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BACKGROUND: The dose-limiting toxicity of doxorubicin on hematopoietic stem cells reduces the maximum benefit from this powerful drug. Melatonin may play a role in reducing this toxicity. MATERIALS AND METHODS: Melatonin at 10 microM was used while challenging human peripheral blood stem cells (PBSC) with doxorubicin (0.6 microM and 1 microM),and colony formation was used to evaluate the protective effect of melatonin. RESULTS: Melatonin was protective for the myeloid and erythroid series when given during or 1 hour after,but not before,doxorubicin,as measured by colony assay. This protection was independent from its antioxidant function as measured by 2',7'-dichlodihydro-fluorescein diacetate and was selective for PBSC when compared to the MCF-7 cancer cell line. CONCLUSION: The results suggest the importance of the time sequence for melatonin administration to exert its protective effect in relation to doxorubicin treatment,as well as its protective effect on both erythroid and myeloid elements independent from its antioxidant function. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. View Publication

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly,the BCR/ABL fusion gene,which is present in chronic myelogenous leukemia (CML),was also detected in the endothelial cells of patients with CML,suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells,thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells. View Publication

The HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors,and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis,we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner,pre-B-cell leukemia transcription factor-1 (PBX1). Additionally,we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo,HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4,a region frequently deleted in HOXA9-induced AMLs. In vitro,HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1. View Publication

Adult bone marrow-derived cells can participate in muscle regeneration after bone marrow transplantation. In recent studies a single hematopoietic stem cell (HSC) was shown to give rise to cells that not only reconstituted all of the lineages of the blood,but also contributed to mature muscle fibers. However,the relevant HSC derivative with this potential has not yet been definitively identified. Here we use fluorescence-activated cell sorter-based protocols to test distinct hematopoietic fractions and show that only fractions containing c-kit(+) immature myelomonocytic precursors are capable of contributing to muscle fibers after i.m. injection. Although these cells belong to the myeloid lineage,they do not include mature CD11b(+) myelomonocytic cells,such as macrophages. Of the four sources of mature macrophages tested that were derived either from monocytic culture,bone marrow,peripheral blood after granulocyte colony-stimulating factor mobilization,or injured muscle,none contributed to muscle. In addition,after transplantation of bone marrow isolated from CD11b-Cre-transgenic mice into the Cre-reporter strain (Z/EG),no GFP myofibers were detected,demonstrating that macrophages expressing CD11b do not fuse with myofibers. Irrespective of the underlying mechanisms,these data suggest that the HSC derivatives that integrate into regenerating muscle fibers exist in the pool of hematopoietic cells known as myelomonocytic progenitors. View Publication

Immunomodulatory derivative (IMiD) CC-4047,a new analog of thalidomide,directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However,there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047,while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors,including GATA-1,GATA-2,erythroid Kruppel-like factor (EKLF),and growth factor independence-1B (Gfi-1b),were down-regulated in CC-4047-treated CD34+ cells,while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha),C/EBPdelta,and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion,these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore,CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment. View Publication

Internal tandem duplications (ITDs) of the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in approximately 30% of patients with acute myelogenous leukemia (AML) and are associated with a poor prognosis. FLT3 ITD mutations result in constitutive kinase activation and are thought to be pathogenetically relevant,implicating FLT3 as a plausible therapeutic target. MLN518 (formerly CT53518) is a small molecule inhibitor of the FLT3,KIT,and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases with significant activity in murine models of FLT3 ITD-positive leukemia. Given the importance of FLT3 and KIT for normal hematopoietic progenitor cells,we analyzed the effect of MLN518 on murine hematopoiesis under steady-state conditions,after chemotherapy-induced myelosuppression,and during bone marrow transplantation. In these assays,we show that MLN518 has mild toxicity toward normal hematopoiesis at concentrations that are effective in treating FLT3 ITD-positive leukemia in mice. We also demonstrate that MLN518 preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML,at concentrations that do not significantly affect colony formation by normal human progenitor cells. In analogy to imatinib mesylate in BCR-ABL-positive acute leukemia,MLN518-induced remissions may not be durable. Our studies provide the basis for integrating this compound into chemotherapy and transplantation protocols. View Publication

Chromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies,resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98,and more than one half of these are homeobox transcription factors. By contrast,the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1),a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors,evidenced by a competitive growth advantage in liquid culture,increased replating efficient of colony-forming cells (CFCs),and a marked increase in spleen colony-forming cell output. Moreover,in a murine bone marrow transplantation model,NUP98-TOP1 expression led to a lethal,transplantable leukemia characterized by extremely high white cell counts,splenomegaly,and mild anemia. Strikingly,a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings,together with similar biologic effects reported for NUP98-HOX fusions,suggest unexpected,overlapping functions of NUP98 fusion genes,perhaps related to common DNA binding properties. View Publication

We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen alpha1 type I promoter (Col2.3 Delta TK). This construct confers lineage-specific expression in developing osteoblasts,allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition,treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3 Delta TK mice,and we show that after treatment transgenic animals lose lymphoid,erythroid,and myeloid progenitors in the bone marrow,followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis,active extramedullary hematopoiesis was observed in the spleen and liver,as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV,osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow. View Publication

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis,fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro,preferentially promoting B-lymphocyte development,enhancing self-renewal of B-cell precursors,and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However,it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis,TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. View Publication

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However,the molecular mechanisms of this process remain elusive. In this study,we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3,which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells,which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation,and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus,this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3. View Publication

The CCAAT/enhancer binding protein alpha (C/EBPalpha) is an essential transcription factor for granulocytic differentiation. C/EBPalpha mutations are found in approximately 8% of acute myeloid leukemia (AML) patients. Most of these mutations occur in the N-terminal coding region,resulting in a frame shift and the enhanced translation of a dominant-negative 30-kDa protein,which may be responsible for the differentiation block observed in AML. To test this hypothesis,we introduced a cDNA encoding an N-terminal mutated C/EBPalpha (mut10) into primary hematopoietic progenitors using a retroviral vector. Expression of mut10 in human CD34+ cord blood cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated coexpression of both myeloid and erythroid markers in the immature transformed cells. Surprisingly,mut10 did not block myelocytic differentiation in murine progenitors but did alter their differentiation kinetics and clonogenicity. Experiments were performed to confirm that the differential effect of mut10 on murine and human progenitors was not due to species-specific differences in C/EBPalpha protein sequences,expression levels,or inefficient targeting of relevant cells. Taken together,our results underline the intrinsic differences between hematopoietic controls in mouse and human and support the hypothesis that mutations in CEBPA are critical events in the disruption of myeloid differentiation in AMLs. View Publication