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BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47,CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47,higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression,arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated,in part,by increased CD44-cytoskeleton binding. View Publication

Growth factor independence-1B (Gfi-1B) is a transcriptional repressor essential for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI1B in mice leads to embryonic lethality resulting from failure to produce definitive erythrocytes,hindering the study of Gfi-1B function in adult hematopoiesis. We here show that,in humans,Gfi-1B controls the development of erythrocytes and megakaryocytes by regulating the proliferation and differentiation of bipotent erythro-megakaryocytic progenitors. We further identify in this cell population the type III transforming growth factor-beta receptor gene,TGFBR3,as a direct target of Gfi-1B. Knockdown of Gfi-1B results in altered transforming growth factor-beta (TGF-beta) signaling as shown by the increase in Smad2 phosphorylation and its inability to associate to the transcription intermediary factor 1-gamma (TIF1-gamma). Because the Smad2/TIF1-gamma complex is known to specifically regulate erythroid differentiation,we propose that,by repressing TGF-beta type III receptor (TbetaRIotaII) expression,Gfi-1B favors the Smad2/TIF1-gamma interaction downstream of TGF-beta signaling,allowing immature progenitors to differentiate toward the erythroid lineage. View Publication

In ischemic congestive heart failure (CHF),anemia is associated with poor prognosis. Whether anemia develops in nonischemic CHF is uncertain. The hematopoietic inhibitors TNF-alpha and nitric oxide (NO) are activated in ischemic CHF. We examined whether mice with ischemic or nonischemic CHF develop anemia and whether TNF-alpha and NO are involved. We studied mice (n = 7-9 per group) with CHF either due to myocardial infarction (MI) or to overexpression of the Ca(2+)-binding protein calsequestrin (CSQ) or to induced cardiac disruption of the sarcoplasmic reticulum Ca(2+)-ATPase 2 gene (SERCA2 KO). Hematopoiesis was analyzed by colony formation of CD34(+) bone marrow cells. Hemoglobin concentration was 14.0 +/- 0.4 g/dl (mean +/- SD) in controls,while it was decreased to 10.1 +/- 0.4,9.7 +/- 0.4,and 9.6 +/- 0.3 g/dl in MI,CSQ,and SERCA2 KO,respectively (P textless 0.05). Colony numbers per 100,000 CD34(+) cells in the three CHF groups were reduced to 33 +/- 3 (MI),34 +/- 3 (CSQ),and 39 +/- 3 (SERCA2 KO) compared with 68 +/- 4 in controls (P textless 0.05). Plasma TNF-alpha nearly doubled in MI,and addition of anti-TNF-alpha antibody normalized colony formation. Inhibition of colony formation was completely abolished with blockade of endothelial NO synthase in CSQ and SERCA2 KO,but not in MI. In conclusion,the mechanism of anemia in CHF depends on the etiology of cardiac disease; whereas TNF-alpha impairs hematopoiesis in CHF following MI,NO inhibits blood cell formation in nonischemic murine CHF. View Publication

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis,only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34(+) cells. Tal1 expression activated UBE2H expression,whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis. View Publication

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor-mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice,reduced numbers of PMF CD34+ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice,whereas similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4,but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA),but not treatment with small molecule inhibitors of JAK2,resulted in the generation of increased numbers of CD34+CXCR4+ cells,which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment,JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents. View Publication

Forced expression of MN1 in primitive mouse hematopoietic cells causes acute myeloid leukemia and impairs all-trans retinoic acid-induced granulocytic differentiation. Here,we studied the effects of MN1 on myeloid differentiation and proliferation using primary human CD34(+) hematopoietic cells,lineage-depleted mouse bone marrow cells,and bipotential (granulocytic/monocytic) human acute myeloid leukemia cell lines. We show that exogenous MN1 stimulated the growth of CD34(+) cells,which was accompanied by enhanced survival and increased cell cycle traverse in cultures supporting progenitor cell growth. Forced MN1 expression impaired both granulocytic and monocytic differentiation in vitro in primary hematopoietic cells and acute myeloid leukemia cell lines. Endogenous MN1 expression was higher in human CD34(+) cells compared with both primary and in vitro-differentiated monocytes and granulocytes. Microarray and real-time reverse-transcribed polymerase chain reaction analysis of MN1-overexpressing CD34(+) cells showed down-regulation of CEBPA and its downstream target genes. Reintroduction of conditional and constitutive CEBPA overcame the effects of MN1 on myeloid differentiation and inhibited MN1-induced proliferation in vitro. These results indicate that down-regulation of CEBPA activity contributes to MN1-modulated proliferation and impaired myeloid differentiation of hematopoietic cells. View Publication

As a member of the class III histone deacetylases,Sirtuin-2 (SIRT2) is critical in cell cycle regulation which makes it a potential target for cancer therapeutics. In this study,we identified a novel SIRT2 inhibitor,AC-93253,with IC(50) of 6 microM in vitro. The compound is selective,inhibiting SIRT2 7.5- and 4-fold more potently than the closely related SIRT1 and SIRT3,respectively. AC-93253 significantly enhanced acetylation of tubulin,p53,and histone H4,confirming SIRT2 and SIRT1 as its cellular targets. AC-93253 as a single agent exhibited submicromolar selective cytotoxicity towards all four tumor cell lines tested with a therapeutic window up to 200-fold,comparing to any of the three normal cell types tested. Results from high content analysis suggested that AC-93253 significantly triggered apoptosis. Taken together,SIRT2 selective inhibitor AC-93253 may serve as a novel chemical scaffold for structure-activity relationship study and future lead development. View Publication

Tumors that recur following surgical resection of melanoma are typically metastatic and associated with poor prognosis. Using the murine B16F10 melanoma and a robust antimelanoma vaccine,we evaluated immunization as a tool to improve tumor-free survival following surgery. We investigated the utility of vaccination in both neoadjuvant and adjuvant settings. Surprisingly,neoadjuvant vaccination was far superior and provided approximately 100% protection against tumor relapse. Neoadjuvant vaccination was associated with enhanced frequencies of tumor-specific T cells within the tumor and the tumor-draining lymph nodes following resection. We also observed increased infiltration of antigen-specific T cells into the area of surgery. This method should be amenable to any vaccine platform and can be readily extended to the clinic. View Publication

Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However,20-25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown,but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34(+) cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway. View Publication

Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach,we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor,retinoid-inducible nuclear factor (RINF),that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34(+) progenitors. Furthermore,short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly,RINF localizes to 5q31.3,a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene. View Publication

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow-derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans,the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD,which extends over many months,is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45(+)HLA-DR(+) cells: CD1a(+)CD14(-) DC,CD1a(-)CD14(+) DC,and CD1a(-)CD14(+)FXIIIa(+) macrophages. In vitro,each subset has characteristic properties. After transplantation,both CD1a(+) and CD14(+) DC are rapidly depleted and replaced by donor cells,but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4(+) T cells,macrophages induce cytokine expression in memory CD4(+) T cells and activation and proliferation of CD8(+) T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages,although unlikely to initiate alloreactivity,may contribute to GVHD by sustaining the responses of previously activated T cells. View Publication

Focal adhesion kinase (FAK) has been implicated in the development of cancers,including those of the breast. Nevertheless,the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here,we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers,including aldehyde dehydrogenase,CD24,CD29,and CD61,we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their self-renewal and migration in vitro. Finally,through transplantation in NOD-SCID mice,we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together,these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. View Publication