技术资料

OBJECTIVE: Ionizing radiation (IR) and busulfan (BU) are commonly used as preconditioning regimens for bone marrow transplantation (BMT). We examined whether induction of apoptosis in murine bone marrow (BM) hematopoietic cells contributes to IR- and BU-induced suppression of their hematopoietic function. METHODS: The hematopoietic functions of hematopoietic stem cells (HSCs) and progenitors were analyzed by the cobblestone area-forming cell (CAFC) assay. Apoptosis was determined by measuring 3,3'-dihexyloxacarbocyanine iodide (DiCO6) uptake,annexin V staining,and/or sub-G(0/1) cells. Four cell types were studied: murine BM mononuclear cells (BM-MNCs),linage-negative hematopoietic cells (Lin-) cells),Lin- Scal+ c-kit+ cells,and Lin- Scal- c-kit+ cells by flow cytometry. RESULTS: Exposure of BM-MNCs to IR (4 Gy) or incubation of the cells with BU (30 microM) resulted in a significant reduction in CAFC frequency (ptextless0.001). The survival fractions of various day-types of CAFC for the irradiated cells were less than 10%,while that for BU-treated cells was 71.3% on day 7 and progressively declined to 5.3% on day 35. Interestingly,IR significantly induced apoptosis in BM-MNCs,Lin- cells,HSCs,and progenitors,whereas BU failed to increase apoptosis in these cells. In addition,preincubation of BM-MNCs with z-Val-Ala-Asp (OCH3)-fluoromethylketone,methyl ester (z-VAD) attenuated IR-induced reduction in CAFC but not that induced by BU. CONCLUSION: IR and BU differentially suppress the hematopoietic function of HSCs and progenitors by fundamentally different mechanisms. IR inhibits the function primarily by the induction of HSC and progenitor apoptosis. In contrast,BU suppresses HSC and progenitor function via an apoptosis-independent mechanism. View Publication

Protein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA,RAC1,and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized,they have very different properties,and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study,we determined the effects of GGTase-I deficiency on cell viability and K-RAS-induced cancer development in mice. Inactivating the gene for the critical beta subunit of GGTase-I eliminated GGTase-I activity,disrupted the actin cytoskeleton,reduced cell migration,and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover,the absence of GGTase-I activity reduced lung tumor formation,eliminated myeloproliferative phenotypes,and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly,several cell types remained viable in the absence of GGTase-I,and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS-induced malignancies. View Publication

Remarkably,apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice,but not other strains of mice,to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins,Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1,but not Bcl-2,Bcl-X(L),Bax,A1,or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand,Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis. View Publication

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107),a novel,more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF),which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells,including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples,suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly,inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together,adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance,providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML. View Publication

The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs),high-proliferative potential-CFCs (HPP-CFCs),and long-term culture-initiating cells (LTC-ICs). In mice,both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs,whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold,respectively,as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone,suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys,rhPlGF-1 alone had no mobilization effect,whereas rhPlGF-1 (260 microg/kg per day) plus rhG-CSF (100 microg/kg per day) increased rhG-CSF-elicited mobilization of CFCs,HPP-CFCs,and LTC-ICs per milliliter of blood by 5-,7-,and 15-fold,respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion,our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting. 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

Multiple myeloma is a highly radiosensitive skeletal malignancy,but bone-seeking radionuclides have not yet found their place in disease management. We previously reported that the proteasome inhibitor PS-341 selectively sensitizes myeloma cells to the lethal effects of ionizing radiation. To extend these observations to an in vivo model,we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays demonstrated synergistic killing of myeloma cells exposed to both PS-341 and 153-Sm-EDTMP. Using the orthotopic,syngeneic 5TGM1 myeloma model,the median survivals of mice treated with saline,2 doses of PS-341 (0.5 mg/kg),or a single nonmyeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21,22,and 28 days,respectively. In contrast,mice treated with combination therapy comprising 2 doses of PS-341 (0.5 mg/kg),1 day prior to and 1 day following 153-Sm-EDTMP (22.5 MBq) showed a significantly prolonged median survival of 49 days (P textless .001). In addition to prolonged survival,this treatment combination yielded reduced clonogenicity of bone marrow-resident 5TGM1 cells,reduced serum myeloma-associated paraprotein levels,and better preservation of bone mineral density. Myelosuppression,determined by peripheral blood cell counts and clonogenicity assays of hematopoietic progenitors,did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. PS-341 is a potent,selective in vivo radiosensitizer that may substantially affect the efficacy of skeletal-targeted radiotherapy in multiple myeloma. View Publication

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context,little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional,biochemical,and microarray analyses. When deprived of natural ligands,GM-CSF,interleukin-3,or erythropoietin,AP20187 prevented apoptosis of transduced TF1 cells,induced dose-dependent proliferation,and supported long-term growth. In addition,AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably,mRNAs affiliated with heat stress,including Hsp70 and Hsp105,were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence,in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor,or KDR,signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes. View Publication

Here we report the discovery of ON044580,an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases,JAK2 and BCR-ABL,and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly,this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally,ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate,STAT5,and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly,ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients,suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS,imatinib-resistant CML,and myeloproliferative neoplasms that develop resistance to ATP-competitive agents. View Publication

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure,we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition,the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug,resveratrol,maintained Fancd2(-/-) KSL cells in quiescence,improved the marrow microenvironment,partially corrected the abnormal cell cycle status,and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects,and that this model is well suited for pharmacologic screening studies. View Publication

Because of the wide use of pesticides for domestic and industrial purposes,the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice,but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study,an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E,CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors,has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 micro M to 1000 micro M,and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand,human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC(50) values are 305.2 +/- 22.6 micro M [total erythroid colonies] and textgreater500 micro M [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors,as was found for the murine cells,emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose. View Publication