aging/physiology , Alleles , Animals , Bone Marrow Cells/pathology , Cell Differentiation , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Endothelium/blood supply , Endothelium/embryology , Flow Cytometry , Genes, Reporter/genetics , Mice , Mice, Transgenic , Neoplasms/blood supply , Neoplasms/metabolism , Neovascularization, Pathologic , Recombination, Genetic/drug effects , Tamoxifen/pharmacology
20.
Int Immunol ; 14(12): 1383-95, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12456586

ABSTRACT

FTL-1, -3 and -10 are three murine day 14 fetal thymocyte cell lines produced in order to model developmental stages within early (CD3-CD4-CD8-) thymocyte differentiation. In this study, we used the serial analysis of gene expression (SAGE) method to perform a systematic analysis of transcripts present in these three cell lines. A total of 77,313 SAGE tags were sequence identified from the three cell lines, representing 24,645 unique transcripts. Differentially expressed mRNA transcripts representing different gene classes were identified, including T cell functional genes, cytokine receptors, adhesion molecules and transcription factors. These results may serve as a model of the transcriptome of early thymocyte differentiation. A large number of unknown expressed sequence tags were also found to be differentially expressed. In order to validate the SAGE data, selected differentially expressed transcripts identified by SAGE were analyzed by quantitative RT-PCR in normal murine double-negative stage DN1-4 thymocytes. Expression of the transcription factors RUNX2 and PHD finger protein 2 and of the IGF type 1 receptor was shown to have differentially regulated expression patterns in sorted DN1-4 cells. These genes, and others identified by this analysis, are likely to play important roles in the development of T cells.


Subject(s)
Fetus/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Immediate-Early Proteins , Neoplasm Proteins , Thymus Gland/embryology , Thymus Gland/metabolism , Animals , Base Sequence , Cell Line , Cell Lineage , Core Binding Factor Alpha 1 Subunit , DNA-Binding Proteins/genetics , Fetus/cytology , Hypoxia-Inducible Factor-Proline Dioxygenases , Mice , Molecular Sequence Data , Procollagen-Proline Dioxygenase , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, IGF Type 1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Thymus Gland/cytology , Time Factors , Transcription Factors/genetics
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