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

BACKGROUND Although studies have shown that Oct4,Sox2,Klf4,and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs,the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors,including us,have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein,we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells. METHODS Among three miRNAs (miR-200c,miR-302s,and miR-369s) that were previously presented for miRNA-mediated reprogramming,miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302,the cells were incubated in ES medium for 3 weeks and then characterized. RESULTS iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression,low proliferation,high apoptosis,low epithelial-mesenchymal transition marker expression (N-cadherin,TGFBR2),and sensitization to drugs. Several miRNAs were changed (e.g.,low oncomiR miR-21,high miR-29b). cMyc was decreased,and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP,FABP4,and ALB). AOF2 (also known as LSD1 or KDM1),one of the targets for miR-302,was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres,including cMyc down-regulation and H3K4 methylation. In drug-resistant cells,sensitization was achieved through miR-302-mediated reprogramming. CONCLUSIONS miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation,which caused H3K4 methylation and c-Myc repression. View Publication

BACKGROUND: Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells,termed cancer stem cells (CSC),appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed,therefore,that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents,then residual tumors might be expected to contain a higher frequency of CoCSC. METHODS AND FINDINGS: Xenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3),at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover,the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones,we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. CONCLUSIONS: CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC,major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes,thereby suggesting avenues for improving cancer therapy. 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

Identification of cancer stem cells is crucial for advancing cancer biology and therapy. Several markers including CD24,CD44,CD117,CD133,the G subfamily of ATP-binding cassette transporters (ABCG),epithelial specific antigen (ESA) and aldehyde dehydrogenase (ALDH) are used to identify and investigate human epithelial cancer stem cells in the literature. We have now systemically analyzed and compared the expression of these markers in fresh ovarian epithelial carcinomas. Although the expression levels of these markers were unexpectedly variable and partially overlapping in fresh ovarian cancer cells from different donors,we reliably detected important levels of CD133 and ALDH in the majority of fresh ovarian cancer. Furthermore,most of these stem cell markers including CD133 and ALDH were gradually lost following in vitro passage of primary tumor cells. However,the expression of ALDH and CD133,but not CD24,CD44 and CD117,could be partially rescued by the in vitro serum-free and sphere cultures and by the in vivo passage in the immune-deficient xenografts. ALDH+ and CD133+ cells formed three-dimensional spheres more efficiently than their negative counterparts. These sphere-forming cells expressed high levels of stem cell core gene transcripts and could be expanded and form additional spheres in long-term culture. ALDH+,CD133+ and ALDH+ CD133+ cells from fresh tumors developed larger tumors more rapidly than their negative counterparts. This property was preserved in the xenografted tumors. Altogether,the data suggest that ALDH+ and CD133+ cells are enriched with ovarian cancer-initiating (stem) cells and that ALDH and CD133 may be widely used as reliable markers to investigate ovarian cancer stem cell biology. View Publication

STI571 is a 2-phenylalaminopyrimidine derivative that inhibits c-abl,Bcr-Abl,and platelet-derived growth factor receptor tyrosine kinases. Recently,inhibition of stem cell factor (SCF)-induced c-kit phosphorylation and cell proliferation by STI571 was reported in the human myeloid cell line MO7e. Because approximately 70% of acute myelogenous leukemia (AML) cases are c-kit positive,we evaluated in vitro effects of STI571 on c-kit-positive cell lines and primary AML blast cells. At concentrations textgreater5 microM,the drug marginally inhibited SCF-independent proliferation of cell lines and most of AML blasts. Treatment of AML cells with cytarabine and STI571 showed synergistic effect at low concentrations. Western blotting analysis documented a distinct band of M(r) 145,000 specific for c-kit in cell lines and in AML samples. There was no correlation between the level of the c-kit expression evaluated by Western blotting and percentage of c-kit-positive blasts as measured by flow cytometry. Neither in cell lines nor in primary AML cells,c-kit autophosphorylation was detectable under standard growth conditions. SCF-induced phosphorylation of c-kit in MO7e cells was inhibited by STI571. In a c-kit-positive AML-4 cell line,as well as in AML samples,c-kit phosphorylation was not induced by SCF exposure,suggesting that in these cases,the receptor could not be functionally activated. In conclusion,with the exception of MO7e,SCF did not induce phosphorylation of c-kit,and cell proliferation was not modulated in the presence of STI571. We did not detect any SCF-independent c-kit phosphorylation in our experimental systems. Consequently,STI571 exerted only a limited inhibitory effect on the cell growth. View Publication

UNLABELLED Basal-like breast cancer (BLBC) is an aggressive subtype of breast cancer which is often enriched with cancer stem cells (CSC),but the underlying molecular basis for this connection remains elusive. We hypothesized that BLBC cells are able to establish a niche permissive to the maintenance of CSCs and found that tumor cell-derived periostin (POSTN),a component of the extracellular matrix,as well as a corresponding cognate receptor,integrin $$(v)$$(3),are highly expressed in a subset of BLBC cell lines as well as in CSC-enriched populations. Furthermore,we demonstrated that an intact periostin-integrin $$3 signaling axis is required for the maintenance of breast CSCs. POSTN activates the ERK signaling pathway and regulates NF-$$B-mediated transcription of key cytokines,namely IL6 and IL8,which in turn control downstream activation of STAT3. In summary,these findings suggest that BLBC cells have an innate ability to establish a microenvironmental niche supportive of CSCs. IMPLICATIONS The findings reported here indicate that POSTN produced by CSCs acts to reinforce the stem cell state through the activation of integrin receptors and the production of key cytokines. View Publication

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However,the molecular mechanisms of this process remain elusive. In this study,we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3,which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells,which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation,and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus,this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3. View Publication

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells,addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC),we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population,which,despite its clonality,comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC,capable of repopulating secondary liquid cultures of CRA assays,confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle,in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However,quiescent LSC were also detected,capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC,believed to sustain minimal residual disease in vivo,were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines,which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results,we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age,77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600,etoposide 100) and 8A (tipifarnib 400,etoposide 200). In vivo,tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as NCT00112853. View Publication

The metabolism of oxygen,although central to life,produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer,cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny,and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably,subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing,CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that,similar to normal tissue stem cells,subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny,which may contribute to tumour radioresistance. View Publication

The cancer stem cell hypothesis asserts that malignancies arise in tissue stem and/or progenitor cells through the dysregulation or acquisition of self-renewal. In order to determine whether the dietary polyphenols,curcumin,and piperine are able to modulate the self-renewal of normal and malignant breast stem cells,we examined the effects of these compounds on mammosphere formation,expression of the breast stem cell marker aldehyde dehydrogenase (ALDH),and Wnt signaling. Mammosphere formation assays were performed after curcumin,piperine,and control treatment in unsorted normal breast epithelial cells and normal stem and early progenitor cells,selected by ALDH positivity. Wnt signaling was examined using a Topflash assay. Both curcumin and piperine inhibited mammosphere formation,serial passaging,and percent of ALDH+ cells by 50% at 5 microM and completely at 10 microM concentration in normal and malignant breast cells. There was no effect on cellular differentiation. Wnt signaling was inhibited by both curcumin and piperine by 50% at 5 microM and completely at 10 microM. Curcumin and piperine separately,and in combination,inhibit breast stem cell self-renewal but do not cause toxicity to differentiated cells. These compounds could be potential cancer preventive agents. Mammosphere formation assays may be a quantifiable biomarker to assess cancer preventive agent efficacy and Wnt signaling assessment can be a mechanistic biomarker for use in human clinical trials. View Publication

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs,we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently,the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated,stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast,ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results. View Publication