技术资料

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons,astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker,the aldehyde dehydrogenase activity,we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency,BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly,we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting. View Publication

Trib1,Trib2,and Trib3 are mammalian homologs of Tribbles,an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice,whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members,we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia,whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor,CCAAT/enhancer-binding protein-alpha (C/EBPalpha),is important for leukemogenesis. Similar to Trib2,Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast,Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha,which account for their differential ability to induce leukemia. View Publication

Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic,adipogenic,and chondrogenic differentiation properties retrospectively. Here,we report 2 subpopulations of MSCs,separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs,with the upregulation of Flt-1,CXCR4,and Angiopoietin-2. Thus,MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time. View Publication

Determining how normal and leukemic stem cells behave in vivo,in a dynamic and noninvasive way,remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes,which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context,we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI],DiD,DiR,and PKH26) can give rise to microenvironmental contamination,including when used in suboptimal concentration,after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually,we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches. View Publication

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress,cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62),but its role in the regulation of autophagy is controversial. Here,we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK,thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly,p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV,and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy. View Publication

BACKGROUND: Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples. METHODS: HPC-A samples from 44 patients were incubated with Aldefluor with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1(bright)) were analyzed for stem/progenitor markers CD34,CD90,CD117,and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression. RESULTS: In ALDH1(bright)/SSC(low) cells,0.13% of the cells had CD34(+) expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1(bright)/SSC(low)/CD90(dim) cells amongst the nonlineage depleted samples was 0.04%. CD117(dim-bright) expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1(bright)/SSC(low) nonlineage depleted cells with a frequency of 0.28%. The ALDH1(bright)/CD90(dim) cells had the smallest mean electronic volume of 264.9 microm(3) when compared with cells with CD34(bright) expression (270.2 microm(3)) and ALDH1(dim)/CD90(dim) cells (223 microm(3)). CONCLUSIONS: ALDH1(bright)/SSC(low) cells show heterogeneity in expression of the four stem cell markers studied. The CD90 cells in both the ALDH1(bright) and ALDH1(dim) populations had the smallest mean electronic volume when compared with similar cells with CD117 expression. View Publication

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells,and Ras activity enhances the oncogenic ability of Bcr-Abl. However,the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction,resulting in blocking the MAP kinase pathway. In the present study,we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl(+) progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status,resulting in inhibited proliferation of CML cells. Moreover,RKIP up-regulated cell cycle regulator FoxM1 expression,resulting in G(1) arrest via p27(Kip1) and p21(Cip1) accumulation. In colony-forming unit granulocyte,erythroid,macrophage,megakaryocyte,colony-forming unit-granulocyte macrophage,and burst-forming unit erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions,and inhibited colony formation of Bcr-Abl(+) progenitor cells,whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl(+) progenitor cells. Thus,Bcr-Abl represses the expression of RKIP,continuously activates pERK1/2,and suppresses FoxM1 expression,resulting in proliferation of CML cells. View Publication

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However,it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias,we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition,overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover,PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia. View Publication

Hairy enhancer of split 1 (Hes1) is a basic helix-loop-helix transcriptional repressor that affects differentiation and often helps maintain cells in an immature state in various tissues. Here we show that retroviral expression of Hes1 immortalizes common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in the presence of interleukin-3,conferring permanent replating capability on these cells. Whereas these cells did not develop myeloproliferative neoplasms when intravenously administered to irradiated mice,the combination of Hes1 and BCR-ABL in CMPs and GMPs caused acute leukemia resembling blast crisis of chronic myelogenous leukemia (CML),resulting in rapid death of the recipient mice. On the other hand,BCR-ABL alone caused CML-like disease when expressed in c-Kit-positive,Sca-1-positive,and lineage-negative hematopoietic stem cells (KSLs),but not committed progenitors CMPs or GMPs,as previously reported. Leukemic cells derived from Hes1 and BCR-ABL-expressing CMPs and GMPs were more immature than those derived from BCR-ABL-expressing KSLs. Intriguingly,Hes1 was highly expressed in 8 of 20 patients with CML in blast crisis,but not in the chronic phase,and dominant negative Hes1 retarded the growth of some CML cell lines expressing Hes1. These results suggest that Hes1 is a key molecule in blast crisis transition in CML. View Publication

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases,neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore,midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally,a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression,or by BH3-mimetics such as obatoclax,may be an attractive therapy concept in SM. View Publication

Frequent hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) include aberrant NOTCH signaling and deletion of the CDKN2A locus,which contains 2 closely linked tumor suppressor genes (INK4A and ARF). When bone marrow cells or thymocytes transduced with a vector encoding the constitutively activated intracellular domain of Notch1 (ICN1) are expanded ex vivo under conditions that support T-cell development,cultured progenitors rapidly induce CD4+/CD8+ T-ALLs after infusion into healthy syngeneic mice. Under these conditions,enforced ICN1 expression also drives formation of T-ALLs in unconditioned CD-1 nude mice,bypassing any requirements for thymic maturation. Retention of Arf had relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow-derived ICN1+ progenitors in which the locus is epigenetically silenced,and all resulting Arf (+/+) tumors failed to express the p19(Arf) protein. In striking contrast,retention of Arf in thymocyte-derived ICN1+ donor cells significantly delayed disease onset and suppressed the penetrance of T-ALL. Use of cultured thymocyte-derived donor cells expressing a functionally null Arf-GFP knock-in allele confirmed that ICN1 signaling can induce Arf expression in vivo. Arf activation by ICN1 in T cells thereby provides stage-specific tumor suppression but also a strong selective pressure for deletion of the locus in T-ALL. View Publication

Up to now,no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes,which hampers its application in gene therapy and immunotherapy areas. Here,we report that LVs incorporating measles virus (MV) glycoproteins,H and F,on their surface allowed transduction of 50% of quiescent B cells,which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover,the naive and memory phenotypes of transduced resting B cells were maintained. Importantly,H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells,B-cell chronic lymphocytic leukemia cells,blocked in G(0)/G(1) early phase of the cell cycle. Thus,H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells. View Publication