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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.

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

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.

Use of cisplatin, a chemotherapeutic agent, is associated with toxicity as a significant number of patients develop a decline in renal function. The mechanisms by which cisplatin produces renal injury are not well understood. It has been suggested that free radical-catalyzed lipid peroxidation can induce apoptosis or necrosis leading to renal injury. This study examined whether low concentrations of cisplatin induce apoptosis in LLC-PK1 cells and whether caspases 1, 2, 3, 8, and 9 are activated during this event. Our results show a dose- and time-dependent induction of apoptosis by micromolar concentrations of cisplatin. Expression of oncogenes c-myc and p53 was induced, and except for caspase 1, all the other caspases tested were activated. Z-VAD, the broad-spectrum inhibitor of caspases, prevented caspase activation and apoptosis, but not c-myc and p53 induction. On the other hand, N-acetylcysteine prevented cisplatin-induced apoptosis as well as c-myc induction but not p53 induction. The antioxidant trolox also prevented cisplatin-induced apoptosis. The results suggest that antioxidants and caspase inhibitors may alleviate cisplatin-associated nephrotoxicity.

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study, we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs), evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti-vascular endothelial growth factor (anti-VEGF) and/or anti-interleukin 6 (anti-IL-6) antibodies neutralized this inhibitory effect, confirming that VEGF and IL-6, at least in part, mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo, immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor alpha (TNF-alpha) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs, and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6), DCs only (SI = 9.3), or MM lysate-pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM.

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.

CD44 is a multistructural cell-surface glycoprotein that can theoretically generate close to 800 isoforms by differential alternative splicing. At present, several dozen isoforms are known. The polymorphic nature of CD44 might explain its multifunctionality and its ability to interact with many cell-surface and extracellular ligands, the principal one being hyaluronic acid (HA). Of the many CD44 functions, our review focuses on its involvement in cell-cell and cell-matrix interactions, as well as on its implication in the support of cell migration and the presentation of growth factors to their cognate receptors. Cells involved in pathological activities such as cancer cells and destructive inflammatory cells, and also normal cells engaged in physiological functions, use cell-surface CD44 for their localization and expansion at extravascular sites. This article reviews the evidence that the joint synovium of patients with rheumatoid arthritis (RA) contains considerable amounts of various CD44 isoforms as well as the HA ligand. The review also shows that anti-CD44 monoclonal antibody (mAb) directed against constant epitopes, shared by all CD44 isoforms, can markedly reduce the inflammatory activity of arthritis induced by collagen or proteoglycans in mice. Anti-CD44 mAb also interferes with the migration of RA synovial-like fibroblasts in vitro and is able to disturb the destructive interaction between RA synovial-like fibroblasts and the cartilaginous matrix. However, the transition from the experimental model to the patient's bedside is dependent on the ability to target the CD44 of cells engaged in RA pathology, while skipping the CD44 of normal cells.

A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report, we show that marrow cells from WAS protein (WASP)-deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells, including hematopoietic progenitors and stem cells (HSCs), have decreased homing capacities that were associated with a defect in adhesion to collagen. During development, HSCs migrate from the liver to the marrow and the spleen, prompting us to ask if a defect in HSC homing during development may explain the skewed X-chromosome inactivation in WAS carriers. Preliminary evidence has shown that, in contrast to marrow progenitor cells, fetal liver progenitor cells from heterozygous females had a random X-chromosome inactivation. When fetal liver cells from WASP-carrier females were injected into irradiated recipients, a nonrandom inactivation of the X-chromosome was found at the level of hematopoietic progenitors and HSCs responsible for the short- and long-term hematopoietic reconstitution. Therefore, the mechanism of the skewed X-chromosomal inactivation observed in WAS carriers may be related to a migration defect of WASP-deficient HSCs.

We investigated the in vivo role of CD69 by analyzing the susceptibility of CD69-/- mice to tumors. CD69-/- mice challenged with MHC class I- tumors (RMA-S and RM-1) showed greatly reduced tumor growth and prolonged survival compared with wild-type (WT) mice. The enhanced anti-tumor response was NK cell and T lymphocyte-mediated, and was due, at least in part, to an increase in local lymphocytes. Resistance of CD69-/- mice to MHC class I- tumor growth was also associated with increased production of the chemokine MCP-1, diminished TGF-beta production, and decreased lymphocyte apoptosis. Moreover, the in vivo blockade of TGF-beta in WT mice resulted in enhanced anti-tumor response. In addition, CD69 engagement induced NK and T cell production of TGF-beta, directly linking CD69 signaling to TGF-beta regulation. Furthermore, anti-CD69 antibody treatment in WT mice induced a specific down-regulation in CD69 expression that resulted in augmented anti-tumor response. These data unmask a novel role for CD69 as a negative regulator of anti-tumor responses and show the possibility of a novel approach for the therapy of tumors.

BACKGROUND: The most debilitating skeletal complication of stem cell transplantation (SCT) is avascular necrosis (AVN). METHODS: Two hundred seven consecutive patients were evaluated prospectively for AVN. They survived disease free for more than 180 days after autologous or allogeneic SCT for hematologic malignancies. The diagnosis of AVN in suspicious cases was confirmed by magnetic resonance imaging. Possible correlations with treatments, bone mineral density (BMD), graft versus host disease (GVHD), and in vitro growth of fibroblast progenitors were investigated. Bone mineral density was evaluated by dual-energy X-ray absorptiometry in 100 transplanted patients, and the in vitro growth of fibroblast progenitors was monitored by a fibroblast colony-forming unit (CFU-F) assay in 30 patients after allogeneic SCT. RESULTS: Twelve patients developed AVN 3-114 months (median, 26 months) following SCT: 10 (10%) after allogeneic SCT and 2 (1.9%) after autologous SCT (P = 0.04). Twenty-five joints were affected by AVN. All patients had femoral head involvement, which was managed with hip replacement in six of them. All but one patient who developed AVN after allogeneic SCT suffered from chronic GVHD (cGVHD). Avascular necrosis occurred 1-4 months after exacerbation or progression of cGVHD. Cumulative dose of steroids was similar in both SCT groups (including steroids given pretransplant for the basic disease), whereas treatment duration was significantly longer in the allogeneic SCT group. Avascular necrosis was related to the decreased number of bone marrow CFU-F colonies in vitro, but not to BMD values. CONCLUSIONS: Avascular necrosis is a skeletal complication that occurs more often after allogeneic than after autologous SCT. Occurrence of AVN symptoms after clinical follow-up of cGVHD suggests that cGVHD requiring long-term steroid therapy is one of the main risk factors for AVN. Avascular necrosis may be facilitated by a severe deficit in the repopulating capacity of bone marrow stromal stem cells after SCT.

Vascular endothelial growth factor (VEGF) and stem cell factor (SCF) act as growth factors for the hemangioblast, an embryonic progenitor of the hematopoietic and endothelial lineages. Because thrombopoietin (TPO) and its receptor, c-Mpl, regulate primitive hematopoietic populations, including bone marrow hematopoietic stem cells, we investigated whether TPO acts on the hemangioblasts that derive from differentiation of embryonic stem cells in vitro. Reverse transcriptase polymerase chain reaction analysis detected expression of c-Mpl beginning on day 3 of embryoid body differentiation when the hemangioblast first arises. In assays of the hemangioblast colony-forming cell (BL-CFC), TPO alone supported BL-CFC formation and nearly doubled the number of BL-CFC when added together with VEGF and SCF. When replated under the appropriate conditions, TPO-stimulated BL-CFC gave rise to secondary hematopoietic colonies, as well as endothelial cells, confirming their nature as hemangioblasts. Addition of a neutralizing anti-VEGF antibody did not block TPO enhancement of BL-CFC formation, suggesting that TPO acts independently of VEGF. These results establish that Mpl signaling plays a role in the earliest stages of hematopoietic development and that TPO represents a third growth factor influencing hemangioblast formation. View Publication