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

Transforming growth factor beta (TGF-$$) signaling has been implicated in driving tumor progression and metastasis by inducing stem cell-like features in some human cancer cell lines. In this study,we have utilized a novel murine cell line NMuMG-ST,which acquired cancer stem cell (CSC) phenotypes during spontaneous transformation of the untransformed murine mammary cell line NMuMG,to investigate the role of autocrine TGF-$$ signaling in regulating their survival,metastatic ability,and the maintenance of cancer stem cell characteristics. We have retrovirally transduced a dominant-negative TGF-$$ type II receptor (DNRII) into the NMuMG-ST cell to abrogate autocrine TGF-$$ signaling. The expression of DNRII reduced TGF-$$ sensitivity of the NMuMG-ST cells in various cell-based assays. The blockade of autocrine TGF-$$ signaling reduced the ability of the cell to grow anchorage-independently and to resist serum deprivation-induced apoptosis. These phenotypes were associated with reduced levels of active and phosphorylated AKT and ERK,and Gli1 expression suggesting that these pathways contribute to the growth and survival of this model system. More interestingly,the abrogation of autocrine TGF-$$ signaling also led to the attenuation of several features associated with mammary stem cells including epithelial-mesenchymal transition,mammosphere formation,and expression of stem cell markers. When xenografted in athymic nude mice,the DNRII cells were also found to undergo apoptosis and induced significantly lower lung metastasis burden than the control cells even though they formed similar size of xenograft tumors. Thus,our results indicate that autocrine TGF-$$ signaling is involved in the maintenance and survival of stem-like cell population resulting in the enhanced metastatic ability of the murine breast cancer cells. View Publication

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing,highly tumorigenic,drug-resistant tumor cells termed cancer stem cells. Recently,we and others reported a new phenotypic plasticity wherein highly tumorigenic,drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations but also from cancer cells lacking these properties. In the current study,we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β-catenin signaling,pathways previously implicated in carcinogenesis,pluripotency and drug resistance. Using GFP expression,Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines,we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells,whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β,which led to activation and accumulation of β-catenin. Accordingly,pharmacological or genetic perturbation of GSK3β or β-catenin dramatically impacted conversion of NSP to SP. Further downstream,β-catenin's effects on NSP-SP equilibrium were dependent upon its interaction with CBP,a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β-catenin/CBP signaling mediates phenotypic plasticity in and out of a drug-resistant,highly tumorigenic state. Therefore,targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem-like phenotype. View Publication

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL-expressing cells. To extend these observations,we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases,including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation,inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound,the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL-positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome-positive ALL patient. As CGP 57148 inhibits the PDGFR kinase,we also showed that cells expressing an activated PDGFR tyrosine kinase,TEL-PDGFR,are sensitive to this compound. Thus,this compound may be useful for the treatment of a variety of BCR-ABL-positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein. View Publication

Aldehyde dehydrogenase 1 A1 (ALDH1A1) has recently been suggested as a marker for cancer stem or stem-like cancer cells of some human malignancies. The purpose of this study was to investigate the stem cell-related function and clinical significance of the ALDH1A1 in bladder urothelial cell carcinoma. Aldefluor assay was used to isolate ALDH1A1+ cells from bladder cancer cells. Stem cell characteristics of the ALDH1A1+ cells were then investigated by in vitro and in vivo approaches. Immunohistochemistry was done for evaluating ALDH1A1 expression on 22 normal bladder tissues and 216 bladder tumor specimens of different stage and grade. The ALDH1A1+ cancer cells displayed higher in vitro tumorigenicity compared with isogenic ALDH1A1- cells. The ALDH1A1+ cancer cells could generate xenograft tumors that resembled the histopathologic characteristics and heterogeneity of the parental cells. High ALDH1A1 expression was found in 26% (56 of 216) of human bladder tumor specimens and significantly related to advanced pathologic stage,high histologic grade,recurrence and progression,and metastasis of bladder urothelial cell carcinomas (all P textless 0.05). Furthermore,ALDH1A1 expression was inversely associated with cancer-specific and overall survivals of the patients (P = 0.027 and 0.030,respectively). Therefore,ALDH1A1+ cell population could be enriched in tumor-initiating cells. ALDH1A1 may serve as a useful marker for monitoring the progression of bladder tumor and identifying bladder cancer patients with poor prognosis who might benefit from adjuvant and effective treatments. View Publication

Acute myeloid leukemia (AML) is characterized by the block of differentiation,deregulated apoptosis,and an increased self-renewal of hematopoietic precursors. It is unclear whether the self-renewal of leukemic blasts results from the cumulative effects of blocked differentiation and impaired apoptosis or whether there are mechanisms directly increasing self-renewal. The AML-associated translocation products (AATPs) promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha),promyelocytic leukemia zinc finger (PLZF)/RAR alpha (X-RAR alpha),and AML-1/ETO block hematopoietic differentiation. The AATPs activate the Wnt signaling by up-regulating gamma-catenin. Activation of the Wnt signaling augments self-renewal of hematopoietic stem cells (HSCs). Therefore,we investigated how AATPs influence self-renewal of HSCs and evaluated the role of gamma-catenin in the determination of the phenotype of HSCs expressing AATPs. Here we show that the AATPs directly activate the gamma-catenin promoter. The crucial role of gamma-catenin in increasing the self-renewal of HSCs upon expression of AATPs is demonstrated by (i) the abrogation of replating efficiency upon hindrance of gamma-catenin expression through RNA interference,and (ii) the augmentation of replating efficiency of HSCs upon overexpression of gamma-catenin itself. In addition,the inoculation of gamma-catenin-transduced HSCs into irradiated recipient mice establishes the clinical picture of AML. These data provide the first evidence that the aberrant activation of Wnt signaling by the AATP decisively contributes to the pathogenesis of AML. View Publication

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome,formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment,we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method,we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly,we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+),whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations,we found that Ph(+) and Ph(-) candidate CML stem cells could be prospectively separated. In addition,by generating an anti-IL1RAP antibody,we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph(+) from Ph(-) candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML. View Publication

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. 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

Few epitopes are available for vaccination therapy of patients with squamous cell carcinoma of the head and neck (SCCHN). Using a tumor-specific CTL,aldehyde dehydrogenase 1 family member A1 (ALDH1A1) was identified as a novel tumor antigen in SCCHN. Mass spectral analysis of peptides in tumor-derived lysates was used to determine that the CTL line recognized the HLA-A*0201 (HLA-A2) binding ALDH1A1(88-96) peptide. Expression of ALDH1A1 in established SCCHN cell lines,normal mucosa,and primary keratinocytes was studied by quantitative reverse transcription-PCR and immunostaining. Protein expression was further defined by immunoblot analysis,whereas ALDH1A1 activity was measured using ALDEFLUOR. ALDH1A1(88-96) peptide was identified as an HLA-A2-restricted,naturally presented,CD8(+) T-cell-defined tumor peptide. ALDH1A1(88-96) peptide-specific CD8(+) T cells recognized only HLA-A2(+) SCCHN cell lines,which overexpressed ALDH1A1,as well as targets transfected with ALDH1A1 cDNA. Target recognition was blocked by anti-HLA class I and anti-HLA-A2 antibodies. SCCHN cell lines overexpressing ALDH1 had high enzymatic activity. ALDH1A1 protein was expressed in 12 of 17 SCCHN,and 30 of 40 dysplastic mucosa samples,but not in normal mucosa. ALDH1A1 expression levels in target cells correlated with their recognition by ALDH1A1(88-96) peptide-specific CD8(+) T cells. Our findings identify ALDH1A1,a metabolic antigen,as a potential target for vaccination therapy in the cohort of SCCHN subjects with tumors overexpressing this protein. A smaller cohort of subjects with SCCHN,whose tumors express little to no ALDH1A1,and thus are deficient in conversion of retinal to retinoic acid,could benefit from chemoprevention therapy. View Publication

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis,cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation,colony formation),chemokine receptor expression,constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors,but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression,but,compared to CD34- AML cells,CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore,a broad constitutive chemokine release profile was detected for most patients,and the following chemokine clusters could be identified: CCL2-4/CXCL1/8,CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile. View Publication

OBJECTIVE: Animal models have provided evidence for the existence of leukemia stem cells (LSC). However,prospective isolation of human LSC from patients with acute myeloid leukemia (AML),as well as the assessment of their clinical significance,has remained a major challenge. MATERIALS AND METHODS: We have studied the functional characteristics of a subset of leukemia cells that expressed CD34 and high aldehyde dehydrogenase activity (ALDH(br)),which was freshly isolated from the mononuclear cells at the time of diagnosis from the marrow of 68 consecutive patients suffering from AML. RESULTS: The percentage of ALDH(br) cells ranged from 0.01% to 16.0% with a median of 0.5%. Compared to their counterparts with low aldehyde dehydrogenase activity from the same individual patients,the ALDH(br) population showed a significantly higher affinity to human mesenchymal stromal cells (n=12; ptextless0.01),a more than twofold higher proportion of slow-dividing and quiescent cells (n=4; ptextless0.05),higher numbers of long-term culture-initiating cell colonies in vitro (n=25; ptextless0.01),and an enhanced engraftment in the nonobese diabetic/severe combined immunodeficient mouse model (n=3; ptextless0.05). Above all,we found that the frequency of ALDH(br) cells correlated significantly with diminished survival probability (p=0.025) and with adverse cytogenetic factors (ptextless0.05). CONCLUSION: A small proportion of leukemia cells derived from the marrow of patients with AML were ALDH(br) and CD34(+). They demonstrated functional characteristics of LSC and high percentages of these cells among the leukemia cells correlated significantly with poor clinical outcome. View Publication

BACKGROUND: Previous studies have shown that patients with Bcr-Abl-positive acute lymphoblastic leukemia (ALL) either have primary disease that is refractory to imatinib mesylate or develop disease recurrence after an initial response. METHODS: The authors investigated the effects of a newly designed Bcr-Abl inhibitor,AMN107,by comparing its in vitro inhibitory potency on p190 Bcr-Abl ALL cell lines with that of imatinib. RESULTS: In two Philadelphia (Ph)-positive ALL cell lines,AMN107 was found to be 30-40 times more potent than imatinib in inhibiting cellular proliferation. AMN107 was also more effective than imatinib in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. The inhibition of cellular proliferation was associated with the induction of apoptosis in only one of the cell lines. No activity was observed in cell lines lacking the BCR-ABL genotype. CONCLUSIONS: The results of the current study suggest the superior potency of AMN107 compared with imatinib in Ph-positive ALL and support clinical trials of AMN107 in patients with Ph-positive ALL. View Publication