技术资料

OBJECTIVE: The aim of this study was the preclinical evaluation of imatinib mesylate (Gleevec,formerly STI571) in conjunction with arsenic trioxide (As2O3,Trisenox) for the treatment of chronic myelogenous leukemia (CML). MATERIALS AND METHODS: Tetrazolium-based cell line proliferation assays (MTT assays) were performed to determine the cytotoxicity of As2O3 alone and in combination with imatinib. Cell lines tested in this study were Bcr-Abl-expressing cells (K562,MO7p210,32Dp210) and parental cells (MO7e,32D). Isobologram analysis was performed manually and using the median effect method. In vitro cytotoxicity also was determined in colony-forming assays using CML patient cells. Western blot analysis was performed to detect Bcr-Abl protein levels in K562 cells exposed to As2O3 at graded concentrations. Bcr-Abl protein level kinetics were correlated with cell viability (trypan blue count) and activated caspase-3 detected by flow cytometry. RESULTS: We show additive to synergistic cytotoxicity in Bcr-Abl+ cell lines depending on inhibitory concentrations and cell type. Results obtained by colony-forming assays confirmed the findings in cell line proliferation assays. Flow cytometric detection of activated caspase-3 revealed synergistic activity in K562 cells. Treatment of K562 cells with As2O3 alone led to down-regulation of Bcr-Abl protein within 24 hours,even at low doses. The decline of Bcr-Abl preceded activation of caspase-3 and the loss of viable cells. CONCLUSIONS: Favorable cytotoxicity and proapoptotic activity of imatinib in conjunction with As2O3 and specific down-regulation of Bcr-Abl protein levels by As2O3 in K562 cells indicate that As2O3 in combination with imatinib might be useful for circumventing resistance to imatinib monotherapy. View Publication

Hematologic stem cell rescue after high-dose cytotoxic therapy is extensively used for the treatment of many hematopoietic and solid cancers. Gene marking studies suggest that occult tumor cells within the autograft may contribute to clinical relapse. To date purging of autografts contaminated with cancer cells has been unsuccessful. The selective oncolytic property of reovirus against myriad malignant histologies in in vitro,in vivo,and ex vivo systems has been previously demonstrated. In the present study we have shown that reovirus can successfully purge cancer cells within autografts. Human monocytic and myeloma cell lines as well as enriched ex vivo lymphoma,myeloma,and Waldenström macroglobulinemia patient tumor specimens were used in an experimental purging model. Viability of the cell lines or purified ex vivo tumor cells of diffuse large B-cell lymphoma,chronic lymphocytic leukemia,Waldenström macroglobulinemia,and small lymphocytic lymphoma was significantly reduced after reovirus treatment. Further,[35S]-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cellular proteins demonstrated reovirus protein synthesis and disruption of host cell protein synthesis as early as 24 hours. Admixtures of apheresis product with the abovementioned tumor cells and cell lines treated with reovirus showed complete purging of disease. In contrast,reovirus purging of enriched ex vivo multiple myeloma,Burkitt lymphoma,and follicular lymphoma was incomplete. The oncolytic action of reovirus did not affect CD34+ stem cells or their long-term colony-forming assays even after granulocyte colony-stimulating factor (G-CSF) stimulation. Our results indicate the ex vivo use of an unattenuated oncolytic virus as an attractive purging strategy for autologous stem cell transplantations. View Publication

Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo. Here,we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10,25,or 50 mWcm(-2) and,(1)O(2) luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells,the photobleaching,and the pO(2) in the cell suspensions were also monitored. There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However,in all of the cases,cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT,as well as confirming the likely role of (1)O(2) in porphyrin-based PDT. View Publication

Internal tandem duplications (ITDs) of the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in approximately 30% of patients with acute myelogenous leukemia (AML) and are associated with a poor prognosis. FLT3 ITD mutations result in constitutive kinase activation and are thought to be pathogenetically relevant,implicating FLT3 as a plausible therapeutic target. MLN518 (formerly CT53518) is a small molecule inhibitor of the FLT3,KIT,and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases with significant activity in murine models of FLT3 ITD-positive leukemia. Given the importance of FLT3 and KIT for normal hematopoietic progenitor cells,we analyzed the effect of MLN518 on murine hematopoiesis under steady-state conditions,after chemotherapy-induced myelosuppression,and during bone marrow transplantation. In these assays,we show that MLN518 has mild toxicity toward normal hematopoiesis at concentrations that are effective in treating FLT3 ITD-positive leukemia in mice. We also demonstrate that MLN518 preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML,at concentrations that do not significantly affect colony formation by normal human progenitor cells. In analogy to imatinib mesylate in BCR-ABL-positive acute leukemia,MLN518-induced remissions may not be durable. Our studies provide the basis for integrating this compound into chemotherapy and transplantation protocols. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. 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

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells,we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells,whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover,long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage,strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether,these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors,and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis. 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

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However,clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody,we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here,we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells,lymphocytes,or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors,suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely,2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs,melphalan,or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore,administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma. View Publication