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We have investigated the mechanism of inhibition of enoyl-acyl carrier protein reductase of Plasmodium falciparum (PfENR) by triclosan in the presence of a few important catechins and related plant polyphenols. The examined flavonoids inhibited PfENR reversibly with Ki values in the nanomolar range,EGCG being the best with 79 +/- 2.67 nM. The steady-state kinetics revealed time dependent inhibition of PfENR by triclosan,demonstrating that triclosan exhibited slow tight-binding kinetics with PfENR in the presence of these compounds. Additionally,all of them potentiated the binding of triclosan with PfENR by a two-step mechanism resulting in an overall inhibition constant of triclosan in the low picomolar concentration range. The high affinities of tea catechins and the potentiation of binding of triclosan in their presence are readily explained by molecular modeling studies. The enhancement in the potency of triclosan induced by these compounds holds great promise for the development of effective antimalarial therapy. View Publication

OBJECTIVE: Our laboratory has established two unique methods to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of both protocols is the separation of whole bone marrow into small-sized cells by counter-flow elutriation. We sought to provide the scientific community with an alternate approach to acquire our stem cells by replacing elutriation with the use of density-gradient centrifugation. METHODS: The elutriated fraction 25 population was characterized based on density using a discontinuous gradient. The long-term reconstituting potential of whole bone marrow cells collected at each density interface was determined by subjecting the fractions to the two-day homing protocol,transplanting them into lethally irradiated recipient mice,and assessing peripheral blood chimerism. We also investigated the ability of high-density bone marrow cells isolated in conjunction with the ALDH protocol to repopulate the hematopoietic system of myeloablated recipients. RESULTS: Bone marrow cells collected at the high-density interface of 1.081/1.087 g/mL (fraction 3) had the capacity for homing to marrow and the ability to provide long-term hematopoietic reconstitution. Fraction three lineage-depleted ALDH-bright cells could also engraft and provide long-term hematopoiesis at limiting dilutions. CONCLUSIONS: Density-gradient centrifugation can be used in conjunction with either of our stem cell isolation protocols to obtain cells with long-term reconstitution ability. We anticipate that this strategy will encourage and enable investigators to study the biology of HSCs isolated using functional characteristics. View Publication

OBJECTIVE The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors,such as 5-aza-2'-deoxycytidine,can reverse p15INK4B promoter methylation,but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies. METHODS We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples. RESULTS Zebularine treatment of AML193,which has a densely methylated p15INK4B promoter,results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation,arrests cells at G(2)/M,and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis. CONCLUSION Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies. View Publication

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells,addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC),we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population,which,despite its clonality,comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC,capable of repopulating secondary liquid cultures of CRA assays,confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle,in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However,quiescent LSC were also detected,capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC,believed to sustain minimal residual disease in vivo,were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines,which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls,and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy,and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors,patients with vasculitis,and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1,a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor. View Publication

Costimulation by the chemokine,stromal cell-derived factor-1 (SDF-1)/CXCL12,has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however,the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at -1082,-819,and -592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA(-) (memory") phenotype T cells that includes cells expressing the presumed regulatory T cell marker� View Publication

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors,and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions,yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore,we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1,and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors,leading to target cell death. View Publication

Hepatocellular carcinoma (HCC) is a common malignancy in Asia and Africa. We previously reported that overexpression of extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) and ERK1/2 was detected in HCC,and that their activation was required for liver cancer cell proliferation and survival. In the present study,we determined the efficacy of a specific MEK1/2 inhibitor AZD6244 (ARRAY-142886) in treatment of HCC. Treatment of primary HCC cells with AZD6244 led to growth inhibition,elevation of the cleavage of caspase-3 and caspase-7,and cleaved poly(ADP)ribose polymerase,but inhibition of ERK1/2 and p90RSK phosphorylation. Studying the protein expression profile of seven HCC xenografts revealed that their growth rate was positively correlated with the levels of phosphorylated MEK. AZD6244,when given p.o. to mice bearing these xenografts,resulted in a dose-dependent inhibition of tumor growth. AZD6244-induced growth suppression was associated with inactivation of ERK1/2 and p90RSK,and up-regulation of activated caspase-3 and caspase-7,and cleaved poly(ADP)ribose polymerase. Our data suggest that the MEK-ERK pathway plays an important role in the growth and survival of liver cancer cells and that the HCC xenograft models are excellent tools for screening preclinical drugs. Targeted inhibition of the MEK-ERK pathway with AZD6244 may represent an alternative approach for the treatment of this disease. View Publication

The transcription factor PAX5 is a critical regulator of B-cell commitment and development. Although normally not expressed in myeloid progenitors,PAX5 has recently been shown to be frequently expressed in myeloid malignancies and to suppress expression of myeloid differentiation genes,compatible with an effect on the differentiation or maintenance of myeloid progenitors. However,previous studies in which PAX5 was ectopically expressed in normal myeloid progenitors in vivo and in vitro provided conflicting results as to the effect of PAX5 on myeloid development. Herein,we demonstrate that on ectopic expression of PAX5 in bone marrow multipotent stem/progenitor cells,cells with a biphenotypic B220(+)GR-1/MAC-1(+) phenotype are produced. These remain cytokine-dependent,but unlike control-transduced cells they sustain long-term generation of myeloid progenitors in vitro and remain capable of myeloid differentiation. Notably,PAX5(+)B220(+)GR-1/MAC-1(+) myeloid progenitors coexpress,at the single-cell level,myeloid genes and otherwise B-cell-specific PAX5 target genes. These findings establish that ectopic expression of PAX5 introduces extensive self-renewal properties in otherwise short-lived myeloid progenitors. Along with the established ectopic expression of PAX5 in acute myeloid leukemia,this motivates a careful investigation of the potential involvement of ectopic PAX5 expression in myeloid and biphenotypic leukemias. View Publication

Expression of the constitutively activated TEL/PDGFbetaR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia. TEL/PDGFbetaR activates multiple signal transduction pathways in cell-culture systems,and expression of the TEL-PDGFRB fusion gene induces myeloproliferative disease (MPD) in mice. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB-mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays. Fetal liver hematopoietic stem and progenitor cells harboring targeted deletion of both Stat5a and Stat5b (Stat5ab(null/null)) genes were refractory to transformation by TEL-PDGFRB in methylcellulose colony assays. Notably,these cell populations were maintained in Stat5ab(null/null) fetal livers and succumbed to transformation by c-Myc. Surprisingly,targeted disruption of either Stat5a or Stat5b alone also impaired TEL-PDGFRB-mediated transformation. Survival of TPiGFP--textgreaterStat5a(-/-) and TPiGFP--textgreaterStat5a(+/-) mice was significantly prolonged,demonstrating significant sensitivity of TEL-PDGFRB-induced MPD to the dosage of Stat5a. TEL-PDGFRB-mediated MPD was incompletely penetrant in TPiGFP--textgreaterStat5b(-/-) mice. In contrast,Src family kinases Lyn,Hck,and Fgr and the Stat family member Stat1 were dispensable for TEL-PDGFRB disease. Together,these data demonstrate that Stat5a and Stat5b are dose-limiting mediators of TEL-PDGFRB-induced myeloproliferation. View Publication

Current evidence supports a model in which the low-affinity state of the platelet integrin alphaIIbbeta3 results from alphaIIbbeta3 adopting a bent conformation. To assess alphaIIbbeta3 biogenesis and how alphaIIbbeta3 initially adopts the bent conformation,we mapped the conformational states occupied by alphaIIb and beta3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that alphaIIbbeta3 complex formation was not limited by the availability of either free pro-alphaIIb or free beta3,suggesting that other molecules,perhaps chaperones,control complex formation. Five beta3-specific,ligand-induced binding site (LIBS) mAbs reacted with much or all free beta3 but not with beta3 when in complex with mature alphaIIb,suggesting that beta3 adopts its mature conformation only after complex formation. Conversely,2 alphaIIb-specific LIBS mAbs directed against the alphaIIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-alphaIIb,raising the possibility that pro-alphaIIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-alphaIIb adopts a bent conformation soon after synthesis,and then beta3 assumes its bent conformation by virtue of its interaction with the bent pro-alphaIIb. View Publication

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study,we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed,soluble Notch ligands that allows the titration of T cell lineage commitment,we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast,their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically,this is not due to differences in receptor expression,because early T lineage precursors,bone marrow lineage marker-negative,Sca-1-positive,c-Kit-positive and common lymphoid progenitor cells,express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment. View Publication