技术资料

Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23,MYB was a good candidate gene. Our molecular investigations,based on fluorescence in situ hybridization and rapid amplification of cDNA ends,revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together,these results establish,for the first time,a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia. View Publication

The lipocalin mouse 24p3 has been implicated in diverse physiological processes,including apoptosis,iron trafficking,development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However,a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3,we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid,myeloid,and erythroid cells,which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead,apoptotic defects were unique to many mature hematopoietic cell types,including neutrophils,cytokine-dependent mast cells,thymocytes,and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells,thus explaining their resistance to the ensuing cell death. The results of these studies,in conjunction with those of previous studies,reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly,these functions are limited to relatively mature blood cell compartments. View Publication

In addition to the well-recognized role in extracellular matrix remodeling,the tissue inhibitor of metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biologic functions,including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of HSCs,whose biologic behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1(-/-) mice have decreased BM cellularity and,consistent with this finding,TIMP-1(-/-) HSCs display reduced capability of long-term repopulation. Interestingly,the cell cycle distribution of TIMP-1(-/-) stem cells appears distorted,with a dysregulation at the level of the G(1) phase. TIMP-1(-/-) HSCs also display increased levels of p57,p21,and p53,suggesting that TIMP-1 could be intrinsically involved in the regulation of HSC cycling dynamics. Of note,TIMP-1(-/-) HSCs present decreased levels of CD44 glycoprotein,whose expression has been proven to be controlled by p53,the master regulator of the G(1)/S transition. Our findings establish a role for TIMP-1 in regulating HSC function,suggesting a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu. View Publication

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound,SGI-1776 inhibits Pim-1,Pim-2 and Pim-3,and was evaluated in AML-cell line,-xenograft model,and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets,c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47),were both decreased in actively cycling AML cell lines MV-4-11,MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2,Bcl-x(L),XIAP,and proapoptotic Bak and Bax were unchanged; however,a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data,xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly,SGI-1776 was also cytotoxic in AML primary cells,irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment. View Publication

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6,enhances the level of cyclin D-Cdk6 complexes,and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here,we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which,unexpectedly,it was unable to do despite high levels of Cdk6. Instead,reduction of Fbxo7 expression increased proliferation,decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27,and increased levels of S phase cyclins and Cdk2 activity. Also,Fbxo7 protein levels correlated inversely with those of CD43,suggesting direct regulation of its expression and,therefore,of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle,indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however,decreased expression of Cdk6 also enhanced levels of CD43,indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations,consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells. View Publication

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs),mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine,numerous umbilical cord blood banks have been established. In this study,we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs,MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs),slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48 h supernatant transferring,we successfully isolated MSCs which expressed CD13,CD44 and CD90 while CD34,CD45 and CD133 negative,had typical fibroblast-like shape,and was able to differentiate into adipocytes; EPCs which were CD34,and CD90 positive,CD13,CD44,CD45 and CD133 negative,adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium. View Publication

Oligomerization is an important regulatory mechanism for many proteins,including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity,suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges,resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations,which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl,reduced cell proliferation,and increased caspase-3/7 activity and DNA segmentation. Importantly,the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. View Publication

The mixed lineage leukemia (MLL) proto-oncogenic protein is a histone-lysine N-methyltransferase that is produced by proteolytic cleavage and self-association of the respective functionally distinct subunits (MLL(N) and MLL(C)) to form a holocomplex involved in epigenetic transcriptional regulation. On the basis of studies in Drosophila it has been suggested that the separated subunits might also have distinct functions. In this study,we used a genetically engineered mouse line that lacked MLL(C) to show that the MLL(N)-MLL(C) holocomplex is responsible for MLL functions in various developmental processes. The stability of MLL(N) is dependent on its intramolecular interaction with MLL(C),which is mediated through the first and fourth plant homeodomain (PHD) fingers (PHD1 and PHD4) and the phenylalanine/tyrosine-rich (FYRN) domain of MLL(N). Free MLL(N) is destroyed by a mechanism that targets the FYRN domain,whereas free MLL(C) is exported to the cytoplasm and degraded by the proteasome. PHD1 is encoded by an alternatively spliced exon that is occasionally deleted in T-cell leukemia,and its absence produces an MLL mutant protein that is deficient for holocomplex formation. Therefore,this should be a loss-of-function mutant allele,suggesting that the known tumor suppression role of MLL may also apply to the T-cell lineage. Our data demonstrate that the dissociated MLL subunits are subjected to distinct degradation pathways and thus not likely to have separate functions unless the degradation mechanisms are inhibited. View Publication

BACKGROUND Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however,the optimal cell type and long-term efficacy are unknown. In this study,we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34/M-cad BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34/M-cad BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. METHODS AND FINDINGS Colony-forming cell assays and flow cytometry analysis showed that CD34/M-cad BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE/ mice,CD34/M-cad BMCs alleviated ischemia and significantly improved blood flow compared with CD34/M-cad BMCs,CD34/M-cad BMCs,or unselected BMCs. Significantly more arterioles were seen in CD34/M-cad cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore,histologic assessment and morphometric analyses of hindlimbs treated with GFP CD34/M-cad cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP CD34/M-cad cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34/M-cad progenitor cells. A cytokine antibody array revealed that CD34/M-cad cell-conditioned medium contained higher levels of cytokines in a unique pattern,including bFGF,CRG-2,EGF,Flt-3 ligand,IGF-1,SDF-1,and VEGFR-3,than did CD34/M-cad cell-conditioned medium. The proangiogenic cytokines secreted by CD34/M-cad cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34/M-cad cells during hypoxia. CONCLUSION CD34/M-cad BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE/ mice by consistently improving blood flow and promoting arteriogenesis. Additionally,CD34/M-cad BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors. View Publication

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells,identified by binding of the fluoresceinated peptide ligand,APELIN (APLN),or an anti-APLNR mAb,were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs),increased the expression of hematoendothelial genes in differentiating hESCs,and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore,APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells. View Publication

Despite the fact that glucocorticoids and long acting beta agonists are effective treatments for asthma,their effects on human mast cells (MC) appear to be modest. Although MC are one of the major effector cells in the underlying inflammatory reactions associated with asthma,their regulation by these drugs is not yet fully understood and,in some cases,controversial. Using a human immortalized MC line (LAD2),we studied the effects of fluticasone propionate (FP) and salmeterol (SM),on the release of early and late phase mediators. LAD2 cells were pretreated with FP (100 nM),SM (1 µM),alone and in combination,at various incubation times and subsequently stimulated with agonists substance P,C3a and IgE/anti-IgE. Degranulation was measured by the release of β-hexosaminidase. Cytokine and chemokine expression were measured using quantitative PCR,ELISA and cytometric bead array (CBA) assays. The combination of FP and SM synergistically inhibited degranulation of MC stimulated with substance P (33% inhibition compared to control,n = 3,P>05). Degranulation was inhibited by FP alone,but not SM,when MC were stimulated with C3a (48% inhibition,n = 3,P>05). As previously reported,FP and SM did not inhibit degranulation when MC were stimulated with IgE/anti-IgE. FP and SM in combination inhibited substance P-induced release of tumor necrosis factor (TNF),CCL2,and CXCL8 (98%,99% and 92% inhibition,respectively,n = 4,P>05). Fluticasone and salmeterol synergistically inhibited mediator production by human MC stimulated with the neuropeptide substance P. This synergistic effect on mast cell signaling may be relevant to the therapeutic benefit of combination therapy in asthma. View Publication

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with $$-thalassemia ($$-thal) with the aim to generate trangene-free $$-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4,Klf4,Sox2,cMyc,and Lin28 resulted in formation of five iPSC colonies,from which three were picked up and expanded in $$-thal-iPSC lines. After 10 serial passages in vitro,$$-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs,whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%,but with a decreased hematopoietic colony-forming capability. In conclusion,we report herein the generation of transgene-free $$-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover,it was demonstrated that the mRNA-based reprogramming method,used mainly in fibroblasts,is also suitable for reprogramming of human BM-MSCs. View Publication