技术资料

Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation,the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells,including proteins involved in cytoskeletal rearrangement,signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2,syntrophin,and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient,with significantly higher expression in the more aggressive disease subset identified by zeta-chain-associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion,a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin,whereas PH domain-mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin,or treatment with PI3K inhibitors,significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression. View Publication

Increased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. In this study,we report an increase in apoptotic T cells from RCC patients compared with T cells from normal donors that coincided with the detection of T cells staining positive for GM2 and that the apoptosis was predominantly observed in the GM2(+) but not the GM2(-) T cell population. Ganglioside shedding from tumor rather than endogenous production accounts for GM2(+) T cells since there was no detectable level of mRNA for GM2 synthase in RCC patient T cells and in T cells from normal healthy donors after incubation with either purified GM2 or supernatant from RCC cell lines despite their staining positive for GM2. Moreover,reactive oxygen species as well as activated caspase 3,8,and 9 were predominantly elevated in GM2(+) but not GM2(-) T cells. Similarly,increased staining for GD2 and GD3 but not GD1a was detected with patient T cells with elevated levels of apoptosis in the GD2(+) and GD3(+) cells. These findings suggest that GM2,GD2,and GD3 play a significant role in immune dysfunction observed in RCC patient T 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

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

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies,those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here,we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR(+) control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast,54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further,and,although one line,NCI-H661,bound detectable levels of [(125)I]-recombinant human Epo (rHuEpo),none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5,AKT,ERK,or S6RP with rHuEpo. In addition,EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors. View Publication

BACKGROUND: The use of functional genomics has largely increased our understanding of cell biology and promises to help the development of systems biology needed to understand the complex order of events that regulates cellular differentiation in vivo. One model system clearly dependent on the integration of extra and intra cellular signals is the development of B-lymphocytes from hematopoietic stem cells in the bone marrow. This developmental pathway involves several defined differentiation stages associated with specific expression of genes including surface markers that can be used for the prospective isolation of the progenitor cells directly from the bone marrow to allow for ex vivo gene expression analysis. The developmental process can be simulated in vitro making it possible to dissect information about cell/cell communication as well as to address the relevance of communication pathways in a rather direct manner. Thus we believe that B-lymphocyte development represents a useful model system to take the first steps towards systems biology investigations in the bone marrow. RESULTS: In order to identify extra cellular signals that promote B lymphocyte development we created a database with approximately 400 receptor ligand pairs and software matching gene expression data from two cell populations to obtain information about possible communication pathways. Using this database and gene expression data from NIH3T3 cells (unable to support B cell development),OP-9 cells (strongly supportive of B cell development),pro-B and pre-B cells as well as mature peripheral B-lineage cells,we were able to identify a set of potential stage and stromal cell restricted communication pathways. Functional analysis of some of these potential ways of communication allowed us to identify BMP-4 as a potent stimulator of B-cell development in vitro. Further,the analysis suggested that there existed possibilities for progenitor B cells to send signals to the stroma. The functional consequences of this were investigated by co-culture experiments revealing that the co-incubation of stromal cells with B cell progenitors altered both the morphology and the gene expression pattern in the stromal cells. CONCLUSIONS: We believe that this gene expression data analysis method allows for the identification of functionally relevant interactions and therefore could be applied to other data sets to unravel novel communication pathways. View Publication

BACKGROUND: Specific populations of highly tumorigenic cells are thought to exist in many human tumors,including pancreatic adenocarcinoma. However,the clinical significance of these tumor-initiating (ie,cancer stem) cells remains unclear. Aldehyde dehydrogenase (ALDH) activity can identify tumor-initiating cells and normal stem cells from several human tissues. We examined the prognostic significance and functional features of ALDH expression in pancreatic adenocarcinoma. METHODS: ALDH expression was analyzed by immunohistochemistry in 269 primary surgical specimens of pancreatic adenocarcinoma and examined for association with clinical outcomes and in paired primary tumors and metastatic lesions from eight pancreatic cancer patients who had participated in a rapid autopsy program. The clonogenic growth potential of ALDH-positive pancreatic adenocarcinoma cells was assessed in vitro by a colony formation assay and by tumor growth in immunodeficient mice (10-14 mice per group). Mesenchymal features of ALDH-positive pancreatic tumor cells were examined by using quantitative reverse transcription-polymerase chain reaction and an in vitro cell invasion assay. Gene expression levels and the invasive potential of ADLH-positive pancreatic cancer cells relative to the bulk cell population were examined by reverse transcription-polymerase chain reaction and an in vitro invasion assays,respectively. All statistical tests were two-sided. RESULTS: ALDH-positive tumor cells were detected in 90 of the 269 primary surgical specimens,and their presence was associated with worse survival (median survival for patients with ALDH-positive vs ALDH-negative tumors: 14 vs 18 months,hazard ratio of death = 1.28,95% confidence interval = 1.02 to 1.68,P = .05). Six (75%) of the eight patients with matched primary and metastatic tumor samples had ALDH-negative primary tumors,and in four (67%) of these six patients,the matched metastatic lesions (located in liver and lung) contained ALDH-positive cells. ALDH-positive cells were approximately five- to 11-fold more clonogenic in vitro and in vivo compared with unsorted or ALHD-negative cells,expressed genes consistent with a mesenchymal state,and had in vitro migratory and invasive potentials that were threefold greater than those of unsorted cells. CONCLUSIONS: ALDH expression marks pancreatic cancer cells that have stem cell and mesenchymal features. The enhanced clonogenic growth and migratory properties of ALDH-positive pancreatic cancer cells suggest that they play a key role in the development of metastatic disease that negatively affects the overall survival of patients with pancreatic adenocarcinoma. View Publication

PURPOSE: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non-small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed,paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials. EXPERIMENTAL DESIGN: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines,normal tissue,and tumor tissue. RESULTS: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues,suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004,respectively) and drozitumab (P = 0.03 and P textless 0.0001,respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely,with approximately 45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125). CONCLUSIONS: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection. View Publication

Breast cancer stem cells are a group of undifferentiated cells with self-renewal and multidifferentiation potential. Chemotherapeutic and radiotherapeutic resistance,hypoxic resistance,high tumorigenicity,high cell invasion,and metastatic abilities are characteristics of these cells,which are responsible for breast cancer recurrence. Therefore,the correct sorting and identification of breast cancer stem cells is a primary step for research in this field. This article briefly describes the recent progress on sorting and identification technologies for breast cancer stem cells. Sorting technologies include the side population technique,technologies that depend on cell surface markers,ALDEFLUOR assays,and in situ detection. Identification technologies include mammosphere cultures,limited dilution in vitro,and in-vivo animal models. This review provides an important reference for breast cancer stem cell research,which will explore new methods for the treatment of patients with breast cancer. View Publication

We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27,a key regulator of cell cycle,in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly,expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly,when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients,increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition,when CLL cells were induced in vitro to enter cell cycle (eg,with cytosine phosphate guanine oligodeoxynucleotide),a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition. View Publication

The major goal of researchers and oncologists is to develop promising ground for novel therapeutic strategies to prevent recurrence or relapse of cancer. Recent evidences suggest that a subset of cells called cancer stem cells (CSCs) are present within the tumor mass which possess tumorigenic capacity and may be responsible for propagation,relapse,and metastatic dissemination. These cells have certain stem cell-like properties,e.g. quiescence,selfrenewal,asymmetric division,and multidrug resistance which allow them to drive tumor growth and evade conventional therapies. A number of markers and assays have been designed to isolate and characterize the CSC population from the bulk tumor. The objective now is to selectively target the CSCs in order to eliminate the tumor from root,overcoming the emergence of clones capable of evading traditional therapy. This approach may help in increasing the overall disease-free survival in some cancers. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication