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A battery of clonal assays for myeloid progenitor cells (HPP-CFC,CFU-gemm,CFU-gm,CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine,busulfan,melphalan,carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis,mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine,melphalan,carmustine and lomustine. Generally,there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays,the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents. View Publication

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However,because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor,we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells,HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore,a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5,demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly,high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However,neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing. View Publication

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Delta/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition,mast cell numbers in the dorsal skin were increased. Therefore KitV558Delta/+ mice reproduce human familial GISTs,and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly,these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal. View Publication

This study has evaluated the individual control of isotype production and the influence of external signals that can be experimentally provided in vitro,in antibody responses to two different recombinant Schistosoma antigens (Sh28GST and TPx-1). Peripheral blood mononuclear cells or enriched B cell fractions obtained from S. haematobium infected Senegalese adults were induced to terminal differentiation in vitro. The production of antibody to either antigen was donor-dependent and for each donor it was antigen-dependent. Differentiation to IgG1 and IgG3 production,and possibly IgA,specific to these conserved parasite antigens could be regulated differentially in vitro. Exogenous IL-2 and IL-10 or IL-10 and TGF-beta led to the production of specific IgG3 or IgG1 and/or IgA,respectively. This is the first report on such experimentally induced differential regulation of antigen-specific IgG1 and IgG3. This may have implications in designing protocols for protein based-vaccinations aiming at eliciting antibody responses of certain protective-type isotypes. View Publication

Treatment with arsenic trioxide (As(2)O(3)) by inducing apoptosis and partial differentiation of acute promyelocytic leukemia (APL) cells results in clinical remission in APL patients resistant to chemotherapy and all-trans-retinoic acid. As(2)O(3) (iAs(III)) is methylated in the liver to mono- and dimethylated metabolites,including methylarsonic acid,methylarsonous acid,dimethylarsinic acid,and dimethylarsinous acid. Methylated trivalent metabolites that are potent cytotoxins,genotoxins,and enzyme inhibitors may contribute to the in vivo therapeutic effect of iAs(III). Therefore,we compared the potency of iAs(III) and trivalent metabolites using chemical precursors of methylarsonous acid and dimethylarsinous acid to induce differentiation,growth inhibition,and apoptosis. Methylarsine oxide (MAs(III)O) and to a lesser extent iododimethylarsine were more potent growth inhibitors and apoptotic inducers than iAs(III) in NB4 cells,an APL cell line. This was also observed in K562 human leukemia,lymphoma cell lines,and in primary culture of chronic lymphocytic leukemia cells,but not human bone marrow progenitor cells. Apoptosis was associated with greater hydrogen peroxide accumulation and inhibition of glutathione peroxidase activity. MAs(III)O,in contrast to iAs(III),did not induce PML-retinoic acid receptor alpha degradation,or restore PML nuclear bodies or differentiation in NB4 cells. In a cocultivation experiment,hepatoma-derived HepG2 cells,but not NB4 cells,methylate radiolabeled iAs(III). Methylated metabolites released from HepG2 cells are preferentially accumulated by NB4 cells. This experimental model suggests that in vivo hepatic methylation of iAs(III) may contribute to As(2)O(3)-induced apoptosis but not differentiation of APL cells. MAs(III)O as an apoptotic inducer should be considered in the treatment of other hematologic malignancies like lymphoma. View Publication

Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling,we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor,p21Cip1/Waf1 (p21),have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling,we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control,manipulated cells. Further,we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore,lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. View Publication

The mechanisms that regulate the growth and survival of acute myeloid leukemia (AML) cells are largely unknown. We hypothesized that constitutive activation of phosphatidyl-inositide 3 kinase (PI3 kinase) could regulate survival in primary cells from patients with AML. Here we demonstrate that Akt,a critical substrate of PI3 kinase,is activated in AML blasts. In a short-term culture system,most AML patient samples showed a dose-dependent decrease in survival after incubation with the PI3 kinase inhibitor LY294002. This decrease in survival was partially due to the induction of apoptosis. Furthermore,we have shown that p70 S6 kinase and 4EBP-1,downstream mediators of Akt signaling,also are phosphorylated in AML blasts. Phosphorylation of these proteins is inhibited by the mTOR inhibitor RAD001. Incubation of AML blasts with RAD001 induces only a small decrease in survival of the cells; however,when combined with Ara-C,RAD001 enhances the toxicity of Ara-C. These results demonstrate that constitutive activation of the PI3 kinase pathway is necessary for the survival of AML blasts and that targeting of this pathway with pharmacologic inhibitors may be of clinical benefit in treatment of AML. View Publication

OBJECTIVE: We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS: After single cell deposition onto 96-well plates,cells were cocultured for 10 days with the stem cell supporting cell line AFT024,fibronectin (FN),or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS: Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times,a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS: For the first time,we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC),this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential. View Publication

PURPOSE: Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore,we compared morphological,immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture. METHODS: AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days,respectively,in FCS-containing medium (FCS),StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity. RESULTS: Serum-free culture of AML-APCs resulted in comparable morphology,relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture. CONCLUSION: These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological,immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP). View Publication

Vascular endothelial growth factor (VEGF) and its receptors play an essential role in the formation and maintenance of the hematopoietic and vascular compartments. The VEGF receptor-2 (VEGFR-2) is expressed on a population of hematopoietic cells,although its role in hematopoiesis is still unclear. In this report,we have utilized a strategy to selectively activate VEGFR-2 and study its effects in primary bone marrow cells. We found that VEGFR-2 can maintain the hematopoietic progenitor population in mouse bone marrow cultured in the absence of exogenous cytokines. Maintenance of the hematopoietic progenitor population is due to increased cell survival with minimal effect on proliferation. Progenitor survival is mainly mediated by activation of the phosphatidylinositol 3'-kinase/Akt pathway. Although VEGFR-2 also activated Erk1/2 mitogen-activated protein kinase,it did not induce cell proliferation,and blockade of this pathway only partially decreased VEGFR-2-mediated survival of hematopoietic progenitors. Thus,the role of VEGFR-2 in hematopoiesis is likely to maintain survival of hematopoietic progenitors through the activation of antiapoptotic pathways. View Publication

BACKGROUND Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions,ES cells can differentiate into specialized cells,including cardiac myocytes,but the efficiency is typically low and the process is incompletely understood. METHODS AND RESULTS We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific alpha-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP),880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid,also known as vitamin C,markedly increased the number of EGFP-positive cells,which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and alpha-actinin. Furthermore,ascorbic acid induced the expression of cardiac genes,including GATA4,alpha-MHC,and beta-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine,Tiron,or vitamin E. CONCLUSIONS Ascorbic acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells. View Publication

OBJECTIVE: Dysregulation of the apoptotic mechanisms plays a key role in the accumulation of malignant B-chronic lymphocytic leukemia (B-CLL) cells. The transcription nuclear factor (NF)-kappaB is important for cell survival by regulating the expression of anti-apoptotic genes. Several cytokines can modulate leukemic growth and apoptosis in B-CLL. The aim of this study was to determine whether cytokine-mediated regulation of apoptosis occurs via modulation of NF-kappaB activity in peripheral blood mononuclear cells from B-CLL patients. PATIENTS AND METHODS: We evaluated NF-kappaB activity in peripheral blood mononuclear cells from 15 untreated B-CLL patients and 11 controls in resting conditions and in the presence of phorbol-12-myristate-13-acetate (PMA) and different cytokines by electrophoretic mobility shift assay. Apoptosis was studied by spectrophotometric analysis of DNA fragmentation. RESULTS: We found a constitutive high NF-kappaB activity not induced by PMA in B-CLL patients,in contrast with a normal inducible NF-kappaB activity in controls. In B-CLL cultures,addition of interleukin (IL)-4 and IL-13 increased,whereas transforming growth factor (TGF)-beta reduced NF-kappaB activity compared with unstimulated cultures. Accordingly,IL-4 and IL-13 decreased,whereas TGF-beta increased DNA fragmentation compared with unstimulated cultures. IL-13 and IL-4 production was increased,whereas TGF-beta was reduced in PMA-stimulated and unstimulated cultures from B-CLL patients compared with controls. CONCLUSIONS: B-CLL patients have a constitutive high NF-kappaB activity,which is modulated by cytokines. In particular,TGF-beta displays a pro-apoptotic activity,whereas IL-4 and IL-13 have opposite effects. These cytokine alterations could be responsible for a positive autocrine circuit that maintains leukemic cells in a pre-apoptotic state. View Publication