搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

INTRODUCTION: The expression of proinflammatory protein tissue transglutaminase 2 (TG2) is frequently upregulated in multiple cancer cell types. However,the exact role of TG2 in cancer cells is not well-understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted cancer stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells. METHODS: Using various mutant constructs,we analyzed the activity of TG2 that is essential for promoting the EMT-CSC phenotype. RESULTS: Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT-CSC in mammary epithelial cells. In contrast,overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover,TG2-dependent activation of the proinflammatory transcription factor NF-κB is deemed essential for promoting the EMT-CSC phenotype in mammary epithelial cells. CONCLUSIONS: Our results suggest that the transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide a strong rationale for developing small-molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways,reversing drug resistance and inhibiting the metastasis of cancer cells. 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

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

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted,mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC),investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly,aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom,where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma,ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells,which are more numerous and broadly distributed in normal crypts,showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not),(b) generated them after implanting as few as 25 cells,and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus,ALDH1 seems to be a specific marker for identifying,isolating,and tracking human colonic SC during CRC development. These findings also support our original hypothesis,derived previously from mathematical modeling of crypt dynamics,that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development. View Publication

INTRODUCTION Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs),molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However,no currently accepted molecular target exists against TNBC and,moreover,TICs. Therefore,we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs. METHODS A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1),including its expression in breast cancer cell lines,effect on the CD44high/CD24-/low TIC subpopulation,growth inhibition,mammosphere formation,and apoptosis,as well as the activity of the PLK1 inhibitor,BI 2536. RESULTS Of the 85 kinases identified in the screen,28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC),BT474-M1 (ER+/HER2+,a metastatic variant),and HR5 (ER+/HER2+,a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly,12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines,representing all of the breast cancer subtypes,and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth,mammosphere formation,and apoptosis as did PLK1 siRNAs. Finally,whereas paclitaxel,doxorubicin,and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149,subsequent treatment with BI 2536 killed the emergent population,suggesting that it could potentially be used to prevent relapse. CONCLUSION Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus,PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer. View Publication

Aurora kinases play an important role in chromosome alignment,segregation,and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases,and ZM447439,a potent inhibitor of Aurora kinases,effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152,a highly selective inhibitor of Aurora B kinase,on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60,NB4,MOLM13),ALL line (PALL-2),biphenotypic leukemia (MV4-11),acute eosinophilic leukemia (EOL-1),and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM,as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis,as measured by cell-cycle analysis and annexin V staining,respectively. Of note,AZD1152 synergistically enhanced the antiproliferative activity of vincristine,a tubulin depolymerizing agent,and daunorubicin,a topoisomerase II inhibitor,against the MOLM13 and PALL-2 cells in vitro. Furthermore,AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together,AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

TEL2/ETV7 is highly homologous to the ETS transcription factor TEL/ETV6,a frequent target of chromosome translocation in human leukemia. Although both proteins are transcriptional inhibitors binding similar DNA recognition sequences,they have opposite biologic effects: TEL inhibits proliferation while TEL2 promotes it. In addition,forced expression of TEL2 but not TEL blocks vitamin D3-induced differentiation of U937 and HL60 myeloid cells. TEL2 is expressed in the hematopoietic system,and its expression is up-regulated in bone marrow samples of some patients with leukemia,suggesting a role in oncogenesis. Recently we also showed that TEL2 cooperates with Myc in B lymphomagenesis in mice. Here we show that forced expression of TEL2 alone in mouse bone marrow causes a myeloproliferative disease with a long latency period but with high penetrance. This suggested that secondary mutations are necessary for disease development. Treating mice receiving transplants with TEL2-expressing bone marrow with the chemical carcinogen N-ethyl-N-nitrosourea (ENU) resulted in significantly accelerated disease onset. Although the mice developed a GFP-positive myeloid disease with 30% of the mice showing elevated white blood counts,they all died of T-cell lymphoma,which was GFP negative. Together our data identify TEL2 as a bona fide oncogene,but leukemic transformation is dependent on secondary mutations. View Publication

Genes that are strongly repressed after B-cell activation are candidates for being inactivated,mutated,or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4),a gene down-regulated in activated murine B cells,is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this,overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre-B-cell transformation by v-Abl and BCR-ABL,oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death,but not cell-cycle arrest,can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively,our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies. View Publication

After DNA replication,sister chromatids must be untangled,or decatenated,before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation,causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint,and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain,and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance,Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro,purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus,Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors,and Metnase is a potential therapeutic target for small molecule interference. View Publication