技术资料

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

Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease,incurable by standard chemotherapy. NK314,a new anticancer agent possessing inhibitory activity specific for topoisomerase IIα (Top2α),inhibited the growth of various ATL cell lines (50% inhibitory concentration: 23-70nM) with more potent activity than that of etoposide. In addition to the induction of DNA double-strand breaks by inhibition of Top2α,NK314 induced degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs),resulting in impaired DNA double-strand break repair. The contribution of DNA-PK to inhibition of cell growth was affirmed by the following results: NK314 inhibited cell growth of M059J (a DNA-PKcs-deficient cell line) and M059K (a cell line with DNA-PKcs present) with the same potency,whereas etoposide exhibited weak inhibition of cell growth with M059K cells. A DNA-PK specific inhibitor,NU7026,enhanced inhibitory activity of etoposide on M059K as well as on ATL cells. These results suggest that NK314 is a dual inhibitor of Top2α and DNA-PK. Because ATL cells express a high amount of DNA-PKcs,NK314 as a dual molecular targeting anticancer agent is a potential therapeutic tool for treatment of ATL. View Publication

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However,an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study,we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1,side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore,knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice,supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion,but not for CSC maintenance. Taken together,our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus,targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer. View Publication

With regard to human induced pluripotent stem cells (hiPSCs),in which adult cells are reprogrammed into embryonic-like cells using defined factors,their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study,hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method,and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs,hESCs,hiPSC-ECs,hESC-ECs,and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the distances" among hiPSCs� View Publication

Nano- and microscale topographical cues play critical roles in the induction and maintenance of various cellular functions,including morphology,adhesion,gene regulation,and communication. Recent studies indicate that structure and function at the heart tissue level is exquisitely sensitive to mechanical cues at the nano-scale as well as at the microscale level. Although fabrication methods exist for generating topographical features for cell culture,current techniques,especially those with nanoscale resolution,are typically complex,prohibitively expensive,and not accessible to most biology laboratories. Here,we present a tunable culture platform comprised of biomimetic wrinkles that simulate the heart's complex anisotropic and multiscale architecture for facile and robust cardiac cell alignment. We demonstrate the cellular and subcellular alignment of both neonatal mouse cardiomyocytes as well as those derived from human embryonic stem cells. By mimicking the fibrillar network of the extracellular matrix,this system enables monitoring of protein localization in real time and therefore the high-resolution study of phenotypic and physiologic responses to in-vivo like topographical cues. View Publication

The present study shows that alumina nanotopography affects monocyte/macrophage behavior. Human mononuclear cells cultured on alumina membranes with pore diameters of 20 and 200 nm were evaluated in terms of cell adhesion,viability,morphology,and release of proinflammatory cytokines. After 24 hours,cell adhesion was assessed by means of light microscopy and cell viability by measuring LDH release. The inflammatory response was evaluated by quantifying interleukin-1β and tumour necrosis factor-α. Finally,scanning electron microscopy was used to study cell morphology. Results showed pronounced differences in cell number,morphology,and cytokine release depending on the nanoporosity. Few but highly activated cells were found on the 200 nm porous alumina,while relatively larger number of cells were found on the 20 nm porous surface. However,despite their larger number,the cells adhering on the 20 nm surface exhibited reduced pro-inflammatory activity. The data of this paper implies that nanotopography could be exploited for controlling the inflammatory response to implants. View Publication

Pseudo-Pelger-Huët anomaly (PPHA) has been documented in association with transplant medications and other drugs. This iatrogenic neutrophilic dysplasia is reversible with cessation or adjustment of medications but is frequently confused with myelodysplastic syndrome (MDS) based on the conventional concept that PPHA is a marker for dysplasia. We investigated the clinicopathologic features in iatrogenic PPHA and compared them with MDS-related PPHA. The 13 cases studied included 5 bone marrow/stem cell transplantations,3 solid organ transplantations,1 autoimmune disease,3 chronic lymphocytic leukemias,and 1 breast carcinoma. For 12 cases,there was follow-up evaluation,and all demonstrated at least transient normalization of neutrophilic segmentation. All 9 cases of MDS demonstrated at least 2 of the following pathologic abnormalities on bone marrow biopsy: hypercellularity (8/9),morphologic dysplasia (8/9),clonal cytogenetic abnormality (7/9),and increased blasts (3/9),whereas these abnormalities were typically absent in iatrogenic PPHA. Iatrogenic PPHA displayed a higher proportion of circulating PPHA cells than in MDS (mean,47.4%; SD,31.6% vs mean,12.3%; SD,9.8; P textless .01). A diagnostic algorithm is proposed in which isolated PPHA is indicative of transient or benign PPHA unless proven otherwise. View Publication

Human embryonic stem cells (hESCs) offer exciting potential in regenerative medicine for the treatment of a host of diseases including cancer,Alzheimer's and Parkinson's disease. They also provide insight into human development and disease and can be used as models for drug discovery and toxicity analyses. The key properties of hESCs that make them so promising for medical use are that they have the ability to self-renew indefinitely in culture and they are pluripotent,which means that they can differentiate into any of more than 200 human cell types. Since proteins are the effectors of cellular processes,it is important to investigate hESC expression at the protein level as well as at the transcript level. In addition,post-translational modifications,such as phosphorylation,may influence the activity of pivotal proteins in hESCs,and this information can only be determined by studying the proteome. In this review,we summarize the results obtained from several proteomics analyses of hESCs that have been reported in the last few years. View Publication

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However,because most HES patients lack FIP1L1-PDGFRA,we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G,L507P,I562M,H570R,H650Q,N659S,L705P,R748G,and Y849S). When cloned into 32D cells,N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation,clonogenic growth,and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity,we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion,our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients. View Publication

We have used in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells (CSC) and bone marrow-derived mesenchymal stem cells (MSC). We show that both of these cell populations are organized in a cellular hierarchy in which primitive aldehyde dehydrogenase expressing mesenchymal cells regulate breast CSCs through cytokine loops involving IL6 and CXCL7. In NOD/SCID mice,labeled MSCs introduced into the tibia traffic to sites of growing breast tumor xenografts where they accelerated tumor growth by increasing the breast CSC population. With immunochemistry,we identified MSC-CSC niches in these tumor xenografts as well as in frozen sections from primary human breast cancers. Bone marrow-derived MSCs may accelerate human breast tumor growth by generating cytokine networks that regulate the CSC population. View Publication

The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2,α₂δ₂) and fetal hemoglobin (HbF,α₂γ₂) both inhibit the polymerization of hemoglobin S,which results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA,α₂β₂) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to up-regulate δ-globin to increase HbA2 expression,we created a series of EKLF-GATA1 fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATA1,and then tested their effects on hemoglobin expression. EKLF-GATA1 fusion proteins activated δ-,γ-,and β-globin promoters in K562 cells,and significantly up-regulated δ- and γ-globin RNA transcript and protein expression in K562 and/or CD34(+) cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter,and may represent a new genetic therapeutic approach to β-hemoglobinopathies. View Publication

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation,reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization,and the cytoplasmic domain propagates signals that activate β(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly� View Publication