技术资料

RARA (retinoic acid receptor alpha) haploinsufficiency is an invariable consequence of t(15;17)(q22;q21) translocations in acute promyelocytic leukemia (APL). Retinoids and RARA activity have been implicated in hematopoietic self-renewal and neutrophil maturation. We and others therefore predicted that RARA haploinsufficiency would contribute to APL pathogenesis. To test this hypothesis,we crossed Rara(+/-) mice with mice expressing PML (promyelocytic leukemia)-RARA from the cathepsin G locus (mCG-PR). We found that Rara haploinsufficiency cooperated with PML-RARA,but only modestly influenced the preleukemic and leukemic phenotype. Bone marrow from mCG-PR(+/-) × Rara(+/-) mice had decreased numbers of mature myeloid cells,increased ex vivo myeloid cell proliferation,and increased competitive advantage after transplantation. Rara haploinsufficiency did not alter mCG-PR-dependent leukemic latency or penetrance,but did influence the distribution of leukemic cells; leukemia in mCG-PR(+/-) × Rara(+/-) mice presented more commonly with low to normal white blood cell counts and with myeloid infiltration of lymph nodes. APL cells from these mice were responsive to all-trans retinoic acid and had virtually no differences in expression profiling compared with tumors arising in mCG-PR(+/-) × Rara(+/+) mice. These data show that Rara haploinsufficiency (like Pml haploinsufficiency and RARA-PML) can cooperate with PML-RARA to influence the pathogenesis of APL in mice,but that PML-RARA is the t(15;17) disease-initiating mutation. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein,which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis,we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells,as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients,even in the absence of t(8;21). On a functional level,knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly,self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies,serial replating capacity of primary cells,and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML. View Publication

Tristetraprolin (TTP,Zfp36,Nup475,Tis11) dramatically reduces the stability of target mRNAs by binding to AU-rich elements in their 3' untranslated regions. Through this mechanism,TTP functions as a rheostatic,temporal regulator of gene expression. TTP knockout (KO) mice exhibit completely penetrant granulocytic hyperplasia. We have shown that the hematopoietic stem-progenitor cell compartment in TTP KO mice is also altered. Although no change was detected in long-term hematopoietic stem cell (HSC) frequency or function,as assayed by immunophenotypic markers or limiting dilution transplants,we observed increases in the frequencies and numbers of short-term HSCs,multipotent progenitors,and granulocyte-monocyte progenitors. This pattern is consistent with reactive granulopoiesis� View Publication

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene,which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding,induction of endoplasmic reticulum (ER) stress,activation of the unfolded protein response (UPR),and ultimately a block in granulocytic differentiation. To test this model,we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice,though without significant basal UPR activation,are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover,inaction of protein kinase RNA-like ER kinase (Perk),one of the major sensors of ER stress,either alone or in combination with G193X Elane,had no effect on basal granulopoiesis. However,inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN. View Publication

The role of autophagy,a lysosomal degradation pathway which prevents cellular damage,in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis,malignant transformation of HSCs leads to leukemia. Therefore,mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study,we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions,a severe myeloproliferation,and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species,as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded,Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions,the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively,these data show that Atg7 is an essential regulator of adult HSC maintenance. 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

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

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

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