技术资料

Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha,a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation,inhibited proliferation in vitro and in mice,and suppressed leukemogenesis. Moreover,activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells,respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis,as indicated by the effects of p42 C/EBPalpha,which were more potent than those of K298E C/EBPalpha,a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC,including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation,also induced granulocyte differentiation. Thus,these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors,and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation. View Publication

Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers,although antitumor responses are often heterogeneous,and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug,we studied the action of imatinib (STI-571,Gleevec) against murine cells and leukemias expressing BCR-ABL,an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53,which can accompany disease progression in human CML,impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly,p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule,the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals,testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed,there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover,the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor,AG490. View Publication

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However,the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g.,the brain) is higher than for treatment of anemia. Notably,a dose-dependent risk of adverse effects has been associated with rhEPO administration,especially in high-risk groups,including polycythemia-hyperviscosity syndrome,hypertension,and vascular thrombosis. Of note,several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity,primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover,we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here,we demonstrate that rhEPO administration in the rat increases systemic blood pressure,reduces regional renal blood flow,and increases platelet counts and procoagulant activities. In contrast,carbamylated rhEPO,a heteromeric receptor-specific ligand that is fully tissue protective,increases renal blood flow,promotes sodium excretion,reduces injury-induced elevation in procoagulant activity,and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands,which appear to elicit fewer adverse effects,may be especially useful in clinical settings for tissue protection. View Publication

OBJECTIVE: Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. MATERIALS AND METHODS: Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span,granulocyte macrophage colony-stimulating factor,interleukin-4,and calcium ionophore. All B-precursor ALL (N22) and AML (N13),but not T-cell ALL (N3),differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). RESULTS: Upon in vitro culture,donor-derived CTL acquired a memory T phenotype,showing concomitant high CD45RA,CD45RO,CD62L expression. CD8(+) cells,but not CD4(+) cells,were granzyme,perforine,and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%,30:1 E:T ratio),but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%,1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. CONCLUSIONS: Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation. View Publication

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro,bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-),PAX5(+),TdT(+),IgH rearranged)/myeloid (CD11b/Mac1(+),c-fms(+),lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast,young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model." View Publication

Nuclear accumulation of beta-catenin is a key event for the development of colorectal cancer. Little is known,however,about the mechanisms underlying translocation of beta-catenin from the cytoplasm or the membrane to the nucleus. The present study examined whether signal transducers and activators of transcription 3 (STAT3) activation is involved in the nuclear accumulation of beta-catenin in colorectal cancer cells. Of the 90 primary colorectal cancer tissues,40 (44.4%) were positive for nuclear staining of p-STAT3 and 63 (70.0%) were positive for nuclear staining of beta-catenin. The nuclear staining of both p-STAT3 and beta-catenin were observed predominantly in the periphery of the cancer tissues. Importantly,of the 40 tumors with p-STAT3 nuclear staining,37 (92.5%) were also positive for nuclear beta-catenin staining and there was a significant correlation between p-STAT3 and beta-catenin nuclear staining (P textless 0.01). Coexpression of nuclear p-STAT3 and beta-catenin was associated with lower patient survival (P textless 0.01). In an in vitro study using a human colon cancer cell line,SW480,inhibition of STAT3 by dominant negative STAT3 or the Janus kinase inhibitor,AG490,induced translocation of beta-catenin from the nucleus to the cytoplasm or membrane. Luciferase assays revealed that STAT3 inhibition resulted in significant suppression of beta-catenin/T-cell factor transcription in association with significant inhibition of cell proliferation (P textless 0.05). These findings suggest that in colorectal cancer,STAT3 activation is involved in the nuclear accumulation of beta-catenin,resulting in poor patient survival. View Publication

The ability of acute myeloid leukemic (AML) blasts to differentiate into leukemic dendritic cells (DC) thus acquiring the potential to present known and unknown leukemic antigens efficiently,holds promise as a possible new treatment for AML patients with minimal residual disease. Recent advances in culture methods have made the clinical use of leukemic DC feasible. However,additional measures appear to be essential in order to potentiate vaccines and to overcome the intrinsic tolerant state of the patients immune system. This review describes ways to improve AML-DC vaccines and discusses critical aspects concerning the development of clinical vaccination protocols. View Publication

BACKGROUND AND OBJECTIVES: Campath-1H is used in conditioning regimens and more recently as an anti-leukemic therapy in acute lymphoblastic leukemias (ALL). We therefore investigated CD52 expression and campath-1H-mediated lysis of ALL cells in vitro. DESIGN AND METHODS: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells and cell lines using human serum. Antibody-dependent cellular cytotoxicity (ADCC) was performed by calcein-AM release assays. RESULTS: CD52 was expressed in four out of eight ALL cell lines studied. Among 61 freshly isolated ALL samples CD52 was expressed at varying levels in 87% of cases. Whereas ADCC was equivalent in different CD52+ lines,complement-dependent cytotoxicity (CDC) was variable. The REH cell line bearing the t(12;21) translocation showed 47-60% lysis when treated with 10 microg/mL campath-1H compared to 0-6% for the other cell lines expressing equivalent amounts of CD52. Furthermore all nine ALL samples with t(12;21) showed very high CDC (mean 97%) compared to the other 24 CD52+cases (mean 24%)(ptextless0.0001). In t(12;21) samples,efficient CDC was obtained with as little as 1 microg/mL campath-1H. CDC correlated in part with CD52 levels,suggesting that CD52 expression and other yet undefined factors contribute to the particular sensitivity of t(12;21) cells. The resistance of non t(12;21) ALL cases could be overcome to a limited extent by increasing the concentration of campath-1H,blocking the CD55 and CD59 complement inhibitors,and more effectively by combining campath-1H with fludarabine. INTERPRETATION AND CONCLUSIONS: We conclude that most ALL samples express CD52 to a variable level and that campath-1H has cytotoxic activity against CD52+ALL,alone or in combination with cytotoxic drugs. View Publication

An acquired somatic mutation,Jak2V617F,was recently discovered in most patients with polycythemia vera (PV),chronic idiopathic myelofibrosis (CIMF),and essential thrombocythemia (ET). To investigate the role of this mutation in vivo,we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F,but not Jak2wt,resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit,leukocytosis,megakaryocyte hyperplasia,extramedullary hematopoiesis resulting in splenomegaly,and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors,although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity,and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore,we observed strain-specific differences in phenotype,with Balb/c mice demonstrating markedly elevated leukocyte counts,splenomegaly,and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication