技术资料

4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα,leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314,to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314,and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA,a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs,as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit,Ku80,ataxia telangiectasia mutated (ATM),BRCA2,or XRCC3 compared with wild-type cells,indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore,both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314. View Publication

Although natural killer (NK) cells are well known for their ability to kill tumors,few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here,we show that human leukocyte antigen class I(low) tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1,ULBP2,and ULBP3. In line with the NK receptor ligand expression profiles,antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy. View Publication

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy,many of them subsequently relapse,and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A),most frequently at arginine 882 (DNMT3A(R882)),have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3A(R882) AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy,suggesting that DNMT3A(R882) cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion,cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3(ITD)) and the nucleophosmin gene (Npm1(c)) to induce AML in vivo,and promoted resistance to anthracycline chemotherapy. In patients with AML,the presence of DNMT3A(R882) mutations predicts minimal residual disease,underscoring their role in AML chemoresistance. DNMT3A(R882) cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment,which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress,which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3A(R882) mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy. View Publication

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation,a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation,as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data,possibly due to their crosstalk with Notch Signaling. Overall,this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs. View Publication

Chromosomal translocations of the mixed lineage leukemia (MLL) gene are a common cause of acute leukemias. The oncogenic function of MLL fusion proteins is,in part,mediated through aberrant activation of Hoxa genes and Meis1,among others. Here we demonstrate using a tamoxifen-inducible Cre-mediated loss of function mouse model that DOT1L,an H3K79 methyltransferase,is required for both initiation and maintenance of MLL-AF9-induced leukemogenesis in vitro and in vivo. Through gene expression and chromatin immunoprecipitation analysis we demonstrate that mistargeting of DOT1L,subsequent H3K79 methylation,and up-regulation of Hoxa and Meis1 genes underlie the molecular mechanism of how DOT1L contributes to MLL-AF9-mediated leukemogenesis. Our study not only provides the first in vivo evidence for the function of DOT1L in leukemia,but also reveals the molecular mechanism for DOT1L in MLL-AF9 mediated leukemia. Thus,DOT1L may serve as a potential therapeutic target for the treatment of leukemia caused by MLL translocations. View Publication

A relatively rare aldehyde dehydrogenase 1 (ALDH1)-positive stem cell-like" subpopulation of tumor cells has the unique ability to initiate and perpetuate tumor growth; moreover� View Publication

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters,miR-200c-141,miR-200b-200a-429,and miR-183-96-182 were downregulated in human BCSCs,normal human and murine mammary stem/progenitor cells,and embryonal carcinoma cells. Expression of BMI1,a known regulator of stem cell self-renewal,was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly,miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells. View Publication

BACKGROUND: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy,although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. METHODOLOGY/FINDINGS: Treatment of lung tumor cells with doxorubicin,cisplatin,or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133,CD117,SSEA-3,TRA1-81,Oct-4,and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres,maintain self-renewal capacity,and differentiate. Differentiated progenitors lost expression of CD133,gained CK 8/18 and acquired drug sensitivity. In the presence of drugs,differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice,which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF,bFGF,IL-6,IL-8,HGF,PDGF-BB,G-CSF,and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2,FGFR2,CXCR1,2 and 4 receptors. Moreover,human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy. View Publication

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line,metformin blocked the cell cycle progression in G(0)/G(1) phase without inducing significant cell death. In confluent C6 cultures,on the other hand,metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation,mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine),while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride,iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C,an inhibitor of AMP-activated protein kinase (AMPK),and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251,while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. View Publication

The importance of cancer metabolism has been appreciated for many years,but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis,we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway,paced by its rate-limiting enzyme,hydroxymethylglutaryl coenzyme A reductase (HMGCR),is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease,the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway,achieved by ectopic expression of either full-length HMGCR or its novel splice variant,promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and,importantly,cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together,our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. View Publication

Creation of fusion genes by balanced chromosomal translocations is one of the hallmarks of acute myeloid leukemia (AML) and is considered one of the key leukemogenic events in this disease. In t(12;13)(p13;q12) AML,ectopic expression of the homeobox gene CDX2 was detected in addition to expression of the ETV6-CDX2 fusion gene,generated by the chromosomal translocation. Here we show in a murine model of t(12;13)(p13;q12) AML that myeloid leukemogenesis is induced by the ectopic expression of CDX2 and not by the ETV6-CDX2 chimeric gene. Mice transplanted with bone marrow cells retrovirally engineered to express Cdx2 rapidly succumbed to fatal and transplantable AML. The transforming capacity of Cdx2 depended on an intact homeodomain and the N-terminal transactivation domain. Transplantation of bone marrow cells expressing ETV6-CDX2 failed to induce leukemia. Furthermore,coexpression of ETV6-CDX2 and Cdx2 in bone marrow cells did not accelerate the course of disease in transplanted mice compared to Cdx2 alone. These data demonstrate that activation of a protooncogene by a balanced chromosomal translocation can be the pivotal leukemogenic event in AML,characterized by the expression of a leukemia-specific fusion gene. Furthermore,these findings link protooncogene activation to myeloid leukemogenesis,an oncogenic mechanism so far associated mainly with lymphoid leukemias and lymphomas. View Publication

BACKGROUND: Excessive maturation of hematopoietic cells leads to a reduction of long-term proliferative capability during cord blood (CB) expansion. In this study,we report the effects of anit-CD52 (Alemtuzumab,Campath) on both short- and long-term ex vivo expansion of CB hematopoietic stem cells (HSC) by evaluating the potential role of Alemtuzumab in preserving the repopulating capability in CB HSC and nonlymphoid progenitors. METHODS: Ex vivo expansion experiments were carried out using freshly purified CB CD34(+)cells in StemSpantrade mark SFEM medium in the presence of stem cell factor,Flt3-Ligand and thrombopoietin at 50 ng/ml. Alemtuzumab (10 microg/ml) was used to deplete CD52(+) cells during the cultures. Flow cytometry was used to monitor CB HSC and their differentiation. Colony forming unit (CFU) assays and long term culture-initiating cell (LTC-IC) assays were performed on cells obtained from day 0 (before culture) and day 14 after cultures. Secondary cultures was performed using CD34(+) cells isolated at 35 days from primary cultures and further cultured in StemSpantrade mark SFEM medium for another 14 days to confirm the long term effect of alemtuzumab in liquid cultures. RESULTS: Compared to cytokines alone,addition of alemtuzumab resulted in a significant increase in total nucleated cells,absolute CD34(+) cells,myeloid and megakaryocytic progenitors,multi-lineage and myeloid CFU and LTC-IC. CONCLUSION: The results from current study suggested that the use of alemtuzumab for ex vivo expansion of CBHSC maybe advantageous. Our findings may improve current technologies for CBHSC expansion and increase the availability of CB units for transplantation. However,in vivo studies using animal models are likely needed in further studies to test the hematopoietic effects using such expanded CB products. View Publication